Anti-FRα antibodies and antibody-drug conjugates thereof and uses thereof

Abstract

The present invention discloses anti-FRα antibodies and antibody-drug conjugates thereof, as well as the application of the anti-FRα antibodies and antibody-drug conjugates in the treatment of diseases associated with FRα expression.

Description

[Technical field]

[0001] The present invention relates to an anti-FRα antibody, an antibody-drug conjugate comprising the anti-FRα antibody, and uses thereof. [Background technology]

[0002] Folate is an essential vitamin required for DNA synthesis and repair, and cell division processes. By binding to the folate receptor on the cell surface, folate is transported by endocytosis, and the folate receptor is internalized and then recycled back to the cell membrane. The folate receptor is a transmembrane single-chain glycoprotein linked to glycosylated phosphatidylinositol and has a high affinity for folate. Folate receptors include three subtypes, namely folate receptor α (FOLR1, FRα), folate receptor β (FOLR2), and folate receptor γ (FOLR3). The expression of folate receptors is very limited in normal cells, but is significantly overexpressed in tumor cells, of which folate receptor α is overexpressed in various malignant tumors, making it one of the targets of interest for anticancer drugs. Summary of the Invention

[0003] The present invention provides antibodies that specifically recognize and bind to folate receptor alpha (also referred to as "anti-folate receptor alpha antibodies" or "anti-FRα antibodies"). In one or more embodiments, the antibodies of the present invention can recognize and bind to human folate receptor alpha.

[0004] In one or more embodiments, the present invention provides an antibody, or an antigen-binding unit thereof, that specifically binds to the folate receptor alpha and (a) a VH CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:5; (b) a VH CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; (c) a VH CDR3 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 7; (d) a VL CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8; (e) a VL CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:9; (f) a VL CDR3 comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:10.

[0005] In one or more embodiments, the antibody or antigen binding unit thereof specifically binds to the folate receptor alpha and (a) a VH CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:5; (b) a VH CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; (c) a VH CDR3 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:7.

[0006] In one or more embodiments, the antibody or antigen binding unit thereof comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR2 as set forth in SEQ ID NO:6, and a VH CDR3 as set forth in SEQ ID NO:7.

[0007] In one or more embodiments, the antibody or antigen binding unit thereof specifically binds to the folate receptor alpha and (d) a VL CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8; (e) a VL CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:9; (f) a VL CDR3 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:10.

[0008] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VL CDR1 as set forth in SEQ ID NO:8, a VL CDR2 as set forth in SEQ ID NO:9, and a VL CDR3 as set forth in SEQ ID NO:10.

[0009] In one or more embodiments, the antibody or antigen binding unit thereof specifically binds to the folate receptor alpha and (a) a VH CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:5; (b) a VH CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:6; (c) a VH CDR3 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:7; (d) a VL CDR1 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:8; (e) a VL CDR2 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:9; (f) a VL CDR3 comprising, or alternatively consisting of, the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:10.

[0010] In one or more embodiments, the substitutions, deletions or insertions at the above sites are independently one, two or three.

[0011] In one or more embodiments, the antibody or antigen binding unit thereof comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR2 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:7, a VL CDR1 as set forth in SEQ ID NO:8, a VL CDR2 as set forth in SEQ ID NO:9, and a VL CDR3 as set forth in SEQ ID NO:10.

[0012] In one or more embodiments, the antibody or antigen-binding unit thereof comprises: (g) a VH FR1 comprising an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:29, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:29, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:29; (h) a VH FR2 comprising an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30; (i) a VH FR3 comprising an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 31, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 31; (j) a VH FR4 comprising an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 11; (k) a VL FR1 comprising an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 12; (l) a VL FR2 comprising an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 13; (m) a VL FR3 comprising an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 32, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 32, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 32; (n) a VL FR4 comprising an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 14.

[0013] In one or more embodiments, the antibody or antigen binding unit thereof specifically binds to the folate receptor alpha and (g) a VH FR1 comprising an amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:29, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:29, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:29; (h) a VH FR2 comprising an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:30; (i) a VH FR3 comprising an amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:31, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:31, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:31; (j) a VH FR4 comprising an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 11, and / or (k) a VL FR1 comprising an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 12; (l) a VL FR2 comprising an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 13; (m) a VL FR3 comprising an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:32, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:32, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:32; (n) a VL FR4 comprising an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 14.

[0014] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VH FR1 set forth in SEQ ID NO:1, a VH FR2 set forth in SEQ ID NO:2, a VH FR3 set forth in SEQ ID NO:3, and a VH FR4 set forth in SEQ ID NO:11.

[0015] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VL FR1 set forth in SEQ ID NO: 12, a VL FR2 set forth in SEQ ID NO: 13, a VL FR3 set forth in SEQ ID NO: 4, and a VL FR4 set forth in SEQ ID NO: 14.

[0016] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VH FR1 set forth in SEQ ID NO:1, a VH FR2 set forth in SEQ ID NO:2, a VH FR3 set forth in SEQ ID NO:3, a VH FR4 set forth in SEQ ID NO:11, a VL FR1 set forth in SEQ ID NO:12, a VL FR2 set forth in SEQ ID NO:13, a VL FR3 set forth in SEQ ID NO:4, and a VL FR4 set forth in SEQ ID NO:14.

[0017] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VH FR1 set forth in SEQ ID NO:29, a VH FR2 set forth in SEQ ID NO:30, a VH FR3 set forth in SEQ ID NO:31, and a VH FR4 set forth in SEQ ID NO:11.

[0018] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VL FR1 set forth in SEQ ID NO: 12, a VL FR2 set forth in SEQ ID NO: 13, a VL FR3 set forth in SEQ ID NO: 32, and a VL FR4 set forth in SEQ ID NO: 14.

[0019] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a VH FR1 set forth in SEQ ID NO:29, a VH FR2 set forth in SEQ ID NO:30, a VH FR3 set forth in SEQ ID NO:31, a VH FR4 set forth in SEQ ID NO:11, a VL FR1 set forth in SEQ ID NO:12, a VL FR2 set forth in SEQ ID NO:13, a VL FR3 set forth in SEQ ID NO:32, and a VL FR4 set forth in SEQ ID NO:14.

[0020] In one or more embodiments, the present invention provides an antibody or antigen-binding unit thereof that specifically binds to the folate receptor alpha and comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 15; and / or The light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:16.

[0021] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a heavy chain variable region set forth in SEQ ID NO:15 and a light chain variable region set forth in SEQ ID NO:16.

[0022] In one or more embodiments, the antibody or antigen-binding unit thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In one or more embodiments, the light chain constant region is a kappa or lambda chain constant region. In one or more embodiments, the antibody or antigen-binding unit thereof is of one of the following isotypes: IgG, IgM, IgA, IgE, or IgD. In one or more embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In one or more embodiments, the antibody or antigen-binding unit thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0023] In one or more embodiments, the antibody or antigen-binding unit thereof is an scFV, Fab, Fab', or F(ab)2. In one or more embodiments, the antibody or antigen-binding unit thereof is a monoclonal antibody.

[0024] In one or more embodiments, the antibody or antigen-binding unit thereof comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), of which: the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 17; and / or The light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO:18, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:18, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:18.

[0025] In one or more embodiments, the antibody or antigen binding unit thereof comprises a heavy chain constant region set forth in SEQ ID NO:17 and a light chain constant region set forth in SEQ ID NO:18.

[0026] In one or more embodiments, the present invention provides an antibody that specifically binds to folate receptor alpha, wherein the heavy chain of the antibody comprises a heavy chain variable region set forth in SEQ ID NO: 15 and a heavy chain constant region set forth in SEQ ID NO: 17, and the light chain of the antibody comprises a light chain variable region set forth in SEQ ID NO: 16 and a light chain constant region set forth in SEQ ID NO: 18.

[0027] In one or more embodiments, the present invention provides an antibody that specifically binds to the folate receptor alpha and comprises a heavy chain (H) and a light chain (L), wherein: the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence which has substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO: 19; and / or The light chain comprises an amino acid sequence as set forth in SEQ ID NO:20, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in SEQ ID NO:20, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence as set forth in SEQ ID NO:20.

[0028] In one or more embodiments, the antibody comprises a heavy chain set forth in SEQ ID NO:19 and a light chain set forth in SEQ ID NO:20.

[0029] In one or more embodiments, the substitutions are conservative amino acid substitutions.

[0030] In one or more embodiments, the antibody or antigen-binding unit thereof is chimeric, humanized or fully human.

[0031] In one or more embodiments, the antibody or antigen-binding unit thereof is a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody or antigen-binding unit thereof (e.g., a bispecific antibody or antigen-binding unit thereof).

[0032] In one or more embodiments, the antibody has two heavy chains with identical sequences and two light chains with identical sequences. In one or more embodiments, the Fc region pairs to form a disulfide bond. In one or more embodiments, the antibody or antigen-binding unit thereof of the present invention is an isolated antibody or antigen-binding unit thereof.

[0033] In one or more embodiments, the antibody or antigen-binding unit thereof of the present invention is a monoclonal antibody or a fragment thereof.

[0034] In one or more embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: (1) a polynucleotide encoding an antibody or an antigen-binding unit thereof according to the present invention, or a part thereof; (2) an expression vector comprising a polynucleotide encoding the antibody or antigen-binding unit thereof according to the present invention; or (3) We further provide a biological material, which is a cell comprising one or more polynucleotides encoding an antibody or an antigen-binding unit thereof according to the present invention.

[0035] In one or more embodiments, the present invention further provides pharmaceutical compositions comprising the antibodies or antigen-binding units thereof provided by the present invention. These compositions may be included in a reagent kit, such as a diagnostic reagent kit.

[0036] In one or more embodiments, the invention further provides a method of alleviating or treating cancer or other conditions by administering to a patient in need thereof one or more antibodies or antigen binding units thereof that bind to the folate receptor alpha. The amount of antibody administered should be sufficient to alleviate or treat the cancer or other condition in the patient. In one or more embodiments, the patient is a human.

[0037] In one or more embodiments, the anti-FRα antibody according to the present invention is used in combination with one or more other therapies. Suitable other therapies include existing drugs, radiation therapy and / or surgical therapy used in a particular application (e.g., cancer). For example, the anti-FRα antibody is used in combination with one or more other chemotherapy or antitumor reagents. Alternatively, the other chemotherapy is radiation therapy. In one or more embodiments, the chemotherapy agent is a cell death inducer.

[0038] In one or more embodiments, the anti-FRα antibody and other reagents are prepared in a single therapeutic composition, and the anti-FRα antibody and other reagents are administered simultaneously. Alternatively, the anti-FRα antibody and other reagents are prepared independently of each other, for example, as separate therapeutic compositions, and the anti-FRα antibody and other reagents are administered simultaneously, or the anti-FRα antibody and other reagents are administered at different times during a therapeutic regimen. For example, the anti-FRα antibody is administered before the other reagent, the anti-FRα antibody is administered after the other reagent, or the anti-FRα antibody and other reagents are administered alternately. As used herein, the anti-FRα antibody and other reagents are administered in a single dose or multiple doses.

[0039] In one or more embodiments, the present invention further provides an antibody drug conjugate (ADC) comprising an antibody or an antigen-binding unit thereof according to the present invention, or a pharma- ceutically acceptable salt or solvate thereof, coupled to a drug via a linker.

[0040] In one or more embodiments, the linker is a degradable linker.

[0041] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0042] In one or more embodiments, the drug is an anti-cancer drug.

[0043] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0044] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatin, and maytansine.

[0045] In one or more embodiments, the drug is an auristatin and is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or auristatin F (AF).

[0046] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, or anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0047] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, exatecan derivatives, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivatives SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-( 3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0048] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0049] In one or more embodiments, the drug has an amino group or the amino group is substituted with an alkyl group and is linked to the linker via an amide bond.

[0050] In one or more embodiments, the drug is [ka] or its stereoisomers, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q-CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to the linker, y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0051] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0052] In one or more embodiments, the heterocyclyl is azetidine, niverazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0053] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0054] In one or more embodiments, the drug is [ka] or its stereoisomers, among which X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is linked to the linker.

[0055] In one or more embodiments, the drug is [ka] or its stereoisomers, among which X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is linked to the linker.

[0056] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0057] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0058] In one or more embodiments, X 1 and X 2 are F, respectively.

[0059] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0060] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0061] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0062] In one or more embodiments, the antibody drug conjugate provided by the present invention has the structure shown in Formula I, or a stereoisomer thereof, or a pharma- ceutically acceptable salt, or solvate thereof: [ka] Among them Abu is an antibody or an antigen-binding unit thereof that specifically binds to the folate receptor α, D is a drug, M is [ka] where * is linked to Abu, ** is linked to B, and R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -Arylene-(CH2)r-, -(CH2) r -(C3~C8 carbocyclyl)-, -(C3~C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O)r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which, each R m is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; B is [ka] For example, [ka] where * is linked to M, ** is linked to L, and *** is linked to G; L is -(AA) i -(FF) f -, wherein AA is an amino acid or a polypeptide, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and each FF is independently [ka] Of these, each R F are independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, z is 0, 1, 2, 3 or 4, f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in which * is linked to AA and ** is linked to D; G is [ka] wherein n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0063] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0064] In one or more embodiments, the drug is an anti-cancer drug.

[0065] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0066] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0067] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0068] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0069] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, exatecan derivatives, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3 ,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0070] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0071] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or a chlorambucil derivative.

[0072] In one or more embodiments, the drug has an amino group or the amino group is substituted with an alkyl group and is linked to FF via an amide bond.

[0073] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NRn (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is concatenated to L, y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0074] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0075] In one or more embodiments, the heterocyclyl is azetidine, niverazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0076] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0077] In one or more embodiments, the drug is [ka] Of these, X 1and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is linked to L.

[0078] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is linked to L.

[0079] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0080] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0081] In one or more embodiments, X 1 and X 2 are F, respectively.

[0082] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0083] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0084] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0085] In one or more embodiments, R is -(CH) r -It is.

[0086] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0087] In one or more embodiments, each AA is independently selected from the amino acid or peptide sequences Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.

[0088] In one or more embodiments, AA is Val-Cit and i is 1.

[0089] In one or more embodiments, each FF independently [ka] Of these, each R F are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO2, or a halogen, in which * is linked to AA and ** is linked to D.

[0090] In one or more embodiments, the halogen is F and z is 0, 1, 2, 3 or 4.

[0091] In one or more embodiments, R F is -CH3, F, -NO2 or -OCH3.

[0092] In one or more embodiments, z is 0.

[0093] In one or more embodiments, z is 1 or 2.

[0094] In one or more embodiments, each FF independently [ka] where * is linked to AA and ** is linked to D.

[0095] In one or more embodiments, f is 1.

[0096] In one or more embodiments, FF is [ka] where f is 1, in which * is connected to AA and ** is connected to D.

[0097] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0098] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0099] In one or more embodiments, G is [ka] where n is 4 to 12.

[0100] In one or more embodiments, n is 4-8.

[0101] In one or more embodiments, n is 4.

[0102] In one or more embodiments, n is 8.

[0103] In one or more embodiments, p is 2-8.

[0104] In one or more embodiments, p is 4-8.

[0105] In one or more embodiments, p is 6-8.

[0106] In one or more embodiments, p is 7-8.

[0107] In one or more embodiments, p is 7.

[0108] In one or more embodiments, p is 7.4.

[0109] In one or more embodiments, p is 8.

[0110] In one or more embodiments, the Abu is an antibody or an antigen-binding unit thereof provided by the present invention.

[0111] In one or more embodiments, the antibody drug conjugate has the structure shown in I-1, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, R is -(CH2) r -, -(CHR m )r-, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -Arylene-(CH2)r-, -(CH2) r -(C3~C8 carbocyclyl)-, -(C3~C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r-(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which, each R m is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; D is a drug, n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0112] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0113] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0114] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0115] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0116] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0117] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydropyranos ... 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0118] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0119] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, a doxorubicin, a methotrexate, a vincristine, a vinblastine, a daunorubicin, a mitomycin C, a melphalan, or a chlorambucil derivative.

[0120] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q-CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to another moiety of the antibody drug conjugate; y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0121] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0122] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0123] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0124] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0125] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0126] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0127] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0128] In one or more embodiments, X 1 and X 2 are F, respectively.

[0129] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0130] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0131] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0132] In one or more embodiments, R is -(CH) r -It is.

[0133] In one or more embodiments, R is -(CH) r- and r is 1 or 5.

[0134] In one or more embodiments, n is 4-12.

[0135] In one or more embodiments, n is 4-8.

[0136] In one or more embodiments, n is 4.

[0137] In one or more embodiments, n is 8.

[0138] In one or more embodiments, p is 2-8.

[0139] In one or more embodiments, p is 4-8.

[0140] In one or more embodiments, p is 6-8.

[0141] In one or more embodiments, p is 7-8.

[0142] In one or more embodiments, p is 7.

[0143] In one or more embodiments, p is 7.4.

[0144] In one or more embodiments, p is 8.

[0145] In one or more embodiments, the antibody drug conjugate has the structure shown in formula I-2 or I-2-1, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, R is -(CH2) r -, -(CHRm )r-, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -Arylene-(CH2)r-, -(CH2) r -(C3~C8 carbocyclyl)-, -(C3~C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which, each R m is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; D is a drug, n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0146] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0147] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0148] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0149] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0150] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0151] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydropyranos ... 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0152] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0153] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, a doxorubicin, a methotrexate, a vincristine, a vinblastine, a daunorubicin, a mitomycin C, a melphalan, or a chlorambucil derivative.

[0154] In one or more embodiments, the drug is [ka] Of which, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q-CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to another moiety of the antibody drug conjugate; y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0155] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0156] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0157] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0158] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0159] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0160] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0161] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0162] In one or more embodiments, X 1 and X 2 are F, respectively.

[0163] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0164] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0165] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0166] In one or more embodiments, R is -(CH) r -It is.

[0167] In one or more embodiments, R is -(CH) r- and r is 1 or 5.

[0168] In one or more embodiments, n is 4-12.

[0169] In one or more embodiments, n is 4-8.

[0170] In one or more embodiments, n is 4.

[0171] In one or more embodiments, n is 8.

[0172] In one or more embodiments, p is 2-8.

[0173] In one or more embodiments, p is 4-8.

[0174] In one or more embodiments, p is 6-8.

[0175] In one or more embodiments, p is 7-8.

[0176] In one or more embodiments, p is 7.

[0177] In one or more embodiments, p is 7.4.

[0178] In one or more embodiments, p is 8.

[0179] In one or more embodiments, the antibody drug conjugate has the structure shown in formula I-3, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, D is a drug, n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0180] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0181] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0182] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0183] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0184] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0185] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydropyranos ... 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0186] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0187] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, a doxorubicin, a methotrexate, a vincristine, a vinblastine, a daunorubicin, a mitomycin C, a melphalan, or a chlorambucil derivative.

[0188] In one or more embodiments, the drug is [ka] Of which, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q-CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to another moiety of the antibody drug conjugate; y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0189] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0190] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0191] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0192] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0193] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0194] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0195] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0196] In one or more embodiments, X 1 and X 2 are F, respectively.

[0197] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0198] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0199] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0200] In one or more embodiments, n is 4-12.

[0201] In one or more embodiments, n is 4-8.

[0202] In one or more embodiments, n is 4.

[0203] In one or more embodiments, n is 8.

[0204] In one or more embodiments, p is 2-8.

[0205] In one or more embodiments, p is 4-8.

[0206] In one or more embodiments, p is 6-8.

[0207] In one or more embodiments, p is 7-8.

[0208] In one or more embodiments, p is 7.

[0209] In one or more embodiments, p is 7.4.

[0210] In one or more embodiments, p is 8.

[0211] In one or more embodiments, the antibody drug conjugate has the structure shown in formula I-4 or I-4-1, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, D is a drug, n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0212] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0213] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0214] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0215] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0216] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, anthramycin derivatives PBD (pyrrolobenzodiazepine), or a DNA topoisomerase inhibitor.

[0217] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydropyranos ... 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0218] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0219] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, a doxorubicin, a methotrexate, a vincristine, a vinblastine, a daunorubicin, a mitomycin C, a melphalan, or a chlorambucil derivative.

[0220] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q-CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to another moiety of the antibody drug conjugate; y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0221] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0222] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0223] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0224] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0225] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0226] In one or more embodiments, X 1 and X 2 are each independently -CH, F, Cl, Br or I.

[0227] In one or more embodiments, X 1 and X 2 are F, respectively.

[0228] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0229] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0230] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0231] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0232] In one or more embodiments, n is 4-12.

[0233] In one or more embodiments, n is 4-8.

[0234] In one or more embodiments, n is 4.

[0235] In one or more embodiments, n is 8.

[0236] In one or more embodiments, p is 2-8.

[0237] In one or more embodiments, p is 4-8.

[0238] In one or more embodiments, p is 6-8.

[0239] In one or more embodiments, p is 7-8.

[0240] In one or more embodiments, p is 7.

[0241] In one or more embodiments, p is 7.4.

[0242] In one or more embodiments, p is 8.

[0243] In one or more embodiments, the antibody drug conjugate has the structure shown in formula I-5, I-5-1, I-6, I-6-1, I-7, I-7-1, I-8, I-8-1, I-9, I-9-1, I-10, I-10-1, I-11, or I-11-1, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: [ka] [ka] [ka] [ka] [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, D is a drug, p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0244] In one or more embodiments, the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0245] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0246] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0247] In one or more embodiments, the drug is an auristatin, such as MMAE, MMAF, or AF.

[0248] In one or more embodiments, the drug is a DNA damaging agent, such as the calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0249] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydropyranos ... 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0250] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatin, or exatecan.

[0251] In one or more embodiments, the drug is a Tubulysin, a taxane derivative, a leptomycine derivative, CC-1065 and its analogs, an Amatoxin, a spliceosome inhibitor, a benzodiazepine (PBD) dimer, a doxorubicin, a methotrexate, a vincristine, a vinblastine, a daunorubicin, a mitomycin C, a melphalan, or a chlorambucil derivative.

