Antibody-drug conjugate, and preparation method therefor and use thereof

Abstract

A compound represented by formula I or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, and a linker-payload molecule having a specific structure and an antibody-drug conjugate prepared therefrom. The compound of formula I and / or the antibody-drug conjugate exhibits good anti-tumor activity in a variety of tumors (such as colon cancer, pancreatic cancer, lung adenocarcinoma, and breast cancer).

Description

An antibody-drug conjugate and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, to an antibody-drug conjugate and preparation method and use thereof. BACKGROUND

[0002] Cytotoxin, linker and conjugate of the two can be loaded on the targeting molecule including antibody, in the form of antibody-drug conjugates (ADC) targeted delivery to target cells such as cancer cells and other pathological state cells, and then play a killing role to achieve the purpose of treatment.

[0003] There are many small molecules with cytotoxicity for antibody-drug conjugates in the prior art, among which the most notable are camptothecin derivatives, including SN-38, DXD, CPT-11, irinotecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like. They have anti-tumor effect by inhibiting topoisomerase I. At present, several camptothecin drugs have been approved for marketing for tumor treatment.

[0004] Camptothecin drugs or derivatives often have hematotoxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia and the like, and gastrointestinal side effects such as nausea, vomiting, diarrhea and the like. Clinical studies have found that measures to improve the safety and effectiveness of camptothecin compounds include improving their pharmacokinetic properties, adjusting activity, reducing dosage or using conjugates to form antibody-drug conjugates with antibodies. Therefore, there is still a high clinical demand and application value for developing novel camptothecin compounds and their conjugates that can improve effectiveness and safety problems.

[0005] However, how to develop camptothecin drugs or derivatives with greater clinical value, and ADCs with camptothecin as cytotoxin that can meet the needs of different clinical indications, with a larger safety window, still needs further development and exploration. SUMMARY

[0006] To solve the above problems, the present application provides a new payload, a linker-payload molecule containing the toxin and a new ADC formed by the linker-payload and its application.

[0007] In the first aspect of the present application, a compound represented by formula (I) is provided, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof,

[0008] X is selected from the group consisting of -OH, -SH, -NHR m ;

[0009] R m is selected from the group consisting of hydrogen, C1-C6 alkyl;

[0010] R1and R3are each independently selected from the group consisting of hydrogen, C1-C6 alkyl;

[0011] R2is C1-C6 alkyl, and said C1-C6 alkyl is optionally substituted with a group selected from the group consisting of hydroxy, mercapto, carboxy, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkylamino;

[0012] A has the following structure of Formula IV:

[0013] Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O);

[0014] R 4 and R 5 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuterium- substituted alkyl;

[0015] Alternatively, R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C 12 carbocyclic ring, a saturated or unsaturated 5-12 membered heterocyclic ring;

[0016] Unless specifically stated, each of the above-mentioned alkyl, haloalkyl, deuterium-substituted alkyl, alkoxy, alkylthio, and alkylamino groups can be substituted with a substituent selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, mercapto, carboxy, C1-C6 alkyl-amine, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkyl-S-, haloC1-C6 alkyl, haloC2-C6 alkenyl, haloC2-C6 alkynyl, haloC1-C6 alkoxy, allyl, benzyl, C6-C 12 aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl.

[0017] In another preferred embodiment, R 4 and R 5 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 deuterium- substituted alkyl.

[0018] or R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C7carbocyclic ring, a saturated or unsaturated 5-7 membered heterocyclic ring.

[0019] In another preferred embodiment, said R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C7carbocyclic ring, a saturated or unsaturated 5-7 membered heterocyclic ring.

[0020] In a preferred embodiment, said R1is selected from the group consisting of hydrogen, C1-C4alkyl; the configuration of the carbon atom to which R1is attached can be either R or S;

[0021] R2is C1-C4alkyl;

[0022] R3is hydrogen, methyl, ethyl or n-propyl; the configuration of the carbon atom to which R3is attached can be either R or S.

[0023] In another preferred embodiment, said R1is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl.

[0024] In another preferred embodiment, said R2is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl.

[0025] In another preferred embodiment, said A has the structure shown in the following formula:

[0026] In a preferred embodiment, said compound of formula (I) has the structure shown in formula (la):

[0027] wherein X is selected from the group consisting of -OH, -SH, -NHR m ;

[0028] R m is selected from the group consisting of hydrogen, C1-C4alkyl;

[0029] R3is hydrogen, methyl, ethyl or n-propyl;

[0030] R1is selected from the group consisting of hydrogen, C1-C4alkyl;

[0031] R2is C1-C4alkyl;

[0032] Z is selected from the group consisting of a bond, C(O), C(S);

[0033] R 4 and R 5each independently is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C4alkyl group, a C1-C4haloalkyl group, a C1-C4deuteroalkyl group;

[0034] or, R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a C5-C8cycloalkyl group, a saturated or unsaturated 5-7 membered heterocyclic ring.

[0035] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0036] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0037] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0038] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of: pyrimidinyl;

[0039] L is -L1-L2-L3-L4-;

[0040] wherein L1is selected from the group consisting of: p1 C(=O)-, -(CH2CH2O) q1 -, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 (CHR) p3 C(=O)-, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 -, -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-, -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-, -X1-(CHROCHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-, -X1-(CHR) m1-X2-(CHR) m2 -C(O)-, -(CH2CH2O) n3 -C(O)-;

[0041] X1and X2are each independently selected from the group consisting of -O-, -C(O)-, -C(O)-NR-, optionally substituted C6-C 10 aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heteroalicyclyl, and optionally substituted C3-C6alicyclyl;

[0042] wherein each of said R is independently selected from the group consisting of H, D, (CH2) n4 OH, (CH2) n4 NH2, (CH2O) n4 (CH2CH2O) n5 H, (CH2O) n4 (CH2CH2O) n5 CH3, (CH2) n4 OCH3, (CH2CH2O) n5 CH3, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 H, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3;

[0043] wherein each of p1, p2, p3, q1and q2is independently 0, 1, 2, 3, 4, 5, 6, or 7; each of m1, m2, n3, n4and n5is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0044] L2is a peptide residue;

[0045] L3is -L 4a -(NR b ) n6 -R 12 -L 4b -,

[0046] wherein L 4a is absent, or L 4a is an optionally substituted

[0047] n6is 0 or 1;

[0048] R 12 is a bond, CH2, or CD2;

[0049] L4b is absent, or L 4b is optionally substituted

[0050] R a and R b each independently is selected from the group consisting of hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl;

[0051] L4is absent, or is optionally substituted

[0052] wherein Y is selected from the group consisting of O, S, NH;

[0053] v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0054] R 10 and R 11 each independently is selected from the group consisting of hydrogen, deuterium, halogen, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl, or R 10 and R 11 together with the atom to which they are attached form an optionally substituted C3-C8cycloalkyl;

[0055] L1, L2, L3, and L4as described above can also be optionally substituted with a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C1-C6alkyl-NH-, a (C1-C6alkyl)2N-, a C1-C6alkyl, a C2-C6alkenyl, a C2-C6alkynyl, a C1-C6alkoxy, a haloC1-C6alkyl, a haloC2-C6alkenyl, a haloC2-C6alkynyl, a haloC1-C6alkoxy, an allyl group, a benzyl group, a C6-C10aryl, a C1-C6alkoxy-C1-C6alkyl, a C1-C6alkoxy-carbonyl, a phenoxycarbonyl, a C2-C6alkynyl-carbonyl, a C2-C6alkenyl-carbonyl, a C3-C6cycloalkyl-carbonyl, a C1-C6alkyl-sulfonyl, a phenyl, a 5-7 membered heteroaryl, a C3-C8cycloalkyl, a 3-12 membered heterocyclyl; 12 aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C3-C8cycloalkyl, 3-12 membered heterocyclyl;

[0056] said D has a structure represented by formula (I-1):

[0057] wherein X’ is selected from the group consisting of O, S, NR m ;

[0058] R m , R1, R2, R3, and A are as defined above.

[0059] In a preferred embodiment, X1and X2are each independently selected from the group consisting of -0-, -C(O)-, -C(O)-NR-, optionally substituted phenyl, optionally substituted pyridyl, optionally substituted C3-C6cycloalkyl, optionally substituted optionally substituted

[0060] In another preferred embodiment, said L1is a group selected from the group consisting of -(CH2) m1 -X1-(CH2CH2O) n3 -(CH2) m2 -C(O)-;

[0061] wherein X1is -C(O)-NH-; preferably, m1and m2are each independently selected from 1, 2 or 3; n3is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0062] In another preferred embodiment, said L1is a group selected from the group consisting of -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1is optionally substituted or optionally substituted

[0063] wherein X2is -C(O)-NR-; preferably, m1and m2are each independently selected from 0, 1 or 2; n3is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In another preferred embodiment, said L1is a group selected from the group consisting of -X1-(CHROCHR) m2 -C(O)-;

[0064] wherein X1is optionally substituted aryl, optionally substituted heteroaryl; preferably, m2is 0, 1, 2 or 3.

[0065] In a preferred embodiment, said L1is a group selected from the group consisting of -(CHR) p1 -C(O)-, -(CH2CH2O) q1 -, p2 C(=O)-NH-(CH2CH2O) q2 (CHR) p3 C(=O)-, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 -;

[0066] wherein each of p1, p2, p3, q1and q2is independently selected from 0, 1, 2, 3, 4 or 5; R is selected from the group consisting of H, (CH2) n4 OH, (CH2O) n4 (CH2CH2O) n5 H; preferably, each of n4and n5is independently selected from 0, 1, 2 or 3.

[0067] In another preferred embodiment, said L1is a group selected from the group consisting of -(CH2) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-;

[0068] wherein X1is -C(O)-; preferably, each of m1and m2is independently selected from 0, 1, 2 or 3; n3is selected from 0, 1 or 2.

[0069] In another preferred embodiment, said L1is a group selected from the group consisting of -X1-(CH2) m1 -X2-(CHR) m2 -C(O)-;

[0070] wherein X1is optionally substituted aryl or optionally substituted heteroaryl; X2is -C(O)-; preferably, each of m1and m2is independently selected from 0, 1 or 2.

[0071] In another preferred embodiment, said L1is a group selected from the group consisting of -(CHR) m1 -X1-(CHR) m2 -C(O)-;

[0072] wherein X1is optionally substituted 3-8 membered heteroalicyclic or optionally substituted C3-C6alicyclic; preferably, m1is 0, 1 or 2, and m2is 0.

[0073] In another preferred embodiment, said L1is a group selected from the group consisting of -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1is O; preferably, each of m1, n3and m2is independently 0, 1 or 2.

[0074] In another preferred embodiment, said L1is a group selected from the group consisting of -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-; wherein X1is optionally substituted -C(O)-NR-, and X2is O; preferably, each of m1, n3and m2is independently 1, 2 or 3; R is as described above.

[0075] In a preferred embodiment, said L1 is an optionally substituted moiety selected from the group consisting of:

[0076] In a preferred embodiment, said L2 is a peptide residue consisting of one or more amino acids selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, glycine;

[0077] Preferably, said L2 is a peptide residue consisting of one or more amino acids selected from the group consisting of glycine, alanine, lysine, phenylalanine, valine, glutamine, citrulline;

[0078] More preferably, said L2 is a peptide residue selected from the group consisting of -glycine- (-Gly-), -phenylalanine- (-Phe-), -glutamine- (-Gln-), -glycine-glycine- phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -alanine-alanine-alanine- (-Ala-Ala-Ala-), -alanine-alanine-glycine- (-Ala-Ala-Gly-), -glycine-phenylalanine-glycine- (-Gly-Phe-Gly-), -citrulline-valine- (-Cit-Val-), -citrulline-alanine- (-Cit-Ala-), -valine-arginine- (-Val-Arg-), -valine-lysine- (-Val-Lys-), -valine-lysine(Ac)- (-Val-Lys(Ac)-), -lysine-valine- (-Lys-Val-), -leucine-citrulline- (-Leu-Cit-), -isoleucine-citrulline- (-Ile-Cit-), -tryptophan-citrulline- (-Trp-Cit-), -phenylalanine-lysine- (-Phe-Lys-), -phenylalanine-lysine(Ac)- (-Phe-Lys(Ac)-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylalanine-alanine- (-Phe-Ala-), -phenylalanine-arginine- (-Phe-Arg-), -alanine-lysine- (-Ala-Lys-), -alanine-alanine- (-Ala-Ala-), -alanine-alanine-asparagine- (-Ala-Ala-Asn-), -alanine-alanine-aspartic acid- (Ala-Ala-Asp-), -lysine-alanine-alanine-asparagine- (-Lys-Ala-Ala-Asn-), -lysine-alanine-alanine-aspartic acid- (-Lys-Ala-Ala-Asp-), -(D)-valine-leucine-lysine- (-D-Val-Leu-Lys-), -glycine-glycine-arginine- (-Gly-Gly-Arg-), -glycine-glycine-asparagine- (-Gly-Gly-Asn-), -glycine-glycine-phenylalanine- (-Gly-Gly-Phe-), -valine-lysine-glycine- (-Val-Lys-Gly-), -glutamic acid-alanine-alanine- (-Glu-Ala-Ala-), -aspartic acid-alanine-alanine- (-Asp-Ala-Ala-), -valine-lysine-glycine-glycine- (-Val-Lys-Gly-Gly-), -lysine-alanine-asparagine- (-Lys-Ala-Asn-).

[0079] In another preferred embodiment, L2 is a peptide residue selected from the group consisting of -glycine- (-Gly-), -phenylalanine- (-Phe-), -glutamine- (-Gln-), -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -alanine-alanine-alanine- (-Ala-Ala-Ala-), -alanine-alanine-glycine- (-Ala-Ala-Gly-).

[0080] In another preferred embodiment, L2 is -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), i.e.

[0081] In a preferred embodiment, L2 is selected from the group consisting of the following structures:

[0082] In a preferred embodiment, L3 is a chemical bond, or is an optionally substituted group selected from the group consisting of:

[0083] wherein R a and R b are each independently selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 deuterated alkyl;

[0084] Preferably, L3 is a chemical bond, or is an optionally substituted group selected from the group consisting of:

[0085] In a preferred embodiment, L4 is absent, or is an optionally substituted group selected from the group consisting of:

[0086] In a preferred embodiment, the compound of formula (II) is selected from the group consisting of:

[0087] In a third aspect, the present application provides a compound of formula (III), a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, Ab-(M’-L-D) n formula (III)

[0088] wherein n is an integer or decimal number from 2.0 to 16.0, preferably from 2.0 to 8.0, most preferably from 4.0 to 8.0;

[0089] Ab is a polypeptide, antibody, antigen binding fragment or non-antibody scaffold that binds to a target;

[0090] M' is selected from the group consisting of: wherein R d is H, C1-C6alkyl, C1-C6deuteroalkyl, C3-C8cycloalkyl or C3-C8deutero-cycloalkyl, * is the position where M' is attached to Ab, is the position where M' is attached to L;

[0091] L and D are as defined above.

[0092] In another preferred embodiment, M' is selected from the group consisting of:

[0093] In a preferred embodiment, Ab is selected from the group consisting of a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide linked Fc, scFv, single domain antibody, diabody, bispecific antibody or multispecific antibody;

[0094] Preferably, Ab is selected from the group consisting of a diabody, DART, anticalin, affibody, avimer, DARPin, adnectin;

[0095] More preferably, Ab is selected from the group consisting of scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, Duobody, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv.

[0096] In a preferred embodiment, Ab is monospecific, bivalent or bispecific.

[0097] In another preferred embodiment, the average drug loading n in the compound of formula (III) is from about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

[0098] In a preferred embodiment, the Ab is selected from the group consisting of an anti- ADAM9 antibody, an anti-Trop-2 antibody, an anti-CD37 antibody, an anti-PD-1 antibody, an anti-HER2 antibody, an anti-B7H4 antibody, an anti-CD70 antibody, an anti-EGFRvIII antibody, an anti-Mesothelin antibody, an anti-Folate eceoptor1 antibody, an anti-Mucin 1 antibody, an anti-CD138 antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD30 antibody, an anti-SLTRK6 antibody, an anti-Nectin 4 antibody, an anti-Tissue factor antibody, an anti-Mucin 16 antibody, an anti-Endothelin receoptor antibody, an anti-STEAP1 antibody, an anti-SLC39A6 antibody, an anti-Guanylylcyclase C antibody, an anti-PSMA antibody, an anti-CCD79b antibody, an anti-CD22 antibody, an anti-Sodium phosphate cotransporter2B antibody, an anti-GPNMB antibody, an anti-Trophoblast glycoprotein antibody, an anti-AGS-16 antibody, an anti-EGFR antibody, an anti-CD33 antibody, an anti-CD66e antibody, an anti-CD74 antibody, an anti-CD56 antibody, an anti-TACSTD2 antibody, an anti-DR5 antibody, an anti-E16 antibody, an anti-STEAP1 antibody, an anti-0772P antibody, an anti-MPF antibody, an anti-Napi3b antibody, an anti-Sema 5b, an anti-PSCAhlg antibody, anti-ETBR antibody, anti-MSG783 antibody, anti-STEAP2 antibody, anti-TrpM4 antibody, anti-CRIPTO antibody, anti-CD21 antibody, anti-CD79b antibody, anti-FcRH2 antibody, anti-NCA antibody, anti-MDP antibody, anti-IL20Ra antibody, anti-Brevican antibody, anti-EphB2R antibody, anti-ASLG659 antibody, anti-PSCA antibody, anti-GEDA antibody, anti-BAFF-R antibody, anti-CD22 antibody, anti-CD79a antibody, anti-CXCR5 antibody, anti-HLA-DOB antibody, anti-P2X5 antibody, anti-CD72 antibody, anti-LY64 antibody, anti-FcRH1 antibody, anti-IRTA2 antibody, anti-TENB2 antibody, anti-integrin alpha5beta6 antibody, anti-alpha4beta7 antibody, anti-FGF2 antibody, anti-FGFR2 antibody, anti-HER3 antibody, anti-CD70 antibody, anti-CA6 antibody, anti-DLL3 antibody, anti-DLL4 antibody, anti-P-cadherin antibody, anti-EpCAM antibody, anti-pCAD antibody, anti-CD223 antibody, anti-LYPD3 antibody, anti-LY6E antibody, anti-EFNA4 antibody, anti-ROR1 antibody, anti-SLITRK6 antibody, anti-5T4 antibody, anti-ENPP3 antibody, anti-SLC39A6 antibody, anti-CLAUDIN18.2 antibody, anti-BMPR1B antibody, anti-E16 antibody, anti-STEAP1 antibody, anti-Tyro7 antibody, anti-0772P antibody, anti-MPF antibody, anti-Napi3b antibody, anti-Sema 5b antibody, anti-PSCA hlg antibody, anti-ETBR antibody, anti-MSG783 antibody, anti-STEAP2 antibody, anti-TrpM4 antibody, anti-CRIPTO antibody, anti-CD21 antibody, anti-CD79b antibody, anti-FcRH2 antibody, anti-NCA antibody, anti-MDP antibody, anti-IL20Ra antibody, anti-Brevican antibody, anti-EphB2R antibody, anti-ASLG659 antibody, anti-PSCA antibody, anti-GEDA antibody, anti-CD22 antibody, anti-CD79a antibody, anti-CXCR5 antibody, anti-HLA-DOB antibody, anti-P2X5 antibody, anti-CD72 antibody, anti-LY64 antibody, anti-FcRH1 antibody, anti-IRTA2 antibody, anti-c-Met antibody, anti-ApoE antibody, anti-CD1lc antibody, an anti-CD40 antibody, an anti-CD45 (PTPRC) antibody, an anti-CD49D (ITGA4) antibody, an anti-CD80 antibody, an anti-CSF1R antibody, an anti-CTSD antibody, an anti-GZMB antibody, an anti-Ly86 antibody, an anti-MS4A7 antibody, an anti-PIK3AP1 antibody, an anti-PIK3CD antibody, an anti-CCR5 antibody, an anti-IFNG antibody, an anti-IL10RA1 antibody, an anti-IL-6 antibody, an anti-ACTA2 antibody, an anti-COL7A1 antibody, an anti-LOX antibody, an anti-LRRC15 antibody, an anti-MCPT8 antibody, an anti-MMP10 antibody, an anti-NOG antibody, an anti-SERPINEl antibody, an anti-STAT1 antibody, an anti-TGFBR1 antibody, an anti-CTSS antibody, an anti-PGF antibody, an anti-VEGFA antibody, an anti-C1QA antibody, an anti-C1QB antibody, an anti-ANGPTL4 antibody, an anti-EGLN antibody, an anti-ANGPTL4 antibody, an anti-EGLN3 antibody, an anti-BNIP3 antibody, an anti-AIF1 antibody, an anti-CCL5 antibody, an anti-CXCL10 antibody, an anti-CXCL11 antibody, an anti-IFI6 antibody, an anti-PLOD2 antibody, an anti-KISS1R antibody, an anti-STC2 antibody, an anti-DDIT4 antibody, an anti-PFKFB3 antibody, an anti-PGK1 antibody, an anti-PDK1 antibody, an anti-AKR1C1 antibody, an anti-AKR1C2 antibody, an anti-CADM1 antibody, an anti-CDH11 antibody, an anti-COL6A3 antibody, an anti-CTGF antibody, an anti-HMOX1 antibody, an anti-KRT33A antibody, an anti-LUM antibody, an anti-WNT5A antibody, an anti-IGFBP3 antibody, an anti-MMP14 antibody, an anti-CDCP1 antibody, an anti-PDGFRA antibody, an anti-TCF4 antibody, an anti-TGF antibody, an anti-TGFB1 antibody, an anti-TGFB2 antibody, an anti-CD1 lb antibody, an anti-ADGRE1 antibody, an anti-EMR2 antibody, an anti-TNFRSF21 antibody, an anti-UPK1B antibody, an anti-TNFSF9 antibody, an anti-MMP16 antibody, an anti-MFI2 antibody, an anti-IGF-1R antibody, an anti-RNF43 antibody, an anti-NaPi2b antibody, an anti-BCMA antibody, an anti-TENB2 antibody.

