Antibody-drug conjugate targeting adam-9, preparation method therefor, and use thereof

Abstract

The present disclosure provides a compound represented by formula III, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof, wherein Ab is an anti-ADAM-9 antibody or antigen-binding fragment binding to the target ADAM-9. Specifically, the compound of the present invention is an antibody-drug conjugate targeting ADAM-9 that exhibits excellent anti-tumor activity against a variety of tumors (such as colon cancer, pancreatic cancer, lung adenocarcinoma, and breast cancer). Ab-(M'-L-D)n Formula (III)

Description

An antibody-drug conjugate targeting ADAM-9 and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, an antibody-drug conjugate targeting ADAM-9 and preparation method and use thereof. BACKGROUND

[0002] ADAM proteins (ADAM: disintegrin and metalloproteinase) are multifunctional proteins involved in the shedding of extracellular domains of transmembrane proteins, cell adhesion and infiltration. Human genome contains 25 ADAMs, of which ADAM-9 has proteolytic activity, is widely expressed in the human body and regulates various biological functions, and plays an important role in various diseases, including neurodegenerative diseases, retinal diseases, inflammation and tumors.

[0003] It is reported that ADAM-9 is involved in the occurrence, development, invasion and metastasis and prognosis of various malignant tumors such as liver cancer, breast cancer, lung cancer, gastric cancer, kidney cancer and prostate cancer. In addition to tumor metastasis, ADAM-9 plays an important role in tumor proliferation, angiogenesis and even immune escape. Furthermore, overexpression of ADAM9 in solid tumors is associated with invasive tumor phenotype and poor clinical prognosis. For example, ADAM-9 mRNA expression is related to tumor grade and histological type of glioma, and there is a significant correlation between high expression of ADAM-9 and poor clinical outcome in patients with low-grade neoplasia. In addition, studies have shown that ADAM-9 expression can be used as a prognostic indicator for patients with low-grade neoplasia, and is also a potential therapeutic target. Kim et al. found that ADAM-9 plays an important role in the proliferation and invasion of gastric cancer, and ADAM-9 can be an effective therapeutic target for advanced gastric cancer. Based on this, more and more researchers have initiated the development of ADAM9 as a drug for anti-tumor therapy target.

[0004] Antibody-drug conjugates (ADC) are targeted biological agents that couple target-specific monoclonal antibodies with highly lethal cytotoxic small molecule drugs through specific linkers, and use monoclonal antibodies as carriers to transport small molecule cytotoxic drugs to target tumor cells in a targeted manner. Compared with chemotherapy drugs, ADC can more accurately identify diseased cells and reduce the killing of normal cells, thereby expanding the therapeutic window. Compared with traditional antibodies or antibody fragments, ADCs enhance the therapeutic effect due to the carrying of highly active cytotoxic drugs.

[0005] 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, etc. They have anti-tumor effect by inhibiting topoisomerase I. Currently, several camptothecin drugs have been approved for marketing for tumor treatment.

[0006] Camptothecin drugs or derivatives often have hematotoxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, etc., and gastrointestinal side effects, such as nausea, vomiting, diarrhea, etc. 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 developing novel camptothecin compounds and their conjugates that can improve effectiveness and safety issues.

[0007] Currently, nearly 15 ADCs have been approved for marketing, but many ADCs have unsatisfactory clinical results, mostly due to lack of excellent effectiveness and safety. From the design of ADCs, the antibody, linker, and cytotoxin have not yet formed a perfect combination, and there is still much room for improvement in reducing on-target and off-target toxicity, improving drug efficacy, and optimizing in vivo pharmacokinetics.

[0008] For example, the only ADC drug for ADAM-9 in the clinical stage, IMGC936 (developed by AbbVie and MacroGenics), was announced to terminate development in March 2024 because its clinical phase I trial results did not meet the expected safety and effectiveness targets.

[0009] Therefore, how to develop more clinically valuable ADCs containing camptothecin drugs or their derivatives targeting ADAM-9 needs further development and exploration. SUMMARY

[0010] 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 ADAM-9 antibody and its application.

[0011] In the first aspect of the present application, a compound represented by formula (III), a pharmaceutically acceptable salt, a stereoisomer, a solvate, or a solvate of a pharmaceutically acceptable salt thereof is provided, Ab-(M'-L-D) n Formula (III)

[0012] 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;

[0013] Ab is an anti-ADAM-9 antibody or antigen binding fragment that binds to the target ADAM-9;

[0014] 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;

[0015] L is -L1-L2-L3-L4-;

[0016] 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)-;

[0017] X1and X2are each independently selected from the group consisting of -O-, -C(O)-, -C(O)-NR-, optionally substituted C6-C 10aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heteroalicyclyl, and optionally substituted C3-C6 alicyclyl;

[0018] 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;

[0019] 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;

[0020] L2is a peptide residue;

[0021] L3is -L 4a -(NR b ) n6 -R 12 -L 4b -,

[0022] wherein L 4a is absent, or L 4a is an optionally substituted

[0023] n6is 0 or 1;

[0024] R 12 is a bond, CH2, or CD2;

[0025] L 4b is absent, or L 4b is an optionally substituted

[0026] R a and R beach independently selected from the group consisting of hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl;

[0027] L4is absent, or is optionally substituted

[0028] wherein Y is selected from the group consisting of O, S, NH;

[0029] v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0030] R 10 and R 11 each 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;

[0031] L1, L2, L3, and L4described 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;

[0032] said D has a structure represented by formula (I-1):

[0033] wherein X’ is selected from the group consisting of O, S, NR m ;

[0034] R m is selected from the group consisting of hydrogen, C1-C6alkyl;

[0035] R1and R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0036] R2is 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, C1-C6alkylamino;

[0037] A has the structure of Formula IV:

[0038] Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O);

[0039] 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;

[0040] 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, a saturated or unsaturated 5-12 membered heterocyclic ring;

[0041] 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, thiol, 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 In another preferred embodiment, said Ab is selected from the group consisting of a monoclonal antibody, a Fab, a Fab', a F(ab'), a Fv, a scFv, a single domain antibody.

[0042] In a preferred embodiment, said Ab is selected from the group consisting of a monoclonal antibody, a Fab, a Fab', a F(ab'), a Fv, a scFv, a single domain antibody.

[0043] In a preferred embodiment, said Ab comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising the following 3 heavy chain complementarity determining regions:

[0044] HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 6;

[0045] HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 7; and

[0046] HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 8;

[0047] The light chain variable region comprises the following 3 light chain complementarity determining regions:

[0048] LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 3;

[0049] LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 4; and

[0050] LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 5.

[0051] In a preferred embodiment, the Ab has a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 10.

[0052] In another preferred embodiment, the Ab comprises a heavy chain variable region having at least 80% (85%, 90%, 92%, 95%, or 99%) sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9, and / or a light chain variable region having at least 80% (85%, 90%, 92%, 95%, or 99%) sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10.

[0053] In another preferred embodiment, the Ab has a heavy chain as set forth in SEQ ID NO: 1 and a light chain as set forth in SEQ ID NO: 2.

[0054] In another preferred embodiment, the Ab is an IgG antibody.

[0055] In another preferred embodiment, the IgG antibody comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is human IgG1, human IgG2, human IgG3, or human IgG4, and the light chain constant region is selected from human kappa (Kappa) or human lambda (Lambda).

[0056] In another preferred embodiment, the sequence of the light chain constant region of the Ab is as set forth in SEQ ID NO: 11.

[0057] In another preferred embodiment, the heavy chain constant region comprises an Fc fragment.

[0058] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0059] In another preferred embodiment, the sequence of the heavy chain constant region of the Ab is as set forth in SEQ ID NO: 12.

[0060] In another preferred embodiment, the Ab comprises a sequence having at least 80% (85%, 90%, 92%, 95%, or 99%) sequence identity to, or conservative substitutions or mutations of, a sequence as set forth in SEQ ID NO: 11 or 12, which sequence retains at least 90% of the biological function or activity.

[0061] In another preferred embodiment, M' is selected from the group consisting of:

[0062] In a preferred embodiment, X1and X2are each independently selected from the group consisting of -O-, -C(O)-, -C(O)-NR-, optionally substituted phenyl, optionally substituted pyridyl, optionally substituted C3-C6cycloalkyl, optionally substituted optionally substituted

[0063] In another preferred embodiment, L1is a group selected from the group consisting of: m1 -X1-(CH2CH2O) n3 -(CH2) m2 -C(O)-;

[0064] wherein X1is -C(O)-NH-; preferably, m1and m2are each independently selected from 1, 2, or 3; and n3is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0065] In another preferred embodiment, L1is a group selected from the group consisting of: m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1is optionally substituted or optionally substituted

[0066] wherein X2is -C(O)-NR-; preferably, m1and m2are each independently selected from 0, 1, or 2; and n3is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In another preferred embodiment, L1is a group selected from the group consisting of: m2 -C(O)-;

[0067] wherein X1is optionally substituted aryl, optionally substituted heteroaryl; preferably, m2is 0, 1, 2, or 3.