[0252] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently H, Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, Nitro group, Cyano group, Sulfhydryl groups, Alkylthio groups, an amino group, an amino group substituted by an amino-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino moiety by an amino-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino moiety with an amino-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q-CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is linked to another moiety of the antibody drug conjugate; y is 0, 1 or 2; Y is O, S or CR 1 R 2 Of these, R 1 and R 2 are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not simultaneously 0.

[0253] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0254] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0255] In one or more embodiments, the amino-protecting group is a formyl group, an acetyl group, a trityl group, a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyloxycarbonyl group.

[0256] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0257] In one or more embodiments, the drug is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen, or -OH, and ** is linked to other moieties of the antibody drug conjugate.

[0258] In one or more embodiments, the C1-C6 alkyl group is -CH3.

[0259] In one or more embodiments, the halogen is F.

[0260] In one or more embodiments, X 1 and X 2 are each independently -CH3, F, or -OH.

[0261] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0262] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0263] In one or more embodiments, X 1 and X 2 are F, respectively.

[0264] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0265] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0266] In one or more embodiments, p is 2-8.

[0267] In one or more embodiments, p is 4-8.

[0268] In one or more embodiments, p is 6-8.

[0269] In one or more embodiments, p is 7-8.

[0270] In one or more embodiments, p is 7.

[0271] In one or more embodiments, p is 7.4.

[0272] In one or more embodiments, p is 8.

[0273] In one or more embodiments, the antibody drug conjugate has the structure shown in formula I-12, I-12-1, I-13, I-13-1, I-14, I-14-1, I-15, I-15-1, I-16, I-16-1, I-17, I-17-1, I-18, I-18-1, I-19, I-19-1, I-20, I-20-1, I-21, I-21-1, I-22, I-22-1, I-23, I-23-1, I-24, I-24-1, I-25, or I-25-1, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof; [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Among them Abu is an antibody or an antigen-binding unit thereof provided by the present invention, p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9 or 10.

[0274] In one or more embodiments, p is 2-8.

[0275] In one or more embodiments, p is 4-8.

[0276] In one or more embodiments, p is 6-8.

[0277] In one or more embodiments, p is 7-8.

[0278] In one or more embodiments, p is 7.

[0279] In one or more embodiments, p is 7.4.

[0280] In one or more embodiments, p is 8.

[0281] In one or more embodiments, an antibody drug conjugate for use as a drug is provided. In one or more embodiments, the DAR(p) of the antibody drug conjugate in the drug is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one or more embodiments, the average DAR(p) of the antibody drug conjugate in the drug is 2-9. In one or more embodiments, the average DAR(p) of the antibody drug conjugate in the drug is 4-9. In one or more embodiments, the average DAR(p) of the antibody drug conjugate in the drug is 6-8.5. In one or more embodiments, the average DAR(p) of the antibody drug conjugate in the drug is 6-8. In one or more embodiments, the average DAR(p) of the antibody drug conjugate in the drug is 7-8.

[0282] In one or more embodiments, the antibody drug conjugate of the present invention is used in combination with one or more other therapies. Suitable other therapies include existing drugs and / or surgical therapies used in specific applications (e.g., cancer). For example, the antibody drug conjugate is used in combination with one or more other chemotherapeutic agents or antitumor reagents. Or, the other chemotherapeutic agent is radiation therapy. In one or more embodiments, the chemotherapeutic agent is a cell death inducer.

[0283] In one or more embodiments, the antibody drug conjugate and other reagents are prepared as a single therapeutic composition, and the antibody drug conjugate and other reagents are administered simultaneously. Alternatively, the antibody drug conjugate and other reagents are prepared independently of each other, for example, as separate therapeutic compositions, and the antibody drug conjugate and other reagents are administered simultaneously, or the antibody drug conjugate and other reagents are administered at different times during a therapeutic regimen. For example, the antibody drug conjugate is administered before the other reagents, the antibody drug conjugate is administered after the other reagents, or the antibody drug conjugate and other reagents are administered alternately. Herein, the antibody drug conjugate and other reagents are administered in a single dose or multiple doses.

[0284] In one or more embodiments, the present invention further provides a pharmaceutical composition comprising the antibody or antigen-binding unit thereof, or antibody-drug conjugate according to the present invention, and a pharma- ceutical acceptable carrier, excipient and / or additive. In one or more embodiments, the pharmaceutical composition optionally further comprises another anti-cancer drug. The pharmaceutical composition provided by the present invention may be administered by any convenient route, for example by infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Thus, the pharmaceutical composition may be administered intravenously, orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drop or transdermal patch), buccally, or orally or by nasal spray.

[0285] In one or more embodiments, the present invention provides the use of at least one of the above-mentioned antibody or antigen-binding unit thereof, biomaterial (selected from polynucleotide, expression vector, and cell), antibody-drug conjugate, and pharmaceutical composition in the manufacture of a drug for treating and / or preventing a disease. In one or more embodiments, the above-mentioned disease is a disease associated with folate receptor alpha expression. In one or more embodiments, the above-mentioned disease is a disease associated with folate receptor alpha overexpression. In one or more embodiments, the above-mentioned disease is a tumor expressing folate receptor alpha. In one or more embodiments, the above-mentioned disease is a tumor overexpressing folate receptor alpha. In one or more embodiments, the above-mentioned disease is a cancer expressing folate receptor alpha. In one or more embodiments, the above-mentioned disease is a cancer overexpressing folate receptor alpha. In one or more embodiments, the above-mentioned drug for treating a tumor further comprises another anticancer drug.

[0286] In one or more embodiments, the present invention provides the application of the antibody or antigen-binding unit thereof, biomaterial (selected from polynucleotide, expression vector, and cell), antibody-drug conjugate, and / or pharmaceutical composition comprising them described herein in the treatment and / or prevention of a disease. In one or more embodiments, the disease is a disease associated with folate receptor alpha expression. In one or more embodiments, the disease is a disease associated with folate receptor alpha overexpression. In one or more embodiments, the disease is a tumor expressing folate receptor alpha. In one or more embodiments, the disease is a tumor overexpressing folate receptor alpha. In one or more embodiments, the disease is a cancer expressing folate receptor alpha. In one or more embodiments, the disease is a cancer overexpressing folate receptor alpha. In one or more embodiments, the disease is a cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0287] In one or more embodiments, a method for treating and / or preventing a disease is provided, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding unit thereof, biomaterial (selected from a polynucleotide, an expression vector, and a cell), antibody-drug conjugate, and / or pharmaceutical composition comprising the same described herein. In one or more embodiments, the disease is a disease associated with folate receptor alpha expression. In one or more embodiments, the disease is a disease associated with folate receptor alpha overexpression. In one or more embodiments, the disease is a tumor expressing folate receptor alpha. In one or more embodiments, the disease is a tumor overexpressing folate receptor alpha. In one or more embodiments, the disease is a cancer expressing folate receptor alpha. In one or more embodiments, the disease is a cancer overexpressing folate receptor alpha. In one or more embodiments, the effective amount refers to the amount of active compound or drug that produces the biological or pharmacological response in tissues, systems, animals, individuals and humans that is desired by a researcher, veterinarian, physician or other clinician, including the treatment of a disease.

[0288] Examples of cancer include, but are not limited to, solid tumors, hematological cancers, and metastatic lesions. Specific examples of such cancers include, but are not limited to, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, liver cancer, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, testicular cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer.

[0289] In one or more embodiments, the antibody or antigen-binding unit thereof, or antibody-drug conjugate may be prepared as a pharmaceutical composition and administered to a patient in a form suitable for a selected route of administration, such as parenteral, intravenous (iv), intramuscular, topical, or subcutaneous.

[0290] In one or more embodiments, the dosage of the antibody or antigen-binding unit thereof, or antibody-drug conjugate (e.g., a single dose) is 1 mg / kg to 10 mg / kg, or 1.2 mg / kg to 8.0 mg / kg, or 1.2 mg / kg to 6.5 mg / kg, or 1.2 mg / kg to 5.0 mg / kg, or 1.2 mg / kg to 3.5 mg / kg, or 1.2 mg / kg to 2.4 mg / kg, or 2.4 mg / kg to 8.0 mg / kg, or 2.4 mg / kg to 6.5 mg / kg, or 2.4 mg / kg to 5.0 mg / kg, or 2.4 mg / kg to 3.5 mg / kg, or 3.5 mg / kg to 8.0 mg / kg, or 3.5 mg / kg to 6. .5mg / kg, or 3.5mg / kg to 5.0mg / kg, or 5.0mg / kg to 8.0mg / kg, or 5.0mg / kg to 6.5mg / kg, or 6.5mg / kg to 8.0mg / kg, or 1.5mg / kg to 8.5mg / kg, or 1.5mg / kg to 6.5mg / kg, or 1.5mg / kg to 4.5mg / kg, or 1.5mg / kg to 3.0mg / kg, or 3.0mg / kg to 8.5mg / kg, or 3.0mg / kg to 6.5mg / kg, or 3.0mg / kg to 4.5mg / kg, or 4.5mg / kg to 8.5mg / kg, or 4.5mg / kg to 6.5mg / kg, or 6.5mg / kg to 8.5mg / kg. In one or more embodiments, the dosage of the antibody or antigen-binding unit thereof, or antibody-drug conjugate (e.g., a single dose) is about 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, about 2.0 mg / kg, 2.4 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 6.5 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 10 mg / kg, or a range between any two of these values ​​(inclusive), or any value therein.

[0291] In one or more embodiments, the single dose of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is 1 mg / kg to 10 mg / kg, or 1.2 mg / kg to 8.0 mg / kg, or 1.2 mg / kg to 6.5 mg / kg, or 1.2 mg / kg to 5.0 mg / kg, or 1.2 mg / kg to 3.5 mg / kg, or 1.2 mg / kg to 2.4 mg / kg, or 2.4 mg / kg to 8.0 mg / kg, or 2.4 mg / kg to 6.5 mg / kg, or 2.4 mg / kg to 5.0 mg / kg, or 2.4 mg / kg to 3.5 mg / kg, or 3.5 mg / kg to 8.0 mg / kg, or 3.5 mg / kg to 6.5 mg / kg. kg, or 3.5 mg / kg to 5.0 mg / kg, or 5.0 mg / kg to 8.0 mg / kg, or 5.0 mg / kg to 6.5 mg / kg, or 6.5 mg / kg to 8.0 mg / kg, or 1.5 mg / kg to 8.5 mg / kg, or 1.5 mg / kg to 6.5 mg / kg, or 1.5 mg / kg to 4.5 mg / kg, or 1.5 mg / kg to 3.0 mg / kg, or 3.0 mg / kg to 8.5 mg / kg, or 3.0 mg / kg to 6.5 mg / kg, or 3.0 mg / kg to 4.5 mg / kg, or 4.5 mg / kg to 8.5 mg / kg, or 4.5 mg / kg to 6.5 mg / kg, or 6.5 mg / kg to 8.5 mg / kg. In one or more embodiments, the single dose of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is about 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, about 2.0 mg / kg, 2.4 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 6.5 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 10 mg / kg, or a range between any two of these values ​​(inclusive), or any value therein.

[0292] In one or more embodiments, the dosage of the antibody or antigen-binding unit thereof, or antibody-drug conjugate (e.g., a single dose) is 50 mg to 1000 mg, or 50 mg to 600 mg, or 60 mg to 600 mg, or 100 mg to 600 mg, or 72 mg to 480 mg, or 72 mg to 390 mg, or 72 mg to 300 mg, or 72 mg to 210 mg, or 72 mg to 144 mg, or 144 mg to 480 mg, or 144 mg to 390 mg. 0mg, or 144mg to 300mg, or 144mg to 210mg, or 210mg to 480mg, or 210mg to 390mg, or 210mg to 300mg, or 300mg to 480mg, or 300mg to 390mg, or 390mg to 480mg, or 84mg to 560mg, or 84mg to 455mg, or 84mg to 350mg, or 84mg to 245mg, or 84mg to 168mg, or 168mg to 560mg, or 168mg to 4 55mg, or 168mg to 350mg, or 168mg to 245mg, or 245mg to 560mg, or 245mg to 455mg, or 245mg to 350mg, or 350mg to 560mg, or 350mg to 455mg, or 455mg to 560mg, or 90mg to 595mg, or 90mg to 510mg, or 90mg to 390mg, or 90mg to 270mg, or 90mg to 180mg, or 180mg to 510mg, or 180mg to 390mg, or 180mg to 270mg, or 270mg to 510mg, or 270mg to 390mg, or 390mg to 510mg, or 105mg to 595mg, or 105mg to 455mg, or 105mg to 315mg, or 105mg to 210mg, or 210mg to 595mg, or 210mg to 455mg, or 210mg to 315mg, or 315mg to 595mg, or 315mg to 455mg, or 455mg to 595mg.In one or more embodiments, the dosage (e.g., a single dose) of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 72 mg, about 80 mg, about 84 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 144 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 168 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 245 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 80mg, about 290mg, about 300mg, about 310mg, about 315mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 3 80mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 455mg, about 460mg, about 470mg, about 4 80 mg, about 490 mg, about 500 mg, about 510 mg, about 550 mg, about 560 mg, about 595 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, or a range between any two of these values ​​(inclusive), or any value therein.

[0293] In one or more embodiments, the single dose of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is 50 mg to 1000 mg, or 50 mg to 600 mg, or 60 mg to 600 mg, or 100 mg to 600 mg, or 72 mg to 480 mg, or 72 mg to 390 mg, or 72 mg to 300 mg, or 72 mg to 210 mg, or 72 mg to 144 mg, or 144 mg to 480 mg, or 144 mg to 390 mg, or 1 44mg to 300mg, or 144mg to 210mg, or 210mg to 480mg, or 210mg to 390mg, or 210mg to 300mg, or 300mg to 480mg, or 300mg to 390mg, or 390mg to 480mg, or 84mg to 560mg, or 84mg to 455mg, or 84mg to 350mg, or 84mg to 245mg, or 84mg to 168mg, or 168mg to 560mg, or 168mg to 455mg, or 168mg to 350mg, or 168mg to 245mg, or 245mg to 560mg, or 245mg to 455mg, or 245mg to 350mg, or 350mg to 560mg, or 350mg to 455mg, or 455mg to 560mg, or 90mg to 595mg, or 90mg to 510mg, or 90mg to 390mg, or 90mg to 270mg, or 90mg to 180mg, or 180mg to 510mg, or 180mg to 390 mg, or 180mg to 270mg, or 270mg to 510mg, or 270mg to 390mg, or 390mg to 510mg, or 105mg to 595mg, or 105mg to 455mg, or 105mg to 315mg, or 105mg to 210mg, or 210mg to 595mg, or 210mg to 455mg, or 210mg to 315mg, or 315mg to 595mg, or 315mg to 455mg, or 455mg to 595mg.In one or more embodiments, the single dose of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 72 mg, about 84 mg, about 90 mg, about 100 mg, about 105 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 144 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 168 mg, about 170 mg, about 175 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 245 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about g, about 290 mg, about 300 mg, about 310 mg, about 315 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 455 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 595 mg, about 600 mg, or a range between any two of these values ​​(inclusive), or any value therein.

[0294] In one or more embodiments, the method includes at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 treatment periods. In one or more embodiments, a treatment period is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or at least 7 weeks. In one or more embodiments, a treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values ​​(inclusive), or any value therein.

[0295] In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate is administered once. In one or more embodiments, it is administered once every 2 days to once every 6 weeks. In one or more embodiments, it is administered twice a week, or once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks. In one or more embodiments, it is administered once every 2 days, once every 3 days, once every 4 days, once every 5 days, twice a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks.

[0296] In one or more embodiments, the patient is treated for one treatment session. In one or more embodiments, the patient is treated for multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) treatment sessions. In one or more embodiments, the patient is treated until the disease is palliated and no treatment is necessary.

[0297] In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by injection. In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by subcutaneous (sc) injection, intraperitoneal (ip) injection, parenteral injection, intraarterial injection, intravenous (iv) injection, etc. In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by infusion. In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by bolus injection. In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by intravenous injection. In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by intravenous infusion. The dosage of the antibody or antigen-binding unit thereof, or antibody-drug conjugate, will depend on the nature of the drug, the extent of cell surface induced internalization, transport and release of the drug, and the disease being treated and the condition of the patient (e.g., age, sex, weight, etc.).

[0298] In one or more embodiments, the antibody or antigen-binding unit thereof or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by intravenous (iv) infusion (i.e., intravenous infusion). In one or more embodiments, the duration of the intravenous infusion is about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 40 min, about 50 min, about 55 min, about 60 min, about 65 min, about 70 min, about 75 min, about 81 min, about 87 min, about 90 min, about 95 min, or a range between any two of these values ​​(inclusive), or any value therein. In one or more embodiments, the intravenous infusion time is ≦30 min. In one or more embodiments, the intravenous infusion time is ≧60 min. In one or more embodiments, the intravenous infusion time is ≧90 min.

[0299] In one or more embodiments, the present invention provides a pharmaceutical composition suitable for injection, for example, a bolus injection type pharmaceutical composition or drip (infusion) type pharmaceutical composition, comprising the antibody or antigen-binding unit thereof or antibody-drug conjugate. Pharmaceutical compositions suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water or phosphate buffered saline (PBS), ethanol, a solvent or dispersion medium of polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In one or more embodiments, the pharmaceutically acceptable carrier may be realized by including an antibacterial and / or antifungal agent, such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In one or more embodiments, the pharma- ceutically acceptable carrier may include an isotonicity agent, such as a sugar, a polyol (erythritol, sorbitol, etc.), sodium chloride. In one or more embodiments, the pharmaceutical composition comprises at least 0.1% of an antibody or an antigen-binding unit thereof, or an antibody-drug conjugate. The percentage of the antibody may vary and is between about 2% and 90% of the weight of a given dosage form. The amount of the antibody or an antigen-binding unit thereof, or an antibody-drug conjugate in such a pharmaceutical composition may be an effective amount for administration.

[0300] In one or more embodiments, the present invention provides a method for producing a pharmaceutical composition, comprising mixing an antibody or antigen-binding unit thereof, or an antibody-drug conjugate described herein, with a pharma- ceutically acceptable carrier (e.g., water for injection, saline, etc.), respectively. Methods for mixing the antibody or antigen-binding unit thereof, or antibody-drug conjugate with a pharma- ceutically acceptable carrier are well known in the art.

[0301] In one or more embodiments, the present invention provides a reagent kit comprising an antibody or antigen-binding unit thereof, antibody-drug conjugate, or a pharmaceutical composition comprising the same described herein, and instructions for administration to a patient.

[0302] In one or more embodiments, an anti-FRα antibody or an antigen-binding unit thereof, an antibody-drug conjugate, or a pharmaceutical composition comprising the same according to the present invention; A container; Further provided is an article of manufacture comprising an insert, instructions or label for indicating that the anti-FRα antibody, antibody-drug conjugate or pharmaceutical composition containing the same described in the present invention is used to treat and / or prevent diseases associated with folate receptor α expression.

[0303] Those skilled in the art will appreciate that the antibodies of the present invention have a variety of uses, for example, they can be used as therapeutic agents, as reagents or diagnostic tools in diagnostic reagent kits, or as reagents in competitive experiments to generate therapeutic agents. [Brief description of the drawings]

[0304] [Figure 1] A schematic diagram of ADC coupling is shown. [Diagram 2] 1 shows endocytosis of ADC1. [Diagram 3] The bystander effect of ADC1 is shown. [Figure 4] 1 shows the in vivo tumor inhibitory effect of ADC1. [Diagram 5] 1 shows the in vivo tumor inhibitory effect of ADC1. [Figure 6] The graph shows the growth curve (mean ± standard error) of mouse tumor volume in each group in the LU11554 xenograft model. [Figure 7] The graph shows the growth curve (mean ± standard error) of mouse tumor volume in each group in the LU5197 xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0305] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In general, the nomenclature and techniques used in cell culture, molecular biology, and protein purification described herein are those known and commonly used in the art. Standard techniques were used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's instructions or methods commonly used in the art or described herein. The techniques and methods described above are generally used as known in the art and described in several comprehensive and relatively specific publications referenced and described herein. See Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring, NY (1989)).

[0306] definition It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus the terms "a" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

[0307] As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. includes the recited elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that the antibody, composition, method, etc. excludes other elements that substantially affect the properties of the combination, but does not exclude elements that do not substantially affect the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically recited.

[0308] The term "antibody" as used herein refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "against" refers to an antibody that reacts with one or more antigenic determinants of the target antigen without reacting with other polypeptides, or with very low affinity (KD>10 -6 It means that the antibody binds to another polypeptide at a specific concentration (g / mL). Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab- and F(ab')2 fragments, Fv and Fab expression libraries.

[0309] The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that the heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), which further include several subclasses (e.g., γ1 to γ4). The nature of the chain determines the "type" of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively. For example, immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, have already been fully characterized and the functional specificities conferred by them are known. Any of the immunoglobulin types are within the scope of the claims disclosed in the present invention. In one or more embodiments, the type of immunoglobulin molecule is IgG. The two heavy chains and two light chains are linked in a "Y" configuration by disulfide bonds, in which the light chains start at the mouth of the "Y" and surround the heavy chains continuously through the variable region.

[0310] The antibodies, antigen-binding units or derivatives disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., class Fab, class Fab' and class F(ab')2), class single chain Fvs (scFv).

[0311] Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be bound to a κ or λ light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are bound via a covalent bond, and the "tails" of the two heavy chains are bound via a covalent disulfide bond or a non-covalent bond. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The κ light chain variable region of an immunoglobulin is V κ and the lambda light chain variable region of the immunoglobulin is V λ It is.

[0312] The terms "constant" and "variable" are used according to function. The light chain variable region (VL) and the heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and the heavy chain constant region (CH) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement fixation. By convention, the numbering of constant regions increases with increasing distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0313] In naturally occurring antibodies, assuming that the antibody exhibits its three-dimensional configuration in an aqueous environment, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous, antigen-specific binding amino acid sequences from which the antigen-binding domain is formed. The remaining other amino acids in the antigen-binding domain, called "framework" regions, show relatively little intermolecular variability. Most of the framework regions adopt a β-fold conformation and form a ring structure to which the CDRs are connected, or in some cases form part of a β-fold structure. Thus, the framework regions position the CDRs in the correct orientation with non-covalent interactions between the chains by forming a stent. The antigen-binding domain with the CDRs at specific positions forms a surface complementary to the antigen epitope that promotes non-covalent binding of the antibody to the antigen epitope. For a given heavy or light chain variable region, one of skill in the art can identify the amino acids that comprise the CDRs and framework regions by known methods (see Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). As used herein, the term "monoclonal antibody" (mAb) refers to a population of antibody molecules that includes only one type of molecule that is composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen.