[0099] In another preferred embodiment, the antibody is an anti-ADAM9 antibody.

[0100] In another preferred embodiment, the antibody is an anti-TROP-2 antibody.

[0101] In another preferred embodiment, the antibody is an anti-EGFR antibody.

[0102] In another preferred embodiment, the antibody is an anti-PD-1 antibody.

[0103] In a preferred embodiment, the compound of formula (III) is selected from the group consisting of:

[0104] In a fourth aspect, the present application provides a method for preparing a compound of formula III, a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof as described in the third aspect of the present application, comprising the following steps:

[0105] reacting a compound of formula II, a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof as described in the second aspect of the present application with Ab as described in the third aspect of the present application to obtain a compound of formula III, a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof as described in the third aspect of the present application.

[0106] In a fifth aspect, the present application provides a pharmaceutical composition comprising a compound of formula I as described in the first aspect of the present application, a compound of formula II as described in the second aspect of the present application or a compound of formula III as described in the third aspect of the present application, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof; and

[0107] a pharmaceutically acceptable diluent, carrier and / or excipient.

[0108] In a sixth aspect, the present application provides a pharmaceutical preparation comprising a compound of formula I as described in the first aspect of the present application, a compound of formula II as described in the second aspect of the present application or a compound of formula III as described in the third aspect of the present application, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof; and

[0109] a pharmaceutically acceptable diluent, carrier and / or excipient.

[0110] In a seventh aspect, the present application provides use of a substance X in the preparation of a medicament for preventing or treating cancer or inflammation;

[0111] wherein the substance X is a compound of formula I as described in the first aspect of the present application, a compound of formula II as described in the second aspect of the present application or a compound of formula III as described in the third aspect of the present application, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the fifth aspect of the present application, or a pharmaceutical preparation as described in the sixth aspect of the present application;

[0112] Preferably, the cancer is a solid tumor or a non-solid tumor.

[0113] More preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0114] In an eighth aspect of the present application, there is provided use of a substance X in the preparation of a medicament for preventing or treating a disease associated with abnormal cell activity;

[0115] wherein the substance X is a compound of Formula I according to the first aspect of the present application, a compound of Formula II according to the second aspect of the present application, or a compound of Formula III according to the third aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the fifth aspect of the present application, or a pharmaceutical preparation according to the sixth aspect of the present application;

[0116] Preferably, the disease associated with abnormal cell activity is cancer.

[0117] More preferably, the cancer is a solid tumor or a non-solid tumor.

[0118] Further more preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0119] In a ninth aspect of the present application, there is provided use of a substance X in the preparation of a medicament;

[0120] wherein the substance X is a compound of Formula I according to the first aspect of the present application, a compound of Formula II according to the second aspect of the present application, or a compound of Formula III according to the third aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the fifth aspect of the present application, or a pharmaceutical preparation according to the sixth aspect of the present application;

[0121] The medicament is for treating a disease associated with a target A, wherein the target A is a target corresponding to Ab in the substance X.

[0122] Preferably, the disease associated with the target A is cancer.

[0123] More preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0124] It should be understood that, in the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g. in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0125] Figure 1 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-A.

[0126] Figure 2 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-B.

[0127] Figure 3 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-C.

[0128] Figure 4 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-D.

[0129] Figure 5 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-DXD.

[0130] Figure 6 shows the SEC-HPLC profile of antibody-drug conjugate ADC2-A.

[0131] Figure 7 shows the SEC-HPLC profile of antibody-drug conjugate ADC2-C.

[0132] Figure 8 shows the SEC-HPLC profile of antibody-drug conjugate ADC2-D.

[0133] Figure 9 shows the SEC-HPLC profile of antibody-drug conjugate ADC2-DXD.

[0134] Figure 10 shows the SEC-HPLC profile of antibody-drug conjugate ADC3-C.

[0135] Figure 11 shows the SEC-HPLC profile of antibody-drug conjugate ADC3-DXD.

[0136] Figure 12 shows the in vitro inhibition of proliferation of BxPC-3 cell line by each toxin compound.

[0137] Figure 13 shows the in vitro inhibition of proliferation of PA-1 cell line by each toxin compound.

[0138] Figure 14 shows the in vitro inhibition of proliferation of Calu-3 cell line by antibody-drug conjugate targeting ADAM9.

[0139] Figure 15 shows the in vitro inhibition of proliferation of BxPC-3 cell line by antibody-drug conjugate targeting ADAM9.

[0140] Figure 16 shows the in vitro inhibition of proliferation of AGS cell line by antibody-drug conjugate targeting ADAM9.

[0141] Figure 17 shows the in vitro inhibition of proliferation of BxPC-3 cell line by antibody-drug conjugate targeting TROP-2.

[0142] Figure 18 shows the in vivo tumor growth curve of NCI-H1975 tumor model mice by antibody-drug conjugate targeting ADAM9.

[0143] Figure 19 shows the in vivo tumor mass graph of NCI-H1975 tumor model mice by antibody-drug conjugate targeting ADAM9.

[0144] Figure 20 shows the effect of antibody-drug conjugate targeting ADAM9 on body weight of NCI-H1975 tumor model mice.

[0145] Figure 21 shows the in vivo tumor growth curve of Calu-3 tumor model mice by antibody-drug conjugate targeting ADAM9.

[0146] Figure 22 shows the in vivo tumor mass graph of Calu-3 tumor model mice by antibody-drug conjugate targeting ADAM9.

[0147] Figure 23 shows the effect of antibody-drug conjugate targeting ADAM9 on body weight of Calu-3 tumor model mice.

[0148] Figure 24 shows the in vivo tumor growth curve of NCI-H1975 tumor model mice by antibody-drug conjugate targeting TROP-2.

[0149] Figure 25 shows the in vivo tumor mass graph of NCI-H1975 tumor model mice by antibody-drug conjugate targeting TROP-2.

[0150] Figure 26 shows the effect of the targeting TROP-2 antibody-drug conjugate on the body weight of NCI-H1975 tumor model mice.

[0151] Figure 27 shows a graph of the growth curve of BxPC-3 tumor model mice treated with the targeting EGFR antibody-drug conjugate in vivo.

[0152] Figure 28 shows a graph of the tumor mass of BxPC-3 tumor model mice treated with the targeting EGFR antibody-drug conjugate in vivo.

[0153] Figure 29 shows the effect of the targeting EGFR antibody-drug conjugate on the body weight of BxPC-3 tumor model mice.

[0154] Figure 30 shows the change in body weight of rats after administration of the antibody-drug conjugate in a rat toxicity test.

[0155] Figure 31 shows the change in food intake of rats after administration of the antibody-drug conjugate in a rat toxicity test.

[0156] Figure 32 shows the toxicokinetics of total anti in a cynomolgus monkey pre-toxicology test.

[0157] Figure 33 shows the toxicokinetics of small molecule toxin in a cynomolgus monkey pre-toxicology test. DETAILED DESCRIPTION

[0158] After long-term and in-depth research, through a large number of screening, the inventors first developed a new payload, a linker-payload molecule comprising the payload, and a new ADC prepared therefrom. The linker-payload or ADC comprising the payload of the present application has excellent anti-tumor effect, especially on colon cancer, pancreatic cancer, lung adenocarcinoma, breast cancer, gastric cancer, ovarian cancer, etc. Based on this, the inventors completed the present application.

[0159] Part of the payload, linker-payload and ADC of the present application

[0160] In a first aspect of the present disclosure, the present disclosure provides a compound represented by Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof,

[0161] wherein:

[0162] X is selected from the group consisting of hydroxyl, thiol, -NR m R n ;

[0163] R1, R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, thiol, carboxy, C1-C6alkoxy, C1-C6alkylthio, R a R b N-, R a R b N-C(=O)-, imidazolyl (e.g. ), phenyl, hydroxy-substituted phenyl (e.g. ), indolyl (e.g. );

[0164] R2is selected from the group consisting of C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, thiol, carboxy, C1-C6alkoxy, C1-C6alkylthio, R a R b N-, R a R b N-C(=O)-, imidazolyl (e.g. ), phenyl, hydroxy-substituted phenyl (e.g. ), indolyl (e.g. );

[0165] R a , R b are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0166] R m , R n are each independently selected from the group consisting of hydrogen, C1-C6alkyl, and R m , R n at least one of which is hydrogen.

[0167] In some embodiments, R1, R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, thiol, C1-C6alkoxy, C1-C6alkylthio, R a R b N-C(=O)-.

[0168] In some embodiments, R1, R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, C1-C6alkoxy.

[0169] In some embodiments, R1, R3are each independently selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, -CH2CH2OH, -CH2CH2OCH3.

[0170] In some embodiments, R1, R3are each independently selected from hydrogen, C1-C6alkyl.

[0171] In some embodiments, R1is selected from hydrogen, methyl, ethyl, isopropyl.

[0172] In some embodiments, R1is hydrogen.

[0173] In some embodiments, R3is selected from hydrogen, methyl, isopropyl.

[0174] In some embodiments, R3is hydrogen.

[0175] In some embodiments, R2is selected from C1-C6alkyl, and the C1-C6alkyl is optionally substituted with a group selected from hydroxy, thiol, C1-C6alkoxy, C1-C6alkylthio, R a R b N-C(=O)-.

[0176] In some embodiments, R2is selected from C1-C6alkyl, and the C1-C6alkyl is optionally substituted with a group selected from hydroxy, C1-C6alkoxy.

[0177] In some embodiments, R2is selected from methyl, ethyl, isopropyl, -CH2CH2OH, -CH2CH2OCH3.

[0178] In some embodiments, R2is selected from C1-C6alkyl.

[0179] In some embodiments, R2is methyl.

[0180] In some embodiments, R a , R b are each hydrogen.

[0181] In some embodiments, R m , R n are each hydrogen.

[0182] In some embodiments, X is selected from hydroxy, thiol, amino.

[0183] In some embodiments, X is selected from hydroxy, amino.

[0184] In some embodiments, X is hydroxy. In other embodiments, X is amino.

[0185] In some embodiments, the compound of formula I has a structural formula of formula I-1,

[0186] wherein * indicates that the carbon atom at the position shown by * is a chiral carbon atom in S configuration when R1and R3are not hydrogen.

[0187] In some embodiments, the compound is selected from:

[0188] In a second aspect of the present disclosure, the present disclosure provides a compound represented by Formula II, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof, M-L 1 -L 2 -L 3 -D II

[0189] wherein:

[0190] M is

[0191] L 1 selected from -(CH2) p1 C(=O)-, -(CH2CH2O) q1 -, p2 C(=O)-NH-(CH2CH2O) q2 (CH2) p3 C(=O)-, -(CH2) p2 C(=O)-NH-(CH2CH2O) q2 -;

[0192] p1, p2, p3 are each independently selected from any integer between 1-10;

[0193] q1, q2 are each independently selected from any integer between 1-20;

[0194] L 2 is an amino acid residue or a peptide residue formed by 2-10 amino acid residues, which amino acid residue is optionally substituted with a group R x ;

[0195] R x is -(CH2CH2O) q3 -CH3;

[0196] q3 is selected from any integer between 1-20;

[0197] L 3 is absent or -NH-CH2-;

[0198] D is a group formed by removing one hydrogen atom, and the site of removing the hydrogen atom is X;

[0199] X, R1, R2, R3 are each independently described in any technical solution of the first aspect.

[0200] In some implementations, L 1 The site on the left is connected to M, L 1 The site on the right and L 2 Connected.

[0201] In some implementations, L 2 The amino terminus of L 1 Connected, L 3 When it exists, L 2 carbonyl end and L 3 Connected, L 3 When it does not exist, L 2 The carbonyl end is connected to the D phase.

[0202] In some implementations, L 3 When it exists, L 3 The N-terminus and L 2 Connected, L 3 The C end is connected to the D end.

[0203] In some embodiments, D is the group formed by removing a hydrogen atom from the structure shown in I-1', and the site where the hydrogen atom is removed is X.

[0204] Wherein, X, R1, R2, and R3 are each independently described as in any of the technical solutions of the first aspect, and * indicates that when R1 and R3 are not hydrogen, the carbon atom at the position indicated by * is a chiral carbon atom with an S configuration.

[0205] In some implementations, D is selected from R1, R2, and R3 are each independently described as described in any of the technical solutions of the first aspect.

[0206] In some implementations, D is selected from Wherein, R1, R2, and R3 are each independently described as in any of the technical solutions of the first aspect, and * indicates that when R1 and R3 are not hydrogen, the carbon atom at the position indicated by * is a chiral carbon atom with an S configuration.

[0207] In some implementations, D is selected from R1, R2, and R3 are each independently described as described in any of the technical solutions of the first aspect.

[0208] In some implementations, D is selected from Wherein, R1, R2, and R3 are each independently described as in any of the technical solutions of the first aspect, and * indicates that when R1 and R3 are not hydrogen, the carbon atom at the position indicated by * is a chiral carbon atom with an S configuration.

[0209] In some embodiments, D is wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0210] In some embodiments, D is selected from wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect, * indicates that the carbon atom at the position marked by * is a chiral carbon atom in S configuration when R1and R3are not hydrogen.

[0211] In some embodiments, D is wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0212] In some embodiments, D is selected from wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect, * indicates that the carbon atom at the position marked by * is a chiral carbon atom in S configuration when R1and R3are not hydrogen.

[0213] In some embodiments, L 1 is -(CH2) p1 C(=O)-.

[0214] In some embodiments, p1is selected from 1, 2, 3, 4, 5. In some embodiments, p1is selected from 2, 3, 4, 5. In some embodiments, p1is selected from 2, 5.

[0215] In some embodiments, p2is selected from 1, 2, 3, 4, 5. In some embodiments, p2is selected from 2, 3, 4, 5. In some embodiments, p2is selected from 2, 3, 4. In some embodiments, p2is 2.

[0216] In some embodiments, p3is selected from 1, 2, 3, 4, 5. In some embodiments, p3is selected from 2, 3, 4, 5. In some embodiments, p3is selected from 2, 3, 4. In some embodiments, p3is 2.

[0217] In some embodiments, q1is selected from any integer between 1-12. In some embodiments, q1is selected from any integer between 2-12. In some embodiments, q1is selected from any integer between 6-10. In some embodiments, q1is 8.

[0218] In some embodiments, q2is selected from any integer between 1-12. In some embodiments, q2is selected from any integer between 2-12. In some embodiments, q2is selected from any integer between 6-10. In some embodiments, q2is 8.

[0219] In some embodiments, q3 is selected from any integer between 8 and 20. In some embodiments, q3 is selected from any integer between 10 and 16. In some embodiments, q3 is selected from any integer between 10 and 14. In some embodiments, q3 is 12.

[0220] In some embodiments, L 2 is a peptide residue formed from 2-5 (preferably 4 or 5) amino acid residues, which are optionally substituted with a group R x In some embodiments, the amino acid residues are selected from glycine residues, phenylalanine residues, valine residues, citrulline residues, alanine residues, glutamine residues. In some embodiments, the amino acid residues are selected from glycine residues, phenylalanine residues, glutamine residues.

[0221] In some embodiments, L 2 is a peptide residue formed from 2-5 (preferably 4 or 5) amino acid residues selected from glycine residues, phenylalanine residues, valine residues, citrulline residues, alanine residues, glutamine residues (preferably selected from glycine residues, phenylalanine residues, glutamine residues), wherein the glutamine residues are optionally substituted with a group R x (preferably, the side chain amino group of the glutamine residues is optionally substituted with a group R x ).

[0222] In some embodiments, L 2 is selected from glycine residue-glycine residue-phenylalanine residue-glycine residue (GGFG), valine residue-citrulline residue (VC), valine residue-alanine residue (VA), alanine residue-alanine residue-alanine residue (AAA), glutamine residue-glycine residue-glycine residue-phenylalanine residue-glycine residue (QGGFG), the glutamine residue being optionally substituted with a group R x (preferably, the side chain amino group of the glutamine residues is optionally substituted with a group R x ).

[0223] In some embodiments, L 2 is selected from glycine residue-glycine residue-phenylalanine residue-glycine residue (GGFG), glutamine residue-glycine residue-glycine residue-phenylalanine residue-glycine residue (QGGFG), the glutamine residue being optionally substituted with a group R x (preferably, the side chain amino group of the glutamine residues is optionally substituted with a group R 2 is selected from

[0224] In some embodiments, L 3 is -NH-CH2-. In other embodiments, L 3 is absent.

[0225] In some embodiments, D is selected from:

[0226] In some embodiments, the compound is selected from:

[0227] In a third aspect of the present disclosure, the present disclosure provides an antibody drug conjugate represented by Formula III, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof, Ab-(M'-L 1 -L 2 -L 3 -D) n III

[0228] Ab is an antibody or an antigen binding fragment thereof;

[0229] M' is

[0230] L 1 , L 2 , L 3 , D is as described in any of the technical solutions of the second aspect;

[0231] n is any integer or any decimal number between 2 and 8 (e.g., 2, 3, 4, 5, 6, 7, 8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8; or, for example, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, or, for example, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, or 7.5-8).

[0232] In the present disclosure, DAR (drug antibody ratio) refers to the number of drug molecules conjugated to an antibody (e.g., n in Formula III). The number of drug molecules contained in the antibody drug conjugates described herein can be an integer or a decimal number. Whether it is an integer or a decimal number, it refers to the average number of drug molecules conjugated per antibody. "n is any number between 2 and 8" means that n can be any integer selected from 2 to 8 (including the endpoints 2 and 8), or any decimal number selected from 2 to 8. Meanwhile, those skilled in the art can understand that even if the same preparation method is used, the DAR values of antibody drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of not more than 0.5 up and down.

[0233] DAR can be determined by conventional means, such as mass spectrometry, ELISA assay, HIC, and HPLC. The quantitative distribution of ADCs in terms of n can also be determined. In some cases, the separation, purification, and verification of homogenous ADCs with a certain value of n from ADCs with other drug loads can be achieved by means such as HIC, reverse phase HPLC, or electrophoresis.