[0068] In a preferred embodiment, L1is a group 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 -;

[0069] wherein each of pi, p2, p3, qi, and q2 is 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 n4 and n5 is independently selected from 0, 1, 2, or 3.

[0070] In another preferred embodiment, said L1is a group selected from -(CH2) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-;

[0071] wherein X1is -C(O)-; preferably each of mi and m2 is independently selected from 0, 1, 2, or 3; n3 is selected from 0, 1, or 2.

[0072] In another preferred embodiment, said L1is a group selected from -X1-(CH2) m1 -X2-(CHR) m2 -C(O)-;

[0073] wherein X1is optionally substituted aryl or optionally substituted heteroaryl; X2is -C(O)-; preferably each of mi and m2 is independently selected from 0, 1, or 2.

[0074] In another preferred embodiment, said L1is a group selected from -(CHR) m1 -X1-(CHR) m2 -C(O)-;

[0075] wherein X1is optionally substituted 3-8 membered heteroalicyclic or optionally substituted C3-C6alicyclic; preferably mi is 0, 1, or 2, and m2 is 0.

[0076] In another preferred embodiment, said L1is a group selected from -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2-C(O)-; wherein X1is O; preferably, each of m1, n3, and m2 is independently 0, 1, or 2.

[0077] In another preferred embodiment, L1is a group selected from the group consisting of: m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-; wherein X1is optionally substituted -C(O)-NR-, X2is O; preferably, each of m1, n3, and m2 is independently 1, 2, or 3; R is as described above.

[0078] In a preferred embodiment, L1is an optionally substituted group selected from the group consisting of:

[0079] In a preferred embodiment, L2is 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;

[0080] Preferably, L2is a peptide residue consisting of one or more amino acids selected from the group consisting of: glycine, alanine, lysine, phenylalanine, valine, glutamine, citrulline;

[0081] 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-).

[0082] In another preferred embodiment, L2is 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-).

[0083] In another preferred embodiment, L2is -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), i.e.

[0084] In a preferred embodiment, L2is selected from the group consisting of the following structures:

[0085] In a preferred embodiment, L3is a chemical bond, or is an optionally substituted group selected from the group consisting of:

[0086] wherein R a and R b are each independently selected from the group consisting of: hydrogen, optionally substituted C1-C4alkyl, optionally substituted C1-C4deuteroalkyl;

[0087] Preferably, L3is a chemical bond, or is an optionally substituted group selected from the group consisting of:

[0088] In a preferred embodiment, L4is absent, or is an optionally substituted group selected from the group consisting of:

[0089] In another preferred embodiment, R 4 and R 5 are each independently selected from the group consisting of: hydrogen atom, deuterium atom, halogen, C1-C4alkyl, C1-C4haloalkyl, C1-C4deuteroalkyl;

[0090] Alternatively, R 4 and R 5 together with the carbon atom to which they are attached form a group selected from the group consisting of: saturated or unsaturated C5-C7carbocyclic ring, saturated or unsaturated 5-7 membered heterocyclic ring.

[0091] In another preferred embodiment, the R4 and R 5 together with the carbon atom to which they are attached form a

[0092] In a preferred embodiment, R1is selected from the group consisting of hydrogen, C1-C4alkyl; the carbon atom to which R1is attached can be in the R configuration or in the S configuration;

[0093] R2is C1-C4alkyl;

[0094] R3is hydrogen, methyl, ethyl or n-propyl; the carbon atom to which R3is attached can be in the R configuration or in the S configuration.

[0095] In another preferred embodiment, R1is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl.

[0096] In another preferred embodiment, R2is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl.

[0097] In another preferred embodiment, A has the structure shown in the following formula:

[0098] In a preferred embodiment, D has the structure shown in formula (Ia’):

[0099] wherein X’ is selected from the group consisting of -O-, -S-, -NR m -;

[0100] R m is selected from the group consisting of hydrogen, C1-C4alkyl;

[0101] R3is hydrogen, methyl, ethyl or n-propyl;

[0102] R1is selected from the group consisting of hydrogen, C1-C4alkyl;

[0103] R2is C1-C4alkyl;

[0104] Z is selected from the group consisting of a chemical bond, C(O), C(S);

[0105] R 4 and R 5 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C4alkyl, C1-C4haloalkyl, C1-C4deuteroalkyl;

[0106] 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.

[0107] In another preferred embodiment, D is selected from the group consisting of:

[0108] In a preferred embodiment, the compound of formula (III) is selected from the group consisting of:

[0109] In a second aspect, the present application provides a compound of formula (Ia), a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof,

[0110] wherein X is selected from the group consisting of -OH, -SH, -NHR m ;

[0111] R m is selected from the group consisting of hydrogen, C1-C4alkyl;

[0112] R3is hydrogen, methyl, ethyl or n-propyl;

[0113] R1is selected from the group consisting of hydrogen, C1-C4alkyl;

[0114] R2is C1-C4alkyl;

[0115] Z is selected from the group consisting of a bond, C(O), C(S);

[0116] R 4 and R 5 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C4alkyl, C1-C4haloalkyl, C1-C4deuteroalkyl;

[0117] 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 C5-C8cycloalkyl, saturated or unsaturated 5-7 membered heterocycle;

[0118] Preferably, the compound of formula (Ia) is selected from the group consisting of:

[0119] In a third aspect, the present application provides a compound of formula (II), a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof,

[0120] wherein M is selected from the group consisting of: pyrimidinyl;

[0121] L and D are defined as above;

[0122] Preferably, the compound of formula (II) is selected from the group consisting of:

[0123] In a fourth aspect of the present application, there is provided a method for preparing a compound of formula III, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof as described in the first aspect of the present application, comprising the following steps:

[0124] reacting a compound of formula II, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof as described in the third aspect of the present application with Ab as described in the first aspect of the present application to obtain a compound of formula III, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof as described in the first aspect of the present application.

[0125] In a fifth aspect of the present application, there is provided a pharmaceutical composition comprising a compound of formula III as described in the first aspect of the present application, a compound of formula la as described in the second aspect of the present application or a compound of formula II as described in the third aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof; and

[0126] a pharmaceutically acceptable diluent, carrier and / or excipient.

[0127] In a sixth aspect of the present application, there is provided a pharmaceutical preparation comprising a compound of formula III as described in the first aspect of the present application, a compound of formula la as described in the second aspect of the present application or a compound of formula II as described in the third aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof; and

[0128] a pharmaceutically acceptable diluent, carrier and / or excipient.

[0129] In a sixth aspect of the present application, there is provided a use of a substance X in the preparation of a medicament for preventing or treating a disease associated with ADAM-9;

[0130] wherein the substance X is a compound of formula III as described in the first aspect of the present application, a compound of formula la as described in the second aspect of the present application or a compound of formula II as described in 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 as described in the fifth aspect of the present application, or a pharmaceutical preparation as described in the sixth aspect of the present application;

[0131] Preferably, the cancer is a solid tumor or a non-solid tumor;

[0132] 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, thyroid cancer.

[0133] It should be understood that, within the scope of the present application, all the technical features described above and the technical features described in detail hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0134] Figure 1 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-A.

[0135] Figure 2 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-B.

[0136] Figure 3 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-C.

[0137] Figure 4 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-D.

[0138] Figure 5 shows the SEC-HPLC profile of antibody-drug conjugate ADC1-DXD.

[0139] Figure 6 shows the in vitro inhibition of proliferation of BxPC-3 cell line by each toxin compound.

[0140] Figure 7 shows the in vitro inhibition of proliferation of PA-1 cell line by each toxin compound.

[0141] Figure 8 shows the in vitro inhibition of proliferation of Calu-3 cell line by antibody-drug conjugate targeting ADAM-9.

[0142] Figure 9 shows the in vitro inhibition of proliferation of BxPC-3 cell line by antibody-drug conjugate targeting ADAM-9.

[0143] Figure 10 shows the in vitro inhibition of proliferation of AGS cell line by antibody-drug conjugate targeting ADAM-9.

[0144] Figure 11 shows a graph of the tumor growth curve of NCI-H1975 tumor model mice treated with an ADAM-9-targeting antibody-drug conjugate in vivo.

[0145] Figure 12 shows a graph of the tumor mass of NCI-H1975 tumor model mice treated with an ADAM-9-targeting antibody-drug conjugate in vivo.

[0146] Figure 13 shows the effect of an ADAM-9-targeting antibody-drug conjugate on the body weight of NCI-H1975 tumor model mice.

[0147] Figure 14 shows a graph of the tumor growth curve of Calu-3 tumor model mice treated with an ADAM-9-targeting antibody-drug conjugate in vivo.

[0148] Figure 15 shows a graph of the tumor mass of Calu-3 tumor model mice treated with an ADAM-9-targeting antibody-drug conjugate in vivo.