[0314] The term "single chain antibody" (scFv) refers to an antibody in which the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody are linked by a linker of 15 to 20 amino acids. The linker may be rich in glycine to increase flexibility, or rich in serine or threonine to increase solubility, and may be linked to the N-terminus of VH and the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are generally known in the art, for example as described in U.S. Pat. No. 5,892,019.

[0315] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly differentiated branches of the heavy chain variable region (VH) and light chain variable region (VL) (called "hypervariable regions") are located between more conservative branches (called "framework regions" or "FR"). Thus, the term "FR" refers to the amino acid sequence of an immunoglobulin that naturally occurs between or adjacent to the hypervariable regions. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and all three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs". In order of position, the heavy chain variable region comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4. In order of position, the light chain variable region comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4. The alignment of the amino acids of each domain can be in accordance with the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health (1987 and 1991)) or the articles by Chothia and Lesk (J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989)).

[0316] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or shared framework can be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue, so that the CDR or framework residue at that site does not correspond to the donor antibody or shared framework. Usually, at least 80%, at least 85%, more preferably at least 90% or at least 95% of the humanized antibody residues correspond to those residues of the parental FR and CDR sequences. As used herein, the term "shared framework" refers to the framework region in the shared immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed by the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In an immunoglobulin family, each position in the shared sequence is occupied by the amino acid that occurs most frequently at that position within that family. If two amino acids occur equally frequently, the shared sequence may include either one.

[0317] Where a term has two or more definitions as used and / or accepted in the art, the definition of the term as used herein includes all of these meanings unless expressly indicated to the contrary. A specific example uses the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), all of which are incorporated herein by reference.

[0318] Kabat et al. also defined a numbering system that can be applied to any antibody variable region sequence. One of skill in the art can apply the "Kabat numbering" system to any variable region sequence, independent of other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia numbering systems.

[0319] The antibodies disclosed herein may be from any animal, including but not limited to fish, birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, camel, llama, horse or chicken. In other embodiments, the variable region may be from a condricthoid (e.g., shark).

[0320] A "heavy chain constant region" comprises at least one of a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 structural domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In other embodiments, the heavy chain constant region may comprise a hinge region partially derived from an IgG1 molecule and a hinge region partially derived from an IgG3 molecule. In other embodiments, a portion of the heavy chain may comprise a chimeric hinge region partially derived from an IgG1 molecule and a chimeric hinge region partially derived from an IgG4 molecule.

[0321] The "light chain constant region" comprises a portion of the amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. The "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer by disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0322] The "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain. The "CH1 domain" comprises the first constant region of an immunoglobulin heavy chain. The CH2 domain is not tightly paired with other domains and inserts two N-linked branched carbohydrate chains between the two CH2 domains of an intact native IgG molecule. The CH3 domain starts at the CH2 domain and extends to the C-terminus of the IgG molecule and comprises approximately 108 residues. The "hinge region" comprises a portion of the heavy chain region that connects the CH1 domain and the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0323] "Disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of a cysteine ​​can form or bridge a disulfide bond with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0324] "Chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species. In some embodiments, the variable regions are non-human (e.g., murine or primate) and the constant regions are human.

[0325] The term "epitope" as used herein includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof or a T-cell receptor. Epitope determinants generally consist of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0326] As used herein, the term "specific binding" refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and the specific antigen of the immunoglobulin. The strength or affinity of an immunological binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, of which the smaller the KD, the higher the affinity. The immune binding properties of a selected polypeptide may be quantified by methods well known in the art. One method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, of which these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect this rate equally in two directions. Therefore, the "association rate constant" (K on ) and "dissociation rate constant" (K off Both the k and k+ can be determined by calculating the concentration and the actual association and dissociation rates (see Nature 361:186-87 (1993)). off / k on The ratio excludes all parameters unrelated to affinity and can be equal to the equilibrium dissociation constant, KD (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473). Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometric binding assays, or similar assays known to those of skill in the art.

[0327] In the present invention, the term "isolated" DNA, RNA, polypeptide, etc., as used in reference to cells, nucleic acids, polypeptides, etc., refers to molecules, such as DNA or RNA, that are isolated from one or more of the other components of the cell's natural environment. As used in the present invention, the term "isolated" further refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or that are substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" means to include nucleic acid fragments that are not present in the natural state and do not exist in the natural state. In the present invention, the term "isolated" is also used to mean cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are meant to include purified polypeptides and recombinant polypeptides. Isolated polypeptides, etc. are generally produced by at least one purification step. In one or more embodiments, the purity of an isolated nucleic acid, polypeptide, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(inclusive), or any value therein.

[0328] An "isolated" antibody is an antibody that is isolated and / or recovered from a component of its natural environment. Some components of its natural environment may be substances that would interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, molten metals, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the extent that: (1) the antibody is greater than 95%, e.g., greater than 99%, by weight as determined by the Lowry method; (2) at least 15 residues of N-terminal or internal amino acid sequence can be obtained using a spinning cup sequenator; or (3) homogeneity is determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include in situ antibodies in recombinant cells. Ordinarily, isolated antibodies are produced by at least one or more purification steps. In some embodiments, the purity of the isolated antibodies is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(inclusive), or any value therein.

[0329] The term "code", when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide, either in its natural state or when manipulated by methods known to those of skill in the art, is capable of producing that polypeptide and / or fragments thereof by transcription and / or translation.

[0330] The term "recombinant" with respect to a polypeptide or polynucleotide refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example being one that can be combined to produce a polynucleotide or polypeptide that does not normally occur.

[0331] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., identical on a nucleotide-by-nucleotide or residue-by-residue basis) in a comparison window. The term "percent sequence identity" is calculated by the following method: comparing two optimally aligned sequences in a comparison window, determining the number of positions where the same amino acid residue occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the percent sequence identity. "At least 90% sequence identity" refers to about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or a range between any two of these values ​​(inclusive), or any value therein.

[0332] An "amino acid" is an organic compound that contains both an amino group and a carboxyl group, such as an α-amino acid, and can be coded by a nucleic acid either as such or in the form of a precursor. A single amino acid is coded by a nucleic acid consisting of three nucleotides (a so-called codon or base triplet). Each amino acid is coded by at least one codon. The coding of the same amino acid by different codons is called the "degeneracy of the genetic code". Amino acids include natural and unnatural amino acids.

[0333] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd ed., ES Golub and DR Gren, eds., Sinauer Associates, Sunderland 7 Mass. (1991)). Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Conventional (or naturally occurring) amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0334] The term "polypeptide" is intended to encompass a singular "polypeptide" and a plurality of "polypeptides" and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain" or any other term for meaning two or more amino acid chains, and the term "polypeptide" may be used in place of or interchangeably with any one of the above terms. The term "polypeptide" is also intended to refer to products that have been modified after expression of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / closing groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not be translated from a specified nucleic acid sequence and may be produced by any method, including chemical synthesis.

[0335] When applied to polypeptides, the term "essentially identical" means that two peptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity when optimally aligned, such as by the GAP or BESTFIT programs, using default gap weights.

[0336] One of skill in the art will understand that when an amino acid or polypeptide is a component of a molecule (e.g., an antibody or ADC), the amino acid or polypeptide refers to the amino acid or polypeptide residue (whether or not stated), i.e., the portion that remains after a portion of the group (e.g., one hydrogen atom of the amino group and / or a hydroxy group of the carboxy group) is lost through a covalent bond (e.g., an amide bond) that forms with the other portion of the molecule when attached to the other portion of the molecule.

[0337] A polynucleotide consists of a specific sequence of four bases, namely adenine (A), cytosine (C), guanine (G), thymine (T), or, if the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented by the characters of a polynucleotide molecule. The characters can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching.

[0338] The terms "polynucleotide", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides are genes or gene fragments (e.g., probes, primers, EST or SAGE labels), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, modifications to the structure of the nucleotides may be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. After polymerization, the polynucleotide may be further modified, such as by conjugation with a labeling component. The term refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of this disclosure includes the double-stranded form and each of the two known or predicted complementarable single-stranded forms that make up the double-stranded form.

[0339] A nucleic acid or polynucleotide sequence (or a polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" with another sequence means that, upon sequence alignment, that percentage of bases (or amino acids) are the same in the two sequences being compared. The alignment and percentage of identity or sequence identity can be determined by visual inspection or by software programs known in the art, such as those described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, including BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides refer to polynucleotides that have the percentage identity specified above and that encode polypeptides having the same or similar biological activity.

[0340] Unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' direction of addition of the nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand that is identical to the RNA sequence and is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand that is identical to the RNA sequence and is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0341] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0342] Any minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are included in the present disclosure, provided that the identity of the amino acid sequence is maintained at least 90%, for example at least 92%, 95%, 98% or 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly classified into (1) acidic amino acids, which are aspartate and glutamate; (2) basic amino acids, which are lysine, arginine, and histidine; (3) nonpolar amino acids, which are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, which are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine in the aliphatic-hydroxyl family, (ii) asparagine and glutamine in the amide-containing family, (iii) alanine, valine, leucine and isoleucine in the aliphatic family, and (iv) phenylalanine, tryptophan and tyrosine in the aromatic family. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, the individual substitution of leucine with isoleucine or valine, the substitution of aspartate with glutamate, the substitution of threonine with serine, or similar substitutions of amino acids with structurally related amino acids can be reasonably predicted without significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a binding site. Whether an amino acid change results in a functional peptide can be easily determined by measuring the specific activity of the polypeptide derivative. Such measurements are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily produced by one of skill in the art.

[0343] In some embodiments, the amino acid substitutions have the effect of (1) reducing the susceptibility of the protein to hydrolysis, (2) reducing the susceptibility to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, or (5) imparting or improving other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins whose sequences differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not significantly change the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt the helical structure present in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (ed. Creighton, W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (eds. C. Branden and J. Tooze, Garland Publishing, New York, NY (1991)), and Thornton et al., Nature 354:105 (1991).

[0344] The number of amino acids in the conservative amino acid substitutions in VL and VH may be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, or about 15 conservative amino acid substitutions, or a range between any two of these values ​​(including the end values), or any value therein. The number of amino acids in the heavy chain constant region, light chain constant region, heavy chain or light chain conservative amino acid substitutions can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values ​​(inclusive), or any value therein.

[0345] As used herein, the term "reagent" refers to a chemical compound, a mixture of chemical compounds, a biological polymer, or an extract made from biological materials.

[0346] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, for example, by incorporation of a radioactively labeled amino acid or a polypeptide attached to a biotin group moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or having enzymatic activity detected by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the labels are attached via spacer arms of various lengths to reduce possible steric hindrance. The term "pharmaceutical agent" or "drug" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0347] "About" refers to the normal error range of the corresponding numerical value, which is readily known to one of ordinary skill in the art. In some embodiments, "about" as referred to herein refers to the numerical value set forth and a range of ±10%, ±5%, or ±1% thereof.

[0348] The half maximum effective concentration (EC 50 ” (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can produce 50% of the maximum effect.

[0349] "I C 50 " represents the 50% inhibitory concentration, ie, the concentration of a drug or inhibitor required to inhibit a specified biological process by half.

[0350] "Treatment" refers to therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent, alleviate, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to, detectable or undetectable results such as alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or alleviation of disease progression, improvement, amelioration, reduction or elimination (partial or complete) of the disease state, or an increase in expected survival time in the absence of treatment. Patients in need of treatment include those already suffering from a disease condition or disorder, those susceptible to a disease condition or disorder, or those in need of prevention of the disease condition or disorder, those who may benefit or would benefit from administration of an antibody or pharmaceutical composition disclosed by the present invention for detection, diagnostic processes and / or treatment.

[0351] The term "cancer" is meant or intended to describe a physiological condition in mammals typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More specific examples of such cancer include, but are not limited to, colon cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, salivary gland cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, rectal cancer, prostate cancer, vulvar cancer, testicular cancer, squamous cell carcinoma, small cell lung cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer.

[0352] "Overexpression" or "overexpressed" may interchangeably refer to a gene, which is usually transcribed or translated at a detectable higher level in a particular cell, such as a cancer cell, compared to a normal cell. Overexpression may be protein or RNA overexpression (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered proteolysis), as well as localized overexpression (increased nuclear localization) and enhanced functional activity, such as enzymatic hydrolysis of a substrate, due to altered protein transport mode. Compared to normal or control cells, overexpression may be 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the anti-FRα antibodies and antibody drug conjugates of the present invention are used to treat solid tumors that can overexpress folate receptor α.

[0353] As used herein, the term "tumor overexpressing folate receptor alpha" refers to a tumor (including benign tumors and cancers) that overexpresses folate receptor alpha. In some examples, folate receptor alpha expression in a tumor sample above background levels of immune tissue (e.g., as measured by immunohistochemical staining) indicates that the tumor is a tumor that overexpresses folate receptor alpha. Methods for detecting folate receptor alpha expression in tumors are known in the art, for example, immunohistochemical assays. In some examples, "FRα-negative cells" are cells in a cell sample that lack folate receptor alpha above background (e.g., as measured by immunohistochemical techniques).

[0354] As used herein, the term "administration" refers to delivery of a substance (e.g., an anti-folate receptor alpha antibody or an ADC) to achieve a therapeutic objective (e.g., treatment of a folate receptor alpha-associated disorder). Methods of administration may be parenteral, enteral, and topical. Parenteral administration is typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0355] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, e.g., an antibody or ADC, sufficient to reduce or ameliorate the severity and / or duration of a condition (e.g., cancer) or one or more symptoms thereof, prevent progression of a condition, cause regression of a condition, prevent recurrence, progression, onset or progression of one or more symptoms associated with a condition, detect a condition, or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). For example, an effective amount of an antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume), reduce tumor growth (e.g., reduce tumor volume), reduce the number of cancer cells, and / or alleviate to some extent one or more symptoms associated with cancer. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0356] The terms "patient" and "subject" are used interchangeably and refer to any mammal in need of diagnosis, prognosis or treatment, including, but not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc. In some embodiments, the patient is a human.

[0357] As used herein, the term "in need of" refers to a patient being identified as needing a particular method or treatment. In some embodiments, this can be identified by any diagnostic method. The patient may be in need of any of the methods and treatments described herein.

[0358] As used herein, the term "administration" refers to the administration of a substance to achieve a therapeutic goal (eg, treatment of a tumor).

[0359] As used herein, the term "tumor treating drug" refers to an agent that has the functional property of inhibiting the development or progression of tumors in humans, particularly malignant (cancerous) lesions such as carcinomas, sarcomas, lymphomas or leukemias. Inhibition of metastasis is often a property of antitumor drugs.

[0360] The term "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation additive.

[0361] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which an active ingredient can be administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils of animal, vegetable, or synthetic origin, such as petroleum, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions can be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusters such as sodium chloride or dextrose are also foreseeable. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions may be prepared as suppositories with conventional adhesives and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions contain a clinically effective dose of the antibody or antigen-binding unit, preferably in purified form, combined with an appropriate number of carriers to provide a dosage form suitable for the patient. The formulation should be adapted to the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0362] The term "antibody drug conjugate" or "ADC" refers to a binding protein (e.g., an antibody or an antigen-binding unit thereof) linked to one or more chemical entities, which may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, an ADC comprises an antibody, a drug (e.g., a cytotoxic drug), and a linker by which the drug can be attached or attached to the antibody. Non-limiting examples of drugs that may be included in an ADC include mitotic inhibitors, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0363] The terms "antibody drug conjugate" and "ADC" are used interchangeably. The terms "anti-folate receptor alpha antibody drug conjugate", "anti-FRα antibody drug conjugate", "anti-FRα ADC" and "anti-folate receptor alpha ADC" are used interchangeably and refer to an ADC comprising an antibody that specifically binds to folate receptor alpha, wherein the antibody is coupled to one or more drugs. In one or more embodiments, the anti-FRα ADC comprises an antibody coupled to exatecan. In one or more embodiments, the anti-FRα antibody or ADC binds to folate receptor alpha (e.g., human folate receptor alpha).

[0364] The term "drug antibody conjugation ratio" or "DAR" refers to the amount of drug (e.g., exatecan) of an ADC that is conjugated to an antibody. The DAR of an ADC may range from 1 to 10, although higher loadings (e.g., 20) are possible depending on the number of binding sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a set of ADCs. In some embodiments, the value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. When considering the average number of small molecule drugs bound, i.e., the average number of drugs bound to an antibody, or what is referred to as the average drug antibody conjugation ratio, the value is selected from about 0 to about 10, or from about 2 to about 8. In some embodiments, the drug antibody conjugation ratio is about 3 to about 6. In other embodiments, the drug antibody conjugation ratio is about 6 to about 8, or from about 7 to about 8. The DAR value can be represented herein by p. Those skilled in the art understand that the average DAR (p) of different batches of the same ADC may be slightly different. The DAR value of ADC can be measured by ultraviolet-visible absorption spectroscopy (UV-Vis), high performance liquid chromatography-hydrophobic chromatography (HPLC-HIC), high performance liquid chromatography-reverse phase chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. These techniques are described in Ouyang, J.Methods Mol Biol, 2013, 1045: p.275-83.

[0365] Each substituent is defined as follows. In some cases, the number of carbon atoms in a substituent (e.g., alkyl, alkenyl, alkynyl, alkoxy, aminoalkoxy, aminoalkyl, aminoalkylamino, alkylamino, heterocyclyl, heterocyclylamino, and aryl) is indicated by the prefix "Cx-Cy" or "Cx-y", where x is the minimum number of carbon atoms and y is the maximum number of carbon atoms. Thus, for example, a "C1-C6 alkyl group" refers to an alkyl group containing 1 to 6 carbon atoms. When a substituent is described as being "substituted with...", a hydrogen atom on the carbon or nitrogen is replaced with a non-hydrogen group. For example, a substituted alkyl substituent is an alkyl substituent in which at least one hydrogen atom on the alkyl group is replaced with a non-hydrogen group. For purposes of illustration, a monofluoroalkyl group is an alkyl group substituted with one fluorine group, and a difluoroalkyl group is an alkyl group substituted with two fluorine groups. When there is more than one substitution on a substituent, it should be understood that each substitution may be the same or different (unless otherwise specified). When a substituent is described as being "optionally substituted with...", the substituent may be (1) unsubstituted or (2) substituted. Possible substituents include hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 aminoalkoxy, halogen, nitro, cyano, sulfhydryl, alkylthio, amino, C1-C6 aminoalkyl, C1-C6 aminoalkylamino, C1-C6 alkyl linked to a heterocycle, C1-C6 alkylamino linked to a heterocycle, heterocyclyl, heterocyclyl substituted with an amino, heterocyclylamino, carbamoyl, morpholin-1-yl, piperidin-1-yl, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2)q -(C3~C8 carbocyclyl)-CH3, -(C3~C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3~C8 heterocyclyl)-CH3, -(C3~C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3 or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, but is not limited thereto, n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0366] "Alkyl group" refers to a saturated aliphatic hydrocarbon group, the term includes straight-chain and branched-chain hydrocarbon groups. For example, C1-C20 alkyl groups, such as C1-C6 alkyl groups. C1-C20 alkyl groups refer to alkyl groups having 1-20 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and the like. The alkyl group may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxy, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl, and the like.

[0367] The term "alkenyl group," by itself or as part of another substituent, refers to an unsaturated branched, straight chain, or cyclic alkyl group having at least one carbon-carbon double bond derived by the removal of a hydrogen atom from a single carbon atom of a parent alkene. Typical alkyl groups include, but are not limited to, vinyl groups, propenyl groups (such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl), cycloprop-2-en-1-yl, butenyl groups (such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and the like).

[0368] The term "alkynyl group," by itself or as part of another substituent, refers to a branched, straight chain, or cyclic unsaturated alkyl group having at least one carbon-carbon triple bond derived by the removal of a hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl groups, propynyl groups (prop-1-yn-1-yl, prop-2-yn-1-yl, etc.), butynyl groups (but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.), and the like.

[0369] "Carbocyclyl group" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, e.g., 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) carbon atoms, and which is saturated or unsaturated and connected to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. Polycyclic radicals include, for example, adamantyl, norbornyl, decahydronaphthyl, and the like. Where specifically stated, the carbocyclyl group may be optionally substituted by one or more substituents independently selected from alkyl groups, halogens, haloalkyl groups, cyano groups, nitro groups, oxo, aryl groups, aralkyl groups, carbocyclyl groups, carbocyclylalkyl groups, heterocyclyl groups, heterocyclylalkyl groups, heteroaryl groups, and heteroarylalkyl groups.

[0370] "Aryl group" refers to an all-carbon monocyclic or all-carbon fused ring with a fully conjugated pi-electron system, typically having 5 to 14 carbon atoms, e.g., 6, 10, 12, 14 carbon atoms. Aryl groups can be unsubstituted or substituted with one or more substituents, including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carboxy, aryl, aralkyl, amino, halogen, sulfonyl, sulfinyl, and phosphonyl groups. Illustrative examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl groups.

