Drug conjugates and uses thereof

Abstract

The present invention discloses a drug conjugate, such as an antibody drug conjugate, and its use, which belongs to the field of biomedicine.In some embodiments, the drug conjugate is a compound of formula I, or its pharma-ceutically acceptable salt or solvate, and the drug conjugate can be used to treat cancer, autoimmune disease, inflammatory disease, or infectious disease.

Description

[Technical field]

[0001] The present invention relates to drug conjugates, such as antibody drug conjugates, linkers and intermediates for making the drug conjugates, and uses of the drug conjugates. [Background technology]

[0002] Targeted treatment of cancer, immunodeficiency, and infectious diseases is currently the core of precision medicine. The use of cell surface receptor-binding molecules as drug delivery vehicles to form conjugates with cytotoxic molecules and deliver the cytotoxic molecules to attack various pathogenic cells has been reported in many publications for many years (Allen, TM and Cullis, PR, 2004 Science, 303(5665), 1818-22; Hu, QY, et al. (2016), Chem Soc Rev45(6):1691-1719.).

[0003] Antibody-drug conjugates are composed of three parts: an antibody, a cytotoxic molecule, and a linker connecting the two (Thomas, A., et al. (2016), Lancet Oncol 17 (6): e254-e262). Each of the three has a unique function: the antibody needs to bind specifically to tumor cells, the cytotoxic molecule needs sufficient activity and broad spectrum against tumor cells, and the linker needs unique functionality to be stable in blood circulation and effectively release the cytotoxic molecule after reaching tumor cells (Chari, RV (2008), Acc Chem Res 41 (1): 98-107), and the three must be rationally constructed to achieve good clinical efficacy (Singh, SK, et al. (2015), Pharm Res 32 (11): 3541-3571, Hamilton, GS (2015), Biologicals 43 (5): 318-332). Summary of the Invention

[0004] One or more embodiments of the present invention provide a drug conjugate having the structure shown in Formula I, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; D is a drug, such as an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0005] In one or more embodiments, D is an anticancer drug.

[0006] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0007] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0008] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0009] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0010] In one or more embodiments, D 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.

[0011] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0012] In one or more embodiments, D 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.

[0013] In one or more embodiments, D has an amino group or the amino group is replaced by an alkyl group and is linked to FF via an amide bond.

[0014] In one or more embodiments, D 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.

[0015] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0016] In one or more embodiments, the heterocyclyl is azetidine, niverazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0017] 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.

[0018] In one or more embodiments, D 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.

[0019] In one or more embodiments, D 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.

[0020] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0021] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0022] In one or more embodiments, X 1 and X 2 are F or Cl, respectively.

[0023] In one or more embodiments, X 1 and X 2 are F, respectively.

[0024] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0025] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0026] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0027] In one or more embodiments, R is -(CH) r -It is.

[0028] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0029] In one or more embodiments, each AA is independently selected from the following 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.

[0030] In one or more embodiments, i is 1.

[0031] In one or more embodiments, AA is Val-Cit and i is 1.

[0032] In one or more embodiments, each FF independently [ka] In this case, * is connected to AA, ** is connected to D, and each R F are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO2 or a halogen.

[0033] In one or more embodiments, the halogen is F.

[0034] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0035] In one or more embodiments, z is 0.

[0036] In one or more embodiments, z is 1 or 2.

[0037] In one or more embodiments, f is 1.

[0038] In one or more embodiments, each FF independently [ka] where * is connected to AA and ** is connected to D.

[0039] In one or more embodiments, f is 1.

[0040] In one or more embodiments, FF is [ka] where f is 1, in which * is connected to AA and ** is connected to D.

[0041] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0042] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0043] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0044] In one or more embodiments, G is [ka] where n is 4 to 12.

[0045] In one or more embodiments, n is 4-8.

[0046] In one or more embodiments, n is 4.

[0047] In one or more embodiments, n is 8.

[0048] In one or more embodiments, p is 2-8.

[0049] In one or more embodiments, p is 4-8.

[0050] In one or more embodiments, p is 6-8.

[0051] In one or more embodiments, p is 7-8.

[0052] In one or more embodiments, Formula I above is as follows: [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; 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, such as an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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, 8, 9 or 10.

[0053] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0054] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0055] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0056] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0057] In one or more embodiments, D 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 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-dihydro- 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.

[0058] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0059] In one or more embodiments, D 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.

[0060] In one or more embodiments, D 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 a link point, 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.

[0061] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0062] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0063] 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.

[0064] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0065] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0066] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0067] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0068] In one or more embodiments, X 1 and X 2 are F, respectively.

[0069] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0070] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0071] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0072] In one or more embodiments, R is -(CH) r -It is.

[0073] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0074] In one or more embodiments, n is 4-12.

[0075] In one or more embodiments, n is 4-8.

[0076] In one or more embodiments, n is 4.

[0077] In one or more embodiments, n is 8.

[0078] In one or more embodiments, p is 2-8.

[0079] In one or more embodiments, p is 4-8.

[0080] In one or more embodiments, p is 6-8.

[0081] In one or more embodiments, p is 7-8.

[0082] In one or more embodiments, Formula I above is as follows: [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; 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, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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, 8, 9 or 10.

[0083] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0084] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0085] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0086] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0087] In one or more embodiments, D 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 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-dihydro- 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.

[0088] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0089] In one or more embodiments, D 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.

[0090] In one or more embodiments, D 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 a link point, 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.

[0091] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0092] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine. 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.

[0093] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0094] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0095] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0096] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0097] In one or more embodiments, X 1 and X 2 are F, respectively.

[0098] In one or more embodiments, X 1 and X 2are each independently -CH3, F or -OH.

[0099] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0100] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0101] In one or more embodiments, R is -(CH) r -It is.

[0102] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0103] In one or more embodiments, n is 4-12.

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

[0105] In one or more embodiments, n is 4.

[0106] In one or more embodiments, n is 8.

[0107] In one or more embodiments, p is 2-8.

[0108] In one or more embodiments, p is 4-8.

[0109] In one or more embodiments, p is 6-8.

[0110] In one or more embodiments, p is 7-8.

[0111] In one or more embodiments, Formula I above is as follows: [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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, 8, 9 or 10.

[0112] In one or more embodiments, wherein Formula I above is as follows: [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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, 8, 9 or 10. In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor. In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0113] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0114] In one or more embodiments, D is a DNA damaging agent, such as the calicheamicins, duocarmycins, anthramycin derivatives PBD (pyrrolobenzodiazepine), or a DNA topoisomerase inhibitor.

[0115] In one or more embodiments, D 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 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-dihydro- 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.

[0116] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0117] In one or more embodiments, D 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.

[0118] In one or more embodiments, D 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 a link point, 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.

[0119] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0120] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0121] 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.

[0122] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0123] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0124] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0125] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

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

[0127] In one or more embodiments, X1 and X 2 are each independently -CH3, F or -OH.

[0128] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0129] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0130] In one or more embodiments, n is 4-12.

[0131] In one or more embodiments, n is 4-8.

[0132] In one or more embodiments, n is 4.

[0133] In one or more embodiments, n is 8.

[0134] In one or more embodiments, p is 2-8.

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

[0136] In one or more embodiments, p is 6-8.

[0137] In one or more embodiments, p is 7-8.

[0138] In one or more embodiments, Formula I above is as follows: [ka] [ka] [ka] [ka] [ka] Among them, Abu is a polypeptide, such as an antibody or an antigen-binding unit thereof; D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0139] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0140] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0141] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0142] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0143] In one or more embodiments, D 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 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-dihydro- 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.

[0144] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0145] In one or more embodiments, D 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.

[0146] In one or more embodiments, D 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 a link point, 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.

[0147] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0148] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0149] 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.

[0150] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0151] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0152] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0153] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0154] In one or more embodiments, X 1 and X 2 are F, respectively.

[0155] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0156] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0157] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0158] In one or more embodiments, p is 2-8.

[0159] In one or more embodiments, p is 4-8.

[0160] In one or more embodiments, p is 6-8.

[0161] In one or more embodiments, p is 7-8.

[0162] In one or more embodiments, Formula I above is as follows: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Among them, Abu is a drug, e.g., an antibody or an antigen-binding unit thereof; p is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0163] In one or more embodiments, Abu is a polypeptide that contains a cysteine ​​in its sequence and is linked to the other moiety of the drug conjugate (eg, M of formula I) through the sulfur atom of the cysteine.

[0164] In one or more embodiments, Abu is a polypeptide that contains an Fc region. In one or more embodiments, Abu is linked to the other moiety of the drug conjugate (e.g., M in formula I) via the Fc region.

[0165] In one or more embodiments, the target to which the Abu binds is HER2, TROP-2, Nection-4, B7H3, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD20, CD21, CD22, CD30, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R , ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, Fralpha (FRα).

[0166] In one or more embodiments, the target to which the Abu binds is HER2, TROP-2, CLDN18.2, B7H3, or FRα.

[0167] In one or more embodiments, Abu is an antibody or an antigen-binding unit thereof, and the drug conjugate is an antibody drug conjugate.

[0168] In one or more embodiments, Abu is an antibody or an antigen-binding unit thereof that contains an Fc region. In one or more embodiments, the Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc region.

[0169] In one or more embodiments, Abu is linked to the other moiety of the drug conjugate (eg, M of formula I) via the Fc region.

[0170] In one or more embodiments, Abu is a single domain antibody.

[0171] In one or more embodiments, Abu is a Fab fragment.

[0172] In one or more embodiments, Abu is a single chain antibody.

[0173] In one or more embodiments, Abu is a complete antibody.

[0174] In one or more embodiments, the Abu is an anti-HER2 antibody, an anti-Trop2 antibody, an anti-CLDN18.2 antibody, an anti-B7H3 antibody, or an anti-FRα antibody. In one or more embodiments, the Abu is an anti-HER2 monoclonal antibody, an anti-Trop2 monoclonal antibody, an anti-CLDN18.2 monoclonal antibody, an anti-B7H3 monoclonal antibody, or an anti-FRα monoclonal antibody.

[0175] In one or more embodiments, Abu is Trastuzumab, Pertuzumab, Panituzumab, Nimotuzumab, Matuzumab, Rituximab, or Cetuximab.

[0176] In one or more embodiments, Abu is an anti-CLDN18.2 antibody or antigen-binding unit thereof, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprising one or more of (a)-(f), wherein: (a) a VH CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; (b) a VH CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8; (c) a VH CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9; (d) a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; (e) a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (f) a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:12.

[0177] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises the following CDRs: (a) a VH CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; (b) a VH CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8; (c) a VH CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9; (d) a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; (e) a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (f) a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:12.

[0178] In one or more embodiments, the anti-CLDN18.2 antibody or its antigen-binding unit comprises a VH CDR1 set forth in SEQ ID NO: 7, a VH CDR2 set forth in SEQ ID NO: 8, a VH CDR3 set forth in SEQ ID NO: 9, a VL CDR1 set forth in SEQ ID NO: 10, a VL CDR2 set forth in SEQ ID NO: 11, and a VL CDR3 set forth in SEQ ID NO: 12.

[0179] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 13, or an amino acid sequence having one or more conservative amino acid substitutions to the sequence set forth in SEQ ID NO: 13; and / or

[0180] The light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:14, or a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:14, or an amino acid sequence having one or more conservative amino acid substitutions to the sequence set forth in SEQ ID NO:14.

[0181] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:15 or 16.

[0182] In one or more embodiments, the anti-CLDN18.2 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:17.

[0183] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0184] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0185] In one or more embodiments, the anti-CLDN18.2 antibody is an H239H-2b-K-6a-1 antibody, the heavy chain variable region amino acid sequence of which is set forth in SEQ ID NO: 13, the heavy chain constant region amino acid sequence of which is set forth in SEQ ID NO: 15, the light chain variable region amino acid sequence of which is set forth in SEQ ID NO: 14, and the light chain constant region amino acid sequence of which is set forth in SEQ ID NO: 17.

[0186] In one or more embodiments, the antibody is the H239H-2b-K-6a-2 antibody, the heavy chain variable region amino acid sequence of which is set forth in SEQ ID NO: 13, the heavy chain constant region amino acid sequence of which is set forth in SEQ ID NO: 16, the light chain variable region amino acid sequence of which is set forth in SEQ ID NO: 14, and the light chain constant region amino acid sequence of which is set forth in SEQ ID NO: 17.

[0187] In one or more embodiments, the antibody comprises an amino acid sequence having at least 80% identity to the H239H-2b-K-6a-1 antibody, or one or more conservative amino acid substitutions to the H239H-2b-K-6a-1 antibody. In one or more embodiments, the antibody comprises an amino acid sequence having at least 80% identity to the H239H-2b-K-6a-2 antibody, or one or more conservative amino acid substitutions to the H239H-2b-K-6a-2 antibody. In one or more embodiments, Abu is an H239H-2b-K-6a-1 antibody, of which the light chain amino acid sequence is set forth in SEQ ID NO:20 and the heavy chain amino acid sequence is set forth in SEQ ID NO:18.

[0188] In one or more embodiments, Abu is the H239H-2b-K-6a-2 antibody, of which the light chain amino acid sequence is set forth in SEQ ID NO:20 and the heavy chain amino acid sequence is set forth in SEQ ID NO:19.

[0189] In one or more embodiments, Abu is an anti-B7H3 antibody or an antigen-binding unit thereof.