[0234] In some embodiments, the antibody drug conjugate is selected from the group consisting of:

[0235] In the ADC formed in this disclosure, the antibody Ab is linked to the carbon atom of the succinimide at the end of the linker-payload via -S-. This -S- is not an additional thiol group introduced into the Ab, but rather a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0236] In some implementations, n is any integer or decimal between 4 and 8 (e.g., 4.0–4.5, 4.0–5.0, 4.0–5.5, 4.0–6.0, 4.0–6.5, 4.0–7.0, 4.0–7.5, 4.0–8.0, 4.5–5.0, 4.5–5.5, 4.5–6.0, 4.5–6.5, 4.5–7.0, 4.5–7.5, 4.5–8.0, 5.0–5.5, 5.0–6). 0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.0, 6.5~7.0, 6.5~7.5, 6.5~8.0, 7.0~7.5, 7.0~8.0 or 7.5~8.0, as detailed in 7.4).

[0237] In some embodiments, the DAR value of the composition is any integer or decimal selected from 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, 8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8; or, for example, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1). 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or, for example, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0238] In some embodiments, the DAR value of the composition is any integer or any decimal number selected from between 4-8 (e.g., 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 4.5-6.5, 4.5-7.0, 4.5-7.5, 4.5-8.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.5-7.0, 6.5-7.5, 6.5-8.0, 7.0-7.5, 7.0-8.0, or 7.5-8.0, specifically 7.4).

[0239] The terms

[0240] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0241] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, the expression "about 100" includes all values (e.g., 99, 100, 101, etc.) between 99 and 101.

[0242] As used herein, the term "comprising" or "including" can be open-ended, semi- closed, or closed. In other words, the term "comprising" or "including" also includes "consisting essentially of" or "consisting of."

[0243] As used herein, the term "aliphatic group" refers to a non-aromatic carbon hydride group composed of carbon atoms, e.g., a "Ci-6 aliphatic group" refers to a non-aromatic carbon hydride group composed of 1, 2, 3, 4, 5, or 6 carbon atoms.

[0244] As used herein, the term "alkyl" refers to a monovalent straight-chain or branched saturated hydrocarbon group composed of carbon and hydrogen atoms, e.g., a "Ci-6 alkyl" refers to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. 1-6 "alkyl" denotes an alkyl group having 1-6 (e.g., 1, 2, 3, 4, 5, or 6) carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl, and the like. In the present application, alkyl is also meant to include deuterated alkyl groups, examples of deuterated alkyl groups include, but are not limited to, CD3, CD2CD3, CD2CD2CD3.

[0245] As used herein, the term "alkylene" refers to the group resulting from removal of one hydrogen atom from an alkyl group as described above, for example methylene (-CH2-), ethylene (-CH2CH2-), and the like.

[0246] As used herein, the term "alkenyl" refers to a straight or branched chain unsaturated hydrocarbon group having at least one double bond, consisting of carbon and hydrogen atoms, for example "C 2-6 "Alkenyl" refers to an alkenyl group having 2-6 (e.g., 2, 3, 4, 5, or 6) carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, 1 -butenyl, isobutenyl, and 1,3-budadienyl, and the like.

[0247] As used herein, the term "alkynyl" refers to a straight or branched chain unsaturated hydrocarbon group having at least one triple bond, consisting of carbon and hydrogen atoms. For example "C 2-6 "Alkynyl" refers to an alkynyl group having 2-6 (e.g., 2, 3, 4, 5, or 6) carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1 -propynyl, 2-propynyl, and 1 -butynyl, and the like.

[0248] As used herein, the term "carbocyclic" or "carbocyclyl" refers to a saturated or partially saturated all-carbon ring group, for example a monocyclic, bicyclic, or tricyclic ring structure, wherein the ring structure as a whole is not aromatic but which can contain one or more unsaturated structures. The ring can be further substituted with one or more substituents. When there are two or more rings in the carbocyclyl group, the rings can be further fused, bridged, spirocyclic, or any combination thereof.

[0249] As used herein, the term "cycloalkyl" refers to a monovalent saturated carbocyclic ring group consisting of carbon and hydrogen atoms, for example "C 3- "Cycloalkyl" refers to a cycloalkyl group having 3-8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3-8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C

[0250] As used herein, the term "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group having at least one double bond, for example "C 3- "Cycloalkenyl" refers to a cycloalkenyl group having 3-8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C 3-6 "Cycloalkenyl" refers to a cycloalkenyl group having 3-8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C

[0251] As used herein, the term "alkoxy" refers to the group -O-alkyl, where examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, tert-butoxy, and the like.

[0252] As used herein, "halogen" refers to F, CI, Br, I, and isotopes thereof, including but not limited to F, 18 F, CI, 32 CI, Br, I.

[0253] As used herein, the term "alkylamino" refers to the group -NR u R u group, where R u and R u are each independently H or alkyl as defined herein, and R u and R u are not both H. Alkylamino groups can be monoalkylamino or dialkylamino, examples of alkylamino groups include, but are not limited to, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, and the like.

[0254] As used herein, the term "alkylthio" refers to the group -S-R t where R t is H or alkyl as defined herein.

[0255] As used herein, the term "haloalkyl" refers to a group resulting from the replacement of one or more hydrogens on an alkyl group as described above with the same or different halogen. Where "haloC 1-6 alkyl" is preferred, examples of haloalkyl groups include, but are not limited to, -CH2CI, -CH2CF3, -CH2CCI3, perfluoroalkyl (e.g., -CF3-, -CF2CF3), and the like. 1-4

[0256] As used herein, the term "sulfonyl" refers to the group -S(O)2-. Sulfonyl groups are preferably -S(O)2-(C 1-6 alkyl), such as -S(O)2-CH3, -S(O)2-CH2CH3, and the like.

[0257] ​As used herein, the term "heterocyclic" or "heterocyclic group" refers to a fully or partially saturated monocyclic or polycyclic cyclic group with one or more heteroatoms selected from N, S, or O. For example, "4-7 membered heterocyclic group" refers to a group having 4-7 (e.g., 4, 5, 6, or 7) ring members. The nitrogen or sulfur atom may be oxidized, or the nitrogen atom may be quaternized. The heterocyclic group may be attached to any heteroatom or carbon residue in a ring or ring system molecule. Monocyclic heterocyclic groups include, but are not limited to: nitrogen-containing heterocyclic butyl groups, pyrrolyl groups, oxocyclic butyl groups, pyrazolinyl groups, imidazolinyl groups, imidazoalkyl groups, oxazolinyl groups, isoxazolinyl groups, thiazoalkyl groups, isothiazolinyl groups, tetrahydrofuranyl groups, piperidinyl groups, piperazinyl groups, 2-oxopiperidinyl groups, 2-oxopiperidinyl groups, 2-oxopiperidinyl groups, hexahydroacoxaneyl groups, 4-piperidinoneyl groups, tetrahydropyranyl groups, morpholinyl groups, thiomorpholinyl groups, thiomorpholinyl sulfoxide groups, thiomorpholinyl sulfone groups, 1,3-dioxylyl groups, and tetrahydro-1,1-dioxothiophene groups, etc. Polycyclic heterocyclic groups include, but are not limited to, heterocyclic groups of spiroheterocyclic, fused heterocyclic, and bridged heterocyclic groups.

[0258] As used herein, the term "aromatic ring" or "aryl" refers to an aromatic cyclic hydrocarbon group (including monocyclic, bicyclic, or polycyclic groups), such as "C 6-12 "Aryl" refers to an aromatic cyclic hydrocarbon group having 6-12 (6, 7, 8, 9, 10, 11, or 12) ring carbon atoms. It contains two or more aromatic rings (such as bicyclic rings), and the aromatic rings of the aryl group can be linked by single bonds (such as biphenyl) or fused (such as naphthalene, anthracene, etc.). Examples of aryl groups (especially monocyclic and bicyclic groups) include, but are not limited to, phenyl, biphenyl, or naphthyl. Aryl groups can be fused with heterocyclic groups through single bonds or any two adjacent ring carbon atoms, for example: benzotetrahydrofuranyl, benzotetrahydropyranyl, benzodioxane, etc. wait.

[0259] As used herein, the term “heteroaromatic ring” or “heteroaryl” refers to an aromatic cyclic group (including monocyclic, bicyclic, or polycyclic groups) whose ring skeleton contains 1, 2, 3, or 4 heteroatoms selected from N, S, or O. For example, “5-12-membered heteroaryl” refers to a monocyclic, bicyclic, or tricyclic group having 5 to 12 (5, 6, 7, 8, 9, 10, 11, or 12) ring atoms. Examples of heteroaryl groups include, but are not limited to: imidazole, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thiophene, furanyl, pyranyl, pyridinyl, pyrroleyl, pyrazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophene, benzothiaranyl, benzoimidazolyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiazolyl, benzopyranyl, indoleyl, isoindoleyl, triazolyl, triazinyl, quinoxolinyl, purine, quinazolinyl, quinazinyl, naphridinyl, pteridinyl, carbazoleyl, and azazolyl. Bis, diazepine Bis, diazepine

[0260] As used herein, the term "unsaturated" refers to a cyclic structure having at least one double or triple bond between the ring atoms of the ring, but is not intended to include aromatic or heteroaromatic rings as defined herein.

[0261] As used herein, "deuterated" means that one or more hydrogens in a compound or group are replaced by deuterium. Deuterated can be mono-substituted, di-substituted, poly-substituted, or per-substituted. The terms "one or more deuterium substituted" and "one or more deuterium substitutions" are used interchangeably.

[0262] As used herein, the term "substituted" means that one or more hydrogens on a given moiety are replaced by a particular substituent. The particular substituent is as described in the immediately preceding text, or as the substituent appears in each embodiment. Unless otherwise indicated, a given substituted moiety can have at each substitutable position one substituent selected from a particular group, which can be the same or different at each position. Those skilled in the art will appreciate that combinations of substituents contemplated by this application are those stable or chemically feasible combinations.

[0263] Unless otherwise specifically noted, the groups described herein are optionally substituted with a substituent selected from the group consisting of D, halogen, cyano, nitro, hydroxy, amino, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, 3-12 membered heterocyclyl, C3-C 12 Cycloalkyl, 5-10 membered heteroaryl, and C6-C 10 Aryl.

[0264] "Optionally" as used herein means that the subsequently described event or circumstance can or can not occur, and this description includes instances where the event or circumstance occurs, as well as instances where it does not.

[0265] The term "plurality" as used herein means a positive integer of 2, 3, 4, 5, or greater than 5.

[0266] Unless otherwise specifically noted, the term "amino acid" as used herein is intended to include any conventional amino acid such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine (methionine), tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, arginine. It is understood that the term encompasses D-, L-, and DL- forms of the amino acids.

[0267] As used herein, the term "peptide residue" refers to a fragment of one or more amino acids linked by peptide bonds. For example, one or more amino acids in a polypeptide residue can be optionally substituted. For example, a polypeptide residue of the present application can comprise glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0268] As used herein, the term "drug loading" refers to the average number of cytotoxic drugs loaded on each ligand (e.g., Ab) of the present application, and can also be expressed as the ratio of the amount of cytotoxic drug to the amount of Ab. The drug loading can range from 0 to 12, e.g., 1 to 10, cytotoxic drugs per ligand (Ab). In the context of the present application, drug loading is denoted as n, and exemplary values can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The drug loading of each ADC molecule after conjugation reaction can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC profile.

[0269] Abbreviations

[0270] Targeting unit (Ab)

[0271] As used herein, the term "Ab" is a targeting unit, i.e., a polypeptide, antibody, antigen binding fragment, or non-antibody targeting unit that binds to a target. A non-antibody targeting unit can also be referred to as a non-antibody scaffold.

[0272] In some embodiments, the targeting unit specifically binds to a target molecule. As used herein, "specifically binds" refers to the ability of a targeting unit described herein (e.g., an antibody or portion thereof) to bind to a target with a KDof 10 -5 M (10,000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less.

[0273] As used herein, the term "antibody" or "immunoglobulin" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen of interest, which are heterotetrameric glycoproteins of about 150,000 daltons, having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy (H) chains. The light chains are linked to the heavy chains by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains differs depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.

[0274] Each heavy chain has at one end a variable region (VH) followed by a number of constant regions. The heavy chain constant regions can include three domains, CH1, CH2 and CH3, and optionally a fourth domain, CH4. Each light chain has at one end a variable region (VL) and at its other end a constant region. The light chain constant region is a CL domain. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLthus provides three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure for the variable region is well known in the art.

[0275] As used herein, the term "antigen binding fragment" refers to a portion of an antibody that has the VHand / or VLsequences of an antibody or CDRs of an antibody and that specifically binds an antigen of interest. Examples of antigen binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, single domain antibody (also known as VHH, VNAR, sdAb or nanobody), or diabody.

[0276] As used herein, the terms Fab, F(ab')2, and Fv refer to the following:

[0277] (i) Fab is a monovalent fragment consisting of a VL, a VH, a CL, and a CH1 domain;

[0278] (ii) F(ab')2 is a bivalent fragment comprising two Fab fragments that are linked to each other by disulfide bonds under the hinge region; and

[0279] (iii) Fv consisting of a VL and a VH domain.

[0280] Although the two domains of the Fv fragment, i.e., VL and VH, are coded for by separate coding regions, they can also be linked to each other using a synthetic linker, e.g., a poly-G4S amino acid sequence, such that they can be prepared as a single protein chain, where the VL and VH regions combine to form a monovalent molecule (called single chain Fv or scFv). The term "antigen binding fragment" also includes such single chain antibodies. Other forms of single chain antibodies, such as "diabodies" are also included herein.

[0281] Diabodies are bivalent, bispecific antibodies in which VHand VLregions are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.

[0282] A single-domain antibody is an antigen-binding fragment of an antibody comprising a single monomeric variable antibody region. Single-domain antibodies can be derived from the variable region of an antibody heavy chain of a camelid (e.g., a nanobody or VHH moiety). In addition, the term single-domain antibody includes a human articular heavy chain variable domain (AVH) or a VNAR moiety derived from a shark.

[0283] Techniques for producing single-domain antibodies (e.g., DABs or VHHs) are known in the art. Single-domain antibodies can be obtained from, for example, a camel, a llama, or an alpaca by standard immunization techniques. VHHs can have potent antigen-binding ability and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs. Llama serum IgG contains only about 50% of the camelid heavy chain-only IgG antibodies (HCAbs). A llama can be immunized with an antigen, and VHHs that bind to and neutralize the target antigen can be isolated. PCR primers that amplify llama VHH-encoding sequences have been identified and can be used to construct llama VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art.

[0284] In some embodiments, the targeting unit is an antibody or antigen-binding fragment thereof is a bispecific or multispecific binding agent. Bispecific and multispecific antibodies include the following: scFvl-ScFv2, ScFvl2-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv.

[0285] In some embodiments, the IgG-scFv is IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv.

[0286] In some embodiments, the targeting unit is a cancer-associated antigen, such as ADAM9, Trop-2, EGFR, CD19, CD20, CD30, CD33, CD38, CA125, MUC-1, PD-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin (MLSN), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, lp19q, ABL1, AKT1, ALK, APC, AR, ATM, BRAF, BRCA1, BRCA2, cKIT, cMET, CSF1R, CTNNB1, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, JAK2, KDR (VEGFR2), KRAS, MGMT, MGMT-Me, MLH1, MPL, NOTCH1, NRAS, PDGFRA, Pgp, PIK3CA, PR, PTEN, RET, RRM1, SMO, SPARC, TLE3, TOP2A, TOPO1, TP53, TS, TUBB3, VHL, CDH1, ERBB4, FBXW7, HNF1A, JAK3, NPM1, PTPN11, RB1, SMAD4, SMARCB1, STK1, MLH1, MSH2, MSH6, PMS2, ROS1, ERCC1, 5T4 (TPBG), B7-H3, CCR7, CD105, CD22, CD46, CD47, CD56, CD70, CD71, CD79b, CDH6, CLDN6, CLDN18.2, CLEC12A, DLL3, DR5, ERBB3 (HER3), EPCAM, FOLR1, IGF1R, IL2RA (CD25), IL3RA, ITGB6, LIV-1, LRRC15, mesothelin (MSLN), NaPi2b (SLC34A2), Nectin-4, PTK7, ROR1, SEZ6, SLC44A4, SLITRK6, tissue factor (TF), TROP2, or B7-H4.

[0287] According to the present application, the terms “cancer-associated antigen”, “tumor antigen”, “tumor-expressed antigen”, “cancer antigen”, “cancer-associated antigen”, and “cancer-expressed antigen” are equivalent and used interchangeably herein.

[0288] In some embodiments, the targeting unit specifically binds to a target, e.g., ADAM9, Trop-2, EGFR, CD19, CD20, CD30, CD33, CD70, LIV-1, or EGFRv3.

[0289] In some embodiments, non-limiting examples of monoclonal antibodies include rituximab, trastuzumab, pertuzumab, bevacizumab, ranibizumab, cetuximab, alemtuzumab, panitumumab, ibritumomab, tositumomab, ipilimumab, zalutumumab, daratumumab, fetuzumab, ramucirumab, galiximab, fezakinumab, ocrelizumab, ofatumumab, CD antibody 2F2 (HuMax-CD20), 7D8, IgM 2C6, IgG 12C6, 1 IB8, B1, 2H7, LT20, 1FS, or AT80, dalizumab, and anti-LHRH receptor antibodies, e.g., clones A9E4, F1G4, AT2G7, GNRH03, GNRHR2, etc., which can be used, inter alia, with the conjugates according to the present application.

[0290] In some embodiments, the targeting unit is a non-antibody scaffold. In some embodiments, the targeting unit is a non-antibody protein scaffold. Such non-antibody scaffolds include, e.g., Affibodies, Affilins, Anticalins, Atrimers, Avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., Adnectins, Centyrins, Pronectins, and Tn3), Fynomers, Kunitz domains, and OBodies. Such non-antibody protein scaffolds include, e.g., Affibodies, Affilins, Anticalins, Atrimers, Avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., Adnectins, Centyrins, Pronectins, and Tn3), Fynomers, Kunitz domains, and OBodies.

[0291] Non-antibody scaffolds can be considered to belong to two structural classes, domain-sized constructs (in the range of 6 to 20 kDa) and constrained peptides (in the range of 2 to 4 kDa). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins, and OBodies. Peptide-sized non-antibody scaffolds include, for example, Avimers, bicyclic peptides, and cysteine knots. Non-antibody protein scaffolds can be considered to belong to two structural classes, domain-sized constructs (in the range of 6 to 20 kDa) and constrained peptides (in the range of 2 to 4 kDa). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins, and OBodies. Peptide-sized non-antibody scaffolds include, but are not limited to, for example, Avimers, bicyclic peptides, and cysteine knots.

[0292] Advantages of using non-antibody scaffolds include increased affinity, target neutralization, and stability. Various non-antibody scaffolds can also overcome some of the limitations of antibody scaffolds, such as in tissue penetration, smaller size, and thermal stability. Some non-antibody scaffolds can also be easier to engineer, such as when bispecific constructs are desired, without the potential for light chain binding issues. Methods of engineering constructs on non-antibody scaffolds are known to those of ordinary skill in the art and thus, in some embodiments, the targeting unit can comprise a non-antibody scaffold.

[0293] Accordingly, in some embodiments, the targeting unit can comprise a non-antibody scaffold protein. Those skilled in the art will appreciate that, in some embodiments, the targeting unit can comprise, for example, an adnectin scaffold or a moiety derived from the tenth fibronectin type III domain of human (10Fn3); an anticalin scaffold derived from human lipocalin (e.g., those described in, e.g., WO2015 / 104406); an avimer scaffold or protein fragment derived from the A domain of low-density lipoprotein-related protein (LRP) and / or very low-density lipoprotein receptor (VLDLR); a fynomer scaffold or moiety of the SH3 domain of FYN tyrosine kinase; a kunitz domain scaffold or moiety of a Kunitz-type protease inhibitor, such as human pancreatic trypsin inhibitor, aprotinin (bovine pancreatic trypsin inhibitor), amyloid precursor protein in Alzheimer’s, and tissue factor pathway inhibitor; a knottin scaffold (cysteine-knot miniprotein), such as scaffolds based on trypsin inhibitors from E. coli; an affibody scaffold or all or part of the Z domain of staphylococcal protein A; a beta-Hairpin analog scaffold; an ankyrin repeat protein (DARPin) scaffold designed based on ankyrin repeat (AR) proteins or artificial protein scaffolds; or any scaffold derived from or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin. For example, the binding site of human transferrin for human transferrin receptor can be diversified to generate a diverse library of transferrin variants, some of which have acquired affinity for different antigens. The portion of human transferrin not involved in binding the receptor is left unchanged and serves as a scaffold, like the framework regions of an antibody, to provide the variant binding site.