[0149] Figure 16 shows the effect of an ADAM-9-targeting antibody-drug conjugate on the body weight of Calu-3 tumor model mice.

[0150] Figure 17 shows the change in body weight of rats after administration of an antibody-drug conjugate in a rat toxicity test.

[0151] Figure 18 shows the change in food intake of rats after administration of an antibody-drug conjugate in a rat toxicity test.

[0152] Figure 19 shows the toxicokinetics of total antibody in a cynomolgus monkey pre-toxicology test.

[0153] Figure 20 shows the toxicokinetics of a small molecule toxin in a cynomolgus monkey pre-toxicology test. DETAILED DESCRIPTION

[0154] After long-term and in-depth research, through a large number of screenings, the inventors first developed a new payload, a linker-payload molecule comprising the same, and a new ADC prepared from the same and an anti-ADAM-9 antibody. The ADAM-9-targeting ADC comprising the linker-payload of the present application and the anti-ADAM-9 antibody has excellent anti-tumor effects, particularly on colon cancer, pancreatic cancer, lung adenocarcinoma, breast cancer, gastric cancer, ovarian cancer, etc. Based on this, the inventors completed the present application.

[0155] Portions of the payload, linker-payload, and ADC of the present application

[0156] 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,

[0157] wherein:

[0158] X is selected from the group consisting of hydroxy, thiol, -NR m R n ;

[0159] 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 (such as ), phenyl, hydroxy-substituted phenyl (such as ), indolyl (such as );

[0160] 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 (such as ), phenyl, hydroxy-substituted phenyl (such as ), indolyl (such as );

[0161] R a , R b are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0162] 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.

[0163] 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)-.

[0164] In some embodiments, R1, R3are each independently selected from hydrogen, C1-C6 alkyl, and said C1-C6 alkyl is optionally substituted with a group selected from hydroxy, C1-C6 alkoxy.

[0165] In some embodiments, R1, R3are each independently selected from hydrogen, methyl, ethyl, isopropyl, -CH2CH2OH, -CH2CH2OCH3.

[0166] In some embodiments, R1, R3are each independently selected from hydrogen, C1-C6 alkyl.

[0167] In some embodiments, R1is selected from hydrogen, methyl, ethyl, isopropyl.

[0168] In some embodiments, R1is hydrogen.

[0169] In some embodiments, R3is selected from hydrogen, methyl, isopropyl.

[0170] In some embodiments, R3is hydrogen.

[0171] In some embodiments, R2is selected from C1-C6 alkyl, and said C1-C6 alkyl is optionally substituted with a group selected from hydroxy, thiol, C1-C6 alkoxy, C1-C6 alkylthio, R a R b N-C(=O)-.

[0172] In some embodiments, R2is selected from C1-C6 alkyl, and said C1-C6 alkyl is optionally substituted with a group selected from hydroxy, C1-C6 alkoxy.

[0173] In some embodiments, R2is selected from methyl, ethyl, isopropyl, -CH2CH2OH, -CH2CH2OCH3.

[0174] In some embodiments, R2is selected from C1-C6 alkyl.

[0175] In some embodiments, R2is methyl.

[0176] In some embodiments, R a , R b are each hydrogen.

[0177] In some embodiments, R m , R n are each hydrogen.

[0178] In some embodiments, X is selected from hydroxy, thiol, amino.

[0179] In some embodiments, X is selected from hydroxy, amino.

[0180] In some embodiments, X is hydroxyl. In other embodiments, X is amino.

[0181] In some embodiments, the compound of formula I has the structural formula of formula I-1,

[0182] wherein * indicates that the carbon atom at the position marked by * is a chiral carbon atom in S configuration when R1and R3are not hydrogen.

[0183] In some embodiments, the compound is selected from:

[0184] In a second aspect of the present disclosure, the present disclosure provides a compound of formula II, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof, M-L 1 -L 2 -L 3 -D II

[0185] wherein:

[0186] M is

[0187] 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 -;

[0188] p1, p2, p3 are each independently selected from any integer between 1-10;

[0189] q1, q2 are each independently selected from any integer between 1-20;

[0190] 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 by a group R x ;

[0191] R x is -(CH2CH2O) q3 -CH3;

[0192] q3 is selected from any integer between 1-20;

[0193] L3 is absent or is -NH-CH2-;

[0194] D is the group formed after removal of one hydrogen atom, the site of removal of the hydrogen atom being X;

[0195] X, R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0196] In some embodiments, L 1 the left side is connected to M, L 1 the right side is connected to L 2 .

[0197] In some embodiments, L 2 the amino terminus is connected to L 1 , L 3 is present, L 2 the carbonyl terminus is connected to L 3 , L 3 is absent, L 2 the carbonyl terminus is connected to D.

[0198] In some embodiments, L 3 is present, L 3 the N terminus is connected to L 2 , L 3 the C terminus is connected to D.

[0199] In some embodiments, D is the group formed after removal of one hydrogen atom from the structure represented by I-1’, the site of removal of the hydrogen atom being X,

[0200] wherein X, R1, R2, R3are each independently described in any of the technical solutions of the first aspect, * indicates that when R1and R3are not hydrogen, the carbon atom at the position indicated by * is a chiral carbon atom in the S configuration.

[0201] In some embodiments, D is selected from wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0202] 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 when R1and R3are not hydrogen, the carbon atom at the position indicated by * is a chiral carbon atom in the S configuration.

[0203] In some embodiments, D is selected from wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0204] 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.

[0205] In some embodiments, D is wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0206] 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.

[0207] In some embodiments, D is wherein R1, R2, R3are each independently described in any of the technical solutions of the first aspect.

[0208] 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.

[0209] In some embodiments, L 1 is -(CH2) p1 C(=O)-.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] In some embodiments, q1 is selected from any integer between 1-12. In some embodiments, q1 is selected from any integer between 2-12. In some embodiments, q1 is selected from any integer between 6-10. In some embodiments, q1 is 8.

[0214] In some embodiments, q2 is selected from any integer between 1-12. In some embodiments, q2 is selected from any integer between 2-12. In some embodiments, q2 is selected from any integer between 6-10. In some embodiments, q2 is 8.

[0215] In some embodiments, q3 is selected from any integer between 8-20. In some embodiments, q3 is selected from any integer between 10-16. In some embodiments, q3 is selected from any integer between 10-14. In some embodiments, q3 is 12.

[0216] 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.

[0217] 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 are optionally substituted with a group R x ).

[0218] 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), which is optionally substituted with a group R x (preferably, the side chain amino group of the glutamine residues are optionally substituted with a group R x ).

[0219] In some embodiments, L 2 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 optionally substituted with a group R x 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 optionally substituted with a group R 2 selected from

[0220] In some embodiments, L 3 is -NH-CH2-. In other embodiments, L 3 is absent.

[0221] In some embodiments, D is selected from:

[0222] In some embodiments, the compound is selected from:

[0223] 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

[0224] Ab is an antibody or an antigen binding fragment thereof;

[0225] M' is

[0226] L 1 , L 2 , L 3 , D is as described in any of the technical solutions of the second aspect;

[0227] 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).

[0228] 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.

[0229] 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.

[0230] In some embodiments, the antibody drug conjugate is selected from the group consisting of:

[0231] In the ADC formed by the present disclosure, the antibody Ab is connected to the carbon atom of the succinimide at the end of the linker-payload through -S- which is not the thiol group introduced to the Ab additionally, but the thiol group contained in the antibody Ab itself after the antibody Ab is reduced to open the disulfide bond.

[0232] In some embodiments, n is any integer or any decimal number 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, specifically 7.4).

[0233] In some embodiments, the DAR value of the composition is any integer or any decimal number selected from 2-8 (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).

[0234] 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).

[0235] The terms

[0236] 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.

[0237] 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.

[0238] 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."

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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- butadienyl, and the like.

[0243] 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.

[0244] 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.

[0245] 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. The cycloalkyl group can be monocyclic, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like, or bicyclic, for example fused, bridged, or spirocyclic.

[0246] As used herein, the term "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group having at least one double bond, for example "C 3-8 "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. Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. The cycloalkenyl group can be monocyclic or bicyclic, for example fused, bridged, or spirocyclic.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] As used herein, the term "alkylthio" refers to the group -S-R t where R t is H or alkyl as defined herein.

[0251] 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

[0252] 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.

[0253] ​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.

[0254] 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.

[0255] 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

[0256] As used herein, the term "unsaturated" refers to a cyclic structure containing 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.

[0257] 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.

[0258] As used herein, the term "substituted" means that one or more hydrogen atoms on a particular group is replaced with a particular substituent. The particular substituent is the substituent described in the immediately preceding paragraph, or the substituent that appears in each of the embodiments. Unless otherwise indicated, a substituted group can have one substituent at each substitutable position that is permitted by the core structure, and where valencies permit, the substituents on adjacent atoms can be combined to form a ring structure. The skilled artisan will understand that combinations of substituents that are not stable or that are not chemically feasible are not intended within the scope of the application.