[0190] In one or more embodiments, the anti-B7H3 antibody has a heavy chain amino acid sequence set forth in SEQ ID NO:21 and a light chain amino acid sequence set forth in SEQ ID NO:22.

[0191] In one or more embodiments, Abu is an anti-FRα antibody or an antigen-binding unit thereof.

[0192] In one or more embodiments, the anti-FRα antibody has a heavy chain amino acid sequence set forth in SEQ ID NO:23 and a light chain amino acid sequence set forth in SEQ ID NO:24.

[0193] In one or more embodiments, the antibody comprises an amino acid sequence having at least 80% identity to any one of the above antibodies, or having one or more conservative amino acid substitutions to any one of the above antibodies.

[0194] In one or more embodiments, p is 2-8.

[0195] In one or more embodiments, p is 4-8.

[0196] In one or more embodiments, p is 6-8.

[0197] In one or more embodiments, p is 7-8.

[0198] One or more embodiments provide a linker precursor for forming a drug conjugate, such as an antibody drug conjugate, that is a compound of formula II, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof: [ka] Among them, M' is [ka] where * is connected to B and R is -(CH2) r -, -(CHR m )r-, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -, arylene- group, -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] 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 Fare independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, z is 0, 1, 2, 3 or 4, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in which * is linked to AA; G is [ka] wherein n is 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. In one or more embodiments, R is -(CH) r -It is. In one or more embodiments, R is -(CH) r - and r is 1 or 5. In one or more embodiments, each AA is independently selected from the following 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.

[0199] In one or more embodiments, i is 1.

[0200] In one or more embodiments, AA is Val-Cit and i is 1.

[0201] In one or more embodiments, each FF' is independently [ka] In this case, * is connected to AA, and in this case, each R Fare independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO2 or a halogen.

[0202] In one or more embodiments, R F is F.

[0203] In one or more embodiments, z is 0.

[0204] In one or more embodiments, z is 1 or 2.

[0205] In one or more embodiments, f is 1.

[0206] In one or more embodiments, B is [ka] where * is linked to M', ** is linked to L', and *** is linked to G.

[0207] In one or more embodiments, each FF' is independently [ka] [ka] where * is connected to AA.

[0208] In one or more embodiments, f is 1.

[0209] In one or more embodiments, FF′ is [ka] where f is 1, and * is connected to AA.

[0210] In one or more embodiments, L' is [ka] where * is connected to B.

[0211] In one or more embodiments, L' is [ka] where * is connected to B.

[0212] In one or more embodiments, L' is [ka] where * is connected to B.

[0213] In one or more embodiments, R is -(CH) r -It is.

[0214] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0215] In one or more embodiments, n is 4-12.

[0216] In one or more embodiments, n is 4-8.

[0217] In one or more embodiments, n is 4.

[0218] In one or more embodiments, n is 8.

[0219] In one or more embodiments, Formula II above is as follows: [ka] Among them, 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; 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.

[0220] In one or more embodiments, R is -(CH) r -It is.

[0221] In one or more embodiments, R is -(CH)r - and r is 1 or 5.

[0222] In one or more embodiments, n is 4-12.

[0223] In one or more embodiments, n is 4-8.

[0224] In one or more embodiments, n is 4.

[0225] In one or more embodiments, n is 8.

[0226] In one or more embodiments, Formula II above is as follows: [ka] [ka] Among them, 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; 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.

[0227] In one or more embodiments, R is -(CH) r -It is.

[0228] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0229] In one or more embodiments, n is 4-12.

[0230] In one or more embodiments, n is 4-8.

[0231] In one or more embodiments, n is 4.

[0232] In one or more embodiments, n is 8.

[0233] In one or more embodiments, Formula II above is as follows: [ka] In the formula, 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.

[0234] In one or more embodiments, n is 4-12.

[0235] In one or more embodiments, n is 4-8.

[0236] In one or more embodiments, n is 4.

[0237] In one or more embodiments, n is 8.

[0238] In one or more embodiments, Formula II above is as follows: [ka] [ka] In the formula, 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.

[0239] In one or more embodiments, n is 4-12.

[0240] In one or more embodiments, n is 4-8.

[0241] In one or more embodiments, n is 4.

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

[0243] In one or more embodiments, Formula II has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0244] One or more embodiments provide an intermediate for forming a drug conjugate, such as an antibody drug conjugate, that is a compound of formula III, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof: [ka] D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; M' is [ka] where * is connected to B and R is -(CH2) r -, -(CHR m )r-, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r-, arylene- group, -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] 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 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.

[0245] In one or more embodiments, D is an anticancer drug.

[0246] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0247] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0248] In one or more embodiments, D is an auristatin selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0249] In one or more embodiments, D is a DNA damaging agent selected from the calicheamicin class, the duocarmycin class, and the anthramycin derivatives PBD (pyrrolobenzodiazepines).

[0250] In one or more embodiments, D is a DNA topoisomerase inhibitor or a salt thereof, and is selected from the group consisting of irinotecan, irinotecan hydrochloride, 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) ... 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.

[0251] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0252] In one or more embodiments, D 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 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.

[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, D 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.

[0257] In one or more embodiments, D 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.

[0258] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0259] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0260] In one or more embodiments, X 1 and X 2 are F, respectively.

[0261] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0262] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0263] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0264] In one or more embodiments, R is -(CH) r -It is.

[0265] In one or more embodiments, r is 1 or 5.

[0266] In one or more embodiments, B is [ka] where * is linked to M', ** is linked to L, and *** is linked to G.

[0267] In one or more embodiments, each AA is independently selected from the following 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.

[0268] In one or more embodiments, i is 1.

[0269] In one or more embodiments, AA is Val-Cit and i is 1.

[0270] In one or more embodiments, each FF independently [ka] where * is connected to AA, ** is connected to D, and R F is a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO2 or a halogen.

[0271] In one or more embodiments, the halogen is F.

[0272] In one or more embodiments, each R F is independently -CH3, F, -NO2, or -OCH3.

[0273] In one or more embodiments, z is 0.

[0274] In one or more embodiments, z is 1 or 2.

[0275] In one or more embodiments, f is 1.

[0276] In one or more embodiments, FF is [ka] where f is 1, in which * is connected to AA and ** is connected to D.

[0277] In one or more embodiments, FF is [ka] where f is 1, in which * is connected to AA and ** is connected to D.

[0278] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0279] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0280] In one or more embodiments, L is [ka] where * is linked to B and ** is linked to D.

[0281] In one or more embodiments, n is 4-12.

[0282] In one or more embodiments, n is 4-8.

[0283] In one or more embodiments, n is 4.

[0284] In one or more embodiments, n is 8.

[0285] In one or more embodiments, Formula III above is as follows: [ka] Among them, 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, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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.

[0286] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0287] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0288] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0289] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0290] In one or more embodiments, D 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 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-dihydro- 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.

[0291] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0292] In one or more embodiments, D 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.

[0293] In one or more embodiments, D is [ka] and 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 a link point, 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.

[0294] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0295] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0296] 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.

[0297] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0298] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0299] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0300] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0301] In one or more embodiments, X 1 and X 2 are F, respectively.

[0302] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0303] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0304] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0305] In one or more embodiments, R is -(CH) r -It is.

[0306] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0307] In one or more embodiments, n is 4-12.

[0308] In one or more embodiments, n is 4-8.

[0309] In one or more embodiments, n is 4.

[0310] In one or more embodiments, n is 8.

[0311] In one or more embodiments, Formula III above is as follows: [ka] Among them, 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)NRm (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, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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.

[0312] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0313] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0314] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0315] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0316] In one or more embodiments, D 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 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-dihydro- 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.

[0317] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0318] In one or more embodiments, D 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.

[0319] In one or more embodiments, D is [ka] and 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 a link point, 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.

[0320] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0321] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0322] 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.

[0323] In one or more embodiments, D is [ka] Of these, X 1 and X 2are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0324] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0325] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0326] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0327] In one or more embodiments, X 1 and X 2 are F, respectively.

[0328] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0329] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0330] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0331] In one or more embodiments, R is -(CH) r -It is.

[0332] In one or more embodiments, R is -(CH) r - and r is 1 or 5.

[0333] In one or more embodiments, n is 4-12.

[0334] In one or more embodiments, n is 4-8.

[0335] In one or more embodiments, n is 4.

[0336] In one or more embodiments, n is 8.

[0337] In one or more embodiments, Formula III above is as follows: [ka] Among them, D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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.

[0338] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0339] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0340] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0341] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0342] In one or more embodiments, D 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 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-dihydro- 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.

[0343] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0344] In one or more embodiments, D 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.

[0345] In one or more embodiments, D is [ka] and 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 a link point, 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.

[0346] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0347] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0348] 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.

[0349] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0350] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0351] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0352] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0353] In one or more embodiments, X 1 and X 2 are F, respectively.

[0354] In one or more embodiments, X1 and X 2 are each independently -CH3, F or -OH.

[0355] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0356] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0357] In one or more embodiments, n is 4-12.

[0358] In one or more embodiments, n is 4-8.

[0359] In one or more embodiments, n is 4.

[0360] In one or more embodiments, n is 8.

[0361] In one or more embodiments, Formula III above is as follows: [ka] Among them, D is a drug, for example an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; 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.

[0362] In one or more embodiments, D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0363] In one or more embodiments, the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0364] In one or more embodiments, D is an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0365] In one or more embodiments, D is a DNA damaging agent, such as a calicheamicin, a duocarmycin, or an anthramycin derivative PBD (pyrrolobenzodiazepine).

[0366] In one or more embodiments, D 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 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-dihydro- 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.

[0367] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan, or a salt thereof.

[0368] In one or more embodiments, D 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.

[0369] In one or more embodiments, D 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 a link point, 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.

[0370] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0371] In one or more embodiments, the heterocyclyl is azetidine, nibradine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.

[0372] 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.

[0373] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0374] In one or more embodiments, D is [ka] Of these, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0375] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0376] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0377] In one or more embodiments, X 1 and X 2 are F, respectively.

[0378] In one or more embodiments, X 1 and X 2 are each independently -CH3, F or -OH.

[0379] In one or more embodiments, X 1 and X 2 are each independently F or -CH3.

[0380] In one or more embodiments, X 1 is -CH3 and X 2 is F.

[0381] In one or more embodiments, n is 4-12.

[0382] In one or more embodiments, n is 4-8.

[0383] In one or more embodiments, n is 4.

[0384] In one or more embodiments, n is 8.

[0385] In one or more embodiments, Formula III is selected from the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0386] One or more embodiments provide the 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]indolizino[1,2-b]quinoline-10,13, or a pharma- ceutically acceptable salt or solvate thereof.

[0387] One or more embodiments provide a pharmaceutical composition comprising a drug conjugate, such as an antibody drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, and pharma- ceutically acceptable carriers, excipients and / or additives, and optionally other anti-cancer drugs. The pharmaceutical composition 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 with other bioactive agents. Thus, the pharmaceutical composition may be administered intravenously, subcutaneously, orally, rectally, enterogastrically, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drop, or transdermal patch), buccally, or orally or as a nasal spray.

[0388] In some embodiments, 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 and the like.

[0389] 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 fragment, preferably in purified form, combined with an appropriate amount of carrier 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.

[0390] In some embodiments, the composition is prepared by conventional steps as a pharmaceutical composition suitable for intravenous injection into humans. Compositions for intravenous administration are usually solutions in sterile or other water-permeable buffers. The composition may further include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally, the active ingredients are provided singly or mixed in unit dosage form, for example in a sealed container (e.g., an ampoule or sachet) that indicates the amount of active agent, for example in the form of a dry lyophilized powder or water-free concentrate. When the composition is administered by injection, the composition may be dispensed by an infusion bottle containing sterile pharmaceutical grade water or saline. When the 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.

[0391] One or more embodiments provide for the use of the drug conjugate in the manufacture of a medicament for treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0392] In one or more embodiments, the drug conjugates are used in combination with other anti-cancer drugs.

[0393] One or more embodiments provide for the use of the linker or intermediate, or a pharma- ceutically acceptable salt or solvate thereof, in the manufacture of a drug conjugate, such as an antibody drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof.

[0394] Pharmaceutically acceptable salts include drug conjugates, such as antibody drug conjugates, and include pharma- ceutically acceptable salts formed with a variety of organic and inorganic counterions well known in the art; a few exemplary salts include organic or inorganic salts such as 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, and the like. Solvates include hydrates. These salts may generally be prepared by conventional methods, for example by reacting the appropriate acid or base with an ADC of the invention.

[0395] One or more embodiments provide a method of treating cancer comprising administering to a patient an effective amount of an antibody drug conjugate, the effective amount referring to the amount of active compound or drug that produces the biological or pharmaceutical response in tissues, systems, animals, individuals and humans that is desired by a researcher, veterinarian, physician or other clinician, which includes treating a disease.

[0396] Typically, a suitable dosage range may be about 0.1 to 100 milligrams / kilogram, and the administration frequency 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, etc. For example, the administration form may be intravenous infusion, intravenous bolus injection, subcutaneous injection, intramuscular injection, etc.

[0397] One or more embodiments provide a drug conjugate, such as an antibody drug conjugate, for use as a drug.

[0398] One or more embodiments provide for the use of a drug conjugate, such as an antibody drug conjugate, or a pharmaceutical composition comprising a drug conjugate, such as an antibody drug conjugate, in the manufacture of a medicament for treating and / or preventing a disease.