[0294] In some embodiments, the targeting unit is an anti-ADAM9 antibody or antigen-binding fragment thereof that specifically binds ADAM9. In some embodiments, the targeting unit is an anti-TROP-2 antibody or antigen-binding fragment thereof that specifically binds TROP-2. In some embodiments, the targeting unit is an anti-EGFR antibody or antigen-binding fragment thereof that specifically binds EGFR.

[0295] In some embodiments, ADAM9 (e.g., as set forth in SEQ ID NOs: 1 and 2), TROP-2 (e.g., as set forth in SEQ ID NOs: 3 and 4), or EGFR (e.g., as set forth in SEQ ID NOs: 5 and 6) antibodies, antigen-binding fragments thereof, and other binding agents, and conjugates of these antibodies, antigen-binding fragments, and other binding agents are provided. Methods of using ADAM9, TROP-2, or EGFR antibodies, antigen-binding fragments, and other binding agents, and conjugates thereof, to treat cancer and other diseases are also provided. The inventions disclosed herein are based, in part, on ADAM9, TROP-2, or EGFR antibodies, antigen-binding fragments thereof, and other binding agents, and conjugates that specifically bind ADAM9, TROP-2, or EGFR and exhibit improved properties. ADAM9, TROP-2, or EGFR are important and advantageous therapeutic targets for treating certain cancers. ADAM9, TROP-2, or EGFR antibodies, antigen-binding fragments thereof, other binding agents, and conjugates thereof provide compositions and methods based on the use of these antibodies, antigen-binding fragments, and related binding agents, and conjugates thereof, in the treatment of ADAM9, TROP-2, or EGFR+ cancers and other diseases.

[0296] SEQ ID NO: 1

[0297] ADAM9 antibody light chain sequence

[0298] SEQ ID NO: 2

[0299] ADAM9 antibody heavy chain sequence

[0300] SEQ ID NO: 3

[0301] TROP-2 antibody light chain sequence

[0302] SEQ ID NO: 4

[0303] TROP-2 antibody heavy chain sequence

[0304] SEQ ID NO: 5

[0305] EGFR antibody light chain sequence

[0306] SEQ ID NO: 6

[0307] EGFR antibody heavy chain sequence

[0308] The sequence of the DNA molecule of the antibody or fragment thereof of the present application can be obtained by conventional techniques, such as by using PCR amplification or screening a genomic library, etc. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.

[0309] The antibody constituting the antibody drug conjugate of the present application preferably maintains the antigen binding ability thereof in the original wild state. Therefore, the antibody of the present application can, preferably specifically, bind to an antigen.

[0310] Drug

[0311] As used herein, the terms "drug", "toxin", "cytotoxin", "cytotoxic drug" and "payload" are used interchangeably to refer to a chemical molecule that has a strong destructive effect on the normal growth of tumor cells. The cytotoxic drug can kill tumor cells at a sufficiently high concentration. The "cytotoxic drug" can include toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g. At211, I131, I125, Y90, Re18, Rel88, Sm153, Bi212, p32 or Lu radioisotopes), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, for example, can be toxic drugs including but not limited to camptothecin derivatives.

[0312] Camptothecin is a pentacyclic parent nucleus compound isolated from the plant Camptotheca acuminata, which is composed of quinoline ring AB, pyrrole ring C, pyridone ring D and a-hydroxy lactone ring E, wherein the 20 position is S configuration (see below).

[0313] Research data shows that camptothecin can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting the unwinding of DNA, leading to inhibition of DNA replication, and thus causing cell death. Camptothecin and its derivatives have strong antitumor activity in animal models of lung cancer, breast cancer, colorectal cancer, ovarian cancer, etc.

[0314] Clinical studies have found that measures to improve the safety and effectiveness of camptothecin compounds include improving their pharmacokinetic properties, adjusting activity, reducing dosage, or using conjugates with antibodies to form antibody conjugate drugs. Therefore, there is still a high clinical demand and application value for the development of novel camptothecin compounds and their conjugates that can improve effectiveness and safety issues.

[0315] The "drug", "toxin", "cytotoxin", "cytotoxic drug" or "payload" used in the present application refers to a derivative of camptothecin, i.e. a compound as shown in formula (I), a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof,

[0316] wherein X is selected from the group consisting of -OH, -SH, -NHR m ;

[0317] R m is selected from the group consisting of hydrogen, C1-C6alkyl;

[0318] R1and R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0319] R2is C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, mercapto, carboxy, C1-C6alkoxy, C1-C6alkylthio, C1-C6alkylamino;

[0320] A has the structure of Formula IV:

[0321] Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O);

[0322] R 4 and R 5 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C8alkyl, C1-C8haloalkyl, C1-C8deuteroalkyl;

[0323] Alternatively, R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C 12 carbocyclic ring, a saturated or unsaturated 5-12 membered heterocyclic ring;

[0324] Unless specifically indicated, each of the above-mentioned alkyl, haloalkyl, deuteroalkyl, alkoxy, alkylthio, and alkylamino groups can be substituted with a substituent selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, mercapto, carboxy, C1-C6alkyl-amine, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C1-C6alkyl-S-, haloC1-C6alkyl, haloC2-C6alkenyl, haloC2-C6alkynyl, haloC1-C6alkoxy, allyl, benzyl, C6-C10aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl. 12

[0325] In a preferred embodiment, R 4 and R 5 ​each independently is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C4alkyl group, a C1-C4haloalkyl group, a C1-C4deuteroalkyl group;

[0326] or R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C7carbocyclic ring, a saturated or unsaturated 5-7 membered heterocyclic ring.

[0327] In another preferred embodiment, said R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C7carbocyclic ring, a saturated or unsaturated 5-7 membered heterocyclic ring.

[0328] Linker

[0329] As used herein, the term "linker" generally refers to a chemical moiety or bond that links one end of a ligand (e.g., an Ab described herein) to the other end of a cytotoxic drug, and can also be linked to another linker before being linked to the cytotoxic drug.

[0330] According to the mechanism of drug release in cells, "linkers" or "linkers of antibody-drug conjugates" can be divided into two categories: non-cleavable linkers and cleavable linkers. The linker L of the present application is a cleavable linker.

[0331] For antibody-drug conjugates containing non-cleavable linkers, the mechanism of drug release is as follows: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically degraded in the lysosome, releasing an active molecule composed of a small molecule drug, a linker, and antibody amino acid residues. The structural change of the drug molecule thus brought about does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells. Therefore, such active drugs cannot kill adjacent tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect).

[0332] Cleavable linkers, as the name implies, can be cleaved in the target cell and release the active drug (small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzymatically unstable linkers. The linker L of the present application is an enzymatically unstable linker (i.e., an enzyme-cleavable linker).

[0333] Chemically labile linkers can be selectively cleaved due to the differences in the nature of plasma and cytoplasm. Such properties include pH, glutathione concentration, etc. Linkers sensitive to pH, also known as acid-cleavable linkers, are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Linkers sensitive to glutathione, also known as disulfide linkers, are based on the difference in glutathione concentration between the intracellular (millimolar range) and blood (micromolar range).

[0334] Enzymatically labile linkers, such as peptide linkers, can provide better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as Cathepsin B or plasmin (increased levels of which are found in some tumor tissues). Such peptide linkers are believed to be very stable in the plasma circulation because extracellular proteases are generally not active due to the extracellular pH and serum protease inhibitors. Given the high plasma stability and good intracellular cleavage selectivity and efficiency, enzymatically labile linkers are widely used as cleavable linkers for antibody drug conjugates. Typical enzymatically labile linkers include Val-Cit (VC), Phe-Lys, etc.

[0335] The linker of the present application is M-L1-L2-L3-L4-;

[0336] wherein M is for connecting Ab and L1, which is selected from the group consisting of: pyrimidinyl,

[0337] each X3is independently a leaving group, preferably, the X3is selected from the group consisting of Cl, Br, OMs, OTs, OTf;

[0338] R d is H, C1-C6alkyl, C1-C6deuteroalkyl, C3-C8cycloalkyl, or C3-C8deutero-cycloalkyl;

[0339] W1is -(C(R7)(R8))n7-, W2is -(OCH2CH2)n8-On9-, W3is -(C(R9)(R 13 )n10;

[0340] wherein n7, n8, and n10are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0341] n9is 0 or 1;

[0342] When methylene groups are present in W1 and W3, the methylene groups in W1 and W3 are optionally replaced by one or more groups selected from the group consisting of: optionally replaced by one or more R groups. 15 Substituted rings A, -N(R) 14 )C(O)-、-C(O)N(R 14 )-, C(O)-, -OC(O)-, -C(O)O-, -NR 14 -, -O-, -S-, -SO-, -SO2-, -P(R 14 )-、-P(=O)(R 14 )-、-N(R 14 SO2-, -SO2N(R) 14 -, -C(=S)-, -C(=NR) 14 )-, -N=N-, -C=N-, -N=C-, -C(=N2)-;

[0343] The ring A is selected from the following group: C 6-10 arylene, 5-8 membered heteroarylene, 3-10 membered heterocyclic, 3-10 membered saturated or partially unsaturated carbonyl cycloyl;

[0344] Each of R7, R8, R9, and R 13 R 14 and R 15 Each of the following is independently selected: hydrogen, protium, deuterium, tritium, halogen, -NO2, CN, -OR e -SR e -N(R) f (R) g -C(O)R e -CO2R e -C(O)C(O)R e -C(O)CH2C(O)R e -S(O)R e -S(O)2R e -C(O)N(R) e -SO2N(R) f (R) g -OC(O)R e -N(R)SO2R e 、Being R e Optional substitution of C 1-6 Aliphatic groups, optionally substituted C1-C8 alkyl groups, optionally substituted C1-C8 haloalkyl groups, optionally substituted C1-C8 deuterated alkyl groups, optionally substituted C3-C8 cycloalkyl groups, and optionally substituted C4-C8 cycloalkylalkyl groups;

[0345] Among them, each R e Rf and R g each independently is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH-, -OC(O)H, -N(H)SO2H, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl; 1-6 aliphatic, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl;

[0346] L1is selected from the group consisting of an optionally substituted -(CHR) p1 C(=O)-, -(CH2CH2O) q1 -, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 (CHR) p3 C(=O)-, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 -, -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-, -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-, -X1-(CHROCHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-, -X1-(CHR) m1 -X2-(CHR) m2 -C(=O)-, -(CH2CH2O) n3 -C(=O)-;

[0347] X1and X2are each independently selected from the group consisting of -O-, -C(O)-, -C(O)-NR-, optionally substituted C6-C 10 aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heteroalicyclyl, and optionally substituted C3-C6cycloaliphatic;

[0348] wherein each R is independently selected from the group consisting of H, D, (CH2) n4 OH, (CH2) n4 NH2, (CH2O) n4 (CH2CH2O) n5 H, (CH2O) n4 (CH2CH2O) n5 CH3, (CH2) n4 OCH3, (CH2CH2O) n5 CH3, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 H, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3;

[0349] wherein each of pi, p2, p3, qi, and q2 is independently 0, 1, 2, 3, 4, 5, 6, or 7; each of mi, m2, n3, n4, and n5 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0350] L2is a peptide residue or a group selected from the group consisting of C 1-6 alkylene, -N(R6)-, carbonyl,

[0351] R6is selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkyl substituted with one or more -(CH2CH2O)r-;

[0352] r is selected from an integer between 1 and 10; s is selected from an integer between 1 and 20;

[0353] L3is -L 4a -(NR b ) n6 -R 12 -L 4b -,

[0354] wherein L 4a is absent, or L 4a is an optionally substituted

[0355] n6is 0 or 1;

[0356] R 12 is a chemical bond, CH2, or CD2;

[0357] L 4bL4is absent, or is an optionally substituted group selected from the group consisting of: 4b is optionally substituted

[0358] R a and R b each is independently selected from the group consisting of hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl, optionally substituted C1-C4alkylamino, optionally substituted C1-C4alkylthio, optionally substituted -S(O)2-C1-C4alkylthio;

[0359] L4is absent, or is an optionally substituted group selected from the group consisting of:

[0360] wherein Y is selected from the group consisting of O, S, NH;

[0361] v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0362] R 10 and R 11 each is independently selected from the group consisting of hydrogen, deuterium, halogen, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl, or R 10 and R 11 together with the atom to which they are attached form an optionally substituted C3-C8cycloalkyl;

[0363] R 16 and R 17 each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SONH2, -OC(O)H, -N(H)SO2H, optionally substituted C 1-6 aliphatic, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl;

[0364] L1, L2, L3, and L4, as described above, can also be optionally substituted with a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a Ci-C6alkyl-NH-, a (Ci-C6alkyl)2N-, a Ci-C6alkyl group, a C2-C6alkenyl group, a C2-C6alkynyl group, a Ci-C6alkoxy group, a halo-Ci-C6alkyl group, a halo-C2-C6alkenyl group, a halo-C2-C6alkynyl group, a halo-Ci-C6alkoxy group, an allyl group, a benzyl group, a C6-Ci0aryl group, a 5-7 membered heteroaryl group, a C3-C8cycloalkyl group, a 3-12 membered heterocyclyl group, a C6-Ci0aryl-Ci-C6alkyl group, a 5-7 membered heteroaryl-Ci-C6alkyl group, a Ci-C6alkoxy-Ci-C6alkyl group, a Ci-C6alkoxy-carbonyl group, a phenoxycarbonyl group, a C2-C6alkynyl-carbonyl group, a C2-C6alkenyl-carbonyl group, a C3-C6cycloalkyl-carbonyl group, a Ci-C6alkyl-sulfonyl group, a phenyl group, a 5-7 membered heteroaryl group, a C3-C8cycloalkyl group, a 3-12 membered heterocyclyl group. 12 L1, L2, L3, and L4, as described above, can also be optionally substituted with a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a Ci-C6alkyl-NH-, a (Ci-C6alkyl)2N-, a Ci-C6alkyl group, a C2-C6alkenyl group, a C2-C6alkynyl group, a Ci-C6alkoxy group, a halo-Ci-C6alkyl group, a halo-C2-C6alkenyl group, a halo-C2-C6alkynyl group, a halo-Ci-C6alkoxy group, an allyl group, a benzyl group, a C6-Ci0aryl group, a 5-7 membered heteroaryl group, a C3-C8cycloalkyl group, a 3-12 membered heterocyclyl group, a C6-Ci0aryl-Ci-C6alkyl group, a 5-7 membered heteroaryl-Ci-C6alkyl group, a Ci-C6alkoxy-Ci-C6alkyl group, a Ci-C6alkoxy-carbonyl group, a phenoxycarbonyl group, a C2-C6alkynyl-carbonyl group, a C2-C6alkenyl-carbonyl group, a C3-C6cycloalkyl-carbonyl group, a Ci-C6alkyl-sulfonyl group, a phenyl group, a 5-7 membered heteroaryl group, a C3-C8cycloalkyl group, a 3-12 membered heterocyclyl group.

[0365] Antibody-drug conjugate

[0366] As used herein, the terms "antibody drug conjugate", "antibody conjugate", "antibody drug conjugate", "antibody-drug conjugate", and "ADC" are used interchangeably to refer to a targeting unit (e.g., a polypeptide, an antibody, an antigen binding fragment, or a non-antibody targeting unit) linked to a cytotoxic drug having a biological activity via a linker, such as an antibody-drug conjugate of formula (III).

[0367] The antibody drug conjugate provided herein is composed of an antibody, a linker, and a drug, as shown in the following formula (III): n Formula (III)

[0368] wherein the linker M'-L is a cleavable linker, which is defined as described above;

[0369] Ab is a polypeptide, an antibody, an antigen binding fragment, or a non-antibody scaffold that binds to a target, which is defined as described above;

[0370] D is a compound of formula (I-1) as shown below

[0371] wherein X', R1, R2, R3, and A are defined as described above.

[0372] In a preferred embodiment, Ab is a globular protein containing a series of amino acid sites available for conjugation of the drug-linker, and due to its tertiary and quaternary structure, only solvent accessible amino acids are available for conjugation. In fact, conjugation in high yield usually occurs on the epsilon-amino group of lysine residues or the thiol group of cysteine residues.

[0373] The large number of lysine side chains on the surface of antibody proteins leads to a large number of sites available for drug conjugation, resulting in a mixture of antibody drug conjugates with different numbers of drug conjugations (drug / antibody ratio, DAR), e.g. integers or fractions of 2.0 to 16.0 (preferably 2.0 to 8.0, most preferably 4.0 to 8.0) and conjugation sites.

[0374] Active ingredient

[0375] Depending on the context, the "compound of the present invention" can refer to a compound of Formula (I), Formula (II), or Formula (III), and also includes stereoisomers thereof, optical isomers thereof, pharmaceutically acceptable salts thereof, crystalline forms thereof, isotopic derivatives thereof, prodrugs thereof, metabolites thereof, solvates thereof, or hydrates thereof.

[0376] Unless specifically noted, the structural formulas described herein are intended to include all stereoisomers (e.g., cis-trans isomers, enantiomeric, diastereomeric, and conformational isomers): the R, S configurations of asymmetric centers; the (Z), (E) isomers of double bonds; the various rotamers, atropisomers, and the like. Thus, individual stereoisomers of compounds of the present invention or mixtures of stereoisomers, including enantiomeric, diastereomeric, or conformational mixtures, are within the scope of the present invention.

[0377] The compounds of the present invention can contain cis-trans isomers, one or more chiral carbon atoms, and thus can produce stereoisomeric forms such as cis-trans isomers, chiral isomers, enantiomeric, diastereomeric, and other combinations. Cis-trans isomerism refers to the phenomenon of diastereoisomerism in which the spatial arrangement of groups around a molecule differs due to the presence of a restriction on free rotation. This restriction is usually caused by the presence of a functional group in the structure of the organic compound such as a C=C double bond, a C=N double bond, a C=S double bond, a N=N double bond, a heterocycle, or a cycloalkane, which cannot rotate freely. Organic molecules containing such isomers, such as alkenes, azo compounds, cycloalkanes, and the like, are considered cis-trans isomers, with cis referring to the same ligand being in an adjacent position, and trans referring to the same ligand being in a diagonal position. Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms. The preparation of the compounds of the present invention can select a racemate, a cis-trans isomer, a chiral isomer, a diastereomer, or an enantiomer as a starting material or intermediate. The optically active isomers can be prepared using chiral synthons or chiral reagents, or by resolution using conventional techniques, such as crystallization and chiral chromatography.

[0378] Conventional techniques for preparing / isolating individual optical isomers (i.e., cis-trans isomers and chiral isomers) include chiral synthesis from appropriate cis-trans precursors or optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography.

[0379] If one wishes to design a synthesis of a specific stereoisomer of a compound of the application, it can be prepared asymmetrically, or derivatized with a chiral auxiliary, the resulting stereomixture separated, and the chiral auxiliary removed to yield the pure cis-trans monomer, chiral monomer, or mixed stereomonomer. If the molecule contains a cis-trans isomeric center, the pure cis or trans product can be purified by column chromatography (normal phase silica gel column or reverse phase high performance liquid chromatography preparative). Alternatively, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, one can form a diastereomeric salt with an appropriate optically active acid or base, and the diastereomeric salt can be separated by crystallization or chromatography, and the base or acid removed to yield the pure enantiomer.

[0380] The present application also includes isotopically-labelled compounds, i.e., compounds having an atom replaced by an atom having the atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., an atom of 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl. Isotopically-labelled compounds of the present application are within the scope of the present application. Isotopically-labelled compounds, for example those into which radioactive isotopes such as 3 H-labelled compounds and 14 C-labelled compounds are useful in drug and substrate tissue distribution assays. The presence of stable isotopes such as 3 H) and carbon-14 (i.e., 14 C) in a compound makes for easier detection and quantitation of the compound useful in metabolic or other assays. In addition, replacement of protons, i.e., 2 H, with heavier isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Isotopically labelled compounds can generally be prepared by carrying out the procedures disclosed in the Examples by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

[0381] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0382] A "pharmaceutically acceptable acid addition salt" means a salt formed by combination of an inorganic or organic acid with the free base which retains the biological effectiveness of the free base and has no additional adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like. Organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, phenylsulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene- disulfonate, and the like. These salts can be prepared by methods known in the art.