[0259] Unless otherwise specified, the groups described herein are optionally substituted with a substituent selected from the group consisting of D, halogen, cyano, nitro, hydroxyl, 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.

[0260] "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, and instances where it does not.

[0261] The term "plurality" as used herein means a positive integer of 2, 3, 4, 5, or greater than 5.

[0262] Unless otherwise specified, "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.

[0263] 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).

[0264] 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.

[0265] Abbreviations

[0266] Targeting unit (Ab)

[0267] As used herein, the term "Ab" is a targeting unit, i.e., a polypeptide, antibody, or antigen binding fragment that binds to a target. Non-antibody targeting units can also be referred to as non-antibody scaffolds.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] As used herein, the terms Fab, F(ab')2, and Fv refer to the following:

[0273] (i) Fab is a monovalent fragment consisting of a VL, a VH, a CL, and a CH1 domain;

[0274] (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

[0275] (iii) Fv consisting of a VL and a VH domain.

[0276] 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.

[0277] 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.

[0278] 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 humanized heavy chain variable domain (AVH) or a VNAR moiety derived from a shark.

[0279] 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, camels, llamas, or alpacas by standard immunization techniques. VHHs can have potent antigen-binding capacity 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.

[0280] In some embodiments, ADAM-9 (e.g., shown in SEQ ID NOs: 1 and 2) 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 ADAM-9 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 antibodies, antigen-binding fragments thereof, and other binding agents, and conjugates that specifically bind ADAM-9 and exhibit improved properties. ADAM-9 is an important and advantageous therapeutic target for treating certain cancers. ADAM-9 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 treating ADAM-9 cancers and other diseases.

[0281] SEQ ID NO: 1

[0282] ADAM-9 antibody light chain sequence

[0283] SEQ ID NO: 2

[0284] ADAM-9 antibody heavy chain sequence

[0285] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as, for example, PCR amplification or screening of genomic libraries. In addition, the coding sequences of the light and heavy chains can be fused together to form a single-chain antibody.

[0286] The antibody in the antibody drug conjugate of the present application preferably maintains the antigen binding ability of the original wild type. Therefore, the antibody in the present application can, preferably specifically, bind to the antigen.

[0287] drug

[0288] 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.

[0289] Camptothecin is a pentacyclic parent nucleus compound isolated from the plant Camptotheca acuminata, which consists 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).

[0290] Research data shows that camptothecin can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, leading to DNA replication inhibition, 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.

[0291] 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 developing novel camptothecin compounds and their conjugates that can improve effectiveness and safety issues.

[0292] 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 thereof, a stereoisomer thereof, a solvate thereof or a solvate of a pharmaceutically acceptable salt thereof,

[0293] wherein X is selected from the group consisting of -OH, -SH, -NHR m ;

[0294] R mhydrogen, C1-C6alkyl;

[0295] R1and R3are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0296] 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;

[0297] A has the structure shown in Formula IV:

[0298] Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O);

[0299] 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;

[0300] 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, a saturated or unsaturated 5-12 membered heterocyclic ring;

[0301] 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 aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl.

[0302] In a 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-C4alkyl, C1-C4haloalkyl, C1-C4deuteroalkyl;

[0303] or, R 4 and R 5together 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.

[0304] 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.

[0305] Linker

[0306] 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 a cytotoxic drug, and can also be linked to another linker before being linked to a cytotoxic drug.

[0307] According to the mechanism of drug release in cells, "linkers" or "linkers of antibody-drug conjugates" can be classified into two categories: non-cleavable linkers and cleavable linkers. The linker L of the present application is a cleavable linker.

[0308] For antibody-drug conjugates containing non-cleavable linkers, the mechanism of drug release is as follows: after the conjugate binds to an antigen and is endocytosed by a 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 changes in the drug molecule thus brought about do not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Thus, such active drugs cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect).

[0309] Cleavable linkers, as the name implies, can be cleaved in the target cell and release the active drug (the 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).

[0310] Chemically unstable linkers can be selectively cleaved due to the differences in the properties of plasma and cytoplasm. Such properties include pH, glutathione concentration, etc. Linkers sensitive to pH are also commonly referred to as acid-cleavable linkers. Such 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 are also known as disulfide bond linkers. Drug release is based on the difference in the high concentration of glutathione in cells (millimolar range) and the relatively low concentration of glutathione in blood (micromolar range).

[0311] Enzymatically labile linkers, such as peptide linkers, can provide better control of drug release. Peptide linkers can be efficiently cleaved by lysosomal proteases, such as Cathepsin B or plasmin (which are increased in some tumor tissues). Such peptide linkers are believed to be very stable in the plasma circulation because extracellularly the pH is not optimal and serum protease inhibitors render proteases generally inactive. 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.

[0312] The linker of the present application is M-L1-L2-L3-L4-;

[0313] wherein M is for connecting Ab and L1, which is selected from the group consisting of: pyrimidinyl,

[0314] each X3is independently a leaving group, preferably the X3is selected from the group consisting of Cl, Br, OMs, OTs, OTf;

[0315] R d is H, C1-C6alkyl, C1-C6deuteroalkyl, C3-C8cycloalkyl, or C3-C8deutero-cycloalkyl;

[0316] W1is -(C(R7)(R8))n7-, W2is -(OCH2CH2)n8-On9-, W3is -(C(R9)(R 13 )n10;

[0317] wherein each of n7, n8, and n10is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;

[0318] n9is 0 or 1;

[0319] when a methylene group is present in W1and W3, the methylene group in W1and W3is optionally replaced with one or more groups selected from the group consisting of: an optionally substituted ring A, -N(R 15 )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 )-, -N=C(R 14)SO2-, -SO2N(R 14 )-, -C(=S)-, -C(=NR 14 )-, -N=N-, -C=N-, -N=C-, -C(=N2)-;

[0320] said ring A is selected from the group consisting of C 6-10 arylene, 5-8 membered heteroarylene, 3-10 membered heterocyclylene, 3-10 membered saturated or partially unsaturated carbocyclylene;

[0321] wherein each R7, R8, R9, R 13 , R 14 and R 15 are each independently selected from the group consisting of 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 , C e aliphatic optionally substituted with R 1-6 , C1-C8 alkyl optionally substituted, C1-C8 haloalkyl optionally substituted, C1-C8 deuterated alkyl optionally substituted, C3-C8 cycloalkyl optionally substituted, and C4-C8 cycloalkylalkyl optionally substituted;

[0322] wherein each R e , R f and R g is each 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, -SO2NH-, -OC(O)H, -N(H)SO2H, C 1-6aliphatic, optionally substituted C1-C8alkyl, optionally substituted C1-C8haloalkyl, optionally substituted C1-C8deuteroalkyl, optionally substituted C3-C8cycloalkyl, and optionally substituted C4-C8cycloalkylalkyl;

[0323] L1is selected from the group consisting of -(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)-;

[0324] 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-C6cycloalicyclyl;

[0325] 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) n4OCH3, (CH2CH2O) n5 CH3, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 H, (CH2) n3 C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3;

[0326] 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;

[0327] L2is a peptide residue or a group selected from the group consisting of C 1-6 alkylene, -N(R6)-, carbonyl,

[0328] R6is selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkyl substituted with one or more -(CH2CH2O)r-;

[0329] r is selected from an integer between 1 and 10; s is selected from an integer between 1 and 20;

[0330] L3is -L 4a -(NR b ) n6 -R 12 -L 4b -,

[0331] wherein L 4a is absent, or L 4a is an optionally substituted

[0332] n6is 0 or 1;

[0333] R 12 is a bond, CH2, or CD2;

[0334] L 4b is absent, or L 4b is an optionally substituted

[0335] R a and R bEach is independently selected from the group consisting of: hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 deuterated alkyl, optionally substituted C1-C4 alkylamine, optionally substituted C1-C4 alkylthio, optionally substituted -S(O)2-C1-C4 alkylthio.

[0336] L4 is absent, or is an optional substituent of the following group:

[0337] Among them, Y is selected from the following groups: O, S, NH;

[0338] v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0339] R 10 and R 11 Each is independently selected from the group consisting of: hydrogen, deuterium, halogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 deuteralkyl, optionally substituted C3-C8 cycloalkyl and optionally substituted C4-C8 cycloalkylalkyl, or R 10 and R 11 Together with the atoms attached thereto, they form optionally substituted C3-C8 cycloalkyl groups;

[0340] R 16 and R 17 Each of the following is independently selected: 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, -SO2NH2, -OC(O)H, -N(H)SO2H, and optionally substituted 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;

[0341] The L1, L2, L3, and L4 mentioned above can also be optionally substituted by substituents selected from the group consisting of: hydrogen atom, deuterium atom, halogen, nitrile group, nitro group, hydroxyl group, amino group, C1-C6 alkyl-NH-, (C1-C6 alkyl)2N-, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy group, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halo-C1-C6 alkoxy group, allyl group, benzyl group, C6-C 12aryl, 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.