[0399] One or more embodiments provide a drug conjugate, such as an antibody drug conjugate, for treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease, including triple negative breast cancer, glioblastoma, medulloblastoma, urothelial cancer, breast cancer, head and neck cancer, kidney cancer (clear cell renal cell carcinoma and papillary renal cell carcinoma), ovarian cancer (e.g., ovarian adenocarcinoma and ovarian teratoma), pancreatic cancer, gastric cancer, Kaposi's sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary cancer , colorectal cancer, T-cell lymphoma, uterine cancer, liver cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, neuroblastoma, sarcoma (e.g., synovial sarcoma and carcinosarcoma), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer (e.g., basal cell carcinoma and squamous cell carcinoma), pancreatic cancer, malignant melanoma, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, testicular cancer, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma).

[0400] One or more embodiments include a drug conjugate or pharmaceutical composition; A container; and a package insert, instructions, or label indicating that the compound or composition is used to treat cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. [Brief description of the drawings]

[0401] [Figure 1] 1 shows the flow cytometry results of CHO-CLDN18.2 cells, with CHO cells and CHO-CLDN18.2 cells from left to right in the figure. [Diagram 2] 1 shows dose curves of ADC8, ADC10, ADC11 and ADC12 on inhibition of CHO-CLDN18.2 cell proliferation. [Diagram 3] The bystander effect of ADC1 is shown. [Figure 4] The bystander effect of ADC4 is shown. [Diagram 5] The bystander effects of ADC9, ADC11 and ADC12 are shown. [Figure 6] Showing the bystander effect of ADC14. [Figure 7] Showing the bystander effect of ADC16. [Figure 8] ADC4 shows changes in blood drug concentration in rats. [Figure 9] This shows the tumor growth inhibitory effect of ADC4. [Figure 10] 4 shows the tumor growth inhibitory effect of ADC1 and ADC2. [Figure 11] 4 shows the tumor growth inhibitory effect of ADC4 and ADC6. [Figure 12] The tumor growth inhibitory effect of ADC9, ADC11, and ADC12 is shown. [Figure 13] ADC9, ADC11, ADC12 and ADC13 show the tumor weights (mean ± standard error) of mice in each group in the GA0006 xenograft model. [Figure 14]1 shows the in vivo tumor suppressor activity of ADC14. [Figure 15] 1 shows the in vivo tumor suppressor activity of ADC16. [Figure 16] 1 shows the in vivo tumor suppressor activity of ADC16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0402] "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.

[0403] "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 described herein, the carbocyclyl group may be optionally substituted by one or more substituents independently selected from an alkyl group, a halogen, a haloalkyl group, a cyano group, a nitro group, an oxo group, an aryl group, an aralkyl group, a carbocyclyl group, a carbocyclylalkyl group, a heterocyclyl group, a heterocyclylalkyl group, a heteroaryl group, and a heteroarylalkyl group.

[0404] "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.

[0405] A "heterocyclyl group" refers to a stable 3- to 18-membered aromatic or non-aromatic ring substituent which consists of 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, or 8) carbon atoms and 1 to 6 (1, 2, 3, 4, 5, or 6) heteroatoms selected from nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, 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.

[0406] "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.

[0407] "Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0408] An "amino group" refers to -NH2.

[0409] A "cyano group" refers to -CN.

[0410] A "nitro group" refers to --NO.sub.2.

[0411] A "hydroxy group" refers to -OH.

[0412] A "carboxy group" refers to -COOH.

[0413] A "sulfhydryl group" refers to --SH.

[0414] A "carbonyl group" refers to C=O.

[0415] "Polypeptide" refers to a molecule composed of two or more amino acid monomers linearly joined by amide bonds (also called peptide bonds), and may include dipeptides, tripeptides, tetrapeptides, oligopeptides, and the like.

[0416] "Amino acid" refers to organic compounds that contain both amino and carboxy groups, such as α-amino acids and β-amino acids. 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, P ... These include phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), sarcosine (Sar), theanine (Thea), hydroxyproline (Hypro), hydroxylysine (Hylys), β-aminoisobutyric acid (β-AiBA), citrulline (Cit) and β-alanine (β-Ala).

[0417] 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.

[0418] 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 75%, for example at least 80%, 90%, 95% or 99%. In one or more 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. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of skill in the art.

[0419] In one or more 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). The number of amino acids in the conservative amino acid substitutions in VL and VH is 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 is 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.

[0420] 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.

[0421] "Homology," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(inclusive), or any value therein. "At least 90% identity" means about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(inclusive), or any value therein.

[0422] "Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be an intact antibody and any antigen-binding fragment thereof or a single chain thereof. Thus, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule in the molecule that has biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs) of the heavy or light chain or ligand-binding portions thereof, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the frame region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR regions of the light chain variable region (VL CDR1-3) and the CDR regions of the heavy chain variable region (VH CDR1-3). An antibody or an antigen-binding unit thereof can specifically recognize and bind to one or more (e.g., two) antigen polypeptides or polypeptide complexes. An antibody or antigen-binding unit that specifically recognizes and binds multiple (e.g., two) antigens may be referred to as a multispecific (e.g., bispecific) antibody or antigen-binding unit thereof.

[0423] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, and the constitution of the antibody fragment of the present invention can resemble F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. in monospecific antibody fragments. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, enantiomers, and bivalent antibodies. The term "antigen-binding fragment" further includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0424] 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), and 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 immunoglobulin type is 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 continuously surround the heavy chains through the variable region.

[0425] The antibodies, antigen-binding fragments 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).

[0426] 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 sequences run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. The κ light chain variable region of an immunoglobulin is Vκ, and the λ light chain variable region of an immunoglobulin is Vλ.

[0427] Both light and heavy chains are divided into regions of structural and functional homology. 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 the 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.

[0428] 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).

[0429] 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.

[0430] 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.

[0431] The term "antibody drug conjugate" or "ADC" refers to a binding polypeptide (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.

[0432] 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.

[0433] The DAR value of an ADC can be measured using 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.

[0434] Other chemical terms used herein are used according to conventional practice in the art, e.g., The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

[0435] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0436] antibody part The antibody according to the present invention may be any antibody suitable for the preparation of antibody drug conjugates and may have the complete antibody structure or may include antibody fragments such as Fab, Fab', F(ab)'2 and Fv (polyclonal and monoclonal antibodies).

[0437] Antibodies can specifically bind to antigens, such as tumor-specific antigens, which may be used to identify tumor cells and serve as potential indicators or targets for tumor therapy. Thus, the selection of a specific antibody depends primarily on the type of disease and the cells and tissues to be targeted.

[0438] Examples of tumor antigens well known in the art include EGFR, HER2, CD20, CD30, CD33, CD47, CD52, CD133, CEA, VEGF, TROP2, B7H3, FRalpha (FRα), Nectin-4, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD22, FcRH2, NCA, MDP, IL20Rα, Bleoglitazone, and IL20Rα. These include, but are not limited to, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, and the like.

[0439] In one or more embodiments, the antibody may be a humanized monoclonal antibody.

[0440] In some embodiments, the antibody is an anti-EGFR antibody, including but not limited to the anti-EGFR human-mouse chimeric monoclonal antibody cetuximab, the fully humanized monoclonal antibody panim monoclonal antibody, and the anti-EGFR humanized monoclonal antibody nimotuzumab.EGFR is overexpressed in many tumor tissues, such as metastatic colorectal cancer and head and neck cancer.

[0441] In one or more embodiments, the antibody is an anti-EGFR antibody and the heavy and light chain sequences are SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0442] The amino acid sequences corresponding to each SEQ ID NO: are shown in Table 1.

[0443] [Table 1]

[0444] In one or more embodiments, the antibody is an anti-HER2 antibody that can specifically act on human epidermal growth factor receptor 2, such as Trastuzumab, Pertuzumab, Margetuximab, ZW25, etc. The anti-HER2 antibody can be modified, such as by changing, increasing or decreasing one or more amino acid sequences, to achieve a corresponding purpose, such as enhancing antibody-dependent cell-mediated cytotoxicity.

[0445] In one or more embodiments, the anti-HER2 antibody is Trastuzumab and the heavy and light chain sequences are SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0446] The amino acid sequences corresponding to each SEQ ID NO: are shown in Table 2.

[0447] [Table 2]

[0448] In one or more embodiments, the antibody is an anti-Trop2 antibody capable of acting specifically on the Trop2 protein, which is vegetative cell surface glycoprotein antigen 2. In one or more embodiments, the anti-Trop2 antibody is a fully human monoclonal antibody, and in one or more embodiments, the anti-Trop2 antibody is a humanized monoclonal antibody.

[0449] In one or more embodiments, the anti-Trop2 antibody is selected from the group consisting of WO2021147993A1 (e.g., PD3), CN101264325B (e.g., RS7, hRS7), CN105849126B (e.g., hTINA1-H1L1, hTINA1-H2L1, hTINA1-H2L2, hTINA1-H3L3), CN110903395A (e.g., M1, M2, M3), CN113896796A (e.g., 4 These antibodies are disclosed in the following patent documents: US20130089872A (e.g., K5-70, K5-107, K5-116-2-1, T6-16, T5-86), US5840854A (e.g., BR110), US20130122020A (e.g., 3E9, 6G11, 7E6, 15E2, 18B1), and US20120237518A (e.g., 77220, KM4097, KM4590).

[0450] In one or more embodiments, the anti-Trop2 antibodies can be purchased commercially, including LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan BioSciences, Inc., Seattle, WA), 10428-MM01, 10428-MM02, 10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China), MR54 (eBioscience, San Diego, CA), sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, CA), MM0588-49D6 (Novus Biologicals, Littleton, CO), ab79976, and ab89928 (Cambridge, MA).

[0451] In one or more embodiments, the anti-Trop2 antibody is the anti-Trop-2 antibodies 162-25.3 and 162-46.2 disclosed by Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78:5147-50), or the Pr1E11 anti-Trop-2 antibody disclosed by Ikeda et al. (2015, Biochem Biophys Res Comm 458:877-82), which recognizes a unique epitope on Trop-2.

[0452] In one or more embodiments, the antibody is an hRS9 antibody, and the heavy and light chain sequences of the antibody are SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0453] The amino acid sequence corresponding to each SEQ ID NO: is shown in Table 3.

[0454] [Table 3]

[0455] In one or more embodiments, the antibody is an anti-CLDN18.2 antibody or an antigen-binding unit thereof having one or more of the following characteristics: a) specific binding to CLDN18.2, b) high affinity, c) strong ADCC activity, and d) strong CDC activity.

[0456] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof is a murine antibody, a chimeric antibody, or a humanized antibody.

[0457] In one or more embodiments, the antigen-binding fragment or antigen-binding unit of the anti-CLDN18.2 antibody is a Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibody or bibody. In one or more embodiments, the anti-CLDN18.2 antibody is a multispecific antibody (e.g., a bispecific antibody).

[0458] In one or more embodiments, the anti-CLDN18.2 antibody may be a monoclonal antibody.

[0459] 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-CLDN18.2 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-CLDN18.2 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 κ light chain constant region.

[0460] The anti-CLDN18.2 antibodies according to the present invention include humanized antibodies or antigen-binding units thereof. These antibodies or antigen-binding units thereof are suitable for administration to humans without eliciting a human immune response against the administered immunoglobulin. In one or more embodiments, the anti-CLDN18.2 antibodies or antigen-binding units thereof are selected from the anti-CLDN18.2 antibodies or antigen-binding units thereof described in WO2020043044, US2021403552, WO2021238831, WO2021160154, WO2021111003. In one or more embodiments, the anti-CLDN18.2 antibodies or antigen-binding units thereof are selected from zolbetuximab, claudiximab, TST001, AB011, M108, or NBL-015 or antigen-binding units thereof.

[0461] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises one or more of (a) to (f), among which: (a) a VH CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; (b) a VH CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8; (c) a VH CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9; (d) a VL CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; (e) a VL CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (f) a VL CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:12.

[0462] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises or consists of the following CDRs: (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12.

[0463] In one or more embodiments, the anti-CLDN18.2 antibody or its antigen-binding unit comprises or consists of a VH CDR1 set forth in SEQ ID NO: 7, a VH CDR2 set forth in SEQ ID NO: 8, a VH CDR3 set forth in SEQ ID NO: 9, a VL CDR1 set forth in SEQ ID NO: 10, a VL CDR2 set forth in SEQ ID NO: 11, and a VL CDR3 set forth in SEQ ID NO: 12.

[0464] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof is a humanized anti-CLDN18.2 antibody or antigen-binding unit thereof. In one or more embodiments, the heavy chain variable region of the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises an amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having one or more conservative amino acid substitutions to the sequence as set forth in SEQ ID NO: 13; and / or

[0465] The light chain variable region of the above-mentioned anti-CLDN18.2 antibody or its antigen-binding unit comprises an amino acid sequence set forth in SEQ ID NO: 14, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 14, or a sequence having one or more conservative amino acid substitutions to the sequence set forth in SEQ ID NO: 14.

[0466] In one or more embodiments, the anti-CLDN18.2 antibody or antigen-binding unit thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 15 or 16.

[0467] In one or more embodiments, the anti-CLDN18.2 antibody or its antigen-binding unit comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0468] In one or more embodiments, the anti-CLDN18.2 antibody or its antigen-binding unit comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 16 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0469] In one or more embodiments, the humanized anti-CLDN18.2 antibody is the H239H-2b-K-6a-1 antibody, the heavy chain variable region amino acid sequence of which is set forth in SEQ ID NO: 13, the heavy chain constant region amino acid sequence of which is set forth in SEQ ID NO: 15, the light chain variable region amino acid sequence of which is set forth in SEQ ID NO: 14, and the light chain constant region amino acid sequence of which is set forth in SEQ ID NO: 17.