[0383] A "pharmaceutically acceptable base addition salt" means a salt formed by combination of an inorganic or organic base with the free acid which retains the biological effectiveness of the free acid and has no additional adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0384] Metabolites of compounds of Formula (I), Formula (II), or Formula (III), and pharmaceutically acceptable salts thereof, and those compounds which exist in un-ionized enteric form, as well as those which can be converted into the compounds of Formula (I), in vivo, are also within the scope of the present application.

[0385] As used herein, the term "solvate" means a complex of a compound of Formula (I), Formula (II), or Formula (III), with solvent molecules in specific ratios.

[0386] As used herein, the term "hydrate" refers to a compound of Formula (I), Formula (II), or Formula (III) coordinated with water molecules to form a complex in a particular ratio.

[0387] Pharmaceutical compositions and methods of administration

[0388] Because of the antibody-drug conjugates provided by the present application, a particular cell population can be targeted and bound to a specific protein (antigen) on the cell surface, so that the drug is released in an active form into the cell by endocytosis of the conjugate or by permeation of the drug into the cell. Thus, the antibody-drug conjugates of the present application can be used to treat a target disease (e.g., cancer) by administering the above-mentioned antibody-drug conjugates in a therapeutically effective amount to a subject (e.g., a human) by a suitable route. The subject in need of treatment can be a patient at risk for, or suspected of having, a disorder associated with the activity or amount of expression of a particular antigen. Such patients can be identified by routine medical examination.

[0389] Conventional methods, known to those of ordinary skill in the medical arts, can be used to administer the pharmaceutical compositions to a subject, depending on the type of disease to be treated or the site of the disease. The compositions can also be administered by other conventional routes, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implant. The term "parenterally" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered by routes which are subject to administration of injectable depot, e.g., using 1, 3 or 6 month depot injectable or biodegradable materials and methods of the subject.

[0390] The injectable compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethyl alcohol, polyhydric alcohol (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by infusion techniques, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused over a period of time. The physiologically acceptable excipient can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipient. Intramuscular formulations, for example, a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutically acceptable excipient such as water for injection, 0.9% saline, or 5% dextrose solution.

[0391] When treated with the antibody-drug conjugate of the present application, delivery can be performed by methods conventional in the art. For example, it can be introduced into cells by use of liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by use of an infusion pump. Other methods include various transport and carrier systems by use of conjugates and biodegradable polymers.

[0392] The pharmaceutical composition of the present application contains a safe and effective amount of the antibody-drug conjugate of the present application and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present application can be prepared in the form of a solution, for example, by a conventional method using physiological saline or an aqueous solution containing dextrose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of the active ingredient to be administered is a therapeutically effective amount.

[0393] The effective amount of the antibody-drug conjugate of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc.

[0394] The target disease (disease to be treated) of the antibody-drug conjugate of the present application is a tumor.

[0395] In a preferred embodiment, the tumor can be selected from the group of tumors associated with expression of ADAM9, TROP-2, EGFR, HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70.

[0396] In a preferred embodiment, the tumor includes, but is not limited to, lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0397] The selection of the preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) according to various factors. The factors include, but are not limited to, pharmacokinetic parameters of the antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. Generally, satisfactory effects can be obtained when the antibody-drug conjugate of the present application is administered at a dose of about 0.0001 mg to 50 mg / kg of animal body weight (preferably 0.001 mg to 10 mg / kg of animal body weight) per day. For example, several divided doses can be administered daily, or the dose can be proportionally reduced, as required by the therapeutic situation.

[0398] Dosage forms of the antibody-drug conjugate of the present application for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that can be required.

[0399] The antibody-drug conjugate of the present application can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0400] When using the pharmaceutical composition, a safe and effective amount of the antibody-drug conjugate of the present application is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage, and for a 60 kg body weight human, the daily dosage is usually 1-2000 mg, preferably 5-500 mg. Of course, the specific dosage should also consider the administration route, patient health status, etc., which are within the skill of a skilled physician.

[0401] The main advantages of the present application are:

[0402] 1. The payload, linker-payload and / or ADC of the present application has excellent anti-tumor effect, especially on colon cancer, pancreatic cancer, lung adenocarcinoma, breast cancer, etc.

[0403] 2. The ADC of the present application shows excellent biological activity in in vivo and in vitro experiments; it inhibits the proliferation of multiple cell lines in vitro and effectively inhibits the growth of multiple tumor models in vivo, especially in some models, the drug efficacy is significantly better than that of the control positive ADC using the marketed drug (deruxtecan) of the same target.

[0404] 3. The antibody-drug conjugate of the present application has high in vivo tolerance and excellent therapeutic safety window. In rat toxicity tests, compared with the control positive ADC using the marketed drug linker-payload (deruxtecan) of the same target, it shows lower body weight reduction, lower food intake reduction, and lower hematological toxicity.

[0405] 4. The antibody-drug conjugate of the present application has excellent pharmacokinetics, and the total anti in cynomolgus monkey blood shows a long half-life, suggesting that the antibody-drug conjugate of the present application can maintain a steady-state drug concentration, reduce the impact of concentration fluctuations on efficacy, and prolong the drug administration interval to improve patient compliance.

[0406] The application will be further described in connection with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples without specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0407] Example 1: Preparation of antibody

[0408] The preparation method of the humanized antibody H03-2 against ADAM9 used in this application is as described in patent 202410970739.9, specifically as follows:

[0409] I. Antigen, immunization scheme and antibody preparation

[0410] 1. Antigen

[0411] The protein antigen used for immunization is the amino acids 206-297 of human ADAM9 recombinant protein (NCBI Reference Sequence: NP_003807.1), i.e. ADAM9 extracellular domain (ADAM9-ECD), with 6 His tags at the C-terminus, i.e. human ADAM9-ECD-His protein, prepared and provided by Sunny Biotech (Shanghai) Co., Ltd.

[0412] 2. Immunization scheme

[0413] Ten Balb / C mice, female, 10 weeks old, were selected for animal immunization with the aforementioned human ADAM9-ECD-His protein. The first immunization antigen was emulsified with Freund's complete adjuvant, and the antigen amount was 100 μg / mouse. The subsequent immunization adjuvant was Freund's incomplete adjuvant, and the antigen amount was 50 μg / mouse. The injection method was intraperitoneal and subcutaneous multi-point injection. The cell immunization was 1×10 7 cells / mouse for the first time, and 5×10 6 cells / mouse for subsequent immunization. The injection method was intraperitoneal injection. The number of immunizations was 4 times, and the immunization time was set as every other week, i.e. after immunization, the mice were immunized again after one week, and 21 days after the last immunization, the mice were given a booster immunization.

[0414] 3. Serum titer detection

[0415] 3.1. Sample preparation

[0416] The mouse serum after immunization was diluted with 5% PBST at a gradient of 1500, 4500, 13500, and 40500.

[0417] 3.2. ELISA method for detecting serum titer

[0418] 1) Coating: 2 pg / mL antigen, 30 pL per well, 4°C overnight, PBST wash plate 3 times.

[0419] 2) Blocking: 5% PBST, room temperature, 2h, PBST wash plate 3 times.

[0420] 3) Primary antibody: add the above gradient diluted serum, non-immune mouse serum as negative control, 30 pL / well, room temperature, 1h, PBST wash plate 6 times.

[0421] 4) Secondary antibody: serum sample added 1:5000 diluted secondary antibody goat anti-mouse-lgG-HRP (Rockland, cat# 609-103-123), 5% PBST dilution; positive control group added 1:5000 diluted secondary antibody goat anti-human IgG-HRP (Shanghai Enzyme-linked Biotechnology Co., Ltd., ml087062), 5% PBST dilution. 30 pL / well, room temperature, 50min, PBST wash plate 6 times.

[0422] 5) Termination: add 30 pL / well TMB (Suzhou Yake Chemical Reagent Co., Ltd., cat# S0025) room temperature color development 5min-10min, then add 30 pL / well 2M stop solution (Suzhou Yake Chemical Reagent Co., Ltd.) to terminate the reaction, and read the data with a microplate reader at OD450.

[0423] II. Preparation of hybridoma cells and obtaining of mouse-derived antibodies

[0424] As described above, the titer of the above immunized Balb / C mice was detected with human ADAM9-ECD-His protein, the serum titer and the ability to bind cell surface antigens were evaluated, and the control titer detection (more than 100,000-fold dilution) determined the start of cell fusion. The immune mice with high serum titer were sacrificed after the first terminal immunization, and the spleen cells and SP2 / 0 myeloma cells were fused and plated to obtain hybridoma. The target hybridoma was screened by indirect ELISA, and the monoclonal cell strain was established by limiting dilution method. The positive antibody strain obtained was further screened by indirect ELISA, so as to select the hybridoma combined with recombinant protein. The logarithmic growth period hybridoma cells were collected, and the RNA was extracted by Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and finally the mouse-derived antibody was obtained.

[0425] III. Humanization of mouse antibody

[0426] The murine anti-human ADAM9 monoclonal antibody was humanized according to the methods well known in the art. Briefly, the human constant domains were used to replace the constant domains of the parent (murine) antibody, and the human germline antibody sequences were selected according to the homology of the murine antibody and human antibody. In this embodiment, the murine antibody was humanized.

[0427] Specifically, the human germline sequences were used as the acceptor framework for humanizing the murine antibody. To find the closest germline sequences, the most similar expressed light chain and the most similar heavy chain were identified in the germline sequence database by NCBI IgBLAST (ncbi.nlm.nih.gov / igblast / ). In this search, the CDR sequences of the murine antibody were masked. The selection criteria for the most suitable expressed sequences included the sequence identity of the canonical residues and the interface residues, and the similarity of the CDR loop length.

[0428] Based on the obtained murine antibody VH / VL CDR canonical structure, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain the human germline template with high homology.

[0429] The CDR regions of the murine antibody were grafted onto the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the back-mutation was performed on the buried residues, the residues directly interacting with the CDR regions, and the residues having a significant impact on the conformation of VL and VH, and the CDR region chemically unstable amino acid residues were optimized. Through expression testing and comparison of the number of back-mutations, the antibody with the combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences was selected, in which the CDR sequences of the light and heavy chain variable regions are shown in Table 1, and the specific variable region sequence information of the humanized antibody is shown in Table 2.

[0430] Table 1. Humanized Adam-9 antibody heavy and light chain variable region CDR sequences

[0431] Table 2. Humanized Adam-9 antibody heavy and light chain variable region sequences

[0432] The method for preparing the antibody hRS7 against Trop-2 used in this application is described in the patent US2004 / 0001825 A1.

[0433] Example 2: Synthesis of toxin compound A

[0434] 2.1 Synthesis of intermediate compound A2

[0435] To a solution of Exelucin Al (250 mg, 0.57 mmol) in THF (10 mL) was added compound Fmoc-Gly-Osu (226.42 mg, 0.57 mmol) and compound DIEA (222.61 mg, 1.72 mmol) and the reaction was allowed to proceed at room temperature for 3 hours. TLC indicated that the reaction was complete, and the reaction was concentrated and purified by silica gel column to give the target product A2 about 305 mg. LCMS confirmation: [M+H] + = 715.4.

[0436] 2.2 Synthesis of intermediate compound A3

[0437] To a solution of compound A2 (300 mg, 0.42 mmol) in THF (5 mL) was added compound diethylamine (614 mg, 8.39 mmol) and the reaction was allowed to proceed at room temperature for 3 hours. TLC indicated that the reaction was complete, and the reaction was concentrated and purified by silica gel column to give the target product A3 about 163 mg. LCMS confirmation: [M+H] + = 492.9.

[0438] 2.3 Synthesis of compound A

[0439] To a solution of compound A3 (50 mg, 0.1 mmol) in DMA (2 mL) was added glycolic acid (8.5 mg, 0.11 mmol), DIEA (39 mg, 0.3 mmol) and HATU (46.3 mg, 0.12 mmol) and the reaction was allowed to proceed at room temperature for 5 minutes. The reaction was purified by preparative column and lyophilized to give the target product A about 10 mg. 1H NMR (400 MHz, DMSO) δ 8.52 (d, J = 8.9 Hz, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 11.1 Hz, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.57 (t, J = 5.7 Hz, 2H), 5.43 (s, 2H), 5.24 (d, J = 5.5 Hz, 2H), 3.81 (t, J = 12.3 Hz, 4H), 3.16 (s, 2H), 2.40 (s, 3H), 2.22 - 2.03 (m, 2H), 1.92 - 1.80 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0440] Example 3: Synthesis of toxin compound B

[0441] 3.1 Synthesis of intermediate compound B2

[0442] A1 (400 mg, 0.75 mmol), 157 mg B1, 343 mg HATU were dissolved in 10 mL DMF, 389 mg DIPEA was added slowly, and the reaction was carried out at room temperature after completion. Prep-HPLC gave B2: 420 mg of yellow solid product. LCMS confirmation: [M+H] + = 607.

[0443] 3.2 Synthesis of intermediate compound B3

[0444] B2 (420 mg) was dissolved in 15 mL DCM / TFA (V / V = 5 / 1), and the reaction was carried out at room temperature after completion. Prep-HPLC gave B3: 380 mg. LCMS confirmation: [M+H] + = 507.

[0445] 3.3 Synthesis of compound B

[0446] B4 (17.3 mg, 0.23 mmol) was dissolved in 3 mL DMF, and after adding B3 (141 mg, 0.23 mmol), HATU (95.1 mg, 0.25 mmol), DIPEA (118 mg, 0.91 mmol) was slowly added dropwise, and the reaction was carried out at room temperature after completion. Prep-HPLC gave compound B: 100 mg. LCMS confirmation: [M+H] + = 565.

[0447] 1H NMR (400 MHz, DMSO) δ 8.66 (d, J = 8.5 Hz, 0.44H), 8.53 (d, J = 8.6 Hz, 0.65H), 7.80 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.58 (s, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 4.58 (d, J = 5.6 Hz, 0.42H), 4.48 (t, J = 5.6 Hz, 0.61H), 4.18 - 4.07 (m, 2H), 3.96 (ddd, J = 26.1, 14.7, 8.0 Hz, 2H), 3.17 (s, 2H), 2.95 (s, 2H), 2.87 (s, 1H), 2.40 (s, 3H), 2.16 (d, J = 4.9 Hz, 2H), 1.97 - 1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0448] Example 4: Synthesis of toxin compound C

[0449] 4.1 Synthesis of intermediate compound C3

[0450] C1 (600 mg, 1.13 mmol), 252 mg C2, 515 mg HATU were dissolved in 20 mL DMF, 583 mg DIPEA was added slowly at 0 °C, after the addition was completed, the reaction was carried out at room temperature. After the reaction was completed, compound C3 was obtained by reverse phase preparation: 500 mg. LCMS confirmation: [M+H] + = 621.2.

[0451] 4.2 Synthesis of intermediate compound C4

[0452] C3 (500 mg, 0.07 mmol) was dissolved in 25 mL DCM, 5 mL TFA was added slowly, after the addition was completed, the reaction was carried out at room temperature. After the reaction was completed, it was concentrated to dryness, and compound C4 was obtained by reverse phase preparation: 300 mg. LCMS confirmation: [M+H] + = 521.2

[0453] 4.3 Synthesis of compound C

[0454] C4 (26.9 mg, 0.36 mmol) was dissolved in 10 mL DMF, after adding C5 (225 mg, 0.35 mmol), 148 mg HATU, 247 uL DIPEA was added slowly dropwise, after the addition was completed, the reaction was carried out at room temperature. Compound C was obtained by reverse phase preparation and prep-HPLC: 90 mg. LCMS confirmation: [M+H] + = 579.1

[0455] 1H NMR (400 MHz, DMSO-d6) δ 8.46 - 8.34 (m, 1H), 7.75 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 6.32 (s, 1H), 5.59 - 5.54 (m, 1H), 5.46 - 5.36 (m, 2H), 5.17 (s, 2H), 5.04 (br s, 0.6H), 4.34 (br s, 0.4H), 4.27 (br s, 1H), 4.09 (d, J = 4.9 Hz, 2H), 3.19 - 3.16 (m, 2H), 2.86 (s, 3H), 2.40 (s, 3H), 2.16 (d, J = 6.3 Hz, 2H), 1.97 - 1.81 (m, 2H), 1.36 (d, J = 6.2 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).

[0456] Example 5: Synthesis of toxin compound D

[0457] 5.1 Synthesis of intermediate compound D3

[0458] Compound Dl (500 mg, 0.94 mmol) was dissolved in DMF (10 mL), followed by the addition of compound D2 (436 mg, 1.88 mmol), HOBT (255 mg, 1.88 mmol), EDCI (61 mg, 1.88 mmol) and DIEA (366 mg, 2.83 mmol). The reaction was stirred at 25 °C for 18 h. After the reaction was completed, the reaction was concentrated. The resulting crude product was purified by reverse phase chromatography [water (0.1% trifluoroacetic acid) / acetonitrile, 0% to 47%] to give compound D3 (350 mg, yellow solid) with a yield of 57.3%. LCMS: [M+H] + = 648.7.

[0459] 5.2 Synthesis of intermediate compound D4

[0460] Compound D3 (330 mg, 0.51 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was stirred at 25 °C for 1 h. After the reaction was completed, the reaction was concentrated to give compound D4 275 mg of crude product. LCMS: [M+H] + = 549.4

[0461] 5.3 Synthesis of intermediate compound D6

[0462] Compound D4 (220 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of DIEA (155 mg, 1.20 mmol) and compound D5 (109 mg, 0.80 mmol). The reaction was stirred at 25 °C for 1 h. After the reaction was completed, the reaction was concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 20 / 1) to give compound D6 (180 mg, yellow solid). LCMS: [M+H] + = 649.2.

[0463] 5.4 Synthesis of intermediate compound D

[0464] Compound D6 (180 mg, 0.28 mmol) was dissolved in tetrahydrofuran (6 mL), and aqueous lithium hydroxide solution (0.1 M, 6 mL, 0.56 mmol) was added. The reaction solution was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was neutralized to pH 7 with 1 M hydrochloric acid and concentrated. The obtained crude product was purified by pre-HPLC (trifluoroacetic acid) to obtain compound D (28.6 mg, yellow solid), yield: 17.3%. LCMS: [M+H] + = 579.4, 1 H NMR: N241085-113-P1 (400 MHz, DMSO-d6) δ 8.70 - 8.58 (m, 1H), 7.85 - 7.73 (m, 1H), 7.32 - 7.28 (m, 1H), 6.53 (s, 1H), 5.62 - 5.47 (m, 1H), 5.42 (s, 2H), 5.36 - 5.23 (m, 1H), 5.13 - 5.04 (m, 1H), 4.64 (d, J = 10.8 Hz, 1H), 4.24 (d, J = 15.2 Hz, 0.5H), 4.14 (s, 1H), 4.01 (d, J = 15.2 Hz, 0.5H), 3.22 - 3.05 (m, 2H), 2.92 - 2.87 (m, 3H), 2.43 - 2.36 (m, 3H), 2.32 - 2.04 (m, 3H), 1.93 - 1.80 (m, 2H), 0.94 - 0.82 (m, 6H), 0.80 - 0.71 (m, 3H).

[0465] Example 6: Synthesis of toxin compound F

[0466] 6.1 Synthesis of compound F

[0467] C4 (80 mg, 0.15 mmol) and compound F1 (20 mg, 0.23 mmol) were dissolved in DMF (6 mL), and EDCI (44 mg, 0.23 mmol), HOBT (62 mg, 0.46 mmol), and DIEA (89 mg, 0.69 mmol) were sequentially added. The reaction solution was reacted at 25 °C for 18 hours. After the reaction was completed, the reaction solution was purified by prep-HPLC (trifluoroacetic acid system) to obtain compound F (22.4 mg, white solid), yield: 23.5%. LCMS: [(M+H)] + = 593.4, 1H NMR: (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.76 (d, J = 10.8 Hz, 1H), 7.33 (s, 1H), 5.59 - 5.53 (m, 1H), 5.45 - 5.36 (m, 2H), 5.20 (s, 2H), 5.00 - 4.76 (m, 1H), 4.44 (q, J = 6.4 Hz, 1H), 3.18 - 3.14 (m, 2H), 3.06 - 2.97 (m, 2H), 2.89 - 2.79 (m, 1H), 2.42 - 2.38 (m, 3H), 2.21 - 2.12 (m, 2H), 1.94 - 1.83 (m, 2H), 1.41 - 1.30 (m, 3H), 1.19 (d, J = 6.0 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H).