[0342] Antibody-drug conjugate

[0343] 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., an anti-ADAM-9 polypeptide, antibody, antigen binding fragment, or non-antibody targeting unit that binds to a target ADAM-9) linked via a linker to a cytotoxic drug having biological activity, e.g., an antibody-drug conjugate of Formula (III).

[0344] 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)

[0345] wherein the linker M'-L is a cleavable linker, which is defined as above;

[0346] Ab is an anti-ADAM-9 antibody or antigen binding fragment that binds to a target ADAM-9, which is defined as above;

[0347] D is a compound of the following Formula (I-1)

[0348] wherein X', R1, R2, R3, and A are defined as above.

[0349] 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 typically occurs at the epsilon-amino group of lysine residues or the sulfhydryl group of cysteine residues.

[0350] The large number of lysine side chains on the surface of an antibody protein results in a large number of sites available for drug conjugation, resulting in the generation of antibody drug conjugates that are mixtures containing different numbers of drug conjugations (drug / antibody ratio, DAR), e.g., integers or fractions of 2.0-16.0 (preferably 2.0-8.0, most preferably 4.0-8.0) and conjugation sites.

[0351] Active ingredient

[0352] Depending on the context, "a compound of the invention" can refer to a compound of Formula (III), and also includes stereoisomers, optical isomers, pharmaceutically acceptable salts, crystal forms, isotopic derivatives, prodrugs, metabolites, solvates, or hydrates thereof.

[0353] 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 configuration about asymmetric centers; the (Z), (E) configuration about double bonds; and the like. Thus, individual stereochemical isomers or mixtures of enantiomers, diastereomers, or conformational isomers of the compounds of the invention are within the scope of the invention.

[0354] The compounds of the invention can contain cis-trans isomers, one or more chiral carbon atoms, and thus can exist in stereoisomeric forms such as cis-trans isomers, chiral isomers, enantiomeric, diastereomeric, and other combinations. Cis-trans isomerism refers to the non- enantiomeric phenomenon that occurs in a molecule when the spatial arrangement of individual groups is different due to the presence of a restriction on free rotation. This restriction is generally caused by the presence of a functional group in the structure of the organic compound that cannot rotate freely, 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. 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, generally indicated by "cis" or "cis-," and trans referring to the same ligand being in an opposite position, generally indicated by "trans" or "trans-." Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. The invention is intended to include all possible isomers, as well as racemates and optically pure forms. The preparation of the compounds of the invention can select racemates, cis-trans isomers, chiral isomers, diastereomers, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography, and the like.

[0355] 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.

[0356] If a specific stereoisomer of a compound of the application is to be designed for synthesis, it can be prepared by asymmetric synthesis, or derivatized with a chiral auxiliary, the resulting stereomixture separated, and the chiral auxiliary removed to yield the pure syn or anti monomer, chiral monomer, or mixed stereomonomer. If the molecule contains a syn / anti isomeric center, the pure syn or anti product can be purified by column chromatography (normal phase silica gel column or reverse phase HPLC 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, it can be derivatized with an appropriate optically active acid or base to form a diastereomeric salt, which can then be separated by conventional means, and the pure enantiomer recovered by removing the chiral auxiliary.

[0357] The present application also includes isotopically-labeled compounds (i.e., isotopic derivatives), which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that are within the scope of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, and chlorine, such as 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-labeled compounds of the present application are within the scope of the present application. Isotopically-labeled compounds, for example those into which radioactive isotopes such as 3 H-labeled compounds and 14 C-labeled compounds are useful in drug and substrate tissue distribution assays. The preparation of such radiolabeled compounds of interest is well known in the art. For example, radiolabeled compounds of interest can be readily prepared by substituting readily available isotopically-labeled reagents for non-isotopically-labeled reagents in the syntheses as disclosed in the Examples. Alternatively, such radiolabeled compounds of interest can be readily prepared by the techniques of radiohalogenation as described in the literature of the art. 3 H) and carbon-14 (i.e., 14 C) are preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2 H, affords certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of this application can generally be prepared by carrying out the procedures disclosed in the Examples and / or in the Schemes and Preparations below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0358] The term "pharmaceutically acceptable salt" as used herein includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0359] "Pharmaceutically acceptable acid addition salt" refers to salts of the free base which retain the biological effectiveness and non-toxicity of the free amine and which are formed with inorganic acids or with organic acids. Inorganic acids from which salts can be derived include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, but are not limited to, formic acid, acetic acid, 2,2-dichloroacetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, undecylenic acid, glycolic acid, gluconic acid, lactic acid, sebacic acid, adipic acid, glutaric acid, malonic acid, oxalic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, stearic acid, oleic acid, cinnamic acid, lauric acid, malic acid, glutamic acid, pyroglutamic acid, aspartic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, alginic acid, ascorbic acid, salicylic acid, 4-aminosalicylic acid, naphthalene-2-sulfonic acid, and the like. These salts can be prepared by methods known in the art.

[0360] "Pharmaceutically acceptable base addition salt" refers to salts of the free acid which retains the biological effectiveness and non-toxicity of the free acid and which are formed with inorganic or organic bases. 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 the 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.

[0361] Metabolites of the compounds of Formula (III) and pharmaceutically acceptable salts thereof, and the prodrugs of the compounds of Formula (III) and pharmaceutically acceptable salts thereof that can result from in vivo oxidative, hydrolytic, or other metabolic cleavage of a compound of Formula (III) and pharmaceutically acceptable salts thereof, are also within the scope of the present application.

[0362] As used herein, the term "solvate" means a compound of Formula (III) coordinated with solvent molecules in specific ratios.

[0363] As used herein, the term "hydrate" means a compound of Formula (III) coordinated with water molecules in specific ratios.

[0364] Pharmaceutical compositions and methods of administration

[0365] 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, thereby causing the drug to be released in an active form into the cell by endocytosis of the conjugate or diffusion of the drug into the cell. Thus, the antibody-drug conjugates of the present application can be used to treat a disease of interest (e.g., cancer) by administering to a subject (e.g., a human) an antibody-drug conjugate as described above in a therapeutically effective amount by a suitable route. The subject in need of treatment can be one at risk for, or suspected of having, a disorder associated with the activity or amount of expression of a particular antigen. Such a subject can be identified by routine medical examination.

[0366] 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 implanted reservoir. 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 means of an injectable depot formation, e.g., using a reservoir that releases the composition over a period of 1, 3, or 6 months, using biodegradable polymers and methods.

[0367] 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 into the subject. 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.

[0368] When treated with the antibody-drug conjugates of the present application, delivery can be performed by methods conventional in the art. For example, it can be introduced into the cell by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump. Other methods include various transport and carrier systems by using conjugates and biodegradable polymers.

[0369] 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.

[0370] 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.

[0371] The target disease (the disease to be treated) of the antibody-drug conjugate of the present application is a disease associated with ADAM-9, and preferably, the disease associated with ADAM-9 is a tumor.

[0372] 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, stomach cancer, and esophageal cancer.

[0373] The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials). The factors include, but are not limited to, the 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, 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, a satisfactory effect can be obtained. For example, several separate doses per day or proportionally reduced doses can be administered according to the exigencies of the therapeutic situation.

[0374] The dosage form of the antibody-drug conjugate of the present application for topical administration includes ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants, if necessary, under sterile conditions.

[0375] The antibody-drug conjugate of the present application can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0376] When the pharmaceutical composition is used, a safe and effective amount of the antibody-conjugate of the present application is applied to a mammal (e.g. human) in need of treatment, wherein the dosage is pharmaceutically effective dosage, and the daily dosage is usually 1-2000 mg, preferably 5-500 mg for a 60 kg body weight human. Of course, the specific dosage should also consider the administration route, patient health condition, etc., which are within the skill of a skilled physician.

[0377] The main advantages of the present application are:

[0378] 1. The payload, linker-payload and / or ADC prepared from linker-payload and ADAM-9 antibody of the present application has excellent anti-tumor effect, especially on colon cancer, pancreatic cancer, lung adenocarcinoma, breast cancer, etc.

[0379] 2. The ADC targeting ADAM-9 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 completely comparable to the positive control ADC using the marketed linker-payload (deruxtecan) of the same target.

[0380] 3. The antibody-drug conjugate targeting ADAM-9 of the present application has high in vivo tolerance and excellent therapeutic safety window. In rat toxicity tests, compared with the positive control ADC using the marketed linker-payload (deruxtecan) of the same target, it shows lower body weight reduction, lower food intake reduction, and lower hematological toxicity.

[0381] 4. The antibody-drug conjugate targeting ADAM-9 of the present application has excellent pharmacokinetics, and the total anti shows a longer half-life in cynomolgus monkey blood, 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.

[0382] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturer unless otherwise specified. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0383] Example 1: Preparation of humanized antibody H03-2 against ADAM-9

[0384] The preparation method of the humanized antibody H03-2 against ADAM-9 used in the present application is as described in patent 202410970739.9, specifically as follows:

[0385] I. Antigen, immunization scheme and antibody preparation

[0386] 1. Antigen

[0387] The protein antigen used for immunization is the 206-297 amino acids 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.