[0470] In one or more embodiments, the humanized anti-CLDN18.2 antibody is an H239H-2b-K-6a-2 antibody, in which the heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 13, the heavy chain constant region amino acid sequence is set forth in SEQ ID NO: 16, the light chain variable region amino acid sequence is set forth in SEQ ID NO: 14, and the light chain constant region amino acid sequence is set forth in SEQ ID NO: 17.

[0471] In one or more embodiments, the antibody comprises an amino acid sequence having at least 80% identity to any one of the above antibodies, or having one or more conservative amino acid substitutions to any one of the above antibodies.

[0472] In one or more embodiments, the H239H-2b-K-6a-1 antibody has a light chain amino acid sequence set forth in SEQ ID NO:20 and a heavy chain amino acid sequence set forth in SEQ ID NO:18.

[0473] In one or more embodiments, the H239H-2b-K-6a-2 antibody has a light chain amino acid sequence set forth in SEQ ID NO: 20 and a heavy chain amino acid sequence set forth in SEQ ID NO: 19.

[0474] In one or more embodiments, the antibody is an anti-B7H3 antibody or an antigen-binding unit thereof.

[0475] In one or more embodiments, the anti-B7H3 antibody has a heavy chain amino acid sequence set forth in SEQ ID NO:21 and a light chain amino acid sequence set forth in SEQ ID NO:22.

[0476] In one or more embodiments, the antibody is an anti-FRα antibody or an antigen-binding unit thereof.

[0477] In one or more embodiments, the anti-FRα antibody has a heavy chain amino acid sequence set forth in SEQ ID NO:23 and a light chain amino acid sequence set forth in SEQ ID NO:24.

[0478] 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, DL Hacker, 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.

[0479] In one or more embodiments, a DNA encoding an antibody can be designed and synthesized according to conventional methods based on the antibody amino acid sequences described herein, inserted into an expression vector, then transfecting a host cell, and culturing the transfected host cell in a culture medium to produce a monoclonal antibody. In some embodiments, the expression antibody vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing on either side of the insertion sequence. Highly efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and some other cellular promoters such as the actin promoter can also be applied. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0480] In one or more embodiments, the inserted gene fragment should contain a screening marker, and common screening markers include screening genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, and hygromycin resistance to facilitate screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the above genes and cultured in a selective medium, and the successfully transfected cells grow in large quantities and produce the desired target protein. The antibody is purified by one or more purification steps. Purification can be performed by conventional methods, such as first centrifuging the cell suspension, collecting the supernatant, and centrifuging again to further remove impurities. Methods such as Protein A affinity columns and ion exchange columns can be used to purify the antibody protein.

[0481] In one or more embodiments of the present invention, the number of antibodies bound to small molecule drugs in an antibody-drug conjugate, i.e., the number of drugs bound to an antibody, is called the drug-antibody binding ratio (DAR). 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 called the average drug-antibody binding ratio, the value is selected from about 0 to 10, or 2 to 8. In some embodiments, the drug-antibody binding ratio is 3 to 6, and in other embodiments, the drug-antibody binding ratio is about 6 to about 8, or about 7 to about 8.

[0482] Synthesis method The present invention further provides a method for producing drug conjugates, such as antibody drug conjugates, and intermediates. Drug conjugates, such as antibody drug conjugates, and intermediates of the present invention can be produced by known formulations and methods. In some embodiments, the production method is as follows.

[0483] [ka]

[0484] 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.

[0485] In step 2, the compound of general formula 1-2 and the compound of 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] [ka]

[0490] 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.

[0491] 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.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] 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. wherein n, AA, R, i, and f are as described in formula II herein above; 1 teeth [ka] And FF 2 teeth [ka] where * is connected to AA.

[0496] 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.

[0497] 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.

[0498] [ka]

[0499] 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.

[0500] [ka]

[0501] 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.

[0502] wherein n, AA, R, i, f, FF, and D are as described in formula III; 2 teeth [ka] where * is linked to AA.

[0503] 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.

[0504] 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.

[0505] [ka]

[0506] Compounds of general formula 1-8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 1-9.

[0507] [ka]

[0508] Compounds of general formula 8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 9.

[0509] wherein n, AA, R, i, f, FF, D, and Abu are as described in Formula I.

[0510] 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 sulfuric acid, silicic acid, metasilicic acid, phosphoric acid, metaphosphoric acid, sodium bicarbonate, and sodium bisulfite.

[0511] The drug conjugates can be purified using conventional methods, such as preparative high performance liquid chromatography (prep-HPLC). EXAMPLES

[0512] Example 1. Antibody Production The following antibodies were produced according to conventional methods. After vector construction, eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) were transiently transfected or CHO cells were stably transfected, and stable cell lines were selected, purified and expressed.

[0513] Production and purification of Trastuzumab antibody Referring to the method of Wood et al., J Immunol. 145:3011 (1990), a monoclonal antibody anti-HER2 antibody that specifically binds to the HER2 extracellular domain was produced in CHO cells. The expression vector OptiCHO containing the antibody gene TM The Antibody Express System (Invitrogen) was constructed using conventional molecular biology methods, and CHO cells were used as host cells.

[0514] Production and purification of hRS9 antibody Referring to the method of Wood et al., J Immunol.145:3011(1990), anti-Trop2 specific monoclonal antibodies were produced in CHO cells. Expression vectors containing antibody genes were constructed by conventional molecular biology methods, among which the recombinant humanized anti-Trop2 monoclonal antibody hRS9 antibody has heavy and light chain amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0515] Production and purification of anti-CLND18.2 antibody The expression vectors containing the anti-CLDN18.2 antibody genes were constructed by conventional molecular biology methods, transfected into HEK293F cells, cultured and purified to obtain antibodies. The relevant sequences of the anti-CLDN18.2 antibodies are shown in Tables 4 and 5, and the light chain amino acid sequence of the H239H-2b-K-6a-1 antibody is shown in SEQ ID NO: 20, the heavy chain amino acid sequence is shown in SEQ ID NO: 18, and the light chain amino acid sequence of the H239H-2b-K-6a-2 antibody is shown in SEQ ID NO: 20, the heavy chain amino acid sequence is shown in SEQ ID NO: 19.

[0516] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 5]

[0517] Production and purification of anti-B7H3 antibody The expression vectors containing the anti-B7H3 antibody A gene were constructed by conventional molecular biology methods, transfected with HEK293F cells, cultured and purified to obtain the antibody. The relevant sequence of anti-B7H3 antibody A is shown in Table 6, and the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:21, and the light chain amino acid sequence is shown in SEQ ID NO:22.

[0518] [Table 6]

[0519] Production and purification of anti-FRα antibody The expression vectors containing the anti-FRα antibody A gene were constructed by conventional molecular biology methods, transfected with HEK293F cells, cultured and purified to obtain antibodies. The relevant sequences of anti-FRα antibody A are shown in Table 7, and the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:23, and the light chain amino acid sequence is shown in SEQ ID NO:24.

[0520] [Table 7]

[0521] Example 2. Synthesis steps 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]indolizino[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]

[0522] In a nitrogen gas atmosphere, potassium tert-butoxytetrahydrofuran 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 slowly added dropwise to the reaction flask, and then tert-butyl nitrite (4.32 g, 2 eq) was added dropwise (controlling the temperature at 0-5 ° C.), and the mixture was heated to 15-20 ° C. and stirred for 2 hours (h). After the reaction was complete, the temperature was lowered to 0-5 ° C., and acetic acid (25 mL) and acetic anhydride (25 mL) were added dropwise (controlling the temperature below 10 ° C.), and the mixture was stirred for 20 minutes (min) after the completion of the dropwise 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 NaCO3 (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, gradually cooled 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.

[0523] 2. Synthesis of N-(8-amino-5,6-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide [ka]

[0524] 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.

[0525] 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]indolizino[1,2-b]quinolin-1-yl)acetamide [ka]

[0526] 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 pyridinium p-toluenesulfonate (100 mg) was added and the stirring was continued for 20 h. The mixture was cooled to room temperature and stirred for 1 h. The solids were filtered and washed with acetone (10 mL) and cold ethanol (5 mL), respectively. The mixture was dried in vacuum to give a gray-brown powder (1.1 g, 53%). LC-MS: [M+H] + =482.

[0527] 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]indolizino[1,2-b]quinoline-10,13-dione hydrochloride [ka]

[0528] 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]indolizino[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.

[0529] Example 3. 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-octaoxy-27,32,35-triazaheptatriacontane-37-amino)benzyl(4-nitrophenyl)carbonate (CB07) [ka]

[0530] 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]

[0531] 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 the temperature at 0-5 ° C., and after adding for 1 h, the reaction was allowed to proceed for 4 h while stirring at room temperature. 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).

[0532] 2) Synthesis of (S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamate (9H-fluoren-9-yl)methyl (CB02). [ka]

[0533] 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 batches under mechanical stirring, and the reaction was carried out 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 was obtained by silica gel column chromatography (elution solvent was dichloromethane and methanol in a volume ratio of 20:1).

[0534] 3) Synthesis of (S)-2-((S)-2-amino-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CB03). [ka]

[0535] 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).

[0536] 4) Synthesis of (9H-fluoren-9-yl)methyl ((30S,33S,36S)-41-amino-36-((4-(hydroxymethyl)phenyl)carbamoyl)-33-isopropyl-26,31,34,41-tetraoxa-2,5,8,11,14,17,20,23-octaoxa-27,32,35,40-tetraaz-30-yl)carbamate (CB04). [ka]

[0537] 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).

[0538] 5) Synthesis of N-((S)-3-amino-4-(((S)-1-(((S)-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). [ka]

[0539] 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-tetraoxa-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).

[0540] 6) Synthesis of N-((S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)-4-(((S)-1-(((S)-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]

[0541] 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 in an atmosphere of nitrogen gas at 0 to 5 ° C. The mixture was reacted for 4 h while maintaining the temperature at 0 to 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).

[0542] 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-octaoxy-27,32,35-triazaheptatriacontane-37-amino)benzyl(4-nitrophenyl)carbonate (CB07). [ka]

[0543] 9.5 g of N-((S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)-4-(((S)-1-(((S)-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 50 mL of DMF, and 14.0 g of bis(p-nitrobenzene)carbonate ((PNP)2CO) was added in a nitrogen gas atmosphere at 0°C. After dissolution, 8.2 mL of N,N-diisopropylethylamine (DIPEA) was added, and the mixture was reacted for 4 h while maintaining the temperature at 0°C. 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).

[0544] Example 4. 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-tetraxo-15,20,23-triazapentacosane-25-amino)benzyl(4-nitrophenyl)carbonate (CB14) [ka]

[0545] The preparation of CB14 was carried out by referring to the synthesis of CB07 in Example 3, 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, and finally a white solid CB14 was obtained.

[0546] Example 5. Synthesis of intermediate (CB07-Exatecan) Synthesis of 4-(30S,33S,36S)-30-(2-(2,5-dioxa-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxa-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriaminoaconit-37-yl)benzoyl(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]indolizino[1,2-b]quinoline-1-carbamate. [ka]

[0547] 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-octaoxy-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]indolizino[1,2-b]quinoline-10,13-dione mesylate (execan mesylate hydrate, 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 then the washing solution was 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 solvent was removed by concentrating under reduced pressure at 35 °C. The mixture was purified by preparative high performance liquid chromatography (prep-HPLC) and freeze-dried to obtain a white powder (1.6 g, 59%). LC-MS: [1 / 2M+H] + =737.

[0548] Example 6. Synthesis of intermediate (CB07-D-1) Synthesis of 4-((30S,33S,36S)-30-(2-(2,5-dioxa-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxa-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriaminoaconit-37-yl)benzoyl(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]indolizino[1,2-b]quinoline-1-carbamate [ka]

[0549] 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-octaoxy-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]indolizino[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. The product was purified by high performance liquid chromatography (prep-HPLC) and lyophilized to give a white powder (55 mg, 20%). LC-MS: [1 / 2M+H] + =739.

[0550] Example 7. 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]indolizino[1,2-b]quinoline-1-carbamate [ka]

[0551] 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]indolizino[1,2-b]quinoline-10,13-dione mesylate (exatecan mesylate hydrate, 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 reaction flask R2 was added dropwise to reaction flask R1, reaction flask R2 was washed with N,N-dimethylformamide (1 mL), and the washings were added to reaction flask R1. 1-Hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) 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.

[0552] Example 8. Synthesis of ADC1 [ka] According to the amount of antibody material, 4.5 molar equivalents of TCEP (tris(2-carboxyethyl)phosphine) was added to Trastuzumab, and the pH of the system was adjusted to 8 with 1M Tris base. The mixture was reduced by incubating at 25°C for 1.5 h. TCEP was removed by exchanging the liquid with 10 mM succinic acid by ultrafiltration. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0553] During the coupling reaction, 12 molar equivalents of CB07-Exatecan were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 1 h, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin.

[0554] The final concentration of ADC1 was 7.5 mg / mL, and the DAR, i.e., p, was 8 as measured by reverse phase chromatography.