[0468] Example 7: Synthesis of toxin compound G

[0469] 7.1 Synthesis of compound G

[0470] Compound B3 (200 mg, 0.40 mmol) was dissolved in DMF (6 mL), and compound G1 (41 mg, 0.40 mmol), HOBT (107 mg, 0.79 mmol), EDCI (151 mg, 0.79 mmol) and DIEA (204 mg, 1.58 mmol) were added successively. The reaction solution was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was purified by pre-HPLC to obtain compound G: 11.7 mg. LCMS confirmation: [M+H]+=593.4.

[0471] 1H NMR: (400 MHz, DMSO-d6) δ 8.68 - 8.46 (m, 1H), 7.85 - 7.75 (m, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.65 - 5.52 (m, 1H), 5.43 (s, 2H), 5.33 - 5.14 (m, 2H), 4.85 - 4.58 (m, 1H), 4.34 - 4.22 (m, 1H), 4.18 - 4.02 (m, 2H), 3.22 - 3.15 (m, 2H), 3.09 (s, 2H), 2.87 (s, 1H), 2.41 (s, 3H), 2.23 - 2.09 (m, 2H), 1.93 - 1.80 (m, 2H), 1.67 - 1.35 (m, 2H), 0.92 - 0.72 (m, 6H).

[0472] Example 8: Synthesis of toxin compound H

[0473] 8.1 Synthesis of compound H

[0474] Compound B3 (250 mg, 0.49 mmol) was dissolved in DMF (5 mL), and compound H1 (117 mg, 0.99 mmol), HOBT (133 mg, 0.99 mmol), EDCI (189 mg, 0.99 mmol) and DIEA (128 mg, 0.99 mmol) were added successively. The reaction solution was reacted at 25 °C for 16 hours. After the reaction was completed, the reaction solution was purified by pre-HPLC to obtain compound H: 22.21 mg. LCMS confirmation: [M+H]+= 607.4.

[0475] 1H NMR: (400 MHz, DMSO-d6) δ 8.66 - 8.50 (m, 1H), 7.84 - 7.77 (m, 1H), 7.34 - 7.29 (m, 1H), 6.52 (s, 1H), 5.61 - 5.54 (m, 1H), 5.43 (s, 2H), 5.32 - 5.14 (m, 2H), 4.80 (d, J = 7.2 Hz, 0.35H), 4.47 (d, J = 7.6 Hz, 0.65H), 4.24 - 3.87 (m, 3H), 3.22 - 3.14 (m, 2H), 3.09 (s, 2H), 2.89 (s, 1H), 2.40 (s, 3H), 2.24 - 2.08 (m, 2H), 1.95 - 1.77 (m, 3H), 0.92 - 0.77 (m, 9H).

[0476] Example 9: Synthesis of linker-drug conjugate LD-A

[0477] 9.1 Synthesis of compound LD-A1

[0478] Compound A1 (250 mg, 0.57 mmol) was dissolved in THF (10 mL), and compound Fmoc-G-Osu (226.42 mg, 0.57 mmol) and compound DIEA (222.61 mg, 1.72 mmol) were added and reacted at room temperature for 3 hours. TLC detected that the reaction was complete, and the reaction solution was spin-dried and purified by silica gel column to obtain the target product LD-A1 about 305 mg. LCMS confirmation: [M+H]+= 715.4

[0479] 9.2 Synthesis of compound LD-A2

[0480] To a solution of compound LD-A1 (300 mg, 0.42 mmol) in THF (5 mL) was added compound diethylamine (614 mg, 8.39 mmol) and the reaction was allowed to proceed at room temperature for 3 hours. TLC monitoring indicated that the starting material was consumed completely. The reaction was concentrated and purified by silica gel column to give the target molecule LD-A2 about 163 mg. LCMS confirmation: [M+H]+=492.9

[0481] 9.3 Synthesis of final product LD-A

[0482] To a solution of compound LD-A3 (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) (100 mg, 0.16 mmol) in DMF / H2O (2.5 mL / 0.5 mL) was added compound LD-A2 (79.87 mg, 0.16 mmol), DMTMM (71.70 mg, 0.24 mmol) and TEA (49.23 mg, 0.49 mmol) and the reaction was allowed to proceed at room temperature for 1 hour. The reaction was purified by preparative column and lyophilized to give the target molecule LD-A about 20.1 mg. LCMS confirmation: [M+H]+=1091.9.

[0483] Example 10: Synthesis of linker-drug conjugate LD-B

[0484] 10.1 Synthesis of compound LD-B2

[0485] A1 (200 mg, 0.459 mmol), LD-B1 (87 mg, 0.459 mmol) and HATU (174.6 mg, 0.459 mmol) were added to DMF (5 mL), then DIPEA (354 μL, 1.836 mmol) was added dropwise to the reaction system. The reaction was stirred at room temperature for 2 hours. The reaction was extracted with DCM and a large amount of water, concentrated to give the target product LD-B2 about 210 mg. LCMS: [M+H]+=607.25.

[0486] 10.2 Synthesis of compound LD-B3

[0487] LD-B2 (200 mg, 0.329 mmol) was added to DCM (10 mL), then TFA (4 mL) was added dropwise to the reaction system. The reaction was allowed to proceed at room temperature for 1 hour. The reaction was purified by HPLC to give the target product LD-B3 about 140 mg. LCMS: [M+H]+=507.2

[0488] 10.3 Synthesis of final product LD-B

[0489] LD-B3 (130 mg, 0.256 mmol), LD-B4 (158.21 mg, 0.256 mmol) (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) and HATU (97.5 mg, 0.256 mmol) were added to DMF (5 mL), and then DIPEA (198.12 μL, 1.03 mmol) was added dropwise to the reaction system. The reaction was allowed to react at room temperature for 2 hours. The reaction was filtered and purified by preparative liquid phase to obtain the target product LD-B about 21.0 mg. LCMS: [M+H]+=1105.44.

[0490] Example 11: Synthesis of linker-drug conjugate LD-C

[0491] 11.1 Synthesis of compound LD-C2

[0492] LD-C1 (600 mg, 1.13 mmol), 252 mg LD-C2, 515 mg HATU were dissolved in 20 mL DMF, and 583 mg DIPEA was slowly added. After the addition was completed, the reaction was carried out at room temperature. After the reaction was completed, LD-C3 was obtained by direct reverse phase column preparation: 700 mg. Yellow solid. LCMS: [M+H]+=621.26. +

[0493] 11.2 Synthesis of compound LD-C4

[0494] LD-C3 700 mg was dissolved in 25 mL DCM, and 5 mL TFA was slowly added. After the addition was completed, the reaction was carried out at room temperature. After the reaction was completed, LD-C4 was obtained by reverse phase column preparation after concentration to dryness: 660 mg. LCMS: [M+H]+=521.21. +

[0495] 11.3 Synthesis of final product LD-C

[0496] LD-C4 (300 mg, 0.48 mmol), LD-C5 (328 mg, 0.52 mmol), HATU (198 mg, 0.52 mmol) were dissolved in 10 mL NMP, and DIPEA (244 mg, 1.89 mmol) was slowly added. After the addition was completed, the reaction was carried out at room temperature. LD-C was obtained by Prep-HPLC purification: 108 mg. LCMS: [M+H]+=1119.45. +

[0497] ​​​1H NMR (400 MHz, DMSO-d6) δ 8.60 - 8.39 (m, 2H), 8.28 (t, J = 6.0 Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.99 (t, J = 5.5 Hz, 1H), 7.80 - 7.76 (m, 1H), 7.30 (s, 1H), 7.27 - 7.12 (m, 5H), 6.98 (s, 2H), 6.50 (s, 1H), 5.58 - 5.53 (m, 1H), 5.42 (s, 2H), 5.21 - 5.16 (m, 2H), 5.01 (q, J = 7.1 Hz, 1H), 4.56 (d, J = 6.6 Hz, 2H), 4.51 - 4.45 (m, 1H), 4.22 - 4.03 (m, 2H), 3.78 - 3.53 (m, 6H), 3.35 (t, J = 7.1 Hz, 2H), 3.22 - 3.08 (m, 2H), 3.08 - 2.97 (m, 1H), 2.85 - 2.72 (m, 4H), 2.39 (s, 3H), 2.14 - 2.07 (m, 4H), 1.86 (hept, J = 7.1 Hz, 2H), 1.51 - 1.42 (m, 4H), 1.37 - 1.30 (m, 3H), 1.18 (p, J = 7.5, 6.9 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0498] Example 12: Synthesis of linker-drug conjugate LD-D

[0499] 12.1 Synthesis of compound LD-D2

[0500] To a solution of compound LD-D2 (159.3 mg, 0.689 mmol) in NMP (6 mL) was added compound HATU (436.6 mg, 1.15 mmol) and TEA (222.6 mg, 1.72 mmol) and reacted at room temperature for 0.5 hour. To the reaction was added compound LD-D1 (250 mg, 0.574 mmol) and reacted at room temperature for 1 hour. The reaction was purified by reverse phase to give LD-D3: 268 mg. LCMS confirmation: [M+H]+= 649.1.

[0501] 12.2 Synthesis of compound LD-D4

[0502] To a solution of compound LD-D3 (250 mg, 0.385 mmol) in DCM (8 mL) was added compound TFA (2 mL) and the reaction was allowed to proceed at room temperature for 3 hours. The reaction was concentrated and purified by reverse phase HPLC to give LD-D4: 153 mg.

[0503] 12.3 Synthesis of compound LD-D

[0504] To a solution of compound LD-D4 (188.8 mg, 0.306 mmol) in NMP (6 mL) was added compound HATU (194.1 mg, 0.510 mmol) and DIEA (99 mg, 0.766 mmol) and the reaction was allowed to proceed at room temperature for 0.5 hours. To the reaction was added compound LD-D5 (140 mg, 0.255 mmol) and the reaction was allowed to proceed at room temperature for 1 hour. The reaction was purified by Prep-HPLC to give LD-D: 112 mg. LCMS confirmation: [M+H]+= 1147.4.

[0505] Example 13: Preparation of antibody-drug conjugate ADC1-A

[0506] Humanized antibody H03-2 against ADAM9 was diluted with 50 mM EPPS, 10 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration was 5 mg / ml. To the antibody solution was added 4.5 equivalents of TCEP aqueous solution and the reaction was allowed to proceed at 37 °C with shaking for 2 hours. The sample was water bathed using a 25 °C water bath, 16 equivalents of compound LD-A in DMSO (final DMSO concentration was 12%) was added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) was added and the reaction was allowed to proceed at 25 °C with shaking at 30 rpm for 10 minutes to give the conjugation crude product ADC1-A. After desalting column (filler: sephadex G 25) purification, the conjugate ADC1-A was obtained and stored in 20 mM histidine solution containing 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains by RP-HPLC. The DAR value was determined to be 6.9 by analyzing the composition. SEC of purified ADC1-A showed a purity of 98.8%, see Figure 1.

[0507] Example 14: Preparation of antibody-drug conjugate ADC1-B

[0508] Antibody H03-2 was diluted in 20 mM EPPS, 4 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 10 mg / ml. To the antibody solution 7 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 12 equivalents of compound LD-B in DMSO (final DMSO concentration 12%) were added to the antibody mixture and after 60 minutes, 12 equivalents of acetylcysteine (NAC) were added and the reaction was allowed to proceed for 10 minutes at 22 °C on a shaker at 30 rpm to give the crude conjugate ADC1-B. After purification by desalting column (filler: sephadex G 25), the conjugate ADC1-B was obtained and stored in 20 mM histidine with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains by RP-HPLC. The DAR value was determined by analyzing the composition to be 6.7. SEC of purified ADC1-B showed a purity of 98.8%, see Figure 2.

[0509] Example 15: Preparation of antibody-drug conjugate ADC1-C

[0510] Antibody H03-2 was diluted in 50 mM EPPS, 1 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 5 mg / ml. To the antibody solution 6.5 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 13 equivalents of compound LD-C in DMSO (final DMSO concentration 12%) were added to the antibody mixture and after 60 minutes, 13 equivalents of acetylcysteine (NAC) were added and the reaction was allowed to proceed for 10 minutes at 22 °C on a shaker at 30 rpm to give the crude conjugate ADC1-C. After purification by desalting column (filler: sephadex G 25), the conjugate ADC1-C was obtained and stored in 20 mM histidine with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains by RP-HPLC. The DAR value was determined by analyzing the composition to be 6.7. SEC of purified ADC1-C showed a purity of 98.5%, see Figure 3.

[0511] Example 16: Preparation of antibody-drug conjugate ADC1-D

[0512] Antibody H03-2 was diluted with 50 mM EPPS, 1 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 5 mg / ml. To the antibody solution 6.5 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 13 equivalents of compound LD-D in DMSO (final DMSO concentration 12%) were added to the antibody mixture, after 60 minutes, 13 equivalents of acetylcysteine (NAC) were added and the reaction was left for 10 minutes at 22 °C on a shaker at 30 rpm to give the crude product ADC1-D. After desalting column (filler: sephadex G 25) purification, the conjugate ADC1-D was obtained and stored in 20 mM histidine solution with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined by analyzing the composition to be 6.7. SEC of purified ADC1-D showed a purity of 98.5%, see Figure 4.

[0513] Example 17: Preparation of antibody-drug conjugate ADC1-DXD

[0514] Antibody H03-2 was diluted with 50 mM EPPS, 1 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 5 mg / ml. To the antibody solution 4.6 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 12 equivalents of compound deruxtecan (deruxtecan is the linker-drug conjugate of the first sanofi enhertu ADC) in DMSO (final DMSO concentration 12%) were added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) were added and the reaction was left for 10 minutes at 22 °C on a shaker at 30 rpm to give the crude product ADC1-DXD. After desalting column (filler: sephadex G 25) purification, the conjugate ADC1-DXD was obtained and stored in 20 mM histidine solution with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined by analyzing the composition to be 6.9. SEC of purified ADC1-DXD showed a purity of 99.0%, see Figure 5.

[0515] (deruxtecan: linker-drug conjugate of the first sanofi enhertu ADC)

[0516] Example 18: Preparation of antibody-drug conjugate ADC2-A

[0517] The anti-TROP-2 antibody hRS7 was diluted with 50 mM EPPS, 10 mM EDTA was added and the pH was adjusted to 7.0, the antibody concentration was 5 mg / ml. To the antibody solution, 4.5 equivalents of TCEP aqueous solution was added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 16 equivalents of compound LD-A DMSO solution (DMSO final concentration was 12%) was added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) was added, and the reaction was carried out at 25 °C on a shaker at 30 rpm for 10 minutes to obtain the conjugation crude product ADC2-A. After desalting column (filler: sephadex G25) desalting purification, the conjugate ADC2-A was obtained, and stored in 20 mM histidine solution containing 5% sucrose, pH = 5.5. The reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains by RP-HPLC. The DAR value was determined to be 7.1 by analyzing its composition. The SEC of purified ADC2-A showed a purity of 98.4%, and the results are shown in Figure 6.

[0518] Example 19: Preparation of antibody-drug conjugate ADC2-C

[0519] The antibody hRS7 was diluted with 50 mM EPPS, 10 mM EDTA was added and the pH was adjusted to 7.0, the antibody concentration was 5 mg / ml. To the antibody solution, 5.7 equivalents of TCEP aqueous solution was added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 11 equivalents of compound LD-C DMSO solution (DMSO final concentration was 12%) was added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) was added, and the reaction was carried out at 25 °C on a shaker at 30 rpm for 10 minutes to obtain the conjugation crude product ADC2-C. After desalting column (filler: sephadex G25) desalting purification, the conjugate ADC2-C was obtained, and stored in 20 mM histidine solution containing 5% sucrose, pH = 5.5. The reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains by RP-HPLC. The DAR value was determined to be 6.7 by analyzing its composition. The SEC of purified ADC2-C showed a purity of 98.5%, and the results are shown in Figure 7.

[0520] Example 20: Preparation of antibody-drug conjugate ADC2-D

[0521] Antibody hRS7 was diluted with 50 mM EPPS, 10 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 5 mg / ml. To the antibody solution 6.5 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 19 equivalents of compound LD-D in DMSO (final DMSO concentration 12%) were added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) were added and the reaction was carried out at 25 °C on a shaker at 30 rpm for 10 minutes to obtain the conjugation crude ADC 2-D. After desalting column (filler: sephadex G 25) purification, the conjugate ADC 2-D was obtained and stored in 20 mM histidine solution with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined by analyzing the composition and was 7.2. SEC of purified ADC 2-D showed a purity of 98.0%, see Figure 8.

[0522] Example 21: Preparation of antibody-drug conjugate ADC 2-DXD

[0523] Antibody hRS7 was diluted with 50 mM EPPS, 10 mM EDTA was added and the pH was adjusted to 7.0, antibody concentration 5 mg / ml. To the antibody solution 6.5 equivalents of TCEP in water were added and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, 19 equivalents of compound LD-D in DMSO (final DMSO concentration 12%) were added to the antibody mixture, after 60 minutes, 14 equivalents of acetylcysteine (NAC) were added and the reaction was carried out at 25 °C on a shaker at 30 rpm for 10 minutes to obtain the conjugation crude ADC 2-D. After desalting column (filler: sephadex G 25) purification, the conjugate ADC 2-D was obtained and stored in 20 mM histidine solution with 5% sucrose, pH = 5.5. Reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined by analyzing the composition and was 7.2. SEC of purified ADC 2-D showed a purity of 98.0%, see Figure 8.

[0524] Example 22: Preparation of antibody-drug conjugate ADC 3-C

[0525] Cetuximab (a monoclonal antibody against epidermal growth factor receptor (EGFR)) was ultrafiltered into a buffer of 50 mM EPPS 10 mM EDTA pH 7.0, diluted to an antibody concentration of 5 mg / ml. To the antibody solution was added 6.5 equivalents of a TCEP aqueous solution and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, and 13 equivalents of a DMSO solution of compound LD-C (final DMSO concentration of 12%) was added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine (NAC) was added and the reaction was allowed to proceed for 10 minutes at 22 °C on a shaker at 30 rpm to give the conjugation crude ADC 3-C. After desalting column (packing: sephadex G 25) purification, the conjugate ADC 3-C was obtained and stored in a 20 mM histidine solution with 5% sucrose, pH = 5.5. The reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains using RP-HPLC. The DAR value was determined to be 7.6 by analyzing the composition. SEC of purified ADC 3-C showed a purity of 97.6%, the results of which are shown in Figure 10.

[0526] Example 23: Preparation of antibody-drug conjugate ADC 3-DXD

[0527] Cetuximab was ultrafiltered into a buffer of 50 mM EPPS 10 mM EDTA pH 7.0, diluted to an antibody concentration of 5 mg / ml. To the antibody solution was added 6.5 equivalents of a TCEP aqueous solution and the reaction was shaken at 37 °C for 2 hours. The sample was water bathed using a 25 °C water bath, and 12 equivalents of a DMSO solution of compound deruxtecan (deruxtecan is the linker-drug conjugate of the first sanofi enhertu ADC) was added to the antibody mixture (final DMSO concentration of 12%). After 60 minutes, 13 equivalents of acetylcysteine (NAC) was added and the reaction was allowed to proceed for 10 minutes at 22 °C on a shaker at 30 rpm to give the conjugation crude ADC 3-DXD. After desalting column (packing: sephadex G 25) purification, the conjugate ADC 3-DXD was obtained and stored in a 20 mM histidine solution with 5% sucrose, pH = 5.5. The reduced ADC and antibody were analyzed for conjugated and unconjugated antibody light and heavy chains using RP-HPLC. The DAR value was determined to be 7.3 by analyzing the composition. SEC of purified ADC 3-DXD showed a purity of 97.1%, the results of which are shown in Figure 11.

[0528] Example 24: In vitro anti-tumor activity of toxin compounds

[0529] DXD (the drug molecule in deruxtecan, the linker-drug conjugate of Enhertu ADC by Daiichi Sankyo) and different toxin compounds of the present application were tested for in vitro proliferation inhibition activity against human orthotopic pancreatic cancer cell line BxPC-3 and human ovarian teratocarcinoma cell PA-1 expressing ADAM9.