[0388] 2. Immunization scheme

[0389] Animals were immunized with the aforementioned human ADAM9-ECD-His protein, a total of 10 Balb / C mice were selected, female, 10 weeks old, and the mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The first immunization antigen was emulsified with Freund's complete adjuvant, the antigen amount was 100 μg / mouse, and the subsequent immunization adjuvant was Freund's incomplete adjuvant, 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 to be immunized every other week, i.e. immunized again one week after immunization, and 21 days after the last immunization, the booster immunization was performed.

[0390] 3. Serum titer detection

[0391] 3.1. Sample preparation

[0392] The serum of the immunized mouse was taken and diluted with 5% PBST at a gradient of 1500, 4500, 13500, and 40500.

[0393] 3.2. ELISA method for detecting serum titer

[0394] 1) Coating: 2 μg / mL antigen, 30 μL per well, 4°C overnight, PBST washing plate 3 times.

[0395] 2) Blocking: 5% PBST blocking at room temperature for 2 h, PBST washing plate 3 times.

[0396] 3) First antibody: add the serum diluted in the above gradient, the serum of non-immune mice as negative control, 30 μL / well, incubate at room temperature for 1 h, wash the plate with PBST for 6 times.

[0397] 4) Second antibody: add the serum sample to the second antibody goat anti-mouse-lgG-HRP (Rockland, cat# 609-103-123) diluted at 1:5000, diluted with 5% PBST; add the second antibody goat anti-human IgG-HRP (Shanghai Enzyme-linked Biotechnology Co., Ltd., ml087062) diluted at 1:5000, diluted with 5% PBST to the positive control group. 30 μL / well, incubate at room temperature for 50 min, wash the plate with PBST for 6 times.

[0398] 5) Termination: add 30 μL / well of TMB (Suzhou Yake Chemical Reagent Co., Ltd., cat# S0025) to develop color at room temperature for 5 min to 10 min, then add 30 μL / well of 2M termination solution (Suzhou Yake Chemical Reagent Co., Ltd.) to terminate the reaction, and read the data by an enzyme-labeled instrument at OD450.

[0399] II. Preparation of hybridoma cells and obtaining of mouse-derived antibodies

[0400] As described above, the titer of the above-mentioned immunized Balb / C mice was detected using human ADAM9-ECD-His protein, the serum titer and the ability to bind to cell surface antigens were evaluated, and the control titer detection condition (more than 100,000-fold dilution) determined the start of cell fusion. The immune mice with high serum titer were selected for a final immunization, and the mice were sacrificed after the immunization, and the spleen cells and SP2 / 0 myeloma cells were fused and plated to obtain hybridomas. 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 the 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.

[0401] III. Humanization of mouse antibodies

[0402] The humanization of the murine anti-human ADAM9 monoclonal antibody is performed according to the methods reported in many documents in the art. Briefly, the human constant domains are used to replace the constant domains of the parent (murine antibody), and the human antibody sequences are selected according to the homology of the murine antibody and human antibody. In this embodiment, the murine antibody is humanized.

[0403] Specifically, the human germline sequences are used as the acceptor framework for humanizing the murine antibody. In order to find the closest germline sequence, the most similar expressed light chain and the most similar heavy chain are identified in the germline sequence database by NCBI IgBLAST (ncbi.nlm.nih.gov / igblast / ). In this search, the CDR sequences of the murine antibody are masked. The selection criteria of the most suitable expressed sequence include the sequence identity of checking the classic residues and the interface residues, and the similarity of checking the CDR loop length.

[0404] On the basis of the obtained murine antibody VH / VL CDR typical structure, the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain the human germline template with high homology.

[0405] The CDR regions of the murine antibody are grafted onto the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the back-mutation is performed on the buried residues, the residues directly interacting with the CDR regions, and the residues having important influence on the conformation of VL and VH, and the CDR region chemically unstable amino acid residues are optimized. Through expression test and back-mutation number comparison, the antibody with the combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences is selected, in which the CDR sequences of the light and heavy chain variable regions are shown in Table 1, the specific variable region sequence information of the humanized antibody is shown in Table 2, and the constant region sequence information is shown in Table 3.

[0406] Table 1. CDR sequences of heavy and light chain variable regions of humanized ADAM-9 antibody

[0407] Table 2. Sequences of heavy and light chain variable regions of humanized ADAM-9 antibody

[0408] Note: The underlined part represents the CDR sequence of the monoclonal antibody.

[0409] Table 3. Constant region sequences of humanized ADAM-9 antibody

[0410] Example 2: Synthesis of toxin compound A

[0411] 2.1 Synthesis of intermediate compound A2

[0412] 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 the reaction was complete, the reaction was evaporated and the residue was purified by silica gel column to give the target product A2 about 305 mg. LCMS confirmation: [M+H] + = 715.4.

[0413] 2.2 Synthesis of intermediate compound A3

[0414] 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 the reaction was complete, the reaction was evaporated and the residue was purified by silica gel column to give the target molecule A3 about 163 mg. LCMS confirmation: [M+H] + = 492.9.

[0415] 2.3 Synthesis of compound A

[0416] 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 molecule 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).

[0417] Example 3: Synthesis of toxin compound B

[0418] 3.1 Synthesis of intermediate compound B2

[0419] 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 of the reaction. Prep-HPLC gave B2: 420 mg of yellow solid product. LCMS confirmation: [M+H] + = 607.

[0420] 3.2 Synthesis of intermediate compound B3

[0421] 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 of the addition. Prep-HPLC gave B3: 380 mg. LCMS confirmation: [M+H] + = 507.

[0422] 3.3 Synthesis of compound B

[0423] 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 of the addition. Prep-HPLC gave compound B: 100 mg. LCMS confirmation: [M+H] + = 565.

[0424] 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).

[0425] Example 4: Synthesis of toxin compound C

[0426] 4.1 Synthesis of intermediate compound C3

[0427] 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.

[0428] 4.2 Synthesis of intermediate compound C4

[0429] 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

[0430] 4.3 Synthesis of compound C

[0431] 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 and 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

[0432] 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).

[0433] Example 5: Synthesis of toxin compound D

[0434] 5.1 Synthesis of intermediate compound D3

[0435] 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.

[0436] 5.2 Synthesis of intermediate compound D4

[0437] 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 crude product. LCMS: [M+H] + = 549.4

[0438] 5.3 Synthesis of intermediate compound D6

[0439] 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.

[0440] 5.4 Synthesis of intermediate compound D

[0441] 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).

[0442] Example 6: Synthesis of toxin compound F

[0443] 6.1 Synthesis of compound F

[0444] 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, 11H 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).

[0445] Example 7: Synthesis of toxin compound G

[0446] 7.1 Synthesis of compound G

[0447] 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.

[0448] 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).

[0449] Example 8: Synthesis of toxin compound H

[0450] 8.1 Synthesis of compound H

[0451] 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.

[0452] 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).

[0453] Example 9: Synthesis of linker-drug conjugate LD-A

[0454] 9.1 Synthesis of compound LD-A1

[0455] 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

[0456] 9.2 Synthesis of compound LD-A2

[0457] Diethylamine (614 mg, 8.39 mmol) was added to a THF (5 mL) solution of compound LD-A1 (300 mg, 0.42 mmol), and the reaction was carried out at room temperature for 3 hours. TLC monitoring showed that the starting material reacted completely. After the reaction solution was evaporated to dryness, it was passed through a silica gel column to obtain approximately 163 mg of the target molecule LD-A2. LCMS confirmed: [M+H]+ = 492.9 g / L.

[0458] 9.3 Synthesis of the final product LD-A

[0459] Compounds LD-A2 (79.87 mg, 0.16 mmol), DMTMM (71.70 mg, 0.24 mmol), and TEA (49.23 mg, 0.49 mmol) were added to a DMF / H2O (2.5 mL / 0.5 mL) solution of compound LD-A3 (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) (100 mg, 0.16 mmol) and reacted at room temperature for 1 hour. The reaction solution was purified by preparative column chromatography and lyophilized to obtain approximately 20.1 mg of the target molecule LD-A. LCMS confirmed that [M+H]+ = 1091.9.

[0460] Example 10: Synthesis of linker-drug conjugate LD-B

[0461] 10.1 Synthesis of compound LD-B2

[0462] 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), followed by the dropwise addition of DIPEA (354 μL, 1.836 mmol). The reaction mixture was stirred at room temperature for 2 hours. After thorough extraction and concentration with DCM and a large amount of water, approximately 210 mg of the target product LD-B2 was obtained. LCMS: [M+H]+ = 607.25.