[0555] Example 9. Synthesis of ADC2 [ka] According to the amount of antibody material, 4.5 molar equivalents of TCEP were added to Trastuzumab, and the pH of the system was adjusted to 8 with 1M Tris base. The mixture was reduced by incubating at 25°C for 1.5 h. The liquid was exchanged with 10 mM succinic acid by ultrafiltration to remove TCEP. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0556] During the coupling reaction, 12 molar equivalents of CB07-D-1 was added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 hours, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by stirring for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin for replacement.

[0557] The concentration of ADC2 was 3.61 mg / mL and the DAR, p, was determined to be 7.4 by reverse phase chromatography.

[0558] Example 10. Synthesis of ADC3 (Control Drug) [ka] According to the amount of antibody material, 4.5 molar equivalents of TCEP were added to Trastuzumab, and the pH of the system was adjusted to 8 with 1M Tris base. The mixture was reduced by incubating at 25°C for 1.5 h. The liquid was exchanged with 10 mM succinic acid by ultrafiltration to remove TCEP. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0559] During the coupling reaction, 12 molar equivalents of Deuxtecan (MCE, Cat.#HY-13631E) were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 hours, after which 0.1M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by continuing stirring for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin for replacement.

[0560] The concentration of ADC3 was 4.19 g / L and the DAR, p, was determined to be 7.6 by RP-HPLC.

[0561] Example 11. Synthesis of ADC4 [ka] Depending on the amount of antibody material, 2.7 molar equivalents of TCEP were added to the hRS9 antibody, and the pH of the system was adjusted to 7 with 1M Tris base. The mixture was reduced by incubating at 25°C for 2 h. TCEP was removed by exchanging the liquid with 10 mM succinic acid by ultrafiltration. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0562] During the coupling reaction, 6 molar equivalents of CB07-Exatecan were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 h, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin for replacement.

[0563] The protein concentration of ADC4 was 8.46 mg / mL and the DAR, i.e., p, was detected as 4.2 by RP-HPLC.

[0564] Example 12. Synthesis of ADC5 [ka] Depending on the amount of antibody material, 2.7 molar equivalents of TCEP were added to the hRS9 antibody, and the pH of the system was adjusted to 7 with 1M Tris base. The mixture was reduced by incubating at 25°C for 2 h. TCEP was removed by exchanging the liquid with 10 mM succinic acid by ultrafiltration. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0565] During the coupling reaction, 6 molar equivalents of CB07-D-1 were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 h, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin for replacement.

[0566] The protein concentration of ADC5 was 6.5 mg / mL and the DAR, i.e., p, was detected as 4.8 by RP-HPLC.

[0567] Example 13. Synthesis of ADC6 [ka] Depending on the amount of antibody material, 2.7 molar equivalents of TCEP were added to the hRS9 antibody, and the pH of the system was adjusted to 7 with 1M Tris base. The mixture was reduced by incubating at 25°C for 2 h. TCEP was removed by exchanging the liquid with 10 mM succinic acid by ultrafiltration. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0568] During the coupling reaction, 6 molar equivalents of MC-GGFG-Dxd (Deruxtecan, MCE, Cat.#HY-13631E / CS-0045125) were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 h, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter, and then eluted with 10 mM succinic acid on Sephadex G-25 resin for replacement.

[0569] The protein concentration of ADC6 was 2.83 mg / mL, and the DAR, i.e., p, was detected as 4.6 by RP-HPLC.

[0570] Example 14. Synthesis of ADC7 [ka] Depending on the amount of antibody material, 4.5 molar equivalents of TCEP were added to the hRS9 antibody, and the pH of the system was adjusted to 8 with 1M Tris base. The mixture was reduced by incubating at 25°C for 2 h. TCEP was removed by exchanging the liquid with 10 mM succinic acid by ultrafiltration. The sulfhydryl antibody titer was determined by measuring the absorbance, and the concentration of thio groups was determined by reacting thio groups with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0571] During the coupling reaction, 12 molar equivalents of CL2A-SN38 (specifically, refer to Patent US2018161440A1) were added according to the amount of antibody substance, and the mixture was stirred at 25°C for 2 h, after which 0.1 M N-acetylcysteine ​​was added to a final concentration of 2 mM, and the reaction was stopped by continuing stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter, and then eluted and replaced with 10 mM succinic acid on Sephadex G-25 resin.

[0572] The protein concentration of ADC7 was 6.79 mg / mL and the DAR, i.e., p, was detected to be approximately 8.3 by RP-HPLC.

[0573] Example 15. Synthesis of ADC8 [ka] Take 5 mL of 11.5 mg / mL H239H-2b-K-6a-1 antibody, add 100 μL of 0.1 M EDTA aqueous solution, add 2.7 molar equivalents of 10 mM TCEP aqueous solution according to the amount of antibody substance, react for 2 h in an incubator at 25 ° C, exchange the liquid by ultrafiltration, and remove the reducing agent TCEP in the system. Then, add 6 molar equivalents of 10 mM MC-GGFG-DXD (Deruxtecan, MCE, Cat. # HY-13631E / CS-0045125) in dimethylacetamide (DMA) solution according to the amount of antibody substance, add organic solvent DMA until its volume occupies 20% of the coupling system, react for 2 h in an incubator at 25 ° C, add 0.1 M NAC (N-acetylcysteine) aqueous solution until its final concentration is 1.5 mM, react for 15 min, and then stop the reaction.

[0574] The DAR, i.e., p, of purified ADC8 was detected to be approximately 3.43 by RP-UPLC.

[0575] Example 16. Synthesis of ADC9 [ka] Take 5 mL of 11.5 mg / mL H239H-2b-K-6a-1 antibody, add 100 μL of 0.1 M EDTA aqueous solution, add 7 molar equivalents of 10 mM TCEP aqueous solution according to the amount of antibody substance, react for 2 h in an incubator at 25 ° C, exchange the liquid by ultrafiltration, and remove the reducing agent TCEP in the system. Then, add 12 molar equivalents of 10 mM MC-GGFG-DXD (Deruxtecan, MCE, Cat. # HY-13631E / CS-0045125) in dimethylacetamide (DMA) solution according to the amount of antibody substance, and couple. Add organic solvent DMA until its volume occupies 20% of the coupling system, react for 2 h in an incubator at 25 ° C, add 0.1 M NAC (N-acetylcysteine) aqueous solution until its final concentration is 2 mM, react for 15 min, and then stop the reaction.

[0576] The DAR, i.e., p, of purified ADC9 was detected to be approximately 7.67 by RP-UPLC.

[0577] Example 17. Synthesis of ADC10 [ka] Take 5 mL of 11.5 mg / mL H239H-2b-K-6a-1 antibody, add 100 μL of 0.1 M EDTA aqueous solution, add 2.7 molar equivalents of 10 mM TCEP aqueous solution according to the amount of antibody substance, react for 2 h in an incubator at 25 ° C, exchange the liquid by ultrafiltration, and remove the reducing agent TCEP in the system. Then, add 6 molar equivalents of 10 mM CB07-Exatecan dimethylacetamide (DMA) solution according to the amount of antibody substance to couple, add organic solvent DMA until its volume occupies 20% of the coupling system, react for 2 h15 min in an incubator at 25 ° C, add 0.1 M NAC (N-acetylcysteine) aqueous solution until its final concentration is 1.5 mM, react for 15 min, and then stop the reaction.

[0578] The DAR, i.e., p, of purified ADC10 was detected to be approximately 4.05 by RP-UPLC.

[0579] Example 18. Synthesis of ADC11 [ka] Take 15.2 mL of 11.5 mg / mL H239H-2b-K-6a-1 antibody, add 326 μL of 0.1 M EDTA aqueous solution, add 7 molar equivalents of 10 mM TCEP aqueous solution according to the amount of antibody substance, react for 2 h in an incubator at 25 ° C, exchange the liquid by ultrafiltration, and remove the reducing agent TCEP in the system. Then, add 12 molar equivalents of 10 mM CB07-Exatecan dimethylacetamide (DMA) solution according to the amount of antibody substance to couple, add organic solvent DMA until its volume occupies 20% of the coupling system, react for 2.5 h in an incubator at 25 ° C, add 0.1 M NAC (N-acetylcysteine) aqueous solution until its final concentration is 2 mM, react for 15 min, and then stop the reaction.

[0580] The DAR, i.e., p, of purified ADC11 was detected to be approximately 7.49 by RP-UPLC.

[0581] Example 19. Synthesis of ADC12 [ka] 2.5 mL of 11.5 mg / mL H239H-2b-K-6a-1 antibody was taken, 50 μL of 0.1 M EDTA aqueous solution was added, and 6 molar equivalents of 15 mM TCEP aqueous solution were added according to the amount of antibody substance, and the reaction was carried out for 2 h in an incubator at 25 ° C. The liquid was exchanged by ultrafiltration, and the reducing agent TCEP in the system was removed. Then, according to the amount of antibody substance, 12 molar equivalents of 10 mM CB07-D-1 in dimethylacetamide (DMA) solution was added to couple, and the organic solvent DMA was added until its volume occupied 20% of the coupling system, and the reaction was carried out for 2.5 h in an incubator at 25 ° C., and 0.1 M NAC (N-acetylcysteine) aqueous solution was added until its final concentration was 2 mM, and the reaction was stopped after 15 min of reaction.

[0582] The DAR, or p, of purified ADC12 was detected to be approximately 7.65 by RP-UPLC.

[0583] Example 20. Synthesis of ADC13 [ka] 3 mL of 7.89 mg / mL H239H-2b-K-6a-2 antibody was taken, 60 μL of 0.1 M EDTA aqueous solution was added, and 7 molar equivalents of 10 mM TCEP aqueous solution were added according to the amount of antibody substance, and the reaction was carried out for 2 h in an incubator at 25 ° C. The liquid was exchanged by ultrafiltration, and the reducing agent TCEP in the system was removed. Then, according to the amount of antibody substance, 15 molar equivalents of 100 mM CB07-Exatecan dimethylacetamide (DMA) solution was added to couple, and the reaction was carried out for 2 h in an incubator at 25 ° C., and 0.1 M NAC (N-acetylcysteine) aqueous solution was added until the final concentration was 2 mM, and the reaction was stopped after 15 min of reaction.

[0584] The DAR, i.e., p, of purified ADC13 was detected to be approximately 7.19 by RP-UPLC.

[0585] Example 21. Synthesis of ADC14 [ka] 7.0 L of 23.9 g / L anti-B7H3 antibody A solution was taken, and the concentration of antibody V3 was adjusted to 18 g / L with 10 mM succinic acid aqueous solution to obtain an antibody solution. 0.1 M EDTA aqueous solution was added until the final concentration of EDTA was 2 mM, the pH was adjusted to 7, and 5.4 molar equivalents of 0.2 M TCEP (tris(2-carboxyethyl)phosphine) aqueous solution (volume 30.2 mL) was added according to the amount of antibody substance, and the reaction was carried out with stirring at 25°C and 180 rpm for 2 hours. 10 volumes of liquid were exchanged by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 KD with 10 mM succinic acid aqueous solution to remove TCEP.

[0586] During the coupling reaction, 15 molar equivalents of 100 mM CB07-Exatecan in dimethylacetamide (DMA) was added according to the amount of antibody substance, and the reaction was allowed to proceed at 25°C and 180 rpm for 2 h with stirring, and then 0.4 M N-acetylcysteine ​​in water was added until the final concentration reached 2 mM, and the reaction was stopped by stirring for 15 min. The reaction mixture was filtered through a 0.22 micron filter and then eluted with 10 mM succinic acid in water on Sephadex G-25 resin.

[0587] The final concentration of ADC14 was 18.8 mg / mL, and the DAR, i.e., p, measured by reverse phase chromatography was 8.0.

[0588] Example 22. Synthesis of ADC15 (control drug) 5 mL of 3.08 g / L anti-B7H3 antibody A solution was taken, and 0.1 M EDTA aqueous solution was added until the final EDTA concentration was 2 mM. Depending on the amount of antibody substance, 2.7 molar equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) aqueous solution (volume 27.7 μL) was added, the pH was adjusted to 7, and the reaction was allowed to proceed with shaking for 2 hours in a shaker at 25°C. TCEP was then removed by ultrafiltration of 10 volumes of liquid using an ultrafiltration membrane with a molecular weight cutoff of 30 KD with 10 mM succinic acid aqueous solution.

[0589] During the coupling reaction, 6 molar equivalents of MC-GGFG-DXD (MedChemExpress, Lot#41557, CAS:1599440-13-7, product name: Deruxtecan) were added according to the amount of antibody substance, and the reaction was performed by shaking on a shaker at 25°C for 2 hours. An aqueous solution of 0.1M N-acetylcysteine ​​was added to the final concentration of 1.5 mM, and the coupling reaction was stopped by continuing shaking for 15 minutes. The reaction mixture was then filtered through a 0.22 micron filter, and eluted and replaced with a 10 mM aqueous solution of succinic acid on Sephadex G-25 resin.

[0590] The final concentration of ADC15 was 6.3 mg / mL, and the DAR, i.e., p, measured by reverse phase chromatography was 3.7.

[0591] Example 23. Synthesis of ADC16 [ka] The concentration of anti-FRα antibody A was adjusted to 18 g / L using a coupling buffer (10 mM succinic acid aqueous solution), 0.1 M EDTA aqueous solution was added so that the final concentration of EDTA was 2 mM, 5.4 molar equivalents of 0.2 M TCEP aqueous solution were added according to the amount of antibody substance, the pH was adjusted to 7, and the reaction was carried out at 25° C. and 180 rpm for 2 hours to reduce the antibody interchain disulfide bond to a free thiol group. 10 volumes of liquid were exchanged by ultrafiltration using a 30 KD ultrafiltration membrane, and the reducing agent TCEP in the system was removed.