[0371] A "heterocyclyl group" refers to a stable 3- to 18-membered aromatic or non-aromatic ring substituent which consists of two to eight (e.g., 2, 3, 4, 5, 6, 7, or 8) carbon atoms and one to six (one, two, three, four, five, or six) heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which may include fused or bridged ring systems, and the nitrogen, carbon, or sulfur atoms in the heterocyclyl group may be optionally oxidized, the nitrogen atoms may be optionally quaternized, and the heterocyclyl group may be partially or fully saturated. Illustrative examples of such heterocyclyl groups are dioxolanyl, dioxinyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinyl, 4-pyridinyl, 5-pyridinyl, 6-pyridinyl, 7-pyridinyl, 8-pyridinyl, 9-pyridinyl, 10-pyridinyl, 11-pyridinyl, 12-pyridinyl, 13-pyridinyl, 14-pyridinyl, 15-pyridinyl, 16-pyridinyl, 17-pyridinyl, 18-pyridinyl, 19-pyridinyl, 20-pyridinyl, 21-pyridinyl, 22-pyridinyl, 23-pyridinyl, 24-pyridinyl, 25-pyridinyl, 26-pyridinyl, 27-pyridinyl, 28-pyridinyl, 29-pyridinyl, 30-pyridinyl, 31-pyridinyl, 32-pyridinyl, 33-pyridinyl, 34-pyridinyl, 35-pyridinyl, 36-pyridinyl, 37-pyridinyl, 38-pyridinyl, 39-pyridinyl, 40-pyridinyl, 41-pyridinyl, 42-pyridinyl, 43-pyridinyl, 44-py These include, but are not limited to, nonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, 1,2,4-thiadiazol-5(4H)-yl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl. As specifically described herein, heterocyclyl groups may be optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, halogens, haloalkyl groups, cyano groups, oxo, thioxo, nitro groups, aryl groups, aralkyl groups, cycloalkyl groups, cycloalkylalkyl groups, optionally substituted heterocyclyl groups, optionally substituted heterocyclylalkyl groups, optionally substituted heteroaryl groups, and optionally substituted heteroarylalkyl groups.

[0372] "Alkoxy" refers to a group of the formula -O-(alkyl), where alkyl is an alkyl group as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Alkoxy groups may be substituted or unsubstituted.

[0373] "Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0374] An "amino group" refers to -NH2.

[0375] A "cyano group" refers to -CN.

[0376] A "nitro group" refers to --NO.sub.2.

[0377] A "hydroxy group" refers to -OH.

[0378] A "carboxy group" refers to -COOH.

[0379] A "sulfhydryl group" refers to --SH.

[0380] A "carbonyl group" refers to C=O.

[0381] "Stereoisomer" refers to an isomeric compound having the same atomic connection sequence, but with different arrangements of atoms in space. A stereoisomer may have one or more stereocenters, and each center may be present in R or S, and the stereoisomer may be a cis-trans isomer. Stereoisomers of the compounds provided herein include any of their diastereomeric, enantiomeric, and cis-trans isomeric forms, or the appropriate mixtures thereof.

[0382] Pharmaceutically acceptable salts include those formed with the compounds and a variety of organic and inorganic counterions well known in the art, merely exemplary salts include organic or inorganic salts such as lithium, sodium, potassium, calcium, magnesium, ammonium, isopropylamine, trimethylamine, diethylamino, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethyl, piperidine, polyamine resins, and tetraalkylammonium salts when the molecule contains an acidic functional group, and organic or inorganic acid salts such as hydrochloride, hydrobromide, tartrate, methanesulfonate, acetate, maleate, and oxalate when the molecule contains a basic functional group. Other non-limiting examples of acids include sulfuric acid, nitric acid, phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. These salts may generally be prepared by conventional methods, for example by reacting the appropriate acid or base with the compound. Solvates include hydrates.

[0383] Other chemical terms herein are used according to conventional practice in the art, e.g., as used in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

[0384] All publications and patents cited herein are hereby incorporated by reference in their entirety for all purposes.

[0385] Anti-FRα antibody The antibody provided by the present invention is an antibody against folate receptor α or an antigen-binding unit thereof. In one or more embodiments, the antibody of the present invention can specifically bind to human folate receptor α. In one or more embodiments, the present invention provides a humanized anti-FRα antibody or an antigen-binding unit thereof. In the present specification, these antibodies are collectively referred to as anti-FRα antibodies. The anti-FRα antibody or an antigen-binding unit thereof of the present invention has properties including, but not limited to, in vitro binding to FRα (e.g., human FRα), binding to FRα-expressing cells, high affinity, and strong internalization ability.

[0386] In one or more embodiments, the anti-FRα antibody described in the present invention can specifically bind to folate receptor alpha (FOLR1) and does not bind to folate receptor beta (FOLR2) or folate receptor gamma (FOLR3).

[0387] In one or more embodiments, the antigen-binding fragment or antigen-binding unit of the anti-FRα antibody is a Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibody, or diabody. In one or more embodiments, the anti-FRα antibody is a multispecific antibody (e.g., a bispecific antibody).

[0388] In one or more embodiments, the anti-FRα antibody may be a monoclonal antibody.

[0389] The binding specificity of the antibodies or antigen-binding units thereof disclosed by the present invention can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry (Facs) or enzyme-linked immunosorbent assay (ELISA).

[0390] The invention further includes antibodies that bind to the same epitope as the anti-FRα antibodies described herein. For example, the antibodies of the invention specifically bind to an epitope of one or more amino acid residues on human FRα.

[0391] In one or more embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In one or more embodiments, the anti-FRα antibody or antigen-binding unit thereof comprises an immunoglobulin heavy chain constant domain selected from a human IgG constant domain, a human IgA constant domain, a human IgE constant domain, a human IgM constant domain, and a human IgD constant domain. In one or more embodiments, the anti-FRα antibody or antigen-binding unit thereof comprises an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region. In one or more embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In one or more embodiments, the antibody or antigen-binding unit thereof comprises a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. In one or more embodiments, the antibody or antigen-binding unit thereof comprises a kappa light chain constant region.

[0392] The Fc portion of an antibody mediates several important effector functions (e.g., cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), half-life / clearance of antibody and antigen-antibody complexes). In some cases, these effector functions are necessary for therapeutic antibodies, while in other cases, they may be unnecessary or even deleterious depending on the therapeutic objectives. Some human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. The neologous Fc receptor (FcRn) is a key component that determines the circulating half-life of an antibody. In some further embodiments, at least one amino acid residue in the antibody constant region (e.g., the Fc region of an antibody) is substituted to alter the effector function of the antibody. Substitution of amino acid residues in the Fc region of an antibody to alter antibody effector functions is described in US Pat. Nos. 5,648,260 and 5,624,821, incorporated herein by reference.

[0393] In one or more embodiments, the invention includes a labeled anti-FRα antibody, or antigen-binding unit thereof, derived from or linked to one or more functional molecules (eg, another peptide or protein). For example, a labeled antibody is derived by functionally linking (by chemical bond, genetic fusion, non-covalent bonding, or other methods) an antibody of the invention or an antigen-binding unit thereof to one or more other molecular entities, such as another antibody (e.g., a bispecific or bifunctional antibody), a detectable reagent, a drug agent, a protein or peptide capable of mediating binding of the antibody or its antigen-binding unit to another molecule (such as a streptavidin core region or a polyhistidine tag), and / or a cytotoxic or therapeutic agent selected from the group consisting of antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors, radiosensitizers, and combinations thereof.

[0394] In one or more embodiments, the antibody of the invention or an antigen-binding unit thereof is linked to a detectable reagent, such as by incorporation of a radiolabeled amino acid or attachment of a biotin moiety to the polypeptide that is detectable by labeled avidin (e.g., streptavidin that contains a fluorescent label or has enzymatic activity detected by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In one or more embodiments, the labels are attached via spacer arms of various lengths to reduce possible steric hindrance.

[0395] In one or more embodiments, the antibody or antigen-binding unit thereof of the present invention can be used to detect the presence of FRα (e.g., human FRα) in a sample. In one or more embodiments, the antibody comprises a detectable reagent. The antibody is a polyclonal antibody, or more preferably, a monoclonal antibody. A complete antibody or antigen-binding unit thereof (e.g., Fab, scFv, or F(ab')2) can be used. The detection method of the above embodiment can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization, in vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence, and in vitro detection techniques for analyte genomic DNA include Southern hybridization. (ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42, J.R.Crowther Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985). In vivo detection techniques for analyte proteins also include introducing a labeled anti-analyte protein antibody into the patient. For example, the antibody can be labeled with a radioactive label, whose presence and location in the patient can then be detected by standard imaging techniques.

[0396] In one or more embodiments, folate receptor alpha can be detected in a biological sample as part of a clinical trial procedure, for example to determine the efficacy of a given treatment regimen. Detection can be aided by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinyl fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include, 125 I, 131 I, 35 S or 3 Contains H.

[0397] Humanized antibodies The anti-FRα antibodies according to the invention include humanized antibodies or antigen-binding units thereof, which are suitable for administration to humans without eliciting a human immune response against the administered immunoglobulin.

[0398] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises a VH CDR1 set forth in SEQ ID NO:5, a VH CDR2 set forth in SEQ ID NO:6, and a VH CDR3 set forth in SEQ ID NO:7.

[0399] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VL CDR1 shown in SEQ ID NO:8, a VL CDR2 shown in SEQ ID NO:9, and a VL CDR3 shown in SEQ ID NO:10.

[0400] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VH CDR1 set forth in SEQ ID NO:5, a VH CDR2 set forth in SEQ ID NO:6, a VH CDR3 set forth in SEQ ID NO:7, a VL CDR1 set forth in SEQ ID NO:8, a VL CDR2 set forth in SEQ ID NO:9, and a VL CDR3 set forth in SEQ ID NO:10.

[0401] In one or more embodiments, the humanized anti-FRα antibody of the invention or an antigen-binding unit thereof further comprises a framework region. The framework regions of the exemplary antibody VH and VL herein are shown in Table 1.

[0402] [Table 1]

[0403] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VH FR1 set forth in SEQ ID NO: 1, a VH CDR1 set forth in SEQ ID NO: 5, a VH FR2 set forth in SEQ ID NO: 2, a VH CDR2 set forth in SEQ ID NO: 6, a VH FR3 set forth in SEQ ID NO: 3, a VH CDR3 set forth in SEQ ID NO: 7, and a VH FR4 set forth in SEQ ID NO: 11.

[0404] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VH FR1 set forth in SEQ ID NO: 29, a VH CDR1 set forth in SEQ ID NO: 5, a VH FR2 set forth in SEQ ID NO: 30, a VH CDR2 set forth in SEQ ID NO: 6, a VH FR3 set forth in SEQ ID NO: 31, a VH CDR3 set forth in SEQ ID NO: 7, and a VH FR4 set forth in SEQ ID NO: 11.

[0405] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VL FR1 set forth in SEQ ID NO: 12, a VL CDR1 set forth in SEQ ID NO: 8, a VL FR2 set forth in SEQ ID NO: 13, a VL CDR2 set forth in SEQ ID NO: 9, a VL FR3 set forth in SEQ ID NO: 4, a VL CDR3 set forth in SEQ ID NO: 10, and a VL FR4 set forth in SEQ ID NO: 14.

[0406] In one or more embodiments, the humanized anti-FRα antibody or its antigen-binding unit of the present invention comprises a VL FR1 set forth in SEQ ID NO: 12, a VL CDR1 set forth in SEQ ID NO: 8, a VL FR2 set forth in SEQ ID NO: 13, (4) a VL CDR2 set forth in SEQ ID NO: 9, a VL FR3 set forth in SEQ ID NO: 32, a VL CDR3 set forth in SEQ ID NO: 10, and a VL FR4 set forth in SEQ ID NO: 14.

[0407] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises a heavy chain variable region comprising VH FR1 as set forth in SEQ ID NO: 1, VH CDR1 as set forth in SEQ ID NO: 5, VH FR2 as set forth in SEQ ID NO: 2, VH CDR2 as set forth in SEQ ID NO: 6, VH FR3 as set forth in SEQ ID NO: 3, VH CDR3 as set forth in SEQ ID NO: 7, and VH FR4 as set forth in SEQ ID NO: 11, and a light chain variable region comprising VL FR1 as set forth in SEQ ID NO: 12, VL CDR1 as set forth in SEQ ID NO: 8, VL FR2 as set forth in SEQ ID NO: 13, VL CDR2 as set forth in SEQ ID NO: 9, VL FR3 as set forth in SEQ ID NO: 4, VL CDR3 as set forth in SEQ ID NO: 10, and VL FR4 as set forth in SEQ ID NO: 14.

[0408] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises a heavy chain variable region comprising VH FR1 as set forth in SEQ ID NO:29, VH CDR1 as set forth in SEQ ID NO:5, VH FR2 as set forth in SEQ ID NO:30, VH CDR2 as set forth in SEQ ID NO:6, VH FR3 as set forth in SEQ ID NO:31, VH CDR3 as set forth in SEQ ID NO:7, and VH FR4 as set forth in SEQ ID NO:11, and a light chain variable region comprising VL FR1 as set forth in SEQ ID NO:12, VL CDR1 as set forth in SEQ ID NO:8, VL FR2 as set forth in SEQ ID NO:13, VL CDR2 as set forth in SEQ ID NO:9, VL FR3 as set forth in SEQ ID NO:32, VL CDR3 as set forth in SEQ ID NO:10, and VL FR4 as set forth in SEQ ID NO:14.

[0409] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises an amino acid sequence having a suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence shown in SEQ ID NO: 15, in its heavy chain variable region, and / or an amino acid sequence having a suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence shown in SEQ ID NO: 15, in its light chain variable region, and / or an amino acid sequence having a suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence shown in SEQ ID NO: 16. In one or more embodiments, these amino acid sequences having sequence identity have at least the CDRs unchanged.

[0410] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the invention comprises a heavy chain variable region set forth in SEQ ID NO:15 and a light chain variable region set forth in SEQ ID NO:16.

[0411] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 17.

[0412] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the present invention further comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 18.

[0413] In one or more embodiments, the heavy chain of a humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises an amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having an appropriate sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 19.

[0414] In one or more embodiments, the light chain of a humanized anti-FRα antibody or antigen-binding unit thereof of the present invention comprises an amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having an appropriate sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO:20.

[0415] In one or more embodiments, the heavy chain of the humanized anti-FRα antibody of the present invention or its antigen-binding unit further comprises a signal peptide, such as, for example, MDWTWRILFLVAAATGAHS (SEQ ID NO: 21), which has the nucleotide sequence atggattggacctggagaatcctgttcctggtggccgccgccaccggcgctcattct (SEQ ID NO: 25).

[0416] In one or more embodiments, the light chain of the humanized anti-FRα antibody of the present invention or its antigen-binding unit further comprises a signal peptide, such as, for example, MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 22), the nucleotide sequence of which is atggaggcccctgcccagctgctgttcctgctgctgctgtggctgcctgataccaccggc (SEQ ID NO: 26).

[0417] In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the invention has a binding dissociation equilibrium constant (KD) for binding to FRα of about 1 μM or less. In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the invention has a KD for binding to FRα of about 100 nM to about 1 pM or less. In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the invention has a KD for binding to FRα of about 10 nM to about 1 pM or less. In one or more embodiments, the humanized anti-FRα antibody or antigen-binding unit thereof of the invention has a KD for binding to FRα of about 1 nM to about 0.1 nM or less.

[0418] In one or more embodiments, the humanized anti-FRα antibody of the invention or an antigen-binding unit thereof has a KD for binding to human FRα (antigen FRα-His shown in SEQ ID NO:28) of about 1 μM to about 1 pM or less. In one or more embodiments, the humanized anti-FRα antibody of the invention or an antigen-binding unit thereof has a KD for binding to human FRα of about 100 nM to about 1 pM or less, or about 10 nM to about 1 pM or less, or about 1 nM to about 1 pM or less.

[0419] Antibody Drug Conjugates The anti-FRα antibodies or antigen-binding units thereof of the present invention can be coupled to a drug to form an anti-FRα antibody-drug conjugate (anti-FRα ADC). The antibody-drug conjugate (ADC) can selectively deliver one or more drugs to a target tissue (e.g., a tumor expressing FRα), so that the antibody-drug conjugate (ADC) can improve the therapeutic effect of the antibody in treating a disease (e.g., cancer). In one or more embodiments, the antibody-drug conjugate (ADC) of the present invention comprises an anti-FRα antibody or antigen-binding unit thereof described herein and at least one drug (e.g., exatecan). The ADC of the present invention has properties including, but not limited to, in vitro binding to FRα (e.g., human FRα), binding to FRα-expressing cells, high affinity, strong internalization ability, and reduction or inhibition of cancer cell or tumor growth.

[0420] The antigen-binding fragment or antigen-binding unit of the anti-FRα antibody of the invention can be coupled to a drug as described herein. Thus, in one or more embodiments, the antigen-binding fragment or antigen-binding unit of the anti-FRα antibody described herein is coupled to a drug via a linker to form an anti-FRα ADC.

[0421] The anti-FRα ADCs of the invention comprise an antibody or antigen-binding unit thereof that specifically binds to FRα (e.g., human FRα) linked to one or more drugs. The specificity of the ADC can be determined by the specificity of the antibody (e.g., anti-FRα antibody). In one or more embodiments, the anti-FRα antibody is linked to one or more drugs (e.g., DNA topoisomerase inhibitors), and the drugs (e.g., DNA topoisomerase inhibitors) are delivered to cells expressing FRα, particularly cancer cells expressing FRα. In one or more embodiments, the anti-FRα antibody drug conjugate comprises an anti-FRα antibody coupled to a drug (e.g., exatecan) via a linker. The anti-FRα antibodies described herein provide the ADC with the ability to bind to FRα, thereby enabling the drug attached to the antibody to be delivered to cells expressing FRα, particularly cancer cells expressing FRα.

[0422] In one or more embodiments, the ADC has the structure shown in Formula I, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof. [ka] wherein Abu, M, B, G, L, D, and p are as defined herein.

[0423] In one or more embodiments, Abu is Antibody 1.

[0424] In one or more embodiments, the ADC is ADC1 or ADC2, or a pharma- ceutically acceptable salt or solvate thereof.

[0425] In one or more embodiments, the antibody conjugates according to the invention have a significant bystander effect.

[0426] In one or more embodiments, the ADC of the present invention has a binding dissociation equilibrium constant (KD) for binding to FRα of about 1 μM or less. In one or more embodiments, the ADC of the present invention has a KD for binding to FRα of between about 100 nM and about 1 pM or less. In one or more embodiments, the ADC of the present invention has a KD for binding to FRα of between about 10 nM and about 1 pM or less. In one or more embodiments, the ADC of the present invention has a KD for binding to FRα of between about 1 nM and about 1 pM or less.

[0427] In one or more embodiments, the ADC of the present invention has a KD for binding to human FRα (for example, the antigen FRα-His shown in SEQ ID NO: 28) of about 1 μM or less. In one or more embodiments, the ADC of the present invention has a KD for binding to human FRα of between about 100 nM and about 1 pM or less, or between about 10 nM and about 1 pM or less, or between about 1 nM and about 1 pM or less, or between about 1 nM and about 0.1 nM or less, or between about 0.5 nM and about 0.1 nM or less.

[0428] Synthesis method The present invention further provides a method for producing the intermediate and antibody-drug conjugate. The intermediate and antibody-drug conjugate of the present invention can be produced by known formulations and methods. In one or more embodiments, the method for producing intermediate compounds 1 to 7 and compound 7 is as follows: [ka] In step 1, a compound of general formula 1-1 and a compound of general formula 1-1' are reacted under basic conditions to give a compound of general formula 1-2.

[0429] In step 2, the compound of general formula 1-2 and the compound of general formula (AA) i -(FF 1 ) f is reacted in the presence of a condensing agent under basic conditions to give a compound of general formula 1-3.

[0430] In step 3, the amino-protecting group W1 of compounds of general formula 1-3 is removed to give compounds of general formula 1-4.

[0431] In step 4, a compound of general formula 1-4 and a compound of general formula 1-5 are reacted under basic conditions to give a compound of general formula 1-6.

[0432] In step 5, a compound of general formula 1-6 and bis(p-nitrobenzene) carbonate are reacted under basic conditions to give a compound of general formula 1-7. [ka] In step 1, a compound of general formula 1 and a compound of general formula 1′ are reacted under basic conditions to give a compound of general formula 2.

[0433] In step 2, a compound of general formula 2 and a compound of general formula (AA) i -(FF 1 ) f is reacted in the presence of a condensing agent under basic conditions to obtain a compound of general formula 3.

[0434] In step 3, the amino-protecting group W1 of the compound of general formula 3 is removed to give a compound of general formula 4.

[0435] In step 4, a compound of general formula 4 and a compound of general formula 5 are reacted under basic conditions to give a compound of general formula 6.

[0436] In step 5, a compound of general formula 6 and bis(p-nitrobenzene) carbonate are reacted under basic conditions to give a compound of general formula 7.

[0437] Among them W1 is an amino protecting group, such as 9-fluorenylmethyloxycarbonyl, and W2 is a carboxylic acid active ester, such as a succinimide ester.

[0438] wherein n, AA, R, i, and f are as described herein, and each FF 1 are independently [ka] Of these, each R F are independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, z is 0, 1, 2, 3 or 4, in which * is linked to AA, and each FF 2 are independent [ka] Of these, each R F are independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, z is 0, 1, 2, 3 or 4, in which * is linked to AA; In one or more embodiments, R F is F.

[0439] In one or more embodiments, z is 0.

[0440] In one or more embodiments, z is 1 or 2.

[0441] In one or more embodiments, the intermediate is: [ka] wherein R, n are as described herein.

[0442] In one or more embodiments, the intermediate is: [ka] [ka] wherein R, n are as described herein.

[0443] In one or more embodiments, the intermediate is: [ka] wherein n is as described herein.

[0444] In one or more embodiments, the intermediate is: [ka] [ka] wherein n is as described herein.

[0445] In one or more embodiments, the intermediate is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The basic conditions can be provided using reagents including organic and inorganic bases, the organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

[0446] The condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboronate, phosphate, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazo)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.

[0447] In one or more embodiments, the intermediate compounds 1 to 8 and the method for producing compound 8 are as follows. [ka] The compound of the general formula 1-7 and D are reacted in the presence of a condensing agent under basic conditions to give the compound of the general formula 1-8. [ka] The compound of general formula 7 and D are reacted in the presence of a condensing agent under basic conditions to give the compound of general formula 8.

[0448] Among them, n, AA, R, i, f, and FF 2 , FF, D are as described herein.

[0449] In one or more embodiments, the depicted intermediate is: [ka] wherein R, n, and D are as described herein.

[0450] In one or more embodiments, the intermediate is: [ka] wherein R, n, and D are as described herein.

[0451] In one or more embodiments, the intermediate is: [ka] wherein n and D are as described herein.

[0452] In one or more embodiments, the intermediate is: [ka] wherein n and D are as described herein.