[0463] 10.2 Synthesis of compound LD-B3

[0464] LD-B2 (200 mg, 0.329 mmol) was added to DCM (10 mL), followed by the dropwise addition of TFA (4 mL). The reaction mixture was incubated at room temperature for 1 hour. After purification by HPLC, approximately 140 mg of the target product LD-B3 was obtained. LCMS: [M+H]+ = 507.2

[0465] 10.3 Synthesis of the final product LD-B

[0466] 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.

[0467] Example 11: Synthesis of linker-drug conjugate LD-C

[0468] 11.1 Synthesis of compound LD-C2

[0469] 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. +

[0470] 11.2 Synthesis of compound LD-C4

[0471] 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. +

[0472] 11.3 Synthesis of final product LD-C

[0473] 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. +

[0474] ​​​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).

[0475] Example 12: Synthesis of linker-drug conjugate LD-D

[0476] 12.1 Synthesis of compound LD-D2

[0477] 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 h. To the reaction was added compound LD-D1 (250 mg, 0.574 mmol) and reacted at room temperature for 1 h. The reaction was purified by reverse phase to give LD-D3: 268 mg. LCMS confirmation: [M+H]+= 649.1.

[0478] 12.2 Synthesis of compound LD-D4

[0479] 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.

[0480] 12.3 Synthesis of compound LD-D

[0481] 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.

[0482] Example 13: Preparation of antibody-drug conjugate ADC1-A

[0483] Humanized antibody H03-2 against ADAM-9 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.

[0484] Example 14: Preparation of antibody-drug conjugate ADC1-B

[0485] 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.

[0486] Example 15: Preparation of antibody-drug conjugate ADC1-C

[0487] 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.

[0488] Example 16: Preparation of antibody-drug conjugate ADC1-D

[0489] Antibody H03-2 was diluted with 50 mM EPPS, 1 mM EDTA was added and the pH was adjusted to 7.0, the antibody concentration was 5 mg / ml. To the antibody solution, 6.5 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, 13 equivalents of compound LD-D DMSO solution (DMSO final concentration was 12%) was added to the antibody mixture, after 60 minutes, 13 equivalents of acetylcysteine (NAC) was added, and placed in a 22 °C shaker at 30 rpm for 10 minutes to obtain the conjugation crude product ADC1-D. After desalting column (filler: sephadex G 25) desalination purification, the conjugate ADC1-D 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 ADC1-D showed a purity of 98.5%, and the results are shown in Figure 4.

[0490] Example 17: Preparation of antibody-drug conjugate ADC1-DXD

[0491] Antibody H03-2 was diluted with 50 mM EPPS, 1 mM EDTA was added and the pH was adjusted to 7.0, the antibody concentration was 5 mg / ml. To the antibody solution, 6.5 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, 13 equivalents of compound LD-D DMSO solution (DMSO final concentration was 12%) was added to the antibody mixture, after 60 minutes, 13 equivalents of acetylcysteine (NAC) was added, and placed in a 22 °C shaker at 30 rpm for 10 minutes to obtain the conjugation crude product ADC1-D. After desalting column (filler: sephadex G 25) desalination purification, the conjugate ADC1-D 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 ADC1-D showed a purity of 98.5%, and the results are shown in Figure 4.

[0492] (deruxtecan: linker-drug conjugate of first trinity Enhertu ADC)

[0493] Example 18: In vitro anti-tumor activity of toxin compounds

[0494] This embodiment tested the in vitro inhibitory activity of DXD (drug molecule in the linker-drug conjugate deruxtecan of Daiichi Sankyo Enhertu ADC) and different toxic compounds of this application against human in situ pancreatic cancer cell line BxPC-3 and human ovarian teratoma cell line PA-1 expressing ADAM9.

[0495] The cells used in this example were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. or Nanjing Kebai Biotechnology Co., Ltd. (both have STR identification reports) and cultured according to the corresponding instructions, including PA-1, BxPC-3, etc. Cells in the logarithmic growth phase were seeded at a density of 2000 cells per well in 96-well cell culture plates (100 μL / well). After incubation at 37°C and 5% CO2 for approximately 24 hours, different concentrations of the toxin compound were added, with three replicates for each drug concentration, along with corresponding solvent control and blank control wells. After 5 days (120 hours), the culture medium was discarded, and 100 μL / well of complete culture medium containing 10% CCK-8 (purchased from Beyotime, CAT#C0040) was added. The cells were incubated at 37°C for 1-2.5 hours (depending on cell reactivity, until the desired color depth was reached). Cell viability (OD 450 nM) was measured for each group, and cell survival rate was calculated using the following formula: Survival rate = (OD of drug - OD of blank) / (OD of control - OD of blank) × 100%. The data were analyzed using software to calculate the IC50 of DXD and each toxin compound in different cell lines. 50 value.

[0496] The growth curves of various compounds inhibiting BxPC-3 and PA-1 cancer cells are shown in Figures 6 and 7, with corresponding IC50 values. 50 The values ​​are shown in Table 4.

[0497] Table 4: IC50 values ​​of DXD and compounds of this application for inhibiting proliferation in BXPC-3 and PA-1 cell lines. 50 value

[0498] Experimental results showed that compounds DXD, C, D, F, and G had significant IC50 values ​​in inhibiting the proliferation of human ovarian teratoma cells (PA-1). 50 The values ​​were all low (<0.5 ng / mL), indicating that they have excellent inhibitory effects on the proliferation of ovarian cancer cells. In the inhibition of the proliferation of human pancreatic cancer BxPC-3 cells, the IC50 values ​​of compounds DXD, C, D, F, and G were relatively low. 50 The values ​​were all low (<3 ng / mL), indicating that it has an excellent inhibitory effect on the proliferation of pancreatic cancer cells.

[0499] Example 19: In vitro antitumor activity of antibody-drug conjugates targeting ADAM-9

[0500] This embodiment tested the in vitro antitumor activity of the antibody-drug conjugate against ADAM-9-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 18, and the experimental results are shown in Figures 8-10 and Table 5.

[0501] Table 5: Inhibitory activity of different antibody-drug conjugates targeting ADAM-9 against different cancer cell lines

[0502] Note: ADC IC 50 This represents the molar concentration of the toxin contained.

[0503] 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 8), pancreatic cancer cells BxPC-3 (Figure 9) and gastric cancer cells AGS (Figure 10) with high ADAM9 expression.

[0504] 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 18. 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.

[0505] Example 20: In vivo antitumor activity of ADAM-9-targeting antibody-drug conjugate

[0506] NCI-H1975 (human lung adenocarcinoma cells) and Calu-3 cells were cultured in vitro in a monolayer and passaged. Cells were harvested when they reached the exponential growth phase. 5.0 × 10⁶ cells were then cultured. 6 One tumor cell was suspended in 0.1 ml of a 1:1 mixture of PBS and Matrigel and inoculated into the right scapula of five nude mice (P1 generation). Tumors were allowed to grow to 500-800 mm in length. 3 At that time, the tumor-bearing mice were euthanized by CO2 anesthesia, and the tumor was cut into 20-30mm pieces. 3Small tumor fragments were inoculated into a new batch of nude mice (P2 generation). The antitumor activity of the test product was evaluated using stable passaged tumor tissue. Seven days after inoculation, the average tumor volume reached approximately 150 mm². 3 At that time, mice (n=5) were randomly grouped according to tumor volume and drug administration was started.

[0507] In the NCI-H1975 model, the antibody-drug conjugate was converted to a dose of 0.1 mg / kg eciletidine, administered twice weekly. In the Calu-3 model, the antibody-drug conjugate was converted to a dose of 0.02 mg / kg eciletidine, administered as a single dose.

[0508] After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were monitored: animal activity, food and water intake, weight changes (measured twice weekly), and any abnormalities in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume was calculated as follows: tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Around day 35 after tumor inoculation, tumors were harvested from all mice, weighed, and photographed.

[0509] The experimental results are shown in Figures 11-16, where Figures 11 and 14 are statistical curves of tumor-suppressing activity, Figures 12 and 15 are tumor block diagrams of tumor-suppressing effect, and Figures 13 and 16 are corresponding curves of body weight change.

[0510] The in vivo efficacy results of NCI-H1975 show that the antibody-drug conjugates ADC1-B and ADC1-C targeting ADAM-9 of the present invention have excellent antitumor effects, which are significantly better than ADC1-A and ADC1-D. Among them, ADC1-C and the positive control ADC1-DXD both achieved 100% inhibition (Figures 11 and 12).

[0511] The in vivo efficacy results of Calu-3 show that, with a single dose of lower dose, the antibody-drug conjugates targeting ADAM-9 of the present invention exhibit excellent tumor-suppressing effects of ADC1-B and ADC1-C, which are significantly better than ADC1-A and ADC1-D. ADC1-C and the positive control ADC1-DXD both achieved 100% inhibition (Figures 14 and 15).

[0512] In summary, both ADC1-B and ADC1-C exhibit excellent tumor-suppressing effects in vivo, with ADC1-C showing even better tumor-suppressing effects.