[0592] Depending on the amount of antibody substance, 15 times molar equivalent of 100 mM CB07-Exatecan in dimethylacetamide (DMA) was added and reacted for 2 h at 25 ° C. and 180 rpm. 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, ADC16 with a concentration of 23.56 mg / mL was obtained, and the DAR, i.e., p, measured by reverse chromatography was 8.

[0593] Example 24. Synthesis of ADC17 [ka] The concentration of anti-FRα antibody A was adjusted to 18 g / L using a coupling buffer (10 mM succinic acid aqueous solution), a 0.1 M EDTA aqueous solution was added so that the final concentration of EDTA was 2 mM, 2.5 molar equivalents of a 10 mM TCEP aqueous solution were added according to the amount of 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 thiol groups. 10 volumes of liquid were exchanged by ultrafiltration to remove the reducing agent TCEP in the system.

[0594] Depending on the amount of antibody substance, 6-fold molar equivalent of 100 mM CB07-Exatecan in dimethylacetamide (DMA) was added and the reaction was allowed to proceed for 1 h on a shaker at 25° C. Finally, 0.1 M N-acetylcysteine ​​aqueous solution was added to the final concentration of 1.5 mM, the reaction was continued for 15 min, and the coupling reaction was stopped. After purifying the reaction mixture, ADC17 with a concentration of 1.34 mg / mL was obtained, and the DAR, i.e., p, measured by reverse chromatography was 4.

[0595] Active Examples Example 1 In vitro biological activity of ADC1 The present inventors evaluated the inhibition of tumor cell proliferation by ADC1 antibody using HER2-positive breast tumor cell lines NCI-N87, MDA-MB-453, SK-BR-3, BT474, and HER2-negative cell line MDA-MB-468 (purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences). Briefly, after the cells were grown to the logarithmic growth phase, they were digested with trypsin, detached, and then suspended in 100 μL of complete medium. 4000 to 8000 cells were seeded and cultured in a 96-well plate, and allowed to adhere and grow at 37 °C for 3 to 5 h or overnight. Then, 100 μL of medium containing different concentrations of anti-ADC1 was added, and after 120 h, the medium in the culture dish was removed, and relative cell proliferation analysis was performed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) reagent. As can be seen from the results, ADC1 had a proliferation inhibitory effect on all HER2-positive cells, and had a proliferation inhibitory effect on HER2-negative cells, MDA-MB-468, with an EC50 of >10,000 ng / mL.

[0596] [Table 8]

[0597] Example 2 In vitro biological activity of ADC4 The inventors evaluated tumor cell proliferation inhibition by ADC4 antibody using Trop2-positive breast tumor cell lines MDA-MB-453, MDA-MB-468, MX-1, and Trop2-negative cell line HGC27 (purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences). Briefly, after the cells were grown to logarithmic growth phase, they were digested with trypsin, detached, and then suspended in 100 μL of complete medium. 4000-8000 cells were seeded and cultured in a 96-well plate, and grown at 37 °C for 3-5 h or overnight. Then, 100 μL of medium containing different concentrations of anti-ADC4 was added, and after 120 h, the medium in the culture dish was removed, and relative cell proliferation analysis was performed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) reagent. As can be seen from the results, ADC4 had a proliferation inhibitory effect on all Trop2-positive cells, and had a proliferation inhibitory effect on the Trop2-negative cell line HGC27 with an EC50 of >15,000 ng / mL.

[0598] [Table 9]

[0599] Example 3 1. Construction of CHO-CLDN18.2 stabilized cell line Human CLDN18.2 full length amino acid sequence (from NCBI, NM_001002026.3): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV (sequence number 25).

[0600] The corresponding nucleic acid sequence is the following.

[0601] The nucleic acid sequence corresponding to the above human CLDN18.2 was synthesized, and the enzyme cleavage sites HindIII and EcoRI sequences were added to both ends of the sequence, and then constructed into pcDNA3.1 expression vector (Invitrogen, product number V79020). Then, under electroporation conditions of voltage 300V, time 17ms, and 4mm electric rotating cup, it was transfected into CHO cells (Life technologies, product number A13696-01) by electroporation method, and after 48 hours, 50μM MSX (methionine iminosulfone) was added for pressure screening, and after 2 weeks, positive cells were screened. High expression cell lines were screened by FACS detection. The cells were collected, washed once with PBS (phosphate buffer, 1L PBS contains 8.0g NaCl, 0.9g Na2HPO4, 0.156g KH2PO4, 0.125g KCl, pH 7.2-7.4), added with 3μg / mL anti-CLDN18.2 antibody (IMAB362, the sequence of which is identical to that of the antibody expressed by hybridoma cell 175D10 in patent US20090169547), incubated for 1h at 4℃, washed twice with PBS, added with 100μL of goat anti-human IgG-Fc PE fluorescent secondary antibody (product number: 12-4998-82, eBioscience) diluted 1:500, incubated for 1h at 4℃, washed twice with PBS, and analyzed by C6 flow cytometer (BD, product number C6 Flow cytometer). The finally obtained cells were named CHO-CLDN18.2 cells, and the FACS detection results are shown in FIG.

[0602] 2. Growth inhibition experiments of ADC8, ADC10, ADC11 and ADC12 The present inventors evaluated the inhibition of cell proliferation by anti-CLDN18.2 ADC using CHO-CLDN18.2 cells. Logarithmic growth phase CHO-CLDN18.2 cells were collected, centrifuged at 800 rpm for 5 min, the supernatant was removed, washed once with CD-CHO-AGT (product number: 12490, life technologies) medium, centrifuged at 800 rpm for 5 min, and the supernatant was removed. The cells were resuspended in CD-CHO-AGT medium containing 0.5% FBS (product number: FSP500, Excell Bio) to adjust the cell density to 5 × 10 4 The cells were adjusted to 1 / mL and inoculated into a 96-well cell culture plate (product number: 3599, Costar) at 100 μl / well. ADCs gradient-diluted with CD-CHO-AGT containing 0.5% FBS were added at 100 μl / well, and the final concentrations of ADC8, ADC10, ADC11, and ADC12 were 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.9, and 1.95 nM. After culturing for 5 days in a 37°C, 5% CO2 incubator, 20 μl of CCK-8 (product number: CK04, DojinDo) was added per well, and the plate was left at 37°C for 1 h. The absorbance value at OD450 nm was read using a plate reader (model number: SpectraMax M3, Molecular Devices), and the cell inhibition rate was calculated. Inhibition rate %=(1−absorbance value of sample well / absorbance value of control well)×100.

[0603] As can be seen from FIG. 2, ADC8, ADC10, ADC11 and ADC12 had a clear growth-suppressing effect on CHO-CLDN18.2 cells.

[0604] Example 4 Bystander effect of ADC1 and ADC2 HER2 positive cells SK-BR-3 and HER2 negative cells MDA-MB-468 were inoculated into a 6-well plate at a density of (75,000 / well: 200,000 / well), and after culturing for 3-5 h, ADC1 and control drug ADC3 with final concentrations of 0.1 nM, 0.5 nM, and 5 nM were added, and ADC2 with final concentrations of 0.3 nM, 2 nM, and 20 nM was added. After culturing for 120 h at 37 ° C, the cells were digested with pancreatin, washed once with PBS after stopping, and the cells were counted. Then, the cells were labeled with FITC-labeled anti-HER2 antibody (different from the binding epitope of Trastuzumab) at 4 ° C for 30 min to 1 h, and the proportion of different cells was detected by a flow cytometer, and the proportion of each cell in the culture dish after different treatments was calculated, which is clear from Figure 3. A bystander effect was observed for all of ADC1, ADC2 and ADC3, and the bystander effect of ADC1 was stronger than that of ADC2 and the control drug ADC3.

[0605] Example 5 Bystander effect of ADC4 and ADC5 Trop2 positive cells MDA-MB-468 and Trop2 negative cells HGC27 were inoculated into a 6-well plate at a density of 3:1 (150,000 + 50,000), and after 3-5h of incubation, ADC4 and ADC5 with final concentrations of 0.1nM, 5nM and 50nM, respectively, and the control drug ADC6 were added. After 120h of incubation at 37°C, the cells were digested with pancreatin, washed once with PBS after stopping, and the cells were counted. Then, the cells were labeled with FITC-labeled anti-HER2 antibody at 4°C for 30min-1h, and the proportion of different cells was detected by flow cytometry, and the proportion of each cell in the culture dish after different treatments was calculated, as shown in Figure 4. ADC4, ADC5 and ADC6 all showed bystander effects, among which the bystander effect of ADC4 was stronger than that of ADC5 and the control drug ADC6.

[0606] Example 6 Bystander effect of ADC9, ADC11 and ADC12 Under the condition of co-culturing CLDN18.2 positive cells KATO3 and negative cells HGC-27 in vitro, the killing status of these two cells by different ADCs was observed. Logarithmic growth phase KATO3 (CLDN18.2 positive cells) and HGC-27 (CLDN18.2 negative cells) were collected, the cells were resuspended in RPMI1640 containing 2% FBS, and seeded into a 6-well plate (product number 3516, costar), KATO3 at 120,000 cells per well, and HCG-27 at 80,000 cells per well. The cells were incubated overnight in a 37℃, 5% CO2 incubator, and the next day, 3mL of ADC9, ADC11, and ADC12 were added, and the final concentrations of ADC were 10nM, 1nM, and 0.1nM, and wells without ADC were used as negative controls. The cells were cultured in a 37°C, 5% CO2 incubator for 5 days, digested with trypsin (product number 25200-072, Gibco), harvested, and the total number of viable cells was calculated.

[0607] FITC (3326-32-7, Sigma) dissolved in DMSO (D2650, Sigma) was gradually added to a 1 mg / mL hRS9 antibody solution at a ratio of protein:FITC = 1 mg:150 μg, gently shaken while adding to mix evenly with the antibody, and reacted for 8 h at 4 ° C in the dark. 5 M NH4Cl (A501569, Seiko Seibu) was added to a final concentration of 50 mM, and the reaction was stopped at 4 ° C for 2 h. The crosslinked product was dialyzed with PBS (B548117-0500, Seiko Seibu) more than four times until the dialysate became clear, and the hRS9-FITC antibody was obtained.

[0608] To determine the percentage of KATO3 and HGC-27 in the total viable cells, cells were stained with hRS9-FITC antibody (KATO3 is Trop2 positive cell) and incubated on ice for 30 min, washed, and then the fluorescent signal of the stained cells was measured using a CytoFLEX flow cytometer (model number AOO-1-1102, Beckman). Based on the quantity and percentage of KATO3 positive and HGC-27 negative cells in each treatment well, the number of KAOT3 and HGC-27 cells was calculated, and the results are shown in Figure 5.

[0609] As can be seen from FIG. 5, ADC9, ADC11 and ADC12 have killing effects on both positive and negative cells, and ADC11 has the strongest bystander effect on negative cells.

[0610] Example 7 1. In vitro cytotoxicity of ADC14 The in vitro cytotoxicity of ADC14 against tumor cells was evaluated using B7-H3 positive cells Calu-6 and CHO-K1-B7-H3 and B7-H3 negative cells CHO-K1. Briefly, for Calu-6, cells were resuspended in DMEM medium containing 2% fetal bovine serum, seeded in 96-well plates at 100 μL / well (5000 cells), and allowed to adhere overnight in a 37°C, 5% CO2 incubator. 100 μL of different concentrations of ADC (initial concentration 200 μg / mL, diluted 4-fold gradient, solvent DMEM medium containing 10% fetal bovine serum) were added, and the culture was continued for 7 days.

[0611] For CHO-K1 or CHO-K1-B7-H3, resuspend in CD CHO AGT medium (gibco, product number: 12490-003) containing 2% fetal bovine serum, inoculate into a 96-well plate at 100 μL / well (4000 cells), and allow to adhere overnight in a 37°C, 5% CO2 incubator. Add 100 μL of ADC at different concentrations (initial concentration is 200 μg / mL, diluted 4-fold gradient, solvent is CD CHO AGT medium (gibco, product number 12490-003) containing 2% fetal bovine serum) and continue culturing for 6 days. Add 100 μL of CCK8 solution (Cell Counting Kit-8 (purchased from DOJINDO, product number CK04) and incubate at 37°C, 5% CO2 incubator for 30 min. Measure absorbance at 450 nm using a plate reader to determine IC 50 The value was calculated.

[0612] [Table 10]

[0613] As shown in FIG. 8, ADC14 exhibited strong in vitro cytotoxicity against B7-H3-positive cells Calu-6 and CHO-K1-B7-H3, whereas ADC14 had no significant killing effect against B7-H3-negative cells CHO-K1.