[0530] The cells used in this example were purchased from Shanghai Zhijiao Xin Zhou Biological Technology Co., Ltd. or Nanjing Kebai Biological Technology Co., Ltd. (both have STR identification reports) and cultured according to the corresponding instructions, including PA-1, BxPC-3, etc. The above cells in the logarithmic growth phase were inoculated into 96-well cell culture plates at a density of 2000 cells per well, 100 μL / well, 37°C, 5% CO2, and cultured for about 24h. Different concentrations of toxin compounds were added, 3 replicate wells were set for each drug concentration, and the corresponding solvent control group and blank control well were added. After 5 days (120h) of action, the culture solution was poured off, 100 μL / well of complete culture medium containing 10% CCK-8 (purchased from Biyun Tian, cat#C0040) was added, and the reaction was carried out at 37°C for 1-2.5h (depending on the reaction activity of different cells, the reaction was carried out to the expected color depth). The activity (OD 450nM) of each group was determined, and the cell survival rate was calculated according to the following formula: survival rate = (OD drug-OD blank) / (OD control-OD blank) x 100%. The above data were analyzed by software to calculate the IC 50 values of DXD and each toxin compound of the present application on different cell lines.

[0531] The growth curves of each compound inhibiting BxPC-3 and PA-1 cancer cells are shown in Figures 12 and 13, and the corresponding IC 50 values are shown in Table 3.

[0532] Table 3: IC 50 values of DXD and compounds of the present application for inhibiting the proliferation of BXPC-3 and PA-1 cell lines

[0533] The experimental results show that in the inhibition of the proliferation of human ovarian teratocarcinoma cell PA-1 cells, the IC 50 values of compounds DXD, C, D, F, and G are all low (<0.5 ng / mL), indicating that they have excellent effects on inhibiting the proliferation of ovarian cancer cells. In the inhibition of the proliferation of human pancreatic cancer BxPC-3 cells, the IC 50 values of compounds DXD, C, D, F, and G are all low (<3 ng / mL), indicating that they have excellent effects on inhibiting the proliferation of pancreatic cancer cells.

[0534] Example 25: In vitro antitumor activity of antibody-drug conjugates targeting ADAM9

[0535] This embodiment tested the in vitro antitumor activity of the antibody-drug conjugate against ADAM9-expressing gastric cancer cells AGS, non-small cell lung cancer cells Calu-3, and pancreatic cancer cells BxPC-3. The experimental method was the same as in Example 24, and the experimental results are shown in Figures 14-16 and Table 4.

[0536] Table 4: Inhibitory activity of different antibody-drug conjugates targeting ADAM9 against different cancer cell lines

[0537] Note: ADC IC 50 This represents the molar concentration of the toxin contained.

[0538] Experimental results showed that in vitro, ADC1-A, ADC-1B, ADC1-C, ADC1-D and ADC1-DXD targeting ADAM9 could effectively inhibit the growth of non-small cell lung cancer cells Calu-3 (Figure 14), pancreatic cancer cells BxPC-3 (Figure 15) and gastric cancer cells AGS (Figure 16) with high ADAM9 expression.

[0539] However, it can also be seen that the in vitro inhibitory activity of the same ADC differs in different cell models; and the in vitro inhibitory activity of different ADCs also differs in the same cell model. This is in conjunction with the IC50 of the toxin in Example 24. 50 The values ​​show that in in vitro experiments, the activity of the toxin and the ADC are not positively correlated (for example, compound D has very good activity as a toxin, but the corresponding ADC molecule ADC1-D has an effect comparable to ADC1-C, and is even worse than ADC1-C in some cell lines). That is, high toxin activity does not necessarily mean high ADC activity in inhibiting cancer cells. Overall, ADC1-C and ADC1-DXD have comparable cell growth inhibitory activities.

[0540] Example 26: In vitro antitumor activity of an antibody-drug conjugate targeting TROP-2

[0541] This embodiment tested the in vitro antitumor activity of the antibody-drug conjugate against TROP-2-expressing pancreatic cancer cells BxPC-3. The experimental method was the same as in Example 24, and the experimental results are shown in Figure 17.

[0542] Experimental results showed that ADC2-A, ADC2-C, ADC2-D and ADC2-DXD, which target TROP-2 in vitro, could effectively inhibit the growth of BxPC-3 tumor cells with high TROP-2 expression. Among them, the inhibitory effect of ADC2-C was comparable to that of ADC2-DXD.

[0543] Example 27: Anti-tumor activity of the antibody-drug conjugate targeting ADAM9 in vivo

[0544] NCI-H1975 (human lung adenocarcinoma cells), Calu-3 cells were cultured in monolayer in vitro and passaged. When the cells were in exponential growth phase, the cells were harvested. 5.0 x 10 6 cells were suspended in 0.1 ml PBS mixed with Matrigel (1:1) and inoculated into the right scapular of 5 nude mice (P1 generation). When the tumors grew to 500-800 mm 3 , the tumor-bearing mice were sacrificed by CO2 anesthesia, and the tumor mass was cut into small tumor pieces of 20-30 mm 3 , which were inoculated into a new batch of nude mice (P2 generation). The tumor tissue stably passaged was used to evaluate the anti-tumor activity of the test product. When the average tumor volume reached about 150 mm 3 after 7 days of tumor mass inoculation, the mice (n=5) were randomly grouped according to the tumor volume and the drug administration was started.

[0545] In the NCI-H1975 model, the antibody-drug conjugate was converted into an exatecan dose of 0.1 mg / kg, once a week for a total of two times. In the Calu-3 model, the antibody-drug conjugate was converted into an exatecan dose of 0.02 mg / kg, a single administration.

[0546] After the tumor cells were inoculated, in addition to observing the tumor growth, the effect of drug treatment on the behavior of the animals was monitored: the activity of the experimental animals, food and water intake, body weight changes (body weight was measured twice a week), eyes, fur and other abnormalities. The clinical symptoms observed during the experiment were recorded in the raw data. The tumor volume calculation method was: tumor volume (mm 3 ) = 1 / 2 x (a x b 2 ) (where a represents the long diameter and b represents the short diameter). About 35 days after the tumor mass was inoculated, all the mice were taken out of the tumor and weighed, and photographed.

[0547] The experimental results are shown in Figures 18-23, in which Figures 18 and 21 are the statistical curves of the tumor inhibition activity, Figures 19 and 22 are the tumor mass graphs of the tumor inhibition effect, and Figures 20 and 23 are the corresponding body weight change curves.

[0548] From the in vivo efficacy results of NCI-H1975, it can be seen that the tumor inhibition effects of the antibody conjugate drugs ADC1-B and ADC1-C targeting ADAM-9 of the present application are excellent, both of which are significantly better than ADC1-A and ADC1-D, in which ADC1-C and the positive control ADC1-DXD both achieve 100% inhibition effect (Figures 18, 19).

[0549] From the in vivo pharmacodynamic results of Calu-3, it can be seen that the anti-tumor effects of ADC1-B and ADC1-C are excellent at a lower dose of single administration of the antibody-drug conjugate targeting ADAM-9, and are obviously superior to ADC1-A and ADC1-D, wherein ADC1-C and positive control ADC1-DXD both achieve 100% inhibition effect (Figures 21 and 22).

[0550] In summary, ADC1-B and ADC1-C both have excellent anti-tumor effects in vivo, and the anti-tumor effect of ADC1-C is more excellent.

[0551] Example 28: In vivo anti-tumor activity of antibody-drug conjugate targeting TROP-2

[0552] NCI-H1975 cells were cultured in monolayer in vitro, and subcultured, and the test method referred to Example 27.

[0553] In the NCI-H1975 model, the antibody-drug conjugate was converted into 0.1 mg / kg exatecan dose, and the single administration scheme was used.

[0554] The experimental results are shown in Figures 24-26, wherein Figure 24 is a statistical curve of anti-tumor activity, Figure 25 is a tumor block diagram of anti-tumor effect, and Figure 26 is a corresponding body weight change curve.

[0555] From the in vivo pharmacodynamic results of NCI-H1975, it can be seen that the anti-tumor effects of antibody-drug conjugates ADC2-A, ADC2-C and ADC2-D targeting TROP-2 of the present application and positive control ADC2-DXD are excellent, wherein ADC2-C achieves 100% inhibition effect (Figures 24 and 25), and the tumor blocks of one animal in each group do not completely disappear.

[0556] Example 29: In vivo anti-tumor activity of antibody-drug conjugate targeting EGFR

[0557] BxPC-3 cells were cultured in monolayer in vitro, and subcultured, and the test method referred to Example 27.

[0558] In the BxPC-3 model, the antibody-drug conjugate was converted into 0.1 mg / kg exatecan dose, and the scheme of once a week for a total of two administrations was used.

[0559] The experimental results are shown in Figures 27-29, wherein Figure 27 is a statistical curve of anti-tumor activity, Figure 28 is a tumor block diagram of anti-tumor effect, and Figure 29 is a corresponding body weight change curve.

[0560] From the in vivo efficacy results of BxPC-3, the anti-tumor effects of the EGFR-targeted antibody-drug conjugate ADC3-C and the positive control ADC3-DXD of the present application are excellent, wherein the anti-tumor effect of ADC3-C is superior to that of the positive control ADC3-DXD (Figures 27 and 28).

[0561] Example 30: Rat toxicity test of Anti-ADAM9 antibody-drug conjugate

[0562] SD rats were given tail vein injection of ADC1-C (prepared separately, DAR value of 7.8) and ADC1-DXD (prepared separately, DAR value of 7.5), once a week for a total of 3 times, without a recovery period. The nature, degree and possible death of the toxic reactions possibly caused by the drug were observed, the target organ or target tissue of toxicity was preliminarily judged, and the toxicokinetic characteristics were studied.

[0563] Methods

[0564] 18 SD rats were randomly divided into solvent control group, ADC1-C dose group and positive control group ADC1-DXD according to body weight, 6 rats per group, half male and half female. The dosages of ADC1-C dose group and positive control group ADC1-DXD were 200 mg / kg and 160 mg / kg respectively, the dosing volume was 10 mL / kg, and the corresponding dosing concentrations of each group were 20 mg / mL and 16 mg / mL respectively. The solvent control group was given 10 mL / kg of sodium chloride injection. The tail vein was given a bolus injection, once a week for 3 weeks, a total of 3 times.

[0565] (1) General state observation: the animals were observed by a veterinarian for 3 days after receiving the drug, once a day. On the day of administration, observation was performed before, after and in the afternoon, once on the day of dissection, and once a day on the remaining non-administration days in the morning and afternoon, and the observation content included but was not limited to general performance, behavior state, eyes, mouth, nose, ears, hair and skin, feces, urine, genital abnormalities, etc.

[0566] (2) Detailed clinical state observation: observation was performed once a week during the test period, and the content included observing the general performance, behavior state, hair and skin, feces, urine, etc. of the animals at the cage edge, and then moving the animals outside the cage to perform palpation examination on the subcutaneous superficial tissues of the head, face, trunk, limbs, etc., abdominal organs and mucous membranes of the eyes, mouth and genital area.

[0567] (3) Infusion site observation: observation was performed once before administration and once after administration on the day of administration, and once a day during the administration period.

[0568] (4) Body weight: Measured twice during the adaptation period, twice a week during the drug administration period, and once before dissection. The results are shown in Figure 30.

[0569] (5) Food intake: Food intake was measured during the adaptation period from P3 to P4, and average food intake was measured during the drug administration period from D1 to D3, D3 to D7, D7 to D10, D10 to D14, D14 to D17, and D17 to D21. The results are shown in Figure 31.

[0570] (6) Clinical pathology: Blood sample D22 was tested once, and the results are shown in Table 5.

[0571] (7) Toxicokinetics: Detection of ADC, total antibody and small molecule toxin.

[0572] (8) Gross anatomy, organ weighing and histopathological examination: Histopathological examination of the brain, thymus, heart, liver, spleen, kidneys, adrenal glands and tissues / organs that appear abnormal to the naked eye.

[0573] Table 5. Results of Detection of Hematological Indicators of Toxicity in Rats

[0574] Note: Control group data are averages, while ADC1-C and ADC1-DXD group data are percentage differences (%) compared to the control group. Statistical significance is based on actual data (not percentage differences).

[0575] *D=Dunnett LSD Test Significant at the 0.05level.

[0576] +D=Dunnett LSD Test Significant at the 0.01level.

[0577] in conclusion

[0578] Under the conditions of this experiment, SD rats were intravenously administered 200 mg / kg of ADC1-C and 160 mg / kg of ADC1-DXD. During the experiment, all animals survived to the planned dissection, and no animals were near death or died.

[0579] After administration, the weight loss of rats in the ADC1-C group was significantly lower than that in the ADC1-DXD group (Figure 30), and the food intake loss of rats in the ADC1-C group was significantly lower than that in the ADC1-DXD group (Figure 31). Similar shrinkage of the thymus and spleen was observed in both groups, but the shrinkage was less severe in the ADC1-C group than in the ADC1-DXD group.

[0580] The results of the two ADC groups in rat hematology (Table 5) showed that ADC1-C showed lower hematological toxicity in terms of lymphocyte count (LYMPH), white blood cell count (WBC), %LYMPH (lymphocytes), and %MONO (monocytes). The values ​​of the solvent control group are the average values ​​of the measurements, and the data of the ADC1-C and ADC1-DXD groups are the percentage differences (%) from the control group. Positive numbers indicate higher values ​​than the solvent group, and negative numbers indicate lower values ​​than the solvent group.

[0581] In summary, considering hematological, pathological changes, and body weight and feeding, it is suggested that compared with the positive control ADC1-DXD, the ADC1-C of this invention has more stable feeding, better health indicators, and lower toxicity. The linker-payload of this invention maintains good efficacy while having better safety and tolerability, and the animals' condition and compliance are better when administered.

[0582] Example 31: Pretoxicology and toxicokinetics of anti-ADAM9 antibody-drug conjugate in cynomolgus monkeys

[0583] Cynomolgus monkeys were intravenously administered ADC1-C (from the same batch as the rat toxicity test, with a DAR value of 7.8), with escalating doses administered every 2 weeks for a total of 3 doses. The nature, severity, and time-dependent effects of the potential toxic reactions were observed to preliminarily determine the target organs or tissues for toxicity.

[0584] method

[0585] One female cynomolgus macaque (from Hainan Xinzhengyuan Biotechnology Co., Ltd.) was administered ADC1-C intravenously. The first dose was 75 mg / kg; a second dose was given two weeks later at a dose increased to 80 mg / kg; a third dose was given two weeks after the second dose at a dose increased to 85 mg / kg; and the macaque was dissected one week after the third dose. The monitored and observed indicators included:

[0586] (1) General condition observation: On the day of administration, animals are observed once before, once after administration, and once in the afternoon. On other days, they are observed once in the morning and once in the afternoon. On the day of dissection, animals to be dissected are observed once. Observe the general appearance, behavior, eyes, mouth, nose and mouth, ears, hair and skin, feces, urine, genitals and other abnormal symptoms.

[0587] (2) Detailed clinical observation: During the adaptation period of the experiment, observe once, and observe 1 to 2 times a week thereafter. The observation includes observing the animal’s general appearance, behavior, hair and skin, feces, urine and other abnormalities at the cage. After that, move the animal out of the cage and perform palpation examination on the subcutaneous superficial tissues of the animal’s head, face, trunk, limbs, abdominal organs and mucous membranes of the eyes, mouth and genitals.

[0588] (3) Observation of infusion site: Observe once before and after administration on the administration day, and once a day on non-administration days during the administration period.

[0589] (4) Weight: once during the adaptation period, once or twice a week during the drug administration period, and once before dissection.

[0590] (5) Electrocardiogram: Measured once during the adaptation period, and once 6h and 24h after the first and second administrations.

[0591] (6) Food intake / food consumption: Food intake was quantitatively measured once during the adaptation period, twice a week during the drug administration period, and food consumption was estimated at other times.

[0592] (7) Toxicokinetics – Total Antibodies: Blood samples were collected from cynomolgus monkeys at different time points (10 min, 4 h, 24 h, 48 h, 72 h, 96 h, 120 h and 168 h, 240 h) after the first and third administrations for the detection of total toxicokinetic antibodies (only the first blood sample was collected at 240 h). The total antibody content in cynomolgus monkey serum samples was detected by enzyme-linked immunosorbent assay (ELISA). The antigen was bound to a solid-phase carrier. During detection, the antibody conjugate in the serum to be tested was incubated with the antigen bound to the solid-phase carrier. After washing, the detection working solution was added and TMB color development was performed. The total antibody content in the sample was determined by quantitatively detecting the amount of colorimetric product. The results are shown in Figure 32. The half-lives of the first and third administrations were calculated using PKSolver (Computer Methods and Programs in Biomedicine 2010; 99: 306-14). The results showed that the total antibody half-lives calculated using PKSolver (Computer Methods and Programs in Biomedicine 2010; 99: 306-14) were 217 h and 269 h, respectively, for the first and third times.

[0593] (7) Toxicokinetics – Small Molecule Toxins: Blood samples were collected from cynomolgus monkeys at different time points (10 min, 4 h, 24 h, 48 h, 72 h, 96 h, 120 h and 168 h, 240 h) after the first and third administrations for the detection of total toxicotropic antibodies (blood was collected only at 240 h for the first administration). The blood concentration of the small molecule toxin compound was detected by LC / MS, and the results are shown in Figure 33. The half-lives for the first and third administrations were calculated using PKSolver to be 107 h and 174 h, respectively.

[0594] (13) Bone marrow cytology examination.

[0595] (8) Gross dissection, organ weighing and histopathological examination: All dissected animal organs / tissues were fixed. Histopathological examination was performed on the heart, liver, spleen, lungs, kidneys, thymus, brain, eyeballs, optic nerve, bladder, ureters (females only) and any tissues / organs that appeared abnormal to the naked eye.

[0596] in conclusion

[0597] Under the conditions of this experiment, cynomolgus monkeys were intravenously administered ADC1-C at a dose of 75-85 mg / kg. During the experiment, the animals survived until the end of the administration period or the planned dissection date, and no near-death or death was observed.

[0598] Following ADC1-C administration, gross observation of the animals revealed only slight weight loss, decreased food intake, and one small amount of soft, yellow stool. Clinical pathology showed mild decreases in erythroid cells (RBC, HCT, HGB), WBC, #LYMPH, and %LYMPH (Table 6), and an increase in AST (Table 7), which recovered before the next administration. At the end of the administration period, the main pathological changes were a decrease in lymphocytes in the thymus and spleen; no drug-related pathological changes were detected in vital organs (heart, liver, kidneys, lungs, etc.).

[0599] Table 6: Results of hematological markers for pretoxicology in cynomolgus monkeys

[0600] Table 7: Results of Blood Biochemical Indicators Detection in Pre-Toxicological Treatment of Crab-Eating Mammals

[0601] MacroGenics' IMGC-936 is the world's first ADAM9-ADC to enter clinical trials, with the conjugated toxin being the tubulin inhibitor DM50. In its monkey toxicology studies, 22.5 mg / kg of IMGC-936 was observed to cause corneal toxicity (Mol Cancer Ther 2022; 21:1047-59). In contrast, in monkey toxicology studies, ADC1-C, with three dose escalations (75, 80, 85 mg / kg), significantly exceeded the safe dose of IMGC-936 (15 mg / kg), and no corneal toxicity or other serious pathological changes were observed in gross examination or pathological sections, suggesting that the antibody-drug conjugate of this invention has an excellent therapeutic safety window.

[0602] The HNSTD of the cynomolgus monkeys in this invention can be preliminarily determined to be 85 mg / kg, which is much higher than the 30 mg / kg of DS-8201, which uses deruxtecan as a linker toxin, by Daiichi Sankyo (Clin Cancer Res, 2016.22(20):p.5097-5108). Moreover, in this embodiment, the dosing frequency of this invention is once every two weeks for three times, which is higher than the once every three weeks dosing frequency of DS-8201. Therefore, the ADC using the linker technology of this invention has higher safety.