[0453] In one or more embodiments, the intermediate is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0454] The basic conditions can be provided using reagents including organic and inorganic bases, the organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

[0455] The condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboronate, phosphate, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazo)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.

[0456] In one or more embodiments, the intermediate compounds 1 to 9 and the method for producing compound 9 are as follows. [ka] Compounds of general formula 1-8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 1-9. [ka] Compounds of general formula 8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 9.

[0457] wherein n, AA, R, i, f, FF, D, and Abu are as described herein.

[0458] Specifically, the coupling scheme is shown in Figure 1, in which the interchain disulfide bond of the anti-FRα antibody provided by the present invention is reductively cleaved with a thiol-based reducing agent to generate a free sulfhydryl group, which is then subjected to a coupling reaction with a compound of general formula 8 to obtain an antibody-drug conjugate shown in general formula 9.

[0459] The weak acidic conditions can be provided by reagents including organic acids and inorganic acids, the organic acids including, but not limited to, acetic acid, benzoic acid, tartaric acid, oxalic acid, malic acid, citric acid, ascorbic acid, citric acid, salicylic acid, caffeic acid, sorbic acid, quinic acid, oleanolic acid, succinic acid, chlorogenic acid, formic acid, and propionic acid, and the inorganic acids including, but not limited to, carbonic acid, nitrous acid, acetic acid, hypochlorous acid, hydrofluoric acid, sulfurous acid, hydrogen sulfide, silicic acid, metasilicic acid, phosphoric acid, metaphosphoric acid, sodium bicarbonate, and sodium bisulfite.

[0460] The drug conjugates can be purified using conventional methods, such as preparative high performance liquid chromatography (prep-HPLC).

[0461] Pharmaceutical Compositions The anti-FRα antibody or antigen-binding unit thereof, or an antibody-drug conjugate coupled with an anti-FRα antibody according to the present invention can be incorporated into a pharmaceutical composition suitable for administration. The principles and considerations involved in the preparation of such compositions and guidance for the selection of ingredients are well known in the art, see, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th Edition, Mack Pub.Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; Peptide And Protein Drug Delivery, Advances In Parenteral Sciences, Vol. 4, 1991, M. Dekker, New York.

[0462] Such compositions generally comprise an antibody or an antigen-binding unit thereof or an antibody-drug conjugate and a pharma- ceutically acceptable carrier. In one or more embodiments, the antibody fragment or antigen-binding unit is used as the smallest inhibitory fragment of specific binding to the target protein binding domain. For example, a peptide based on the variable region sequence of the antibody and retaining the ability to bind to the target protein sequence. In one or more embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0463] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, stabilizers, buffers, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, etc., compatible with drug administration. Suitable carriers are described in Remington's Pharmaceutical Sciences. Such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin.

[0464] In one or more embodiments, formulations to be used for in vivo administration must be sterile, which can be readily accomplished by filtration through sterile filtration membranes.

[0465] Pharmaceutical composition is prepared in dosage unit form, easy to administer and uniform dosage.As used herein, dosage unit form refers to a physically separable unit suitable for use as a unitary dose for the patient to be treated, each unit contains a predetermined amount of one or more of the above-mentioned antibodies that are combined with required drug carrier by calculation to produce desired therapeutic effect.

[0466] The pharmaceutical compositions can be contained in a container or dispenser and packaged together with instructions for administration.

[0467] The pharmaceutical compositions according to the present invention may also contain other active ingredients depending on the specific condition requiring treatment, preferably with complementary activities that do not adversely affect each other. In one or more embodiments, the composition may contain an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such active ingredients are present in suitable combinations in amounts effective for the intended purpose.

[0468] The pharmaceutical compositions provided by the present invention may be administered by any convenient route, for example by infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Thus, the pharmaceutical compositions may be administered intravenously, orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drops or transdermal patch), buccally, or by oral or nasal spray.

[0469] In one or more embodiments, the pharmaceutical composition is prepared by conventional steps as a pharmaceutical composition suitable for intravenous injection into humans. Pharmaceutical compositions for intravenous administration are usually a solution in a sterile or other water-permeable buffer. The pharmaceutical composition may further include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the injection site. Generally, the active ingredients are provided singly or mixed in unit dosage form, for example in a hermetically sealed container (e.g., an ampoule or sachet) capable of indicating the amount of active agent in the form of a dry lyophilized powder or water-free concentrate. When the pharmaceutical composition is administered by injection, the composition may be dispensed by an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients can be mixed before administration.

[0470] The compositions of the present invention may be formulated in a neutral or salt form.

[0471] Treatment Methods and Uses The antibodies or antigen-binding units thereof or ADCs described herein can be used in a variety of applications, including, but not limited to, therapeutic methods, such as treating tumors. In one or more embodiments, the antibodies or antigen-binding units thereof or ADCs can be used to inhibit tumor growth, reduce tumor volume, and / or reduce tumorigenicity of tumors. The method of use can be an in vitro, ex vivo, or in vivo method. In one or more embodiments, the antibodies or antigen-binding units thereof or ADCs described herein are antagonists of the FRα to which they are bound.

[0472] In one or more embodiments, the method for inhibiting tumor growth comprises contacting a tumor with an anti-FRα antibody or its antigen-binding unit or ADC, or a pharmaceutical composition comprising them, in vitro. For example, an immortalized cell line or cancer cell expressing FRα is cultured in a medium to which an anti-FRα antibody or its antigen-binding unit or ADC, or a pharmaceutical composition comprising them, is added. In one or more embodiments, tumor cells are isolated from a patient sample, and cultured in a medium containing an anti-FRα antibody or its antigen-binding unit or ADC, or a pharmaceutical composition comprising them. In one or more embodiments, the method for inhibiting tumor growth comprises contacting a tumor or tumor cells with an anti-FRα antibody or its antigen-binding unit or ADC, or a pharmaceutical composition comprising them, in vivo.

[0473] The antibody or its antigen-binding unit or ADC provided by the present invention, or pharmaceutical compositions comprising them, can be used for diagnosis, prognosis, monitoring, treatment, alleviation and / or prevention of diseases and conditions associated with abnormal expression of FRα in patients. When the presence of diseases and conditions associated with abnormal expression of FRα in patients is identified by using conventional methods, the antibody or its antigen-binding unit or ADC described in the present invention, or pharmaceutical compositions comprising them, can be administered. In one or more embodiments, the level of FRα expression is detected by immunohistochemistry (IHC), flow cytometry, nucleic acid hybridization, etc. WO2014 / 036495 and WO2015 / 031815 provide exemplary antibodies, detection methods and reagent kits for detecting FRα, all of which are incorporated herein by reference.

[0474] In one or more embodiments, the present invention relates to a method for treating an associated disease in which FRα is a therapeutic target, and relates to a method for improving, alleviating, inhibiting, treating or preventing any disease or condition associated with abnormal expression of FRα (e.g., overexpression of FRα), including a method for treating a tumor (including benign tumors and cancer) in a patient, a method for alleviating symptoms of a tumor (including benign tumors and cancer) in a patient, and a method for avoiding recurrence of a tumor (including benign tumors and cancer) in a patient, comprising administering to the patient an effective amount of any of the antibodies or antigen-binding units thereof or ADCs described herein.

[0475] In one or more embodiments, the present invention provides a method for preventing, treating, or ameliorating a disease, comprising administering to a patient in need thereof an effective amount of an anti-FRα antibody, or an antigen-binding unit thereof, or an ADC, or a pharmaceutical composition comprising the same. In one or more embodiments, the present invention provides use of the antibody, or an antigen-binding unit thereof, or an ADC in the manufacture of a medicament for preventing, treating, or ameliorating a disease. In one or more embodiments, the present invention provides use of the antibody, or an antigen-binding unit thereof, or an ADC in the prevention, treatment, or amelioration of a disease. In one or more embodiments, the disease is a disease associated with abnormal expression of FRα. In one or more embodiments, the disease is a disease associated with overexpression of FRα. In one or more embodiments, the disease is a tumor expressing FRα. In one or more embodiments, the disease is a tumor overexpressing FRα. In one or more embodiments, the disease is a cancer overexpressing FRα. In one or more embodiments, the disease is a cancer overexpressing human FRα.

[0476] In one or more embodiments, the present invention provides a method of treating tumors (including benign tumors and cancers), comprising administering to a patient in need thereof an effective amount of an anti-FRα antibody, or antigen-binding unit thereof, or ADC, or a pharmaceutical composition comprising same. In one or more embodiments, the present invention provides use of the antibody, or antigen-binding unit thereof, or ADC in the manufacture of a medicament for treating tumors (including benign tumors and cancers). In one or more embodiments, the present invention provides use of the antibody, or antigen-binding unit thereof, or ADC in the treatment of tumors (including benign tumors and cancers).

[0477] Examples of cancer include, but are not limited to, solid tumors, hematological cancers, and metastatic lesions. Specific examples of such cancer include, but are not limited to, colon cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, liver cancer, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, testicular cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer. More specific examples of such cancer include ovarian cancer, epithelial ovarian cancer, ovarian primary peritoneal cancer, or fallopian tube cancer. In one or more embodiments, the cancer is a metastatic or progressive cancer. In one or more embodiments, the patient is a patient with an increased FRα expression level. In one or more embodiments, the patient is a human. The patient may also be a mammal into which FRα has been introduced (e.g., by administration of FRα or expression of FRα transgenesis). The antibodies or antigen-binding units thereof or ADCs of the invention can be administered to human patients for therapeutic purposes. The antibodies or antigen-binding units thereof or ADCs of the invention can also be administered to non-human mammals expressing FRα (for veterinary purposes or as animal models of human disease). The animal models of human disease can be used to assess the therapeutic efficacy (e.g., dose testing and administration time courses) of the antibodies or antigen-binding units thereof or ADCs of the invention.

[0478] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody or derivative (e.g., ADC or pharmaceutical composition) used, the age and weight of the patient, general health, sex and diet, and administration time, excretion frequency, drug combination, and the severity of the particular disease being treated. These factors are determined by medical personnel within the scope of the art. The dosage will further depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art. The effective amount refers to the amount of active compound or drug that will produce the biological or pharmacological response in tissues, systems, animals, individuals, and humans that is desired by a researcher, veterinarian, physician, or other clinician, which includes the treatment of a disease. In one or more embodiments, the effective dose ranges from about 0.1 mg / kg to about 100 mg / kg, and the frequency of administration may be, for example, once a month, once every two weeks, once every three weeks, twice every three weeks, three times every four weeks, once a week, twice a week. For example, the administration form may be an intravenous infusion, an intravenous bolus injection, a subcutaneous injection, an intramuscular injection, and the like. It should be noted that dosage values ​​can vary depending on the type and severity of the condition to be alleviated. Furthermore, it should be understood that for any particular patient, a particular dosing regimen can be adjusted from time to time according to the needs of the patient and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the composition as required.

[0479] Methods of administration of antibodies or antigen-binding units thereof or ADCs include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, spinal epidural and oral. Pharmaceutical compositions may be administered by any convenient route, for example by infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Thus, pharmaceutical compositions comprising the antibodies of the invention can be administered orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drop or transdermal patch), buccally, or by spray.

[0480] The term "parenteral" as used herein includes modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0481] The mode of administration may be systemic or local. It may also be necessary to introduce the antibody or antigen-binding unit or ADC of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be assisted by being connected to a reservoir such as an intraventricular catheter (which may be an Ommaya reservoir). Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and by using an atomized formulation.

[0482] The antibodies or antigen-binding units thereof or ADCs of the invention can be administered locally to the area to be treated, which can be achieved by, but is not limited to, local infusion during surgery, topical application in conjunction with, for example, a wound dressing after surgery, by injection, by catheter, as a suppository or by an implant, which can be a porous, non-porous or gel-like material, including a membrane (e.g., a silicone rubber membrane) or a fiber. Preferably, when administering the antibodies or antigen-binding units thereof or ADCs of the invention, care must be taken to use materials that do not absorb proteins.

[0483] Typically, the method of in vitro testing to treat a disease involves administering an antibody or ADC according to the invention, then in vivo testing for the desired therapeutic or prophylactic activity in an acceptable animal model, and finally administering to humans. Suitable animal models (including transgenic animals) are known to those skilled in the art. For example, in vitro assays to confirm the therapeutic use of an antibody or antigen-binding unit thereof or ADC according to the invention include the effect of the antibody or antigen-binding unit thereof or ADC on a cell line or a patient tissue sample. The effect of the antibody or antigen-binding unit thereof or ADC on a cell line and / or tissue sample can be detected by techniques known to those skilled in the art, for example, techniques disclosed in other parts of the present invention. In vitro assay experiments that can be used to determine whether to administer an antibody or antigen-binding unit thereof or ADC according to the present invention include in vitro cell culture experiments, in which a patient tissue sample is grown in culture and exposed or otherwise administered an antibody or antigen-binding unit thereof or ADC, and the effect of such antibody or antigen-binding unit thereof or ADC on the tissue sample is observed.

[0484] A variety of known delivery systems can be used to administer the antibodies or antigen-binding units thereof of the invention, or polynucleotides or ADCs encoding them, such as encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of the nucleic acid as part of a retrovirus or other vector, etc.

[0485] Combination therapy In one or more embodiments, the antibodies or antigen-binding units thereof or ADCs of the invention can be combined with other therapeutic or prophylactic regimens, including the combined or combined use of one or more antibodies or antigen-binding units thereof or ADCs of the invention with one or more other therapeutic agents or methods. In the case of combined treatment, the antibodies or antigen-binding units thereof or ADCs can be administered simultaneously or separately with the other therapeutic agent. In the case of separate administration, the antibodies or antigen-binding units thereof or ADCs of the invention can be administered before or after the administration of another other therapeutic agent.

[0486] In one or more embodiments, when an antibody or antigen-binding unit thereof, or ADC of the invention is administered to a patient, the antibody or antigen-binding unit thereof, or ADC, or pharmaceutical composition or immunoconjugate disclosed herein may also be co-administered to the patient with one or more other therapies, e.g., therapeutic methods and / or other formulations (e.g., therapeutic agents).

[0487] In one or more embodiments, the antibodies or antigen-binding units or ADCs of the invention may be used in combination with immune checkpoint inhibitors. In one or more embodiments, the antibodies or antigen-binding units or ADCs of the invention may be used in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

[0488] Such combination therapy includes simultaneous administration (wherein two or more formulations are in the same preparation or in separate preparations) and separate administration, where an antibody or antigen-binding unit thereof or ADC of the invention can be administered before, during, and / or after administration of another therapy, e.g., a therapeutic method and / or agent. The antibody or antigen-binding unit thereof or ADC and / or the other therapy, e.g., a therapeutic agent or method, can be administered during active disease or during remission or less active disease. The antibody or antigen-binding unit thereof or ADC can be administered before the other treatment, simultaneously with the other treatment, after treatment, or during disease remission.

[0489] Methods for producing antibodies In one or more embodiments, the antibody, antigen-binding unit, or derivative disclosed by the present invention is modified by art-recognized techniques to reduce its immunogenicity. For example, the antibody may be humanized, primatized, deimmunized, or produced as a chimeric antibody. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, and retain or essentially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved by several methods, including: (a) grafting the entire non-human variable region onto a human constant region to produce a chimeric antibody. Methods for producing chimeric antibodies are known in the art, see U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference. (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into human framework and constant regions, with or without retaining important framework residues, or (c) grafting the entire non-human variable region, but "hiding" them by replacing surface residues with human-like portions. Usually, frame residues in the human framework regions are replaced with corresponding residues from the CDR donor antibody, residues that can improve antigen binding, etc. These frame substitutions can be identified by methods known in the art, for example, simulating the interaction of CDRs with frame residues to identify frame residues that play an important role in antigen binding, and identifying unusual frame residues at specific positions by sequence alignment (see U.S. Pat. No. 5,585,089, the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400, WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or resurfacing (EP 592,106, EP 519,596) and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0490] The production of antibodies with reduced immunogenicity can also be achieved by humanization, chimerization and display techniques using appropriate libraries. It should be understood that mouse antibodies or antibodies derived from other species can be humanized or primatized using techniques well known in the art. See, for example, Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al, Crit.Reviews in Immunol.12125-168 (1992). Antibodies can be engineered by recombinant DNA techniques to replace CH1, CH2, CH3, hinge domain and / or backbone domain with the corresponding human sequence (see WO92102190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350 and 5,777,085). The use of Ig cDNA to construct chimeric immunoglobulin genes is also known in the art (Liu et al., PNAS 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from hybridomas or other cells producing the antibodies and used to generate cDNA. The cDNA can be amplified by polymerase chain reaction using specific primers (U.S. Patents 4,683,195 and 4,683,202). Alternatively, the sequence of interest is isolated by constructing and screening libraries. The DNA sequences encoding the variable regions of the antibody are then fused to human constant region sequences. The sequences of human constant region genes are described in Sequences of Proteins of immunological Interest published by Kabat et al. (NIH publication no. 91-3242 (1991)). Human C region genes can be readily obtained from known clones. The choice of isotype is guided by the desired effector functions, such as complement fixation or antibody-dependent cellular cytotoxicity activity. Preferred isotypes are IgG1 and IgG2. Any human light chain constant region, i.e., k or lambda, can then be used to express the chimeric, humanized antibody by conventional methods.

[0491] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering an antibody to modify T cell epitopes (see, for example, WO / 9852976 A1 and WO / 0034317 A2). For example, the heavy and light chain variable region sequences from a starting antibody are analyzed to generate a human T cell epitope "map" from each variable region, showing the location of the epitope relative to the complementarity determining regions (CDRs) and other important residues in the sequence. By analyzing a single T cell epitope from the T cell epitope map, selectable amino acid substitutions with a low risk of altering antibody activity are identified. A set of selectable heavy and light chain variable region sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into a set of binding polypeptides. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into an expression vector, and the plasmids are then introduced into a cell line to produce the complete antibody. The antibodies are then compared in appropriate biochemical and biological experiments to identify the most suitable antibody.

[0492] Fully human antibodies that recognize a selective epitope can be produced by a technique referred to as "guided selection," in which a selected non-human monoclonal antibody is used to guide screening for fully human antibodies that recognize the same epitope (see U.S. Patent 5,565,332, the entire contents of which are incorporated herein by reference).

[0493] For single chain Fv (scFv), refer to the technology for producing a single chain unit (U.S. Patent 4,694,778). A single chain unit is formed by amino acid crosslinking to the heavy and light chain fragments of the Fv region to form a single chain fusion peptide. A technology for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).

[0494] Examples of techniques that can be used to produce scFvs and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498.

[0495] In one or more embodiments, antigen-binding fragments such as Fab, F(ab')2 and Fv can be produced by complete proteolysis, for example by protease or chemical degradation, including but not limited to: (i) digestion of antibody molecules with pepsin to obtain F(ab')2 fragments, (ii) reduction of disulfide bonds of F(ab')2 fragments to obtain Fab fragments, (iii) treatment of antibody molecules with papain and a reducing agent to generate Fab fragments, and (iv) Fv fragments.

[0496] A humanized antibody can be constructed by inserting one or more CDRs of an antibody of the invention into a frame region, for example, a human frame region, using standard recombinant DNA techniques. The frame region may be naturally occurring or a common frame region, but a human frame region is preferred (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a range of human frame regions). Some polynucleotides can encode an antibody that specifically binds to at least one epitope of a target antigen produced by the combination of the frame region and the CDRs. One or more amino acid substitutions can be made within the frame region, and amino acid substitutions can be selected that can improve binding of the antibody to its antigen. Also, antibody molecules in which one or more interchain disulfide bonds are deleted can be produced in this manner by substituting or deleting cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds. Other modifications made to the polynucleotide within the skill of the art are also included in the present invention.

[0497] Antibodies can be produced by conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured by techniques known to those of skill in the art. All of these techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DLHacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including any supplementary content, are incorporated herein by reference in their entirety.

[0498] In one or more embodiments, the anti-FRα antibody of the present invention or its antigen-binding unit is glycosylation-modified. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be produced. By altering the glycosylation, the affinity of the antibody for the antigen can be increased, for example. Such modification can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions are made to remove one or more variable region glycosylation sites, thereby removing the glycosylation at the site. Such deglycosylation can increase the affinity of the antibody for the antigen. Such methods are described in further detail in PCT Publication WO2003016466 A2 and US Patents 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

[0499] The antibody (e.g., anti-FRα antibody) gene can be inserted into an expression vector by standard methods (e.g., ligation of the antibody gene fragment into complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). The expression vector can be a plasmid, retrovirus, YAC, EBV-induced adduct, etc. To express an antibody (e.g., anti-FRα antibody), DNA encoding the full-length light and heavy chains can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. By "operably linked" it is meant that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their expected function of regulating the transcription and translation of the antibody gene.

[0500] The J regions of the heavy and light chain consensus sequences may be used to design oligonucleotides to be used as primers to link the V region fragment to the human C region fragment after introducing a useful restriction site into the J region. The cDNA of the C region may be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence. In one or more embodiments, the expression vector already carries the antibody constant region sequence prior to the insertion of the light or heavy chain gene sequence associated with the antibody. For example, one way to convert the VH and VL sequences associated with an anti-FRα antibody into a full-length antibody gene is to insert them into an expression vector that already encodes the heavy and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment in the vector, and the VL segment is operably linked to the CL segment in the vector.

[0501] The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody heavy and light chains by a host cell. Alternatively, the antibody heavy and light chain genes can be cloned into a vector that encodes a signal peptide that facilitates secretion of the antibody heavy and light chains by a host cell such that the signal peptide is linked in-frame to the amino terminus of the antibody heavy and light chain genes. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein), such as MDWTWRILFLVAAATGAHS (SEQ ID NO: 21), MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 22).

[0502] In addition to the antibody heavy and light chain genes, the recombinant expression vector can also carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, 1990. Those skilled in the art will recognize that the design of the expression vector, including the selection of regulatory sequences can depend on factors such as the choice of the host cell to be transformed, the expression level of protein desired, and the like. Suitable regulatory sequences for use in mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (such as the adenovirus major processive promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patents 5,168,062, 4,510,245 and 4,968,615.