[0614] 2. Bystander effect of ADC14 The bystander effect of ADC14 was evaluated using B7-H3-positive cells CHO-K1-B7-H3 and B7-H3-negative cells CHO-K1-FRα. Briefly, B7-H3 positive cells CHO-K1-B7-H3 or B7-H3 negative cells CHO-K1-FRα were resuspended in CD CHO AGT medium (gibco, product number 12490-003) containing 2% fetal bovine serum, inoculated into a 96-well plate at 100 μL / well (10,000 cells), and allowed to adhere overnight in a 37°C, 5% CO2 incubator. Different concentrations of ADC14 (initial concentration was 6.25 μg / mL, diluted 4-fold gradient, CD CHO AGT medium (gibco, product number 12490-003) containing 2% fetal bovine serum) were added at 100 μL, and incubated at 37°C, 5% CO2 incubator for 3 days. The day before the end of ADC14 treatment, B7-H3 negative cells CHO-K1-FRα were resuspended in CD CHO AGT medium (gibco, product number 12490-003) containing 2% fetal bovine serum, inoculated into a 96-well plate at 100 μL / well (10,000 cells), and allowed to adhere overnight in a 37°C, 5% CO2 incubator. The cells were resuspended in AGT medium (gibco, product number 12490-003), inoculated into a 96-well plate at 100 μL / well (6000 cells), and incubated overnight in a 37°C, 5% CO2 incubator. The culture supernatant of B7-H3 positive cells CHO-K1-B7-H3 or B7-H3 negative cells CHO-K1-FRα treated with different concentrations of ADC14 was added directly to the wells of B7-H3 negative cells CHO-K1-FRα inoculated the day before, and the culture was continued for 4 days. The culture supernatant was discarded, and 100 μL of CCK8 solution (Cell Counting Kit-8 (purchased from DOJINDO, product number CK04)) was added, and the cells were incubated at 37°C, 5% CO2 incubator for 30 min. The absorbance at 450 nm was measured using a plate reader.

[0615] As shown in Figure 6, the culture supernatant of B7-H3-positive cells CHO-K1-B7-H3 treated with ADC14 has a significant killing effect on B7-H3-negative cells CHO-K1-FRα, whereas the culture supernatant of B7-H3-negative cells CHO-K1-FRα treated with ADC14 does not have a significant killing effect on B7-H3-negative cells CHO-K1-FRα.

[0616] Example 8 1. In vitro cytotoxicity of ADC16 ADC16-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 ADC16, 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.

[0617] 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 and 5% CO2 for 3 hours.

[0618] 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 and placed in a Series II Water Jacketed CO2 Incubator and left to stand at 37°C, 5% CO2 for 2 hours.

[0619] 2) ADC16 was diluted in the medium of the corresponding cells. JEG-3 cells: ADC16 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.

[0620] MCF-7 cells: ADC16 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.

[0621] The lethal control was selected as Exatecan at 3000 nM, and the blank control was the medium of the corresponding cells.

[0622] Three replicate wells were set up for each concentration.

[0623] 3) The cells were cultured for 5 days in a Series II Water Jacketed CO2 Incubator at 37°C and 5% CO2.

[0624] 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 and 5% CO2 for approximately 1 hour.

[0625] 5) The plate was read using a SpectraMax M3 plate reader, and the absorption wavelength was 450 nm.

[0626] 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

[0627] 7) Data processing and analysis were performed using GraphPad Prism. The results are shown in Table 9. As can be seen from the results, ADC16 had high in vitro cytotoxicity against FRα-positive cell lines.

[0628] [Table 11]

[0629] 2. Bystander effect of ADC16 To demonstrate the bystander effect induced by ADC16, an in vitro co-culture cell killing assay and a conditioned medium (CM) cytotoxicity assay were performed.

[0630] We selected FRα-positive JEG-3 cells and FRα-negative A549 cells (ADC16 has virtually no killing effect on A549 cells) to conduct experiments. JEG-3 cells were treated with ADC16 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.

[0631] 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 and 5% CO2 for 2 hours.

[0632] 2) ADC16 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.

[0633] 3) Steps 1) and 2) were repeated on the second and third days.

[0634] 4) On the fifth day, the cell density of A549 was adjusted to 40,000 cells / mL with DMEM + 2% FBS medium, inoculated into a 96-well plate at 100μL / well, and placed in a Series II Water Jacketed CO2 Incubator at 37℃ and 5% CO2 for 2h. Culture supernatants from steps 1), 2), and 3) were added at 100μL / well. 100μL of 3000nM Exatecan was added to column 1 as a lethal control, and 100μL of DMEM + 2% FBS medium was added to column 12 as a blank control.

[0635] 5) The cells were cultured in a Series II Water Jacketed CO2 Incubator for 3 days at 37°C and 5% CO2.

[0636] 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 absorption wavelength was 450 nm.

[0637] 7) The data was analyzed. The formula for calculating cell viability is as follows: Cell viability (%) = (experimental wells - lethal wells) / (blank wells - lethal wells)*100

[0638] 8) Data analysis was performed using GraphPad Prism. As shown in FIG. 7, the culture supernatant obtained by co-culturing ADC16 and FRα-positive JEG-3 cells in vitro had a significant killing effect on A549 cells.

[0639] Example 9 Pharmacokinetic detection in rats Healthy adult Sprague Dawley rats aged 6-8 weeks were taken, and ADC4 was bolus-injected into the tail vein for approximately 1 min ± 10 s, with an administration volume of 5 mL / kg and an administration concentration of 20 mg / kg. Blood was collected at 0.083 h, 1 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after the end of administration, respectively, and serum was centrifuged within 30-120 min. The concentrations of total antibody (Tab) and ADC in the blood samples were measured by conventional ELISA methods.

[0640] The detection method of all antibodies is briefly described as follows: Trop2-His is coated overnight at 4°C, the concentration is 0.75μg / mL, and it is blocked with 5% skimmed milk powder at 37°C for 2h, then the standard curve and quality control points are added and incubated for 2h, the detection range of the standard curve is 64ng / mL-0.5ng / mL, starting from 64ng / mL, diluted with a 2-fold gradient, the quality control concentration points are set to 60ng / mL, 6ng / mL, 0.6ng / mL, and the recovery rate of the quality control points should be 80%-120%, followed by the addition of anti-human kappa light chain peroxidase antibody (manufacturer: sigma, product number: A7164-1ML) produced in goat diluted at 1:8000, the secondary antibody is added, incubated for 1h, washed 8 times with PBST, then added TMB to develop the color, stopped with 0.1M sulfuric acid, and the plate is read with an OD450 plate reader, and the plate reader analysis software SoftMax Pro was used to calculate blood sample concentrations at different time points.

[0641] The detection method of ADC is briefly described as follows: Trop2-His is coated overnight at 4 °C, the concentration is 0.75 μg / mL, and it is blocked with 5% skim milk powder at 37 °C for 2 h, and the standard curve and quality control points are added and incubated for 2 h. The detection range of the standard curve is 64 ng / mL to 0.5 ng / mL, starting from 64 ng / mL, diluted with a 2-fold gradient, the quality control concentration points are set to 60 ng / mL, 6 ng / mL, and 0.6 ng / mL, and the recovery rate of the quality control points should be 80% to 120%, followed by rabbit polyclonal antibody small molecule (immunized by GenScript) secondary antibody (1:5000 dilution), after 1 h incubation, diluted 1:8000, Fc fragment specific peroxidase affinity pure goat anti-rabbit IgG (Jackson immunono After adding 100μL of single component TMB color development solution (Huzhou Yingchuang, TMB-S-001), the color was developed, and the plate was stopped with 0.1M sulfuric acid. The plate was read with an OD450 plate reader, and the blood sample concentrations at different time points were calculated using the plate reader analysis software SoftMax Pro. The change curve of the drug concentration in the blood was plotted using ELISA, as shown in Figure 8. The ADC concentration basically overlaps with the total antibody concentration, with very low shedding and very stable in the blood.

[0642] Example 10 In vivo efficacy of ADC4 in Capan-1 This study evaluated the pharmacodynamics of ADC4 in a female BALB / c nude mouse animal model with subcutaneous xenografts of human pancreatic cancer Capan-1 cell line. For 10 mice per group, the study design was as follows: the test drug was ADC4, and the control drugs were ADC6 and ADC7.

[0643] [Table 12]

[0644] All drugs were administered after diluting with PBS to the prescribed concentration. The test animals were 7-9 week old BALB / c nude female mice (Beijing Ankai Yibo Biotechnology Co., Ltd.).

[0645] Capan-1 cells were cultured in IMDM medium containing 20% ​​fetal bovine serum. Exponentially growing Capan-1 cells were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation in nude mice. Experimental mice were inoculated with 5 × 10 6 Capan-1 cells were inoculated subcutaneously and resuspended in PBS (0.1 mL / mouse) mixed 1:1 with Matrigel. Tumor growth was monitored periodically and tumors grew to an average volume of 159.77 mm. 3 When the tumors grew to 100 μg / kg, the mice were randomly divided into groups according to the tumor size and mouse body weight, and the administration was started on the same day.

[0646] After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, specifically the activity of the experimental animals, feeding and drinking status, weight gain or loss (body weight was measured twice a week), eyes, coat and other abnormalities. Any clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the long diameter and b represents the short diameter). TM Data were collected using Studylog System (Version No. 3.1.399.19, Studylog System, Inc., San Francisco, CA, USA) software.

[0647] [Table 13]

[0648] As shown in Figure 9, at a dose of 5 mg / kg, the efficacy of ADC4 was significantly stronger than that of the control drug ADC7 and also stronger than that of ADC6; ADC7 had almost no tumor growth inhibitory effect in the model at a concentration of 5 mg / kg, and ADC4 had a high TGI against the tumor at 114% on day 25 after a single dose.

[0649] Example 11 Pharmacodynamic study of drugs ADC1, ADC2, and ADC3 in a human ovarian cancer SK-OV-3 cell line subcutaneous xenografted female BALB / c nude mice animal model Test drug: ADC1 (5mg / kg) ADC2 (5mg / kg) ADC3 (5mg / kg) Arrangement method: All diluted in PBS Test animals: BALB / c nude, 6 per group (Jiangsu Jiyakang Biotechnology Co., Ltd.). Test method: SK-OV-3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum. SK-OV-3 cells in the exponential growth phase were harvested and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. Experimental mice were inoculated with 1 × 10 cells in the anterior scapula of the right dorsal region. 7 SK-OV-3 cells were inoculated subcutaneously and resuspended in PBS (0.1 mL / mouse) mixed 1:1 with Matrigel. Tumor growth was monitored periodically and tumors grew to an average volume of 129.98 mm. 3 When the tumors grew to 100 mm Hg, the mice were randomly divided into groups according to the size of the tumors and the weight of the mice, and the mice were administered the drug. The day of grouping was set as day 0, and administration began on day 0. A single intravenous administration was used, and the tumor volume and weight were measured twice a week, and the data were recorded.

[0650] The test results are as follows: The changes in tumor volume are shown in Table 12 and Figure 10. The test results show that single administration of ADC1 and ADC2 has significantly stronger efficacy than the control drug ADC3. During the experiment, the mice in each group did not show any obvious abnormalities or weight loss.

[0651] [Table 14]

[0652] Example 12 Pharmacodynamics of ADC4 and ADC6 in a female BALB / c nude mouse animal model subcutaneously xenografted with triple-negative breast cancer MX-1 cell line Test drug: ADC4 (2.5mg / kg and 5mg / kg) ADC6 (2.5 mg / kg and 5 mg / kg) Arrangement method: All diluted in PBS Test animals: BALB / c nude, 6 per group (Beijing Ankai Yibo Biological Technology Co., Ltd.). Test method: MX-1 cells were cultured in RPM1640 medium containing 10% fetal bovine serum. MX-1 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation in nude mice. 5 × 10 6 MX-1 cells were inoculated subcutaneously, the cells were resuspended in PBS (0.1 mL / mouse), and the tumor growth was monitored periodically. The tumors reached an average volume of 149.03 mm. 3 When the tumors grew to 100 μg / kg, the mice were randomly divided into groups according to the tumor size and mouse body weight, and the administration was started on the same day.

[0653] After tumor inoculation, conventional monitoring includes tumor growth and the effect of treatment on the normal behavior of the animals.

[0654] The test results are as follows: The changes in tumor volume are shown in Table 13 and Figure 11. The test showed that ADC4 and ADC6 had similar efficacy after single administration, with the TGI at the administration concentration of 5 mg / kg being 109.48% and 108.84%, respectively. During the experiment, the mice in each group did not show any obvious abnormalities or weight loss.

[0655] [Table 15]

[0656] Example 13 Antitumor Effects of ADC9, ADC11, ADC12, and ADC13 in HuPrime® Gastric Cancer GA0006 Xenograft Female BALB / c Nude Mice Animal Model

[0657] Tumor masses with a diameter of 2–3 mm were inoculated subcutaneously into the right front scapula of BALB / c nude mice. The average tumor volume of tumor-bearing mice was approximately 139.15 mm. 3 When the mice reached the age of 18 days, they were randomly assigned to groups. The day of group assignment was set as day 0, and administration began on day 0, with a single administration. The body weight and tumor growth of the mice were monitored. The tumor growth of each treatment group and the control group in the GA0006 xenograft model is shown in Figure 12.

[0658] Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major axis and b represents the minor axis). TGI TV %=[1-(Ti-T0) / (Ci-C0)]×100 (where T0 and C0 are the average tumor volumes on the day (day 0) in the treatment group and the solvent control group, respectively, and Ti and Ci are the average tumor volumes on day 14 in the treatment group and the solvent control group, respectively). ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle control tumor volume.

[0659] The control group (group 1) of this model had a mean tumor volume of 628.62 mm 14 days after administration. 3 reached.

[0660] After 14 days of treatment with ADC11 (i.e., Groups 2 and 3) at 10 mg / kg and 5 mg / kg, respectively, the mean tumor volume was 78.17 mm 3 and 91.47 mm 3 and the relative tumor inhibition rates (TGI) were 112.45% (P=3.43e-09***) and 109.75% (P=6.53e-08***), respectively.