[0603] The half-lives for the first and third administrations, calculated based on the total antibody blood concentration (Figure 32), were 217 h and 269 h, respectively. The half-lives for the first and third administrations, calculated based on the small molecule toxin blood concentration after ADC administration (Figure 33), were 107 h and 174 h, respectively. This indicates that the antibody-drug conjugate of the present invention has a long half-life, suggesting that the antibody-drug conjugate of the present invention can maintain a steady-state blood drug concentration, reduce the impact of concentration fluctuations on efficacy, prolong the dosing interval, and improve patient compliance.

[0604] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of Formula (I), a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, wherein X is selected from the group consisting of -OH, -SH, -NHR m ; R m selected from the group consisting of hydrogen, C1-C6alkyl; R1and R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl; R2is C1-C6alkyl, and said C1-C6alkyl is optionally substituted with a group selected from the group consisting of hydroxy, mercapto, carboxy, C1-C6alkoxy, C1-C6alkylthio, C1-C6alkylamino; A has the structure shown in Formula IV below: Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O); R 4 and R 5 each independently is selected from the group of hydrogen atom, deuterium atom, halogen, C1-C8alkyl, C1-C8haloalkyl, C1-C8deuteroalkyl; or R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated C5-C 12 carbocyclic ring, saturated or unsaturated 5-12 membered heterocyclic ring; Unless otherwise specified, each of the above-mentioned alkyl, haloalkyl, deuteralkyl, alkoxy, alkylthio, and alkylamine groups may be substituted by substituents selected from the group consisting of: halogen, nitrile, nitro, hydroxyl, amino, mercapto, carboxyl, C1-C6 alkyl-amine, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkoxy, C1-C6 alkyl-S-, haloC1-C6 alkyl, haloC2-C6 alkenyl, haloC2-C6 alkoxy, allyl, benzyl, C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl.

2. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 1, wherein, said R1is selected from the group consisting of hydrogen, C1-C4alkyl; the carbon atom to which R1is attached can be in the R form or in the S form; R2is C1-C4alkyl; R3is hydrogen, methyl, ethyl or n-propyl; the carbon atom to which R3is attached can be in the R form or in the S form.

3. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 1, wherein, The compound of formula (I) has the structure shown in formula (la): wherein X is selected from the group consisting of -OH, -SH, -NHR m ; R m selected from the group consisting of hydrogen, C1-C4alkyl; R3is hydrogen, methyl, ethyl or n-propyl; R1is selected from the group consisting of hydrogen, C1-C4alkyl; R2is C1-C4alkyl; Z is selected from the group consisting of a bond, C(O), C(S); R 4 and R 5 each independently is selected from the group of hydrogen atom, deuterium atom, halogen, C1-C4alkyl, C1-C4haloalkyl, C1-C4deuteroalkyl; or R 4 and R 5 together with the carbon atom to which they are attached form a structure selected from the group consisting of C5-C8cycloalkyl, saturated or unsaturated 5-7 membered heterocycle.

4. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 1, wherein, The compound of formula (I) is selected from the group consisting of:

5. A compound of Formula (II), a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, M-L-D Formula (II) wherein M is selected from the group consisting of: pyrimidinyl; L is -L1-L2-L3-L4-; wherein L1is selected from the group consisting of -(CHR) p1 C(=0)-, -(CH2CH20) q1 -, -(CHR) p2 C(=0)-NH-(CH2CH20) q2 (CHR) p3 C(=0)-, -(CHR) p2 C(=0)-NH-(CH2CH20) q2 -, -(CHR) m1 -X1-(CH2CH20) n3 -(CHR) m2 -C(O)-, -(CHR) m1 -X1-X2-(CH2CH20) n3 -(CHR) m2 -C(O)-, -X1-(CHROCHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) m2 -C(O)-, -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-, -X1-(CHR) m1 -X2-(CHR) m2 -C(O)-, -(CH2CH20) n3 -C(O)-; X1 and X2 are each independently selected from the following groups: -O-, -C(O)-, -C(O)-NR-, and optionally substituted C6-C. 10 Aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heterocyclic group and optionally substituted C3-C6 alicyclic group; wherein each R is independently selected from the group consisting of H, D, (CH2) n4 OH, (CH2) n4 NH2, (CH2O) n4 (CH2CH2O) n5 H, (CH2O) n4 (CH2CH2O) n5 CH3, (CH2) n4 OCH3, (CH2CH2O) n5 CH3, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 H, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3; wherein each of p1, p2, p3, q1, and q2 is independently 0, 1, 2, 3, 4, 5, 6, or 7; each of m1, m2, n3, n4, and n5 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; L2is a peptide residue; L3 is -L 4a -(NR b ) n6 -R 12 -L 4b - wherein L 4a is absent, or L 4a is optionally substituted n6is 0 or 1; R 12 is a chemical bond, CH2, or CD2; L 4b is absent, or L 4b is optionally substituted R a and R b each independently is selected from the group consisting of hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl; L4 is absent, or is optionally substituted wherein Y is selected from the group consisting of O, S, NH; v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R 10 and R 11 are each independently selected from the group consisting of hydrogen, deuterium, halogen, optionally substituted Ci-C8alkyl, optionally substituted Ci-C8haloalkyl, optionally substituted Ci-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl, or R 10 and R 11 together with the atom to which they are attached form an optionally substituted C3-C8cycloalkyl; L1, L2, L3and L4as defined above can also be optionally substituted with a substituent selected from the group consisting of hydrogen atom, deuterium atom, halogen, nitrile group, nitro group, hydroxyl group, amino group, Ci-C6alkyl-NH-, (Ci-C6alkyl)2N-, Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6alkoxy, halo-Ci-C6alkyl, halo-C2-C6alkenyl, halo-C2-C6alkynyl, halo-Ci-C6alkoxy, allyl, benzyl, C6-Ci0aryl, 5-7 membered heteroaryl, C3-C8cycloalkyl, 3-12 membered heterocyclyl; and 12 Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, Ci-C6alkoxy, halo-Ci-C6alkyl, halo-C2-C6alkenyl, halo-C2-C6alkynyl, halo-Ci-C6alkoxy, allyl, benzyl, C6-Cioaryl, 5-7 membered heteroaryl, C3-C8cycloalkyl, 3-12 membered heterocyclyl; The D has a structure shown in formula (I-1): wherein X' is selected from the group consisting of O, S, NR m ; R m , R1, R2, R3, and A are as defined in claim 1.

6. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, X1and X2are each independently selected from the group consisting of -0-, -C(O)-, -C(O)-NR-, optionally substituted phenyl, optionally substituted pyridyl, optionally substituted C3-C6cycloalkyl, optionally substituted optionally substituted 7. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, L1is a radical selected from the group consisting of -(CHR) C(=0)-NH-, p1 -(CH2CH20)2-, q1 -(CH2CH20)2-, p2 -(CH2CH20)2-, q2 -(CH2CH20)2-, p3 -(CH2CH20)2-, p2 -(CH2CH20)2-, q2 -(CH2CH20)2-; wherein pi, p2, p3, qi and q2 are each independently selected from 0, 1, 2, 3, 4 or 5; R is selected from the group consisting of H, (CH2) n4 OH, (CH2O) n4 (CH2CH2O) n5 H; preferably, n4 and n5 are each independently selected from 0, 1, 2 or 3.

8. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, L1is an optionally substituted structure selected from the group consisting of:

9. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, said L2is a peptide residue composed of one or more amino acids selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, glycine; Preferably, said L2is a peptide residue composed of one or more amino acids selected from the group consisting of glycine, alanine, lysine, phenylalanine, valine, glutamine, citrulline; More preferably, said L2 is a peptide residue selected from the group consisting of -glycine- (-Gly-), -phenylalanine- (-Phe-), -glutamine- (-Gln-), -glycine-glycine- phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -alanine-alanine-alanine- (-Ala-Ala-Ala-), -alanine-alanine-glycine- (-Ala-Ala-Gly-), -glycine-phenylalanine-glycine- (-Gly-Phe-Gly-), -citrulline-valine- (-Cit-Val-), -citrulline-alanine- (-Cit-Ala-), -valine-arginine- (-Val-Arg-), -valine-lysine- (-Val-Lys-), -valine-lysine(Ac)- (-Val-Lys(Ac)-), -lysine-valine- (-Lys-Val-), -leucine-citrulline- (-Leu-Cit-), -isoleucine-citrulline- (-Ile-Cit-), -tryptophan-citrulline- (-Trp-Cit-), -phenylalanine-lysine- (-Phe-Lys-), -phenylalanine-lysine(Ac)- (-Phe-Lys(Ac)-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylalanine-alanine- (-Phe-Ala-), -phenylalanine-arginine- (-Phe-Arg-), -alanine-lysine- (-Ala-Lys-), -alanine-alanine- (-Ala-Ala-), -alanine-alanine-asparagine- (-Ala-Ala-Asn-), -alanine-alanine-aspartic acid- (Ala-Ala-Asp-), -lysine-alanine-alanine-asparagine- (-Lys-Ala-Ala-Asn-), -lysine-alanine-alanine-aspartic acid- (-Lys-Ala-Ala-Asp-), -(D)-valine-leucine-lysine- (-D-Val-Leu-Lys-), -glycine-glycine-arginine- (-Gly-Gly-Arg-), -glycine-glycine-asparagine- (-Gly-Gly-Asn-), -glycine-glycine-phenylalanine- (-Gly-Gly-Phe-), -valine-lysine-glycine- (-Val-Lys-Gly-), -glutamic acid-alanine-alanine- (-Glu-Ala-Ala-), -aspartic acid-alanine-alanine- (-Asp-Ala-Ala-), -valine-lysine-glycine-glycine- (-Val-Lys-Gly-Gly-), -lysine-alanine-asparagine- (-Lys-Ala-Asn-).

10. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, L2 is selected from the group of structures:

11. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, said L3is a chemical bond, or is an optionally substituted group selected from the group consisting of: wherein R a and R b are each independently selected from the group of: hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl; Preferably, said L3is a chemical bond, or is an optionally substituted structure selected from the group consisting of:

12. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, L4 is absent, or is an optionally substituted structure selected from the group consisting of:

13. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 5, wherein, The compound of formula (II) is selected from the group consisting of:

14. A compound of Formula (III), a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, Ab-(M'-L-D) n Formula (III) wherein, n is an integer or decimal number from 2.0 to 16.0, preferably from 2.0 to 8.0, most preferably from 4.0 to 8.0; Ab is a polypeptide, an antibody, an antigen binding fragment or a non-antibody scaffold binding to a target; M' is selected from the group consisting of: wherein R is H, C1-C6alkyl, C1-C6deuteroalkyl, C3-C8cycloalkyl, or C3-C8deutero cycloalkyl, * is the position of attachment of M’ to Ab, d wherein R is H, C1-C6alkyl, C1-C6deuteroalkyl, C3-C8cycloalkyl, or C3-C8deutero cycloalkyl, * is the position of attachment of M’ to Ab, is the position of attachment of M' to L; L and D are as defined in any one of claims 5 to 13.

15. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 14, wherein, said Ab is selected from the group consisting of a monoclonal antibody, a Fab, a Fab', a F(ab'), a Fv, a disulfide linked Fc, a scFv, a single domain antibody, a diabody, a bispecific antibody or a multispecific antibody; Preferably, said Ab is selected from the group consisting of a diabody, a DART, an anticalin, an avimer, an avimer, a DARPin, an adnectin; More preferably, said Ab is selected from the group consisting of a scFv1-ScFv2, a ScFv12-Fc-scFv22, an IgG-scFv, a Duobody, a DVD-Ig, a triomab / quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, a scFv-HSA-scFv.

16. The compound of claim 14, wherein Said Ab is monospecific, bivalent or bispecific.

17. The compound of claim 14, wherein The Ab is selected from the group consisting of an anti-ADAM9 antibody, an anti-Trop-2 antibody, an anti-CD37 antibody, an anti-PD-1 antibody, an anti-HER2 antibody, an anti-B7H4 antibody, an anti-CD70 antibody, an anti-EGFRvIII antibody, an anti-Mesothelin antibody, an anti-Folate eceoptor1 antibody, an anti-Mucin 1 antibody, an anti-CD138 antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD30 antibody, an anti-SLTRK6 antibody, an anti-Nectin 4 antibody, an anti-Tissue factor antibody, an anti-Mucin 16 antibody, an anti-Endothelinreceoptor antibody, an anti-STEAP1 antibody, an anti-SLC39A6 antibody, an anti-Guanylylcyclase C antibody, an anti-PSMA antibody, an anti-CCD79b antibody, an anti-CD22 antibody, an anti-Sodium phosphate cotransporter2B antibody, an anti-GPNMB antibody, an anti-Trophoblast glycoprotein antibody, an anti-AGS-16 antibody, an anti-EGFR antibody, an anti-CD33 antibody, an anti-CD66e antibody, an anti-CD74 antibody, an anti-CD56 antibody, an anti-TACSTD2 antibody, an anti-DR5 antibody, an anti-E16 antibody, an anti-STEAP1 antibody, an anti-0772P antibody, an anti-MPF antibody, an anti-Napi3b antibody, an anti-Sema5b, an anti-PSCAhlg antibody, anti-ETBR antibody, anti-MSG783 antibody, anti-STEAP2 antibody, anti-TrpM4 antibody, anti-CRIPTO antibody, anti-CD21 antibody, anti-CD79b antibody, anti-FcRH2 antibody, anti-NCA antibody, anti-MDP antibody, anti-IL20Ra antibody, anti-Brevican antibody, anti-EphB2R antibody, anti-ASLG659 antibody, anti-PSCA antibody, anti-GEDA antibody, anti-BAFF-R antibody, anti-CD22 antibody, anti-CD79a antibody, anti-CXCR5 antibody, anti-HLA-DOB antibody, anti-P2X5 antibody, anti-CD72 antibody, anti-LY64 antibody, anti-FcRH1 antibody, anti-IRTA2 antibody, anti-TENB2 antibody, anti-integrin alpha5beta6 antibody, anti-alpha4beta7 antibody, anti-FGF2 antibody, anti-FGFR2 antibody, anti-HER3 antibody, anti-CD70 antibody, anti-CA6 antibody, anti-DLL3 antibody, anti-DLL4 antibody, anti-P-cadherin antibody, anti-EpCAM antibody, anti-pCAD antibody, anti-CD223 antibody, anti-LYPD3 antibody, anti-LY6E antibody, anti-EFNA4 antibody, anti-ROR1 antibody, anti-SLITRK6 antibody, anti-5T4 antibody, anti-ENPP3 antibody, anti-SLC39A6 antibody, anti-CLAUDIN18.2 antibody, anti-BMPR1B antibody, anti-E16 antibody, anti-STEAP1 antibody, anti-Tyro7 antibody, anti-0772P antibody, anti-MPF antibody, anti-Napi3b antibody, anti-Sema 5b antibody, anti-PSCA hlg antibody, anti-ETBR antibody, anti-MSG783 antibody, anti-STEAP2 antibody, anti-TrpM4 antibody, anti-CRIPTO antibody, anti-CD21 antibody, anti-CD79b antibody, anti-FcRH2 antibody, anti-NCA antibody, anti-MDP antibody, anti-IL20Ra antibody, anti-Brevican antibody, anti-EphB2R antibody, anti-ASLG659 antibody, anti-PSCA antibody, anti-GEDA antibody, anti-CD22 antibody, anti-CD79a antibody, anti-CXCR5 antibody, anti-HLA-DOB antibody, anti-P2X5 antibody, anti-CD72 antibody, anti-LY64 antibody, anti-FcRH1 antibody, anti-IRTA2 antibody, anti-c-Met antibody, anti-ApoE antibody, anti-CD1LC antibody, anti-CD40 antibody, anti-CD45 (PTPRC) antibody, anti-CD49D (ITGA4) antibody, anti-CD80 antibody, anti-CSF1R antibody, anti-CTSD antibody, anti-GZMB antibody, anti-Ly86 antibody, anti-MS4A7 antibody, anti-PIK3AP1 antibody, anti-PIK3CD antibody, anti-CCR5 antibody, anti-IFNG antibody, anti-IL10RA1 antibody, anti-IL-6 antibody, anti-ACTA2 antibody, anti-COL7A1 antibody, anti-LOX antibody, anti-LRRC15 antibody, anti-MCPT8 antibody, anti-MMP10 antibody, anti-NOG antibody, anti-SERPINE1 antibody, anti-STAT1 antibody, anti-TGFBR1 antibody, anti-CTSS antibody, anti-PGF antibody, anti-VEGFA antibody, anti-C1QA antibody, anti-C1QB antibody, anti-ANGPTL4 antibody, anti-EGLN antibody, anti-ANGPTL4 antibody, anti-EGLN3 antibody, anti-BNIP3 antibody, anti-AIF1 antibody, anti-CCL5 antibody, anti-CXCL10 antibody, anti-CXCL11 antibody, anti-IFI6 antibody, anti-PLOD2 antibody, anti-KISS1R antibody, anti-STC2 antibody, anti-DDIT4 antibody, anti-PFKFB3 antibody, anti-PGK1 antibody, anti-PDK1 antibody, anti-AKR1C1 antibody, anti-AKR1C2 antibody, anti-CADM1 antibody, anti-CDH11 antibody, anti-COL6A3 antibody, anti-CTGF antibody, anti-HMOX1 antibody, anti-KRT33A antibody, anti-LUM antibody, anti-WNT5A antibody, anti-IGFBP3 antibody, anti-MMP14 antibody, anti-CDCP1 antibody, anti-PDGFRA antibody, anti-TCF4 antibody, anti-TGF antibody, anti-TGFB1 antibody, anti-TGFB2 antibody, anti-CD1 lb antibody, anti-ADGRE1 antibody, anti-EMR2 antibody, anti-TNFRSF21 antibody, anti-UPK1B antibody, anti-TNFSF9 antibody, anti-MMP16 antibody, anti-MFI2 antibody, anti-IGF-1R antibody, anti-RNF43 antibody, anti-NaPi2b antibody, anti-BCMA antibody, anti-TENB2 antibody.

18. The compound, pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of claim 14, wherein, The compound of formula (III) is selected from the group consisting of:

19. A process for preparing a compound of Formula III, a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, according to any one of claims 14-18, characterized in that, comprising the steps of: reacting a compound of formula II as defined in any one of claims 5 to 13, a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof, with an Ab as defined in any one of claims 14 to 18 to obtain a compound of formula III as defined in any one of claims 14 to 18, a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof.

20. A pharmaceutical composition comprising a compound of formula I as defined in any one of claims 1 to 4, a compound of formula II as defined in any one of claims 5 to 13 or a compound of formula III as defined in any one of claims 14 to 18, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier and / or excipient.

21. A pharmaceutical preparation comprising a compound of formula I as defined in any one of claims 1 to 4, a compound of formula II as defined in any one of claims 5 to 13 or a compound of formula III as defined in any one of claims 14 to 18, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier and / or excipient.

22. Use of a substance X for the manufacture of a medicament for the prevention or treatment of cancer or inflammation; wherein said substance X is a compound of formula I as defined in any one of claims 1 to 4, a compound of formula II as defined in any one of claims 5 to 13 or a compound of formula III as defined in any one of claims 14 to 18, or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 20, or a pharmaceutical preparation as defined in claim 21; Preferably, said cancer is a solid tumor or a non-solid tumor. More preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. neuroglioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

23. Use of a substance X in the manufacture of a medicament for preventing or treating a disease associated with abnormal cell activity; wherein the substance X is a compound of Formula I according to any one of claims 1-4, a compound of Formula II according to any one of claims 5-13, or a compound of Formula III according to any one of claims 14-18, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 20, or a pharmaceutical preparation according to claim 21; Preferably, the disease associated with abnormal cell activity is cancer. More preferably, the cancer is a solid tumor or a non-solid tumor. Further more preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. neuroglioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

24. Use of a substance X in the manufacture of a medicament; wherein, the substance X is a compound of Formula I according to any one of claims 1-4, a compound of Formula II according to any one of claims 5-13, or a compound of Formula III according to any one of claims 14-18, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 20, or a pharmaceutical preparation according to claim 21; the medicament is for treating a disease associated with a target point A, wherein the target point A is the target point corresponding to Ab in the substance X; Preferably, the medicament is for treating a cancer as the disease associated with a target point A. More preferably, the cancer is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal cancer, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumor (e.g. neuroglioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

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