[0503] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors can carry other sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665 and 5,179,017). For example, one can usually select a marker gene that confers resistance to drugs (e.g., G418, hygromycin or methotrexate) on the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR- host cells with methotrexate selection / amplification), the neo gene (for G418 selection) and the GS gene. For expression of the heavy and light chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques.

[0504] The antibody of the present invention (e.g., anti-FRα antibody) can be produced by recombinantly expressing antibody heavy and light chain genes in a host cell. For example, a host cell is transfected with one or more recombinant expression vectors carrying heavy and light chain DNA fragments encoding the antibody, whereby the heavy and light chains are expressed in the host cell, and the expressed antibody can be secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. "Transfection" refers to various techniques, such as electroporation, lipotransfection, calcium phosphate precipitation, and DEAE-glucanotransfection, that are commonly used to introduce foreign DNA into eukaryotic host cells. Standard recombinant DNA methods for obtaining antibody heavy and light chain genes, incorporating these genes into expression vectors, and introducing the vectors into host cells are well known in the art and are described, for example, in Molecular Cloning, A Laboratory Manual, 2nd Edition (Sambrook, Fritsch and Maniatis (eds.), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989) and U.S. Patent 4,816,397. The DNAs expressing the antibody heavy and light chains may be located in the same or different vectors.When placed in different vectors, the antibody heavy chain expressing vector and the light chain expressing vector can be transfected into the host cell in an appropriate ratio (e.g., Tihomir S. Dodev et al., A tool kit for rapid cloning and expression of recombinant antibodies, Scientific Reports volume 4, Article number: 5885 (2014); Stefan Schlatter et al., On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells, Biotechnol Progress, 21: 122-133 (2005); Hadi Bayat et al., Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells, Preparative Biochemistry & Biotechnology, 48 (8): 822-829 (2018)). In one or more embodiments, the antibody expression vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements may include enhancers, a Kozak sequence (GCCACC as shown in SEQ ID NO: 33) and donor and acceptor sites for RNA splicing on either side of the inserted sequence. Highly efficient transcription can be obtained with the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and several other cellular promoters such as the actin promoter can also be applied.Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2, or pCHO1.0, and the like.

[0505] The antibody of the present invention (e.g., anti-FRα antibody) can be expressed in eukaryotic host cells. In some embodiments, the expression of the antibody is carried out in eukaryotic cells, such as mammalian host cells. Exemplary host cells for expressing the antibody of the present invention include Chinese hamster ovary cells (CHO cells) (e.g., CHO-K1 cells) or modified CHO cells CHO-S, CHO-dhfr-, CHO / DG44 or ExpiCHO, NSO bone marrow cells, COS cells, Cos1 cells, Cos7 cells, SP2 cells, CV1 cells, mouse L cells, human embryonic kidney cells HEK293 or modified HEK293 cells HEK293T, HEK293F or HEK293E cells. After introducing the recombinant expression vector encoding the antibody chain gene into the host cell, the host cell is cultured in a medium to produce the antibody, during which the antibody is expressed in the host cell or the antibody is secreted into the medium. The antibody can be recovered from the medium using standard protein purification methods.

[0506] Antibody-producing cell lines can be selected, constructed and cultured by techniques known to those of skill in the art, all of which are described in a variety of laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DLHacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including any supplements, are incorporated by reference in their entirety.

[0507] For recombinant expression of an antibody of the present invention (e.g., an anti-FRα antibody), a host cell can be co-transfected with two recombinant expression vectors, a first recombinant expression vector encoding an antibody heavy chain and a second recombinant expression vector encoding an antibody light chain. The two recombinant expression vectors can contain the same selectable marker, or they can each contain a separate selectable marker. Alternatively, a host cell can be transfected with recombinant expression vectors encoding the antibody heavy and light chains.

[0508] The antibodies of the present invention (e.g., anti-FRα antibodies) can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd Edition, 1984 The Pierce Chemical Co., Rockford, Ill.). Mutant antibodies can also be produced using cell-free platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0509] The antibody of the present invention (e.g., anti-FRα antibody) produced by recombinant expression can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (such as ion exchange, affinity chromatography, and fractional column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. For example, affinity chromatography with protein A or protein G provides mainly the IgG fraction in immune serum. Also, the specific antigen targeted by the immunoglobulin or its epitope can be immobilized on a column to purify the immune specific antibody by immune affinity chromatography. The antibody of the present invention (e.g., anti-FRα antibody) can be fused to a heterologous polypeptide sequence known in the art to facilitate purification. For the purification of immunoglobulins, see D. Wilkinson's article (The Scientist, The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0510] Furthermore, mutations, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions, may be introduced into the nucleotide sequence encoding the antibody of the present invention by standard techniques known to those skilled in the art. Mutants (including derivatives) encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original heavy chain variable region VH CDR1, VH CDR2, VH CDR3 and light chain variable region VL CDR1, VL CDR2, VL CDR3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and mutants that retain activity can be identified by screening the biological activity of the resulting mutants. In one or more embodiments, the substitutions described herein are conservative amino acid substitutions.

[0511] The amino acid sequence of an exemplary Antibody 1 heavy chain of the invention is shown in SEQ ID NO:19 and the gene sequence encoding it is shown in SEQ ID NO:23.

[0512] SEQ ID NO:19 is as follows: QVQLVQSGVEVKKPGASVKVSCKASGYSFTGYFMNWVRQAPGQGLEWIGRIHPYDGDTFYNQNFKDKATLTVDKSTTTAYMELKSLQFDDTAVYYCTRYDGSRAMDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0513] SEQ ID NO:23 is as follows:

[0514] The amino acid sequence of an exemplary Antibody 1 light chain of the invention is shown in SEQ ID NO:20 and the gene sequence encoding it is shown in SEQ ID NO:24.

[0515] SEQ ID NO:20 is as follows: EIVLTQSPATLLSPGERATLSCKASQSVSFAGTSLMHWYQQKPGQAPRLLIYRASNLEAGVPARFSGSGSKTDFTLTISSLEPEDFAVYYCQQSREYPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0516] SEQ ID NO:24 is as follows: GAGATCGTGCTGACCCAGTCCCCCGCCACCCTGAGCCTGAGCCCAGGAGAGAGGGCCACCCTGTCCTGCAAGGCCTCCCAGAGCGTGAGCTTTGCCGGCACCAGCCTGATGCACTGGTATCAACAGAAGCCCGGCCAGGCCCCTAGGCTGCTGATCTACAGAGC CAGCAACCTGGAGGCCGGCGTGCCTGCTAGGTTTTCCGGCAGCGGCTCCAAGACCGATTTCACCCTGACCATCAGCAGCCTGGAGCCTGAGGATTTTGCCGTGTACTACTGCCAGCAGTCCAGGGAGTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGA TCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

[0517] Citation of publications and patent documents herein is not an admission that any of the above is relevant prior art, nor is it an admission of the contents or date thereof. While the present invention has been described in the form of a written specification, those skilled in the art will recognize that the invention can be embodied in various embodiments, and that the above specification and the following examples are intended to illustrate, but not limit, the scope of the invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0518] Working Example Materials, reagents and the like used in the following examples are all obtainable from commercial sources or known methods unless otherwise specified.

[0519] Example 1: Antigen Production Preparation of antigen FRα-His: The gene sequence encoding antigen FRα-His (the amino acid sequence of antigen FRα-His is shown in SEQ ID NO:28, constructed by adding 8×HIS tag (HHHHHHHH) to the C-terminus of human FRα extracellular domain (shown in SEQ ID NO:27, underlined part is signal peptide)) was cloned into an expression vector to construct a recombinant expression vector, which was then stably transfected into CHO-K1 cells, cultured and purified to obtain antigen FRα-His.

[0520] Amino acid sequence of human FRα extracellular domain: MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAM (SEQ ID NO: 27)

[0521] Amino acid sequence of antigen FRα-His: MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMHHHHHHHH (SEQ ID NO: 28)

[0522] Example 2: Anti-FRα antibody The heavy chain of the anti-FRα antibody consists of VH and CH, and the light chain consists of VL and CL, with the amino acid sequence of CH shown in SEQ ID NO: 17 and the amino acid sequence of CL shown in SEQ ID NO: 18. Among them, the VH CDR and VL CDR are shown in Table 2, the framework regions of the heavy and light chains are shown in Table 3, the amino acid sequences of the variable regions are shown in Table 4, and the amino acid sequence of the constant region is shown in Table 5. The amino acid sequences of the heavy and light chains of the control antibody huMOV19 are shown in Table 6.

[0523] The gene sequences encoding the antibody heavy and light chains were cloned into expression vectors, respectively, to construct recombinant expression vectors, which were then transiently transfected into HEK293F cells, cultured and purified to obtain antibodies, which were sequenced and found to be consistent with the desired sequence.

[0524] [Table 2]

[0525] [Table 3]

[0526] [Table 4]

[0527] [Table 5]

[0528] [Table 6]

[0529] Example 3: Detection of antibody affinity and species specificity 3.1 Antibody affinity specificity Experimental Reagents and Consumables: 1. Antigen: FRα-His (ie, FOLR1), obtained by the preparation of Example 1.

[0530] FOLR2 was purchased from Acro, product number FO2-H5223, lot number 2792C-88HF1-K6. FOLR3 was purchased from R&D with product number 5319-FR-050 and lot number RBK0319041.

[0531] 2. Probe: Protein A Chip sensor was purchased from GE Healthcare, Cas#29127556.

[0532] 3. Reagents: HBS-EP+(10×): purchased from GE Healthcare, cat#BR-1006-69; Glycine pH 1.5: purchased from GE Healthcare, cat#BR100354; Running Buffer: 50 mL of HBS-EP+ (10x) was measured, and 450 mL of ultrapure water was added and mixed uniformly.

[0533] Experimental equipment: Biacore:T200 was purchased from GE Healthcare.

[0534] Testing Procedure: Detection was performed using a Protein A chip. The antibody was diluted to 5 μg / mL using running buffer, passed through the experimental channels (Fc2, Fc4) at a flow rate of 10 μL / min, and captured for 15 s until the capture amount was approximately 440 RU. Then, the flow rate was adjusted to 30 μL / min, and antigen dilutions of different concentrations of the analyte (0 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, diluted with running buffer) were added in sequence, and passed simultaneously through the surfaces of the experimental channels (Fc2, Fc4) and the reference channels (Fc1, Fc3). The binding time was 180 s, and the dissociation time was 450 s. Finally, the chip was regenerated with Glycine pH 1.5 for 60 s, and then the next cycle was started.

[0535] Result processing: The test results were analyzed using the data analysis software Evaluation Software 3.1, and the sensing signals collected by the test flow path of the sample were subtracted from the reference flow path and the sample blank twice, and the kinetic "1:1" model was fitted to obtain the kinetic parameters (Ka: binding rate, Kd: dissociation rate, KD: binding dissociation equilibrium constant) of the same antigen of each sample. The results are shown in Table 7.

[0536] [Table 7]

[0537] The results in Table 7 demonstrate that Antibody 1 can specifically bind to FOLR1.

[0538] 3.2 Antibody species specificity The antigen was diluted to 1 μg / mL with PBS and added to a plate reader (manufacturer: Corning, product number: 42592) at a volume of 100 μL / well and placed in a refrigerator (2 °C to 8 °C) for overnight coating. The coating solution was discarded, and 200 μL of blocking solution (containing 5% nonfat powder, prepared with PBS) was added, and the plate was placed in a 37 °C thermostatic incubator and left for 2 h. The blocking solution was discarded, and the plate was washed three times with 0.05% PBST. Different concentrations of antibody dilutions (initial concentration 6 μg / mL, diluted 3-fold, total 8 concentration gradient, diluted with PBS) were added at 100 μL / well. The plate was placed in a 37 °C thermostatic incubator and left for 2 h. The antibody dilutions were discarded, and the plate was washed eight times with 0.05% PBST, and then tapped dry. Anti-Human Kappa Light Chains (Bound and Free) peroxidase antibody produced in goat (manufacturer: sigma, product number: A7164-1ML) diluted 1:10000 was added at 100μL / well. The plate was placed in a thermostatic incubator at 37℃ and left to stand for 1h. The antibody dilution solution was discarded, washed 8 times with 0.05% PBST, and tapped dry. Single-component TMB color development solution I (manufacturer: Huzhou Eichuang Biotechnology Co., Ltd., product number: TMB-S-001) was added at 100μL / well, and the plate was placed in a thermostatic incubator at 37℃ and left to stand in the dark for 10min. The reaction was terminated by adding 0.1M H2SO4 at 100μL / well. The absorbance wavelength was 450nm as read by SpectraMax Plus Absorbance Microplate Reader.

[0539] [Table 8]

[0540] Table 8 shows that antibody 1 can bind to FRα-His and cynomolgus monkey FOLR1, but does not bind to mouse FOLR1 or rat FOLR1.

[0541] Example 4: Steps for the synthesis of compound (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione hydrochloride (D-1) 1. Synthesis of N,N'-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide [ka] In a nitrogen gas atmosphere, potassium tert-butoxide tetrahydrofuran solution (42 mL, 1 M) was added to a dry reaction flask, stirred, and cooled to 0-5 ° C. N-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (CAS number: 143655-49-6, 5 g, 21 mmol) dissolved in tetrahydrofuran (25 mL) was gradually added dropwise to the reaction flask, and then tert-butyl nitrite (4.32 g, 2 eq) was added dropwise (temperature was controlled at 0-5 ° C.), and the mixture was heated to 15-20 ° C. and stirred for 2 h. After the reaction was completed, the temperature was lowered to 0-5 ° C., and acetic acid (25 mL) and acetic anhydride (25 mL) were added dropwise (temperature was controlled below 10 ° C.), and the mixture was stirred for 20 min after the completion of the addition. The mixture was kept at 5-10°C, and zinc powder (8eq) was added according to the amount of N-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide, and stirred at 20-25°C for 1h. It was filtered, the solid was rinsed with ethyl acetate (50mL), cooled to 0-5°C, washed three times by dropwise addition of 15% aqueous Na2CO3 (50mL), extracted with ethyl acetate (25mL), and the organic phases were combined and washed with saturated aqueous NaCl. Ethyl acetate (10mL) was added, stirred at 40°C for 30min, cooled gradually to 0-5°C, and stirred for 2h. It was filtered, and the solid was washed with ethyl acetate / petroleum ether (1 / 2, 10mL). It was dried in vacuum to give a grey powder (2.1g, 33.7%). LC-MS: [M+H] + =297.

[0542] 2. Synthesis of N-(8-amino-5,6-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide [ka] N,N'-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (500 mg, 1.68 mmol) was added to a 2M hydrochloric acid ethanol solution (5 mL), stirred at 50°C for 4 h, and after detecting complete reaction, water (7.5 mL) was added, the temperature was lowered to 0-5°C, triethylamine (1.03 g) was added dropwise, and stirred for 3 h. The mixture was filtered and washed with 40% cold aqueous ethanol solution (3 mL) and water (3 mL). The mixture was dried in vacuum to obtain a gray powder (320 mg, 74.6%). LC-MS: [M+H] + =255.

[0543] 3. Synthesis of N-((9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)acetamide [ka] In a nitrogen atmosphere, N-(8-amino-5,6-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1.1 g, 1 eq), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1.15 g, 1 eq) and toluene (50 mL) were added to a reaction flask, heated to reflux and stirred for 1 h, then 4-methylbenzenesulfonic acid pyridine (100 mg) was added and stirring was continued for 20 h. The mixture was cooled to room temperature and stirred for 1 h. The solid was filtered and washed with acetone (10 mL) and cold ethanol (5 mL), respectively. The mixture was dried in vacuum to obtain a gray-brown powder (1.1 g, 53%). LC-MS: [M+H] + =482.

[0544] 4. Synthesis of (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione hydrochloride (D-1) [ka] N-((9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)acetamide (1.1 g, 2.28 mmol) and 6M aqueous hydrochloric acid (44 mL) were added to the reaction flask and stirred under reflux for 4 h under nitrogen gas. The solvent was concentrated to remove and purified by HPLC to give a white powder (200 mg, 18%). LC-MS: [M+H] + =440.

[0545] Example 5: Steps for the synthesis of 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxo-27,32,35-triazaheptatriacontane-37-amino)benzyl (4-nitrophenyl)carbonate (CB07) [ka] 1) Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-aza-hentriacontan-31-oic acid (CB01) [ka] Under nitrogen gas protection at 0-5 ° C, 8.14 g of N2-fluorenylmethyloxycarbonyl-L-2,4-diaminobutyric acid (CB00-2) was dissolved in 40 mL of dimethylformamide (DMF), 10 g of 4,7,10,13,16,19,22,25-octaoxahexacosanoic acid-N-succinimidyl (CB00-3) and 10 mL of DMF were added, and 6.5 mL of DIPEA was added dropwise while maintaining at 0-5 ° C., and 1 h after the addition was completed, the reaction was allowed to proceed for 4 h while stirring at room temperature, and after the reaction was completed, DMF was removed under reduced pressure, and 14.2 g of pale yellow oily liquid CB01 was obtained by silica gel column chromatography (dichloromethane and methanol in a volume ratio of 20:1 were used as elution solvent).

[0546] 2) Synthesis of (S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamic acid (9H-fluoren-9-yl)methyl ester (CB02) [ka] At room temperature and under nitrogen gas protection, 11 g of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (CB00-4) and 5.5 g of p-aminobenzyl alcohol (CB00-5) were dissolved in 400 mL of dichloromethane and 200 mL of methanol, and 17 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) was added in several portions under mechanical stirring, and the reaction was allowed to proceed in the dark for 15 h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a paste-like solid, and 11.9 g of a gray-white solid CB02 was obtained by silica gel column chromatography (elution solvent was dichloromethane and methanol in a volume ratio of 20:1).

[0547] 3) Synthesis of (S)-2-((S)-2-amino-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CB03) [ka] At room temperature and under nitrogen gas protection, 11.9 g of [(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamic acid (9H-fluoren-9-yl)methyl (CB02) was added to 300 mL of acetonitrile, and 18 mL of piperidine was added dropwise while stirring. After the dropwise addition was completed, the reaction was allowed to proceed for 2 hours at room temperature. After the reaction was completed, the solvent and piperidine were removed by distillation under reduced pressure, and 7.5 g of white solid CB03 was obtained by silica gel column chromatography (dichloromethane and methanol in a volume ratio of 20:1 were used as the elution solvent).

[0548] 4) Synthesis of (9H-fluoren-9-yl)methyl ((30S,33S,36S)-41-amino-36-((4-(hydroxymethyl)phenyl)carbamoyl)-33-isopropyl-26,31,34,41-tetraoxo-2,5,8,11,14,17,20,23-octaoxa-27,32,35,40-tetraaz-30-yl)carbamate (CB04) [ka] In a nitrogen gas atmosphere at 0°C, 14.2g of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-aza-hentriacontan-31-oic acid (CB01) was dissolved in 100mL of DMF, and 11g of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (HATU) was added in several portions, and the mixture was stirred and reacted for 30 minutes. After that, 7.5g of (S)-2-((S)-2-amino-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CB03) was added, and the mixture was reacted for 2.5h while maintaining the temperature at 0°C. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 9.66 g of solid CB04 was obtained by silica gel column chromatography (elution solvent was dichloromethane and methanol in a volume ratio of 10:1).

[0549] 5) Synthesis of N-((3S)-3-amino-4-(((2S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxy)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB05) [ka] At room temperature and under nitrogen gas protection, 9.66 g of (9H-fluoren-9-yl)methyl ((30S,33S,36S)-41-amino-36-((4-(hydroxymethyl)phenyl)carbamoyl)-33-isopropyl-26,31,34,41-tetraoxo-2,5,8,11,14,17,20,23-octaoxa-27,32,35,40-tetraaz-30-yl)carbamate (CB04) was dissolved in 50 mL of DMF, 12 mL of diethylamine was added, and the mixture was stirred and reacted for 1.5 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 7.7 g of pale yellow solid CB05 was obtained by silica gel column chromatography (using dichloromethane and methanol in a volume ratio of 7.5:1 as the elution solvent).

[0550] 6) Synthesis of N-((3S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)-4-(((2S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-butanon-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB06) [ka] 7.7 g of N-((3S)-3-amino-4-(((2S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB05) was dissolved in 40 mL of DMF, and maleimidoacetic acid N-hydroxysuccinimide ester (CB00-1) was added in several portions at 0-5°C in a nitrogen gas atmosphere, and the mixture was reacted for 4 h while maintaining the temperature at 0-5°C. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 9.5 g of pale yellow solid CB06 was obtained by silica gel column chromatography (dichloromethane and methanol in a volume ratio of 10:1 were used as elution solvent).

[0551] 7) Synthesis of 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxo-27,32,35-triazaheptatriacontane-37-amino)benzyl(4-nitrophenyl)carbonate (CB07) [ka] 9.5g of N-((3S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)-4-(((2S)-1-((1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-butanon-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB06) was dissolved in 50mL of DMF, and 14.0g of bis(p-nitrobenzene)carbonate ((PNP)2CO) was added in a nitrogen gas atmosphere at 0℃. After dissolution, 8.2mL of N,N-diisopropylethylamine (DIPEA) was further added, and the mixture was reacted for 4h while maintaining the temperature at 0℃. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 2.6 g of a white solid, CB07, was obtained by silica gel column chromatography (elution solvent: dichloromethane and methanol in a volume ratio of 8:1).

[0552] Example 6: Synthesis of 4-((18S,21S,24S)-18-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxo-15,20,23-triazapentacosane-25-amino)benzyl(4-nitrophenyl)carbonate (CB14) [ka]

[0553] CB14 was prepared by following the synthesis of CB07 in Example 5, except that 4,7,10,13,16,19,22,25-octaoxahexacosanoic acid-N-succinimidyl ester was replaced with 4,7,10,13-tetraoxatetradecanoic acid-N-succinimidyl ester, to finally obtain a white solid CB14.