[0661] After 14 days of treatment with ADC13 (i.e., Groups 4 and 5) at 10 mg / kg and 5 mg / kg, respectively, the mean tumor volume was 25.77 mm 3 and 78.07 mm 3 and the relative tumor inhibition rates TGI were 123.17% (P=7.58e-12***) and 112.48% (P=1.23e-08***), respectively.

[0662] After 14 days of treatment with ADC12 (i.e., Groups 6 and 7) at 10 mg / kg and 5 mg / kg, respectively, the mean tumor volume was 185.69 mm 3 and 284.02 mm 3 The relative tumor inhibition rates (TGI) were 90.50% (P=5.92e-04***) and 70.40% (P=3.51e-01), respectively. ns ) was.

[0663] After administration of ADC9 (i.e., Group 8) at 5 mg / kg for 14 days, the mean tumor volume was 296.86 mm 3 The relative tumor inhibition rate (TGI) was 67.79% (P=3.51e-0 ns ). Consistent with the trends in the tumor volume data, each treatment group similarly demonstrated different degrees of antitumor efficacy, as measured by change in tumor weight (see FIG. 13).

[0664] Example 14 In vivo tumor suppressor activity of ADC14 Pharmacodynamic evaluation of ADC14 in a human liver cancer Hep3B cell line subcutaneous xenograft BALB / c nude mouse model. The number of animals in each group and detailed administration route, dose and method are shown in Table 14.

[0665] [Table 16]

[0666] Hep3B cells were cultured in MEM medium (Hyclone, product number SH30024.01) containing 10% FBS and 1% NEAA (GIBCO, product number 11140050). Hep3B cells in the exponential growth phase were harvested and resuspended in PBS mixed with Matrigel at a 1:1 ratio. Male BALB / c nude mice aged 6-8 weeks were selected, with 10 mice per group. 5 × 10 6 Hep 3B cells (0.1 mL / mouse) were subcutaneously inoculated, and tumor growth was monitored periodically. 3 (100~200mm 3 When the tumors had grown to 100 μg / kg, the mice were randomly divided into groups according to the tumor size and mouse weight, and the administration was started on Day 0.

[0667] After tumor cell inoculation, conventional monitoring includes tumor growth and the effect of treatment on the normal behavior of the animals, specifically the activity of the experimental animals, feeding and drinking status, weight gain or loss, eyes, hair coat and other abnormalities. After the start of administration, the weight of the mice and the size of the tumor were measured twice a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (wherein, a represents the long diameter of the tumor, and b represents the short diameter of the tumor). The relative tumor inhibition rate (TGI%) was calculated on day 25 after inoculation using the formula: TGI% = (1 - mean relative tumor volume of the treatment group / mean relative tumor volume of the vehicle group) x 100%.

[0668] [Table 17]

[0669] The tumor inhibition results are shown in Tables 15 and 14. ADC14 had a very strong dose-dependent growth inhibitory effect on Hep 3B tumors, and at the same small molecular weight, ADC14 had a significantly stronger tumor growth inhibitory effect than ADC15 (group 5 vs. group 3).

[0670] Example 15 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 16.

[0671] [Table 18]

[0672] 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.

[0673] 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.

[0674] 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).

[0675] [Table 19]

[0676] The tumor growth status of each treatment group and control group of the OV3756 xenograft model is shown in Table 17 and Figure 15. As can be seen from Table 17 and Figure 15, ADC16 and ADC17 can significantly inhibit the tumor growth of the OV3756 xenograft model.

[0677] 2. Study of the in vivo antitumor therapeutic effect of experimental drugs in the Balb / c nude mouse JEG-3 subcutaneous model was aimed at investigating the in vivo efficacy of ADC16 in the JeG-3 model. The grouping and administration method are shown in Table 18.

[0678] [Table 20]

[0679] 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 drug.

[0680] 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).

[0681] As shown in Figure 16, treatment with ADC16 had a significant tumor suppression 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. Notably, ADC16 completely regressed the tumors, which was maintained until the end of the experiment.

Claims

1. A drug conjugate having the structure as shown in Formula I, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 1】 Among them, Abu is a polypeptide, D is a drug, M is 【Chemistry 2】 In which * is linked to Abu, ** is linked to B, and R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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 【Chemistry 3】 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 【Chemistry 4】 and each R Ｆ are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO ２ or halogen, in which * is linked to AA, ** is linked to D, z is 0, 1, 2, 3 or 4, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G is 【Chemistry 5】 wherein n is an integer from 1 to 24; p is 1 to 10 Drug conjugates.

2. A drug conjugate having the structure shown in formula I-1, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof, wherein said formula I-1 is as follows: 【Chemistry 6】 Among them, Abu is a polypeptide, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; p is 1 to 10 Drug conjugates.

3. A drug conjugate having a structure as shown in Formula I-2 or Formula I-2-1, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof, wherein: The formula I-2 is as follows: 【Chemistry 7】 Formula I-2-1 is as follows: 【Chemistry 8】 Among them, Abu is a polypeptide, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; p is 1 to 10 Drug conjugates.

4. A drug conjugate having the structure shown in formula I-3, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof, wherein said formula I-3 is as follows: 【Chemistry 9】 Among them, Abu is a polypeptide, D is a drug, n is an integer from 1 to 24; p is 1 to 10 Drug conjugates.

5. A drug conjugate having the structure shown in Formula I-4 or Formula I-4-1, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof, wherein: The formula I-4 is as follows: 【Chemistry 10】 The formula I-4-1 is as follows: 【Chemistry 11】 Among them, Abu is a polypeptide, D is a drug, n is an integer from 1 to 24; p is 1 to 10 Drug conjugates.

6. A drug conjugate having a structure as 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 thereof or a pharma- ceutically acceptable salt or solvate thereof, wherein said formulas 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, I-11-1 are as follows: 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 Among them, Abu is a polypeptide, D is a drug, p is 1 to 10 Drug conjugates.

7. A structure represented by 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 thereof or a pharma- ceutically acceptable form thereof.

1. A drug conjugate having a salt or solvate thereof, wherein said 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-22, I-22-1, I-23, I-24, I-24-1, I-25 or I-25-1 is as follows: 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 Among them, Abu is a polypeptide, p is 1 to 10 Drug conjugates.

8. The amino acid sequence of Abu contains one or more cysteines and is linked to other moieties of the drug conjugate through the sulfur atoms of the cysteines. The drug conjugate of claim 1.

9. An antibody drug conjugate, in which Abu is an antibody or antigen-binding unit. The drug conjugate of claim 8.

10. Abu binds to the following targets: HER2, TROP-2, Nectin-4, B7H3, B7H4, CLDN18, BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD20, CD21, CD22, CD30, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, and ASLG6. 59, PSCA, GEDA, BAFF-R, CD79a, CD79b, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, EpCAM, ROR1, GPR172A, FRalpha The drug conjugate of claim 9.

11. A compound having the structure shown in Formula II, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: 【Chemical 27】 Among them, M' is 【Chemistry 28】 In which * is connected to B and R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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 【Chemical 29】 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 【Chemistry 30】 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, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in which * is linked to AA; G is 【Chemistry 31】 and n is an integer from 1 to 24. compound.

12. A compound having the structure shown in Formula II-1A or Formula II-1B, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 32】 Among them, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; n is an integer from 1 to 24 compound.

13. A compound, or a stereoisomer or a pharma- ceutically acceptable salt or solvate thereof, wherein said compound is 【Chemical 33】 【Chemical 34】 Among them, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; n is an integer from 1 to 24 A compound, or a stereoisomer thereof, or a pharma- ceutically acceptable salt or solvate thereof.

14. A compound, or a pharma- ceutically acceptable salt or solvate thereof, wherein said compound is 【Chemistry 35】 In this case, n is an integer from 1 to 24. A compound, or a pharma- ceutically acceptable salt or solvate thereof.

15. A compound, or a pharma- ceutically acceptable salt or solvate thereof, wherein said compound is 【Chemical 36】 【Chemical 37】 In this case, n is an integer from 1 to 24. A compound, or a pharma- ceutically acceptable salt or solvate thereof.

16. A compound, or a pharma- ceutically acceptable salt or solvate thereof, wherein said compound is 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 A compound, or a pharma- ceutically acceptable salt or solvate thereof.

17. A compound of formula III, or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 48】 D is a drug, M' is 【Chemistry 49】 In which * is connected to B and R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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 【Chemistry 50】 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 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 【Chemistry 51】 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, 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; G is 【Chemistry 52】 wherein n is an integer from 1 to 24, or n is an integer from 4 to 12. A compound, or a pharma- ceutically acceptable salt or solvate thereof.

18. A compound of formula III-1, or a pharma- ceutically acceptable salt or solvate thereof, wherein said formula III-1 is: 【Chemistry 53】 Among them, D is a drug, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; n is an integer from 1 to 24 A compound, or a pharma- ceutically acceptable salt or solvate thereof.

19. Compounds of formula III-2 and III-2-1, or pharma- ceutically acceptable salts or solvates thereof, wherein: The formula III-2 is as follows: 【Chemical 54】 The formula III-2-1 is as follows: 【Chemistry 55】 Among them, D is a drug, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3 to C8 carbocyclyl group, -O-(CH ２ ) ｒ -, an 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 -, wherein 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; n is an integer from 1 to 24 A compound, or a pharma- ceutically acceptable salt or solvate thereof.

20. A compound of formula III-3, or a pharma- ceutically acceptable salt or solvate thereof, wherein said formula III-3 is: 【Chemistry 56】 Among them, D is a drug, n is an integer from 1 to 24 A compound, or a pharma- ceutically acceptable salt or solvate thereof.

21. Compounds of formula III-4 and III-4-1, or pharma- ceutically acceptable salts or solvates thereof, wherein The formula III-4 is as follows: 【Chemistry 57】 The formula III-4-1 is as follows: 【Chemistry 58】 Among them, D is a drug, n is an integer from 1 to 24 A compound, or a pharma- ceutically acceptable salt or solvate thereof.

22. Each AA is 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-Ly s, 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 The drug conjugate of claim 1.

23. AA is Val-Cit and i is 1 The drug conjugate of claim 1.

24. L is 【Chemistry 59】 where * is linked to B and ** is linked to D; The drug conjugate of claim 1.

25. L' is 【Chemistry 60】 where * is connected to B 12. The compound of claim 11, or a pharma- ceutically acceptable salt or solvate thereof.

26. D is an anticancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; The drug conjugate of claim 1.

27. D is an anticancer drug The drug conjugate of claim 1.

28. D is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor. The drug conjugate of claim 1.

29. D is MMAE, MMAF, AF, calicheamicins, duocarmycins, anthramycin derivatives PBD (pyrrolobenzodiazepine), irinotecan, ecandecane derivatives, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacetylene, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-pyranylamino)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-pyrogalosyl-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, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, or a pharma- ceutically acceptable salt thereof. The drug conjugate of claim 1.

30. D is 【Chemistry 61】 Among them, X １ and X ２ are each independently H. Hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted with one or more hydroxy groups, halogens, nitro groups or cyano groups; C2 to C6 alkenyl group, A 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 optionally substituted by 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 optionally substituted by a C1-C6 alkoxy group, and the amino group is optionally substituted by an amino group-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; amino-substituted heterocyclyl groups optionally substituted with a protecting group or one or more C1-C6 alkyl groups at the nitrogen atom or amino portion of the heterocyclyl moiety; a heterocyclylamino group optionally substituted with a protecting group or a C1-C6 alkyl group at the nitrogen atom or amino group portion of the heterocyclyl moiety; 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 to C6 alkyl group, X ４ is H, -(CH ２ ) ｑ -CH ３ , -(CHR ｎ ) ｑ -CH ３ , C3 to 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 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 a connection point, 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 both 0. The drug conjugate of claim 1.

31. D is 【Chemistry 62】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point. The drug conjugate of claim 1.

32. A compound of formula III-5, III-5-1, III-6, III-6-1, III-7, III-7-1, III-8, III-8-1, III-9, III-9-1, III-10, III-10-1, III-11, III-11-1, III-12, III-12-1, III-13, III-13-1, III-14, III-14-1, III-15, III-15-1, III-16, III-16-1, III-17, III-17-1, III-18, or III-18-1, or a pharma- ceutically acceptable salt or solution thereof. A solvate, wherein the formula III-5, III-5-1, III-6, III-6-1, III-7, III-7-1, III-8, III-8-1, III-9, III-9-1, III-10, III-10-1, III-11, III-11-1, III-12, III-12-1, III-13, III-13-1, III-14, III-14-1, III-15, III-15-1, III-16, III-16-1, III-17, III-17-1, III-18, or III-18-1 is as follows: 【Chemistry 63】 【Chemistry 64】 【Chemistry 65】 【Chemistry 66】 【Chemistry 67】 【Chemistry 68】 【Chemistry 69】 【Chemistry 70】 【Chemistry 71】 【Chemical Formula 72】

33. A pharmaceutical composition comprising the drug conjugate according to any one of claims 1 to 10 and 22 to 31 and a pharma- ceutically acceptable vector, excipient and / or additive, and optionally other anticancer drugs. Drug compositions.

34. 32. Use of the antibody-drug conjugate of any one of claims 1 to 10 and 22 to 31 in the manufacture of a medicament for treating cancer, an autoimmune disease, an inflammatory disease or an infectious disease.

35. 33. Use of a compound according to any one of claims 11 to 32, or a pharma- ceutically acceptable salt or solvate thereof, in the manufacture of a drug conjugate.

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