[0513] Example 21: Rat toxicity test of Anti-ADAM9 antibody-drug conjugate

[0514] SD rats were administered ADC1-C (prepared alone, DAR value 7.8) and ADC1-DXD (prepared alone, DAR value 7.5) via tail vein injection once a week for a total of 3 times, with no recovery period. The nature and severity of potential toxic reactions and possible mortality were observed to preliminarily determine the target organs or tissues, and toxicokinetics were studied.

[0515] method

[0516] Eighteen SD rats were randomly divided into three groups according to sex and body weight: a solvent control group, an ADC1-C dosage group, and a positive control group (ADC1-DXD), with six rats per group (half male and half female). The ADC1-C dosage group and the positive control group received ADC1-DXD intravenously at doses of 200 mg / kg and 160 mg / kg, respectively, with an administration volume of 10 mL / kg. The corresponding concentrations in both groups were 20 mg / mL and 16 mg / mL, respectively. The solvent control group received 10 mL / kg of sodium chloride injection. Administered via tail vein bolus injection, once weekly for three weeks, for a total of three administrations.

[0517] (1) General condition observation: The animal shall be observed by a veterinarian for 3 days after receipt, once a day. On the day of administration, the animal shall be observed once before, once after and once in the afternoon. On the day of dissection, the animal shall be observed once in the morning and once in the afternoon. On other days, the animal shall be observed once in the morning and once in the afternoon. The observation content includes, but is not limited to, general appearance, behavior, abnormal symptoms of the eyes, mouth, nose and mouth, ears, hair and skin, feces, urine, genitals and other abnormalities.

[0518] (2) Detailed clinical observation: During the trial, the animals were observed once a week. The observation included observing the animals' general appearance, behavior, hair and skin, feces, urine and other abnormalities at the cage. After that, the animals were moved outside the cage and the subcutaneous tissues of the animal's head, face, trunk, limbs, abdominal organs and mucous membranes of the eyes, mouth and genitals were palpated.

[0519] (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.

[0520] (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 17.

[0521] (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 18.

[0522] (6) Clinical pathology: Blood sample D22 was tested once, and the results are shown in Table 6.

[0523] (7) Toxicokinetics: Detection of ADC, total antibody and small molecule toxin.

[0524] (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.

[0525] Table 6: Results of Detection of Hematological Indicators of Toxicity in Rats

[0526] 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).

[0527] *D=Dunnett LSD Test Significant at the 0.05level.

[0528] +D=Dunnett LSD Test Significant at the 0.01level.

[0529] in conclusion

[0530] 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.

[0531] After administration, the weight loss of rats in the ADC1-C group was significantly lower than that in the ADC1-DXD group (Figure 17), and the food intake loss of rats in the ADC1-C group was significantly lower than that in the ADC1-DXD group (Figure 18). Similar shrinkage of the thymus and spleen was observed in both groups, but the shrinkage was less in the ADC1-C group than in the ADC1-DXD group.

[0532] The results of the two ADC groups in rat hematology (Table 6) showed that ADC1-C showed lower hematological toxicity in indicators such as 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.

[0533] 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.

[0534] Example 22: Pretoxicology and toxicokinetics of anti-ADAM9 antibody-drug conjugate in cynomolgus monkeys

[0535] 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.

[0536] method

[0537] 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:

[0538] (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.

[0539] (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.

[0540] (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.

[0541] (4) Weight: once during the adaptation period, once or twice a week during the drug administration period, and once before dissection.

[0542] (5) Electrocardiogram: Measured once during the adaptation period, and once 6h and 24h after the first and second administrations.

[0543] (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.

[0544] (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 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 19. 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.

[0545] (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 20. The half-lives for the first and third administrations were calculated using PKSolver to be 107 h and 174 h, respectively.

[0546] (13) Bone marrow cytology examination.

[0547] (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.

[0548] in conclusion

[0549] 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.

[0550] 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 7), and an increase in AST (Table 8), 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.).

[0551] Table 7: Results of hematological markers for pretoxicology in cynomolgus monkeys

[0552] Individual Animal Hematology by Parameter Report Hematology: Hematological Metrics Results and Individual Data

[0553] Table 8: Results of Blood Biochemical Indicators Detection in Pre-Toxicological Treatment of Crab-Eating Mammals

[0554] Individual Animal Biochemistry (Serum or Plasma) by Parameter Report Biochemistry (Serum or Plasma) Results of Blood Biochemistry Indicators: Individual Data

[0555] 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.

[0556] 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.

[0557] The half-lives for the first and third doses, calculated based on the total antibody blood concentration (Figure 19), were 217 h and 269 h, respectively. The half-lives for the first and third doses, calculated based on the small molecule toxin blood concentration after ADC administration (Figure 20), were 107 h and 174 h, respectively. This indicates that the ADAM-9-targeting antibody-drug conjugate of the present invention has a long half-life, suggesting that it can maintain a steady-state blood drug concentration, reduce the impact of concentration fluctuations on efficacy, prolong the dosing interval, and improve patient compliance.

[0558] 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

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 an anti-ADAM-9 antibody or antigen-binding fragment that binds to the target ADAM-9; 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 where M is attached to Ab, d wherein R 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 linked to L; L is -L1-L2-L3-L4-; wherein L1is selected from the group consisting of -(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)-; 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; L2 is 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 n6 is 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-Ci0aryl, 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 selected from the group consisting of hydrogen, Ci-C6alkyl; each of R1 and R3 is independently selected from the group consisting of hydrogen, C1-C6 alkyl; R2 is C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with a group selected from the group consisting of hydroxyl, thiol, carboxyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkylamino; 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 consisting 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. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, the Ab is selected from the group consisting of a monoclonal antibody, Fab, Fab', F(ab'), Fv, scFv, a single domain antibody. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, the Ab comprises a heavy chain variable region comprising the following 3 heavy chain complementarity determining regions: HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 6; HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 7; and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 8; the light chain variable region comprises the following 3 light chain complementarity determining regions: LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 3; LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 4; and LCDR3 having an amino acid sequence as set forth in SEQ ID NO:

5. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, the Ab has (1) a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 10; or (2) a heavy chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9, and / or a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 10; or (3) a heavy chain as set forth in SEQ ID NO: 1 and a light chain as set forth in SEQ ID NO:

2. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, 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 The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, said L1is a radical selected from the group consisting of -(CHR) p1 -C(O)-, -(CH2CH2O) q1 - -(CH2CH2O) p2 C(=O)-NH-(CH2CH2O) q2 (CHR) p3 C(=O)-, -(CHR) p2 C(=O)-NH-(CH2CH2O) q2 -; 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. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, L1is an optionally substituted structure selected from the group consisting of: The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, L2 is a peptide residue consisting of one or more amino acids from the group of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, glycine; Preferably, L2 is a peptide residue consisting of one or more amino acids from the group 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-). The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, L2 is selected from the group of structures: The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, 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: The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, L4 is absent, or is an optionally substituted structure selected from the group consisting of: The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, R1is selected from the group consisting of hydrogen, C1-C4alkyl; the carbon atom to which R1is attached can be in the R or S configuration; R2is C1-C4alkyl; R3is hydrogen, methyl, ethyl or n-propyl; the carbon atom to which R3is attached can be in the R or S configuration. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, The D has a structure shown in formula (Ia’): wherein X' is selected from the group consisting of -0-, -S-, -NR 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. The compound, pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt of claim 1, characterized in that, The compound of formula (III) is selected from the group consisting of: A compound of Formula (Ia), a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt of the compound, 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; Preferably, the compound of formula (I) is selected from the group consisting of: 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 and D are as defined in claim 1; Preferably, the compound of formula (II) is selected from the group consisting of: A process for the preparation of a compound of formula III, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 14, characterized in that, comprising the steps of: reacting a compound of formula II as defined in claim 16, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, with Ab as defined in any one of claims 1 to 4, to obtain a compound of formula III as defined in any one of claims 1 to 14, a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a compound of formula III as defined in any one of claims 1 to 14, a compound of formula la as defined in claim 15 or a compound of formula II as defined in claim 16, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier and / or excipient. A pharmaceutical formulation comprising a compound of formula III as defined in any one of claims 1 to 14, a compound of formula la as defined in claim 15 or a compound of formula II as defined in claim 16, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier and / or excipient. Use of a substance X for the manufacture of a medicament for the prevention or treatment of a disease associated with ADAM-9; wherein The substance X is a compound of formula III as defined in any one of claims 1 to 14, a compound of formula la as defined in claim 15 or a compound of formula II as defined in claim 16, or a pharmaceutically acceptable salt, stereoisomer, solvate or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 18, or a pharmaceutical formulation as defined in claim 19; Preferably, the cancer is a solid tumor or a non-solid tumor; Preferably, the cancer is a solid tumor or a non-solid tumor; More preferably, the cancer is selected from the group consisting of oesophageal cancer (e.g. oesophageal adenocarcinoma and oesophageal squamous cell carcinoma), brain tumour, 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 tumour (e.g. glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, thyroid cancer.

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