[0554] Example 7: Synthesis of intermediate (CB07-Exatecan) Synthesis of 4-(30S,33S,36S)-30-(2-(2,5-dioxane-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-triheptaheptyl-37-aconylidenealkyl)-(1s,9s)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoline[1,2-b]quinoline-1-carbamate (CB07-Exatecan) [ka] 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxo-27,32,35-triazaheptatriacontane-37-amino)benzyl(4-nitrophenyl)carbonate (CB07) (2.6 g, 2.21 mmol) and N,N-dimethylformamide (23 mL) were added to reaction flask R1, and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. At the same time, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[3',4':6,7]indoline[1,2-b]quinoline-10,13-dione mesylate (exatecan mesylate, 0.98 g, 1.84 mmol, Advanced ChemBlocks) was added to another reaction flask R2 together with N,N-dimethylformamide (5 mL), and triethylamine (230 mg, 2.27 mmol) was added dropwise at 0-5 ° C. and stirred until completely dissolved. After the solution in reaction flask R2 was added dropwise to reaction flask R1, reaction flask R2 was washed with N,N-dimethylformamide (2 mL), and the washings were added to reaction flask R1. 1-Hydroxybenzotriazole (497 mg, 3.68 mmol) and pyridine (1.45 g, 18.4 mmol) were weighed again and added to reaction flask R1. The mixture was stirred at 0-5°C for 10 min, warmed to room temperature and stirred for 5.5 h. After the reaction was completed, the mixture was concentrated under reduced pressure at 35°C to remove the solvent. The mixture was purified by preparative high performance liquid chromatography (prep-HPLC) and freeze-dried to obtain a white powder (CB07-Exatecan, 1.6 g, 59%). LC-MS: [1 / 2M+H] + =737.

[0555] Example 8: Synthesis of intermediate (CB07-D-1) Synthesis of 4-(30S,33S,36S)-30-(2-(2,5-dioxane-2,5-dihydro-1h-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-triheptaheptyl-37-aconylidenealkyl)-(1s,9s)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoline[1,2-b]quinoline-1-carbamate (CB07-D-1) [ka] 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxo-27,32,35-triazaheptatriacontane-37-amino)benzyl(4-nitrophenyl)carbonate (CB07) (220 mg, 0.189 mmol) and N,N-dimethylformamide (5 mL) were added to reaction flask R1, and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. At the same time, (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione hydrochloride (D-1) (90 mg, 0.189 mmol) was added to another reaction flask R2 together with N,N-dimethylformamide (5 mL), and 3 drops of triethylamine were added dropwise at 0-5 ° C. and stirred until completely dissolved. The solution in the reaction flask R2 was added dropwise to the reaction flask R1, and 1-hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) were further weighed and added to the reaction flask R1. The mixture was stirred at 0-5 ° C. for 10 min, warmed to room temperature and stirred for 3 h. After the reaction was completed, the solvent was removed by vacuum concentration. Purification by preparative high performance liquid chromatography (prep-HPLC) and lyophilization gave a white powder (CB07-D-1, 55 mg, 20%). LC-MS: [1 / 2M+H] + =739.

[0556] Example 9: Synthesis of intermediate (CB14-Exatecan) Synthesis of 4-(18S,21S,24S)-18-(2-(2,5-dioxane-2,5-dihydro-1h-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxo-15,20,23-triazapentacosane-25-amino)-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoline[1,2-b]quinoline-1-carbamate (CB14-Exatecan) [ka] Similarly, 4-((18S,21S,24S)-18-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxo-15,20,23-triazapentacosane-25-amino)benzyl(4-nitrophenyl)carbonate (190 mg, 0.19 mmol) was added to reaction flask R1 together with N,N-dimethylformamide (23 mL), and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. At the same time, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[3',4':6,7]indoline[1,2-b]quinoline-10,13-dione mesylate (exatecan mesylate, 101 mg, 0.19 mmol, Advanced ChemBlocks) was added to another reaction flask R2 together with N,N-dimethylformamide (5 mL), triethylamine (3 drops) was added dropwise at 0-5 ° C, and the mixture was stirred until completely dissolved. After the solution in the reaction flask R2 was added dropwise to the reaction flask R1, the reaction flask R2 was then washed with N,N-dimethylformamide (1 mL), and the washings were added to the reaction flask R1. 1-Hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) were further weighed and added to reaction flask R1. The mixture was stirred at 0-5°C for 10 min, warmed to room temperature and stirred for 5.5 h. After the reaction was completed, the mixture was concentrated under reduced pressure at 35°C to remove the solvent. The mixture was purified by preparative high performance liquid chromatography (prep-HPLC) and freeze-dried to obtain white powder CB14-Exatecan.

[0557] Example 10: Synthesis of ADC [ka] ADC1 coupling process (batch 1): Reduction reaction: The concentration of antibody 1 was adjusted to 18 g / L using a coupling buffer (10 mM aqueous succinic acid solution), a 0.1 M aqueous EDTA solution was added until the final EDTA concentration became 2 mM, 5.4 molar equivalents of a 0.2 M aqueous TCEP solution was added according to the amount of the antibody substance, the pH was adjusted to 7, and the reaction was carried out at 25°C and stirred at 180 rpm for 2 hours to reduce the antibody interchain disulfide bonds to free sulfhydryl groups.

[0558] Ten volumes of liquid were exchanged by ultrafiltration using a 30KD ultrafiltration membrane to remove the reducing agent TCEP in the system.

[0559] Coupling reaction: According to the amount of antibody substance, 15 times molar equivalent of 100 mM CB07-Exatecan in dimethylacetamide (DMA) was added, and the mixture was reacted at 25°C and 180 rpm for 2 h. Finally, 0.2 M N-acetylcysteine ​​aqueous solution was added until the final concentration was 2 mM, and the reaction was continued for 15 min to stop the coupling reaction. After purifying the reaction mixture and ultrafiltration, ADC1 with a concentration of 23.56 mg / mL was obtained, and the DAR, i.e., p, measured by reverse phase chromatography was 8. Unless otherwise stated, in the following examples, ADC1 is batch 1.

[0560] ADC2 coupling process: Reduction reaction: The concentration of antibody 1 was adjusted to approximately 18 g / L using a coupling buffer (10 mM aqueous succinic acid solution), a 0.1 M aqueous EDTA solution was added until the final EDTA concentration became 2 mM, 2.5 molar equivalents of a 10 mM aqueous TCEP solution were added depending on the amount of the antibody substance, the pH was adjusted to 7, and the reaction was carried out with shaking on a shaker at 25°C for 2 hours to reduce the antibody interchain disulfide bonds to free mercapto groups.

[0561] Ten volumes of liquid were exchanged by ultrafiltration to remove the reducing agent TCEP in the system.

[0562] Coupling reaction: Depending on the amount of antibody substance, 6 times molar equivalent of 100 mM CB07-Exatecan in dimethylacetamide (DMA) was added and the reaction was performed for 1 h on a shaker at 25 ° C. Finally, 0.1 M N-acetylcysteine ​​aqueous solution was added until the final concentration was 1.5 mM, and the reaction was continued for 15 min to stop the coupling reaction. After purifying the reaction mixture, ADC2 with a concentration of 1.34 mg / mL was obtained, and the DAR, i.e., p, measured by reverse phase chromatography was 4.

[0563] Example 11: In vitro activity measurement of ADC1 1) Binding activity of ADC1 to JEG-3 cells (FCM) The binding activity of ADC1 and Antibody 1 to JEG-3 cells was detected using flow cytometry.

[0564] Testing Procedure: 1. JEG-3 cells were seeded in a 96-well plate (manufacturer: Corning, product number: 3897) at 300,000 cells / well. The plate was centrifuged at 1500 rpm for 5 minutes using a centrifuge, and the supernatant was removed.

[0565] 2. Diluted ADC1 and Antibody 1 dilutions were added at 200 μL / well, respectively, with 2-fold dilutions starting from a concentration of 25 nM, for a total of 10 concentration gradients. Three duplicate wells were set up for each concentration. A blank control was set up, and a secondary antibody control was incubated. Incubated on ice for 1 h.

[0566] 3. The mixture was centrifuged at 1500 rpm for 5 minutes using a centrifuge, and the supernatant was discarded. 200 μL of PBS was added and blown evenly, followed by centrifugation at 1500 rpm for 5 minutes, and the supernatant was discarded.

[0567] 4. Add 100 μL / well of 1:500 diluted Anti-Hu IgG (Fcγ-specific) PE (manufacturer: eBioscience, product number: 12-4998-82) and incubate on ice in the dark for 0.5 h.

[0568] 5. Centrifugation was performed at 1500 rpm for 5 minutes using a centrifuge, and the supernatant was discarded. 200 μL of PBS was added and blown evenly, and centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. This was repeated once.

[0569] 6. 200 μL of PBS was added and blown evenly. The results were detected by CytoFLEX.

[0570] 7. Data analysis was performed using GraphPad Prism.

[0571] [Table 9]

[0572] The results, as shown in Table 9, showed that both Antibody 1 and ADC1 had strong binding activity to JEG3 cells.

[0573] 2) Binding activity of ADC1 to FRα-His (Biacore) The affinity of ADC1 and Antibody 1 for FRα-His was assessed by Biacore T200 surface plasmon resonance (SPR) technology.

[0574] reagent: HBS-EP+(10×): purchased from GE Healthcare, cat#BR-1006-69; Glycine pH 1.5: purchased from GE Healthcare, cat#BR100354; Running Buffer: 50 mL of HBS-EP+ (10x) was measured, and 450 mL of ultrapure water was added and mixed uniformly.

[0575] Experimental equipment: Biacore:T200 was purchased from GE Healthcare.

[0576] Testing Procedure: Detection was performed using a Protein A chip. The drug (ADC1 or antibody 1) was diluted to 5 μg / mL using the running buffer, passed through the experimental channels (Fc2, Fc4) at a flow rate of 10 μL / min, and captured for 15 s until the capture amount was about 440 RU. Then, the flow rate was adjusted to 30 μL / min, and different concentrations of analyte FRα-His dilutions (0 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, diluted with running buffer) were added in sequence, and passed simultaneously through the surfaces of the experimental channels (Fc2, Fc4) and the reference channels (Fc1, Fc3). The binding time was 180 s, and the dissociation time was 450 s. Finally, the chip was regenerated with Glycine pH 1.5 for 60 s, and then proceeded to the next cycle.

[0577] Result processing: The test results were analyzed using the data analysis software Evaluation Software 3.1, and the sensing signals collected by the test flow path of the sample were subjected to double subtraction of the reference flow path and the sample blank, and then fitted with the kinetic "1:1" model to obtain the kinetic parameters (Ka: binding rate, Kd: dissociation rate, KD: binding dissociation equilibrium constant) of the same antigen of each sample, and the results are shown in Table 10. The results showed that Antibody 1 and ADC1 had similar affinities for FRα-His.

[0578] [Table 10]

[0579] Example 12: Endocytosis of ADC1 in FRα-expressing cell lines The endocytosis of ADC1 in the FRα-expressing cell line KB was observed using a confocal microscope, with the FRα-negative cell line SNU-1 as a control. After overnight culture in a cell culture dish, lysosomal staining LysoBrite Red solution (BBI Life Sciences, Shanghai, China) was added and the cells were incubated for 1.5 h. After washing, the cells were treated with fitc-labeled ADC1 (10 μg / mL) and bound to ADC1 for 20 min at 4 °C, and then washed three times with PBS to remove unbound ADC1. The cells were then cultured at 37 °C to allow ADC1 to be internalized. At each time point, the cells were fixed with 4% paraformaldehyde and observed under a Zeiss LSM710 confocal microscope. As shown in Figure 2, before internalization, there was no fitc-labeled ADC1 in the cells. However, 3 h after internalization, more fitc-labeled ADC1 was detected in the cells, and most of it overlapped with lysosomes.

[0580] Example 13: In vitro cytotoxicity of ADC1 ADC1-mediated in vitro cytotoxicity was evaluated in FRα-positive cell lines JEG-3, MCF-7. Cells were harvested with trypsin and cultured with gradient dilutions of ADC1, followed by incubation at 37°C. Viability was measured after 5 days using CCK-8. IC 50 The results were read and analyzed on a SpectraMax Gemini (Mitani Molecular) to determine the half-maximal inhibitory concentration (HAC) values.

[0581] Testing Procedure: 1) The cell density of JEG-3 cells was adjusted to 60,000 cells / mL with DMEM + 10% FBS, and the cells were seeded into a 96-well plate (manufacturer: Corning, product number: 3599) at 100 μL / well. The plate was then placed in a Series II Water Jacketed CO2 Incubator and left to stand at 37°C, 5% CO2 for 3 hours.

[0582] The cell density of MCF-7 cells was adjusted to 40,000 cells / mL with DMEM + 2% FBS, and the cells were seeded into a 96-well plate (manufacturer: Corning, product number: 3599) at 100 μL / well. The plate was then placed in a Series II Water Jacketed CO2 Incubator and left to stand at 37°C and 5% CO2 for 2 hours.

[0583] 2) ADC1 was diluted with the medium of the corresponding cells.

[0584] JEG-3 cells: ADC1 concentrations were diluted 4-fold from 1000 nM for a total of 8 concentration gradients and added to cells in a volume of 100 μL / well.

[0585] MCF-7 cells: ADC1 concentrations were diluted 4-fold from 250 nM for a total of 8 concentration gradients and added to cells in a volume of 100 μL / well.

[0586] The lethal control was selected as Exatecan at 3000 nM, and the blank control was the medium of the corresponding cells.

[0587] Three replicate wells were set up for each concentration.

[0588] 3) The cells were placed in a Series II Water Jacketed CO2 Incubator at 37°C and 5% CO2 and cultured for 5 days.

[0589] 4) After 5 days, the culture supernatant was discarded, and RPMI basal medium 1640 (1x) containing 10% CCK-8 was added. The cells were then placed in a Series II Water Jacketed CO2 Incubator and left to stand at 37°C, 5% CO2 for approximately 1 hour.

[0590] 5) The plate was read using a SpectraMax M3 plate reader, and the absorption wavelength was 450 nm.

[0591] 6) Data analysis: Data from wells treated with Exatecan were treated as complete kill controls, and the blank controls were treated as zero kill controls. Cell viability was calculated as follows: Cell viability (%) = (experimental group - lethal control group) / (blank control group - lethal control group) x 100

[0592] 7) Data processing and analysis were performed using GraphPad Prism.

[0593] The results are shown in Table 11. As can be seen from the results, ADC1 had high in vitro cytotoxicity against FRα-positive cell lines.

[0594] [Table 11]

[0595] Example 14: Bystander effect of ADC1 To demonstrate the bystander effect induced by ADC1, an in vitro co-culture cell killing assay and a conditioned medium (CM) cytotoxicity assay were performed.

[0596] We selected FRα-positive JEG-3 cells and FRα-negative A549 cells (ADC1 has virtually no killing effect on A549 cells) to conduct experiments. JEG-3 cells were treated with ADC1 for 2, 3, and 4 days, respectively. Then, CM was transferred to A549 cells, and the changes in cell viability were monitored, and it was found that the viability of A549 cells was significantly decreased.

[0597] Testing Procedure: 1) The cell density of JEG-3 cells was adjusted to 100,000 cells / mL with DMEM + 10% FBS, and the cells were seeded into a 96-well plate (manufacturer: Corning, product number: 3599) at 100 μL / well. The plate was then placed in a Series II Water Jacketed CO2 Incubator and allowed to stand at 37°C, 5% CO2 for 2 hours.

[0598] 2) ADC1 was diluted in DMEM + 10% FBS medium, and the concentration was 4-fold diluted from 2000 nM to form a 10 concentration gradient, which was then added to the cells in a volume of 100 μL / well.

[0599] 3) Steps 1) and 2) were repeated on the second and third days.

[0600] 4) On the fifth day, the cell density of A549 was adjusted to 40,000 cells / mL with DMEM + 2% FBS medium, and 100 μL / well of the cells were inoculated into a 96-well plate and placed in a Series II Water Jacketed CO2 Incubator and left at 37°C and 5% CO2 for 2 hours. The culture supernatants of steps 1), 2), and 3) were added at 100 μL / well. 100 μL of 3000 nM Exatecan was added to column 1 as a lethal control, and 100 μL of DMEM + 2% FBS medium was added to column 12 as a blank control.

[0601] 5) The cells were cultured in a Series II Water Jacketed CO2 Incubator for 3 days at 37°C and 5% CO2.

[0602] 6) The culture medium was discarded and DMEM medium containing 10% CCK-8 was added. The plate was placed in a Series II Water Jacketed CO2 Incubator and allowed to develop color for 1 hour in the dark at 37°C. The plate was then read using a SpectraMax M3 plate reader, and the absorbance wavelength was 450 nm.

[0603] 7) The data was analyzed. The formula for calculating cell viability is as follows: Cell viability (%) = (experimental wells - lethal wells) / (blank wells - lethal wells) x 100

[0604] 8) Data analysis was performed using GraphPad Prism.

[0605] The results, as shown in FIG. 3, showed that the culture supernatant obtained by co-culturing ADC1 and FRα-positive JEG-3 cells in vitro had a significant killing effect on A549 cells.

[0606] Example 15: In vivo animal studies of ADC 1) Pharmacodynamic evaluation of test drugs in HuPrime® ovarian cancer OV3756 subcutaneous xenograft BALB / c nude female mouse model. Grouping and administration method are shown in Table 12.

[0607] [Table 12]

[0608] Tumor tissues were harvested from mice bearing HuPrime® ovarian cancer xenograft model OV3756 tumors, cut into tumor masses with a diameter of 2-3 mm, and inoculated subcutaneously into the right front scapula of 6-7 week-old female BALB / c nude mice.

[0609] The average tumor volume of tumor-bearing mice was approximately 137.55 mm 3 When the mice reached the age of 18, they were randomly assigned to groups and administered the treatment. The day of group assignment was set as day 0, and administration began on day 0.

[0610] After tumor inoculation, conventional monitoring includes the effect of tumor growth and treatment on the normal behavior of the animals, specifically, activity of the experimental animals, feeding and drinking status, weight gain or loss (weight was measured twice a week), eyes, hair coat and other abnormalities. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (wherein a represents the long diameter and b represents the short diameter).

[0611] [Table 13]

[0612] The tumor growth status of each treatment group and control group of the OV3756 xenograft model is shown in Table 13 and Figure 4. Table 13 and Figure 4 show that ADC1 and ADC2 can significantly inhibit the tumor growth of the OV3756 xenograft model.

[0613] 2) The study of the in vivo antitumor therapeutic effect of experimental drugs in the Balb / c nude mouse JEG-3 subcutaneous model was aimed at examining the in vivo efficacy of ADC1 in the JeG-3 model. The grouping and administration method are shown in Table 14.

[0614] [Table 14]

[0615] Select 6- to 8-week-old female Balb / c nude mice and incubate them at 1 × 10 6 JEG-3 cells were suspended in 100 μL of EMEM medium containing 50% Matrigel and inoculated subcutaneously into the right side of the mice. The average tumor volume was approximately 118 mm. 3 When the tumor volume reached 100 mg / kg, the animals were randomly assigned to groups of 8 animals each according to their body weight and tumor volume and administered the treatment.

[0616] After the start of administration, the tumor volume was measured twice a week. Tumor volume was calculated using the following formula: tumor volume (mm 3 )=0.5a×b 2 (wherein, a represents the long axis of the tumor and b represents the short axis of the tumor).

[0617] The results, as shown in Figure 5, show that treatment with ADC1 had a significant tumor inhibitory effect compared to the control group and was dose-dependent (p<0.001). By day 8 after administration, the TGI was 98.98% (2.5mg / kg) and 100.00% (5mg / kg), respectively. Of particular note, ADC1 caused complete tumor regression, which was maintained until the end of the experiment.

[0618] 3) Pharmacodynamic evaluation of experimental drugs in the HuPrime® lung cancer LU11554 subcutaneous xenograft NOD / SCID mouse model Tumor tissues were harvested from mice bearing HuPrime® lung cancer xenograft model LU11554 tumors, cut into tumor masses with diameters of 2-3 mm, and inoculated subcutaneously into the right front scapula of NOD / SCID mice.

[0619] The average tumor volume of tumor-bearing mice was approximately 165.15 mm 3 When the mice reached the age of 18, they were randomly assigned to groups according to Table 15. The day of group assignment was set as day 0, and administration began on day 0. Details of the administration method, dose, and administration route are shown in Table 15.

[0620] [Table 15]

[0621] After tumor inoculation, conventional monitoring includes the effect of tumor growth and treatment on the normal behavior of the animals, specifically, activity of the experimental animals, feeding and drinking status, weight gain or loss (weight was measured twice a week), eyes, hair coat and other abnormalities. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (wherein a represents the long axis and b represents the short axis). Mice were euthanized once they had lost more than 20% of their body weight.

[0622] The tumor growth status of each treatment group and the control group in the LU11554 xenograft model is shown in Table 16 and FIG.

[0623] [Table 16]

[0624] Tumor volume was used as an index: the control group (group 1) of the model had an average tumor volume of 1189.51 mm 24 days after administration. 3 The mean tumor volume at 24 days after administration of ADC1 at 1 mg / kg, 3 mg / kg, and 5 mg / kg (i.e., groups 2, 3, and 4) was 1172.94 mm, respectively. 3 , 812.00mm 3 and 672.27 mm 3 The relative tumor inhibition rate (TGI) was 1.60% (P=9.99e-01), respectively. ns ), 36.87% (P = 4.76e-02*) and 50.49% (P = 4.40e-03**).

[0625] 4) Pharmacodynamic evaluation of experimental drugs in the HuPrime® lung cancer LU5197 subcutaneous xenograft NOD / SCID mouse model Tumor tissues were harvested from mice bearing HuPrime® lung cancer xenograft model LU5197 tumors, cut into tumor masses with diameters of 2-3 mm, and inoculated subcutaneously into the right front scapula of NOD / SCID mice.

[0626] The average tumor volume of tumor-bearing mice was approximately 146.50 mm 3 When the mice reached the age of 18, they were randomly assigned to groups according to Table 17. The day of group assignment was set as day 0, and administration began on day 0. Details of the administration method, dose, and administration route are shown in Table 17.

[0627] [Table 17]

[0628] After tumor inoculation, conventional monitoring includes the effect of tumor growth and treatment on the normal behavior of the animals, specifically, activity of the experimental animals, feeding and drinking status, weight gain or loss (weight was measured twice a week), eyes, hair coat and other abnormalities. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (wherein a represents the long axis and b represents the short axis). Mice were euthanized once they had lost more than 20% of their body weight.

[0629] The tumor growth status of each treatment group and the control group in the LU5197 xenograft model is shown in Table 18 and FIG.

[0630] [Table 18]

[0631] Tumor volume was used as an index: the control group (group 1) of the model had an average tumor volume of 595.89 mm2 28 days after administration. 3The mean tumor volume at 28 days after administration for ADC1 at 1 mg / kg, 3 mg / kg, and 5 mg / kg (i.e., groups 2, 3, and 4) was 541.48 mm 3 , 277.93mm 3 and 204.69 mm 3 The relative tumor inhibition rate (TGI) was 12.12% (P=4.56e-01), respectively. ns ), 70.75% (P=6.62e-05***), and 87.04% (P=1.33e-07***).

[0632] Example 16: Pharmacokinetic studies After a single intravenous injection of ADC1 for injection or Exatecan Mesylate was administered to cynomolgus monkeys, blood samples were collected at different time points, and the analyte ADC1 and total antibody concentrations in serum were measured using ELISA analysis method, the analyte Exatecan concentration in plasma was measured using LC-MS / MS analysis method, and its main pharmacokinetic parameters were calculated.

[0633] Twenty-four normal-grade cynomolgus monkeys (half male and half female) aged 3 to 5 years were randomly divided into 4 groups, with 3 males and 3 females. The animals in each group were given a single intravenous injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg of ADC1 for injection and 0.25 mg / kg of Exatecan Mesylate without fasting, and blood samples were taken up to 504 h after administration. Based on the blood drug concentration data, the main pharmacokinetic parameters AUC (0-t) , AUC (0-∞) , T 1 / 2 , C max , T max , CL, MRT, etc. were calculated, and the results are shown in Tables 19, 20 and 21.

[0634] [Table 19]

[0635] [Table 20]

[0636] [Table 21]

[0637] System exposure area under the curve for serum ADC1, total antibody, and plasma Exatecan after single intravenous infusion of injectable ADC1 in nonfasted male and female cynomolgus monkeys at doses ranging from 1 to 10 mg / kg. (0-t) There was no significant difference between sexes in the system exposure, and the system exposure increased with increasing dose. After a single intravenous infusion of 0.25 mg / kg Exatecan Mesylate in fasted male and female cynomolgus monkeys, the plasma system exposure AUC (0-t) There was no significant difference between the sexes.

[0638] For Injectable ADC1 and Exatecan Mesylate administered in a single intravenous infusion to nonfasted cynomolgus monkeys at a dose ratio of 1:0.025 (a dose level of Exatecan Mesylate corresponding to 10 mg / kg of Injectable ADC1 of approximately 0.25 mg / kg), the ratios of Exatecan system exposure in the plasma of male and female animals were 1:5.20 and 1:2.37, respectively, indicating that relatively little Exatecan was dissociated after Injectable ADC1 was administered to the body.

[0639] Example 17: Clinical Study of ADC1 This study was a multicenter, open-label, dose-escalation, dose-expansion Phase I clinical study exploring the safety, tolerability, and PK properties of ADC1 in patients with advanced solid tumors.

[0640] The study was divided into two parts.

[0641] Part 1: Dose Escalation Study. A "3+3" dose escalation rule was used to explore the safety, tolerability and pharmacokinetic properties of ADC1.

[0642] A total of five dose groups were established, namely 1.5 mg / kg, 3.0 mg / kg, 4.5 mg / kg, 6.5 mg / kg, and 8.5 mg / kg, respectively, and the dose escalation study was conducted according to the standard "3+3" rule.

[0643] Part 2: A dose expansion study to further explore the safety and clinical efficacy of ADC1 in the treatment of advanced solid tumors.

[0644] Or, Part 1: Dose Escalation Study. Rapid titration and a "3+3" dose escalation rule were used to explore the safety, tolerability and pharmacokinetic properties of ADC1.

[0645] A total of six dose groups were established, namely 1.2 mg / kg group, 2.4 mg / kg group, 3.5 mg / kg group, 5.0 mg / kg group, 6.5 mg / kg group, and 8.0 mg / kg group, of which the 1.2 mg / kg group was dose-escalated using the rapid titration method, and the 2.4 mg / kg group, 3.5 mg / kg group, 5.0 mg / kg group, 6.5 mg / kg group, and 8.0 mg / kg group were dose-escalated according to the standard "3+3" rule.

[0646] Administration method: Administered by intravenous infusion once every 3 weeks (Q3W). The infusion time for the first cycle is recommended to be ≥ 90 min. If no infusion reaction occurs, subsequent cycles can be completed within 30 min.

[0647] Definition of Dose-Limiting Toxicity (DLT): AEs were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. DLT refers to any study drug-related toxicity of grade 3 or higher occurring within 21 days after first dose in a subject, according to the following definition:

[0648] ■Definition of liver toxicity DLT: Grade 4 elevation of AST or ALT Subjects with hepatocellular carcinoma or liver metastases, e.g., AST or ALT ≤ 3x ULN at baseline and elevation of AST or ALT > 5x ULN and sustained for > 7 days during the DLT evaluation period Subjects with hepatocellular carcinoma or liver metastases, e.g., AST or ALT >3x ULN at baseline and elevation of AST or ALT >8x ULN and sustained for >7 days during the DLT evaluation period Subjects without liver metastases, AST or ALT elevation >5x ULN and sustained for >7 days AST or ALT > 5x the upper limit of normal (ULN) with ≥ Grade 2 elevated blood bilirubin

[0649] Definition of hematological toxicity DLT: Grade 4 neutropenia lasting >7 days Grade ≥ 3 neutropenia accompanied by fever Grade 4 anemia Grade 4 thrombocytopenia ≥ Grade 3 thrombocytopenia lasting > 7 days Grade 3 or higher thrombocytopenia with associated bleeding Grade 4 lymphopenia lasting ≥ 14 days

[0650] ■ Definition of non-hepatic and non-hematological toxicity DLTs: Other ≥ Grade 3 non-hepatic, non-hematological toxicities

[0651] The following TEAEs are not considered DLTs: Grade 3 fatigue <7 days Grade 3 nausea, vomiting, diarrhea, or loss of appetite improves to ≦grade 2 within 3 days No clinically relevant symptoms or signs of laboratory abnormalities occurred, including elevated ALP, uric acid, hepaginase, or lipase reaching grade 3 or grade 4, and hyponatremia of grade 1 at baseline was upgraded to grade 3 but persisted for <72 hours Grade 3 lymphopenia

[0652] Tolerance and safety assessment: Tolerance evaluation index: Dose-limiting toxicity (DLT) events and their incidence.

[0653] Safety evaluation indicators: vital signs, physical examination, clinical tests (blood tests, blood biochemistry, cardiac function, thyroid function, blood coagulation tests, urine tests, stool tests, pregnancy tests), ECOG score, electrocardiogram, adverse events (including immune-related adverse events), etc.

[0654] Pharmacokinetic evaluation: Subjects in all dose groups were required to collect blood samples at designated time points during treatment (first six treatment cycles) to monitor plasma concentrations (Ctrough) within 2 h before dosing. Serum concentration levels of ADC1, Exatecan, and total antibody were detected, and the pharmacokinetic (PK) properties of ADC1 were investigated.

[0655] The following PK parameters were calculated using the actual dose, actual sampling time, and non-compartmental model:

[0656] Single dose: C max T max , T 1 / 2 , CL, Vd, Ke, MRT, AUC (0-τ) , AUC (0-∞)

[0657] Multiple doses: C max、ss , C avg、ss , C min、ss , AUC (0-τ)ss , AUC (0-∞)ss , T max、ss , T 1 / 2、ss , C.L., V. ss , Ke, MRT, accumulation index (R ac ), volatility index DF.

[0658] Immunogenicity assessment: Subjects in all dose groups will be required to have blood samples collected at specific time points during treatment to detect anti-drug and neutralizing antibodies in the serum.

[0659] The immunogenicity evaluation indexes were as follows.

[0660] Sample and individual positivity rates of anti-ADC1 antibody (ADA), Continue to detect whether ADA-positive samples have neutralizing antibodies (Nab) Number and percentage of ADA antibody and Nab positive subjects.

[0661] Pharmacodynamic evaluation: 1. Detection of serum FRα levels before administration 2. Folate receptor expression levels in archived tumor tissue samples or freshly collected tumor tissue samples during the screening period.

[0662] Clinical Efficacy Evaluation: Objective response rate (ORR) defined by RECIST 1.1, duration of response (DOR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS).

[0663] [Table 22]

Claims

1. An antibody or an antigen-binding unit thereof, which specifically binds to folate receptor alpha and (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9; (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; Selectively, (g) a VH FR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 29, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 29, or an amino acid sequence having substitutions, deletions, or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 29; (h) a VH FR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 30, or an amino acid sequence having substitutions, deletions, or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 30; (i) a VH FR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 31, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 31, or an amino acid sequence having substitutions, deletions, or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 31; (j) a VH FR4 comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having substitutions, deletions, or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 11; (k) a VL FR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 12; (l) a VL FR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 13; (m) a VL FR3 comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 32, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 32, or an amino acid sequence having substitutions, deletions, or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 32; (n) a VL FR4 comprising the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 14; An antibody or an antigen-binding unit thereof characterized by:

2. The antibody or antigen-binding unit thereof comprises a VH CDR1 set forth in SEQ ID NO: 5, a VH CDR2 set forth in SEQ ID NO: 6, and a VH CDR3 set forth in SEQ ID NO:

7. The antibody or antigen-binding unit thereof according to claim 1 .

3. The antibody or antigen-binding unit thereof comprises a VL CDR1 set forth in SEQ ID NO: 8, a VL CDR2 set forth in SEQ ID NO: 9, and a VL CDR3 set forth in SEQ ID NO:

10. The antibody or antigen-binding unit thereof according to claim 1 .

4. The antibody or antigen-binding unit thereof comprises a VH CDR1 shown in SEQ ID NO: 5, a VH CDR2 shown in SEQ ID NO: 6, a VH CDR3 shown in SEQ ID NO: 7, a VL CDR1 shown in SEQ ID NO: 8, a VL CDR2 shown in SEQ ID NO: 9, and a VL CDR3 shown in SEQ ID NO:

10. The antibody or antigen-binding unit thereof according to claim 1 .

5. An antibody or an antigen-binding unit thereof, which specifically binds to folate receptor alpha and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 15; and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 16; An antibody or an antigen-binding unit thereof characterized by:

6. the antibody or antigen-binding unit thereof is of an IgG isotype selected from the group consisting of an IgG1 subtype, an IgG2 subtype, an IgG3 subtype, and an IgG4 subtype; The antibody or antigen-binding unit thereof according to claim 1 .

7. The antibody or antigen-binding unit thereof comprises: a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 17; and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 18; The antibody or antigen-binding unit thereof according to claim 1 .

8. An antibody or an antigen-binding unit thereof, which specifically binds to folate receptor alpha and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO: 19; and / or a light chain comprising the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having substitutions, deletions or insertions at one or more sites compared to the amino acid sequence set forth in SEQ ID NO:20; An antibody or an antigen-binding unit thereof characterized by:

9. The substitutions are conservative amino acid substitutions. The antibody or antigen-binding unit thereof according to claim 1 .

10. (1) A polynucleotide encoding the antibody or antigen-binding unit thereof according to any one of claims 1 to 9, or (2) An expression vector comprising a polynucleotide encoding the antibody or antigen-binding unit thereof according to any one of claims 1 to 9; or (3) A cell comprising one or more polynucleotides encoding the antibody or antigen-binding unit thereof according to any one of claims 1 to 9. A biomaterial.

11. The antibody or antigen-binding unit thereof according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, is coupled to a drug via a linker, or the linker is a degradable linker. An antibody-drug conjugate characterized in that:

12. An antibody drug conjugate having the structure shown in Formula I, or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof: 【Chemistry 1】 Among them, Abu is an antibody or antigen-binding unit thereof that binds to folate receptor alpha, or Abu is the antibody or antigen-binding unit thereof of claim 1; D is a drug, M is 【Chemistry 2】 wherein * is linked to Abu, ** is linked to B, and R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3-C8 carbocyclyl group, -O-(CH ２ ) ｒ -, arylene group, -(CH ２ ) ｒ -arylene-, -arylene-(CH ２ ) ｒ -, -(CH ２ ) ｒ -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH ２ ) ｒ -, C3-C8 heterocyclyl group, -(CH ２ ) ｒ -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH ２ ) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ ) ｒ -, -(CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ - and - (CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ ) ｒ -, among which each R ｍ are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or R is —(CH ２ ) ｒ - or r is 1 or 5; B is 【Transformation 3】 where * is connected to M, ** is connected to L, and *** is connected to G; L is -(AA) ｉ - (FF) ｆ -, wherein AA is an amino acid or polypeptide and i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or each AA is independently Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala , Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly, or AA is Val-Cit and i is 1; 【Chemistry 4】 Of these, each R Ｆ are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO ２ or halogen, z is 0, 1, 2, 3, or 4, wherein * is connected to AA and ** is connected to D, or each FF independently 【Transformation 5】 and f is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, in which * is linked to AA and ** is linked to D, or FF is 【Transformation 6】 and f is 1, in which * is linked to AA and ** is linked to D, or L is 【Transformation 7】 where * is connected to B and ** is connected to D, G is 【Transformation 8】 wherein n is 1 to 24, alternatively n is 4 to 12, alternatively n is 4 to 8, alternatively n is 4 or 8; p is 1 to 10, alternatively p is 2 to 8, alternatively p is 4 to 8, alternatively p is 6 to 8, alternatively p is 7 to 8, alternatively p is 7.4, alternatively p is 7, alternatively p is 8; An antibody-drug conjugate characterized in that:

13. An antibody-drug conjugate having the structure shown in Formula I-1, I-2, I-2-1, I-3, I-4, I-4-1, I-5, I-5-1, I-6, I-6-1, I-7, I-7-1, I-8, I-8-1, I-9, I-9-1, I-10, I-10-1, I-11, or I-11-1, or a stereoisomer, or a pharmaceutically acceptable salt, or solvate thereof, The formula I-1 is: 【Chemistry 9】 The formulas I-2 and I-2-1 are: 【Chemistry 10】 The formula I-3 is: 【Chemistry 11】 The formulas I-4 and I-4-1 are: 【Chemistry 12】 The formulas I-5 and I-5-1 are: 【Chemistry 13】 The formulas I-6 and I-6-1 are: 【Chemistry 14】 The formulas I-7 and I-7-1 are: 【Chemistry 15】 The formulas I-8 and I-8-1 are: 【Chemistry 16】 The formulas I-9 and I-9-1 are: 【Chemistry 17】 The formulas I-10 and I-10-1 are: [Chemistry 18] The formulae I-11 and I-11-1 are: 【Chemistry 19】 Eventually Abu is the antibody or antigen-binding unit thereof according to claim 1, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3-C8 carbocyclyl group, -O-(CH ２ ) ｒ -, arylene group, -(CH ２ ) ｒ -arylene-, -arylene-(CH ２ ) ｒ -, -(CH ２ ) ｒ -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH ２ ) ｒ -, C3-C8 heterocyclyl group, -(CH ２ ) ｒ -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH ２ ) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ ) ｒ -, -(CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ - and - (CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ ) ｒ -, among which each R ｍ are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or R is —(CH ２ ) ｒ - or r is 1 or 5; D is a drug, n is an integer from 1 to 24, alternatively n is 4 to 12, alternatively n is 4 to 8, alternatively n is 4 or 8; p is 1 to 10, alternatively p is 2 to 8, alternatively p is 4 to 8, alternatively p is 6 to 8, alternatively p is 7 to 8, alternatively p is 7, alternatively p is 8, or alternatively p is 7.4; An antibody-drug conjugate characterized in that:

14. The drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; or the drug is an anti-cancer drug; or the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor; or the tubulin inhibitor is selected from dolastatins, auristatins, and maytansines; or the drug is an auristatin selected from MMAE, MMAF, or AF; or the drug is a calicheamicin, dexamethasone, or a cyclohexyl 1-(2-methyl-2-methyl-2-propanol), or a cyclohexyl 1-(2-methyl-2-propanol), or a cyclohexyl 1-(2-methyl-2-propanol), or a cyclohexyl 1-(2-methyl-2-propanol), or a cyclohexyl 1-(2-methyl-2-propanol), or a cyclohexyl 1-(2-methyl-2-propanol, ... a DNA damaging agent selected from the group consisting of tuocarmycins and anthramycin derivatives PBD, or the drug is selected from the group consisting of irinotecan, irinotecan hydrochloride, exatecan derivatives, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivatives SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosiratecan, 6,8-dibromo-2-methyl-3-[2-( D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3, a DNA topoisomerase inhibitor selected from N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, or a salt thereof, or the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a pharmaceutically acceptable salt or solvate thereof; The antibody-drug conjugate of claim 11 .

15. The drug 【Chemistry 20】 and among them X １ and X ２ are each independently H. hydroxy groups, C1 to C6 alkyl groups, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, a C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, nitro group, cyano group, sulfhydryl groups, alkylthio groups, an amino group, an amino group substituted by an amino group-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino group moiety by an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino group moiety with an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocyclyl, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocyclyl, the heterocyclyl being optionally substituted with a C1-C6 alkyl group, a C1-C6 alkoxy group, and the amino group being optionally substituted with an amino-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, wherein the nitrogen atom or amino group in the heterocyclyl moiety is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl protecting group or a carbamoyl group optionally substituted with a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X ３ is a C1 to C6 alkyl group, X ４ is H, -(CH ２ ) ｑ -CH ３ , -(CHR ｎ ) ｑ -CH ３ , a C3-C8 carbocyclyl group, —O—(CH ２ ) ｑ -CH ３ , arylene-CH ３ , -(CH ２ ) ｑ -arylene-CH ３ , -arylene-(CH ２ ) ｑ -CH ３ , -(CH ２ ) ｑ -(C3-C8 carbocyclyl)-CH ３ , -(C3-C8 carbocyclyl)-(CH ２ ) ｑ -CH ３ , C3-C8 heterocyclyl group, —(CH ２ ) ｑ -(C3-C8 heterocyclyl)-CH ３ , -(C3-C8 heterocyclyl)-(CH ２ ) ｑ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ ) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ -CH ２ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ２ -CH ３ , -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ２ -CH ３ , or -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ ) ｑ -CH ３ Of these, each R ｎ are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or X ４ is H or a C1-C6 alkyl group, ** is linked to another moiety of the antibody drug conjugate, y is 0, 1 or 2; Y is O, S or CR １ R ２ Among them, R １ and R ２ are each independently H or a C1-C6 alkyl group; s and t are each independently 0, 1 or 2, but are not both 0; Alternatively, the drug 【Chemistry 21】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or said halogen is F, and ** is linked to another moiety of said antibody drug conjugate; Alternatively, the drug 【Chemistry 22】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or said halogen is F and ** is linked to another moiety of said antibody drug conjugate; The antibody-drug conjugate of claim 11 .

16. having a structure shown in Formula I-12, I-12-1, I-13, I-13-1, I-14, I-14-1, I-15, I-15-1, I-16, I-16-1, I-17, I-17-1, I-18, I-18-1, I-19, I-19-1, I-20, I-20-1, I-21, I-21-1, I-22, I-22-1, I-23, I-23-1, I-24, I-24-1, I-25, or I-25-1, or a stereoisomer, or a pharmaceutically acceptable salt, or solvate thereof; Among them, the above I-12, I-12-1, I-13, I-13-1, I-14, I-14-1, I-15, I-15-1, I-16, I-16-1, I-17, I-17-1, I-18, I-18 -1, I-19, I-19-1, I-20, I-20-1, I-21, I-21-1, I-22, I-22-1, I-23, I-23-1, I-24, I-24-1, I-25 or I-25-1: 【Chemistry 23(1)】 【Chemistry 23(2)】 【Chemistry 23(3)】 【Chemistry 23(4)】 【Chemistry 23(5)】 【Chemistry 23(6)】 【Chemistry 23(7)】 【Chemistry 23(8)】 【Chemistry 23(9)】 【Chemistry 23(10)】 Eventually Abu is the antibody or antigen-binding unit thereof according to claim 1, p is 1 to 10, alternatively p is 2 to 8, alternatively p is 4 to 8, alternatively p is 6 to 8, alternatively p is 7 to 8, alternatively p is 8; An antibody-drug conjugate characterized in that:

17. The antibody or antigen-binding unit thereof, or antibody-drug conjugate according to claim 1, and a pharmaceutically acceptable carrier, excipient, and / or additive, The antibody-drug conjugate comprises the antibody or an antigen-binding unit thereof, or a pharmaceutically acceptable salt or solvate thereof, coupled to a drug via a linker. A pharmaceutical composition comprising:

18. The antibody or antigen-binding unit thereof or antibody-drug conjugate of claim 1 and one or more other anticancer drugs, The antibody-drug conjugate comprises the antibody or an antigen-binding unit thereof, or a pharmaceutically acceptable salt or solvate thereof, coupled to a drug via a linker. A pharmaceutical composition comprising:

19. Use of the antibody or antigen-binding unit thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to any one of claims 11 to 16, or the pharmaceutical composition according to claim 17 or 18 in the treatment and / or prevention of a disease or in the manufacture of a medicament for treating and / or preventing a disease, Alternatively, the disease is a disease associated with folate receptor alpha expression, or the disease is a disease associated with folate receptor alpha overexpression, or the disease is a tumor, or the disease is cancer, or the drug for treating the tumor further comprises another anti-cancer drug.

20. The cancer is colorectal cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, liver cancer, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, testicular cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, or head and neck cancer.

20. The use according to claim 19.

21. the single dose of the antibody or antigen-binding unit thereof, or antibody-drug conjugate is 1 mg / kg to 10 mg / kg, or 50 mg to 1000 mg, or 100 mg to 600 mg; the administration cycle is once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks; the administration method is injection; the administration method is intravenous injection; or the administration method is intravenous infusion; 20. The use according to claim 19.

22. The antibody or antigen-binding unit thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to any one of claims 11 to 16, or the pharmaceutical composition according to claim 17 or 18, and instructions for administering the same to a patient. A reagent kit comprising:

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