Anti-CLDN6 antibody, antibody-drug conjugate thereof, and medical use thereof

Abstract

Provided are an anti-CLDN6 antibody, an antibody-drug conjugate thereof, and a medical use thereof.

Description

Anti-CLDN6 antibodies, antibody-drug conjugates thereof and medical uses thereof

[0001] This application claims priority to the Chinese patent application filed on February 1, 2024 (application number: 2024101420189). Technical Field

[0002] The present disclosure belongs to the field of biotechnology and relates to anti-CLDN6 antibodies, antibody-drug conjugates thereof, and medical uses thereof. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Claudin (hereafter referred to as CLDN) is a tight junction protein. Human CLDN6 (hCLDN6), a member of the CLDN family, is a tetraspanin protein consisting of 220 amino acid residues. CLDN6 is not expressed in adult tissues but is expressed at high levels in various solid tumors, including testicular, ovarian, endometrial, and non-small cell lung cancers. CLDN9, to which it has the highest sequence similarity, is partially expressed in normal tissues.

[0005] Among the currently available CLDN6 antibody-drug conjugates (ADCs), two utilize the pyrrolobenzodiazepine (PBD) toxin: Abbive's SC-004 (WO2017096163A1) and Daiichi's DS9606a (WO2019065964A1, WO2020196474A1, WO2020196712A1). Both ADCs utilize bispecific monoclonal antibodies that simultaneously target CLDN6 and 9. SC-004 was discontinued in clinical trials due to high toxicity and low efficacy. Given the extremely low maximum tolerated dose (MTD) of SC-004, at only 0.2 mg / kg, it is impossible to rule out the possibility of CLDN9-mediated off-target toxicity at the effective dose. The antibody portion of another drug, DS9606a, has better cell binding activity, can better recognize the CLDN6 variant I143V, and has good endocytosis activity; its Fc has been silenced and the PBD has been attenuated, but its clinical safety is currently unknown.

[0006] When coupled with the more toxic PBD toxin, simultaneously targeting CLDN9 may bring the risk of off-tumor effects. Therefore, the development of antibodies and ADC drugs that specifically bind to CLDN6 is urgently needed in clinical practice. Summary of the Invention

[0007] The present disclosure relates to an anti-CLDN6 antibody comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 12, and LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 32, 31, 30, 33, 34 or 35.

[0008] In some embodiments, the anti-CLDN6 antibodies described above, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the same numbering convention selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, they are defined according to the Kabat numbering convention. In some embodiments, they are defined according to the IMGT numbering convention. In some embodiments, they are defined according to the Chothia numbering convention. In some embodiments, they are defined according to the AbM numbering convention. In some embodiments, they are defined according to the Contact numbering convention.

[0009] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, 25, 17, 27, 28 or 29, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

[0010] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26 or 25, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

[0011] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

[0012] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19.

[0013] In some embodiments, the anti-CLDN6 antibody as described in any of the preceding items is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; preferably, it is a humanized antibody.

[0014] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32, 31, 30, 33, 34, or 35.

[0015] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32 or 31.

[0016] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32.

[0017] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31.

[0018] In some embodiments, the anti-CLDN6 antibody as described in any of the preceding items, wherein the anti-CLDN6 antibody is an antibody fragment; preferably, the antibody fragment is Fab, Fab′, F(ab′)2, Fd, Fv, scFv, dsFv or dAb.

[0019] In some embodiments, the anti-CLDN6 antibody as described in any of the preceding items, wherein the anti-CLDN6 antibody comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain constant region is a light chain constant region of human κ, λ or a variant thereof; more preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 21.

[0020] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the anti-CLDN6 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 38, 37, 36, 39, 40 or 41.

[0021] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 38 or 37.

[0022] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 38.

[0023] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 37.

[0024] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 38.

[0025] In some embodiments, the anti-CLDN6 antibody of any of the preceding items, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 37.

[0026] In another aspect, the present disclosure relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is represented by formula (I):

[0027] wherein Ab is an anti-CLDN6 antibody as described in any of the preceding items;

[0028] L is a linker connecting Ab and D;

[0029] Ab binds to L directly from its amino acid, or Ab binds to L from its sugar chain or reconstructed sugar chain;

[0030] y is 1 to 10;

[0031] D is represented by the general formula (D1), formula (D1-S) or formula (D1-R):

[0032] in:

[0033] X is (CR a R b ) s ;

[0034] Y and Z are the same or different and are each independently selected from an oxygen atom, a sulfur atom and NR c ;

[0035] R 1 is selected from hydrogen, halogen and alkyl; or R 1 Together with the carbon atom to which it is attached, it forms C=O;

[0036] R 2 is a hydrogen atom or an alkyl group;

[0037] or R 1 and R 2 Together with the carbon and nitrogen atoms to which they are attached, they form C=N;

[0038] R 3 and R 4 The same or different, and each independently selected from hydrogen atom, alkyl, OR d , halogen, haloalkyl, hydroxyalkyl, SH, S-alkyl and NR e R f ;

[0039] R 5 is selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, and a haloalkyl group;

[0040] R 6 is selected from hydrogen, halogen, alkyl and haloalkyl; or R 6 Together with the carbon atom to which it is attached, it forms C=O;

[0041] R 7 and R 8 are the same or different and are each independently selected from hydrogen, halogen, alkyl and haloalkyl; or R 7 and R 8 Together with the carbon atom to which it is attached, it forms C=O; or R 7 and R 8 Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently selected from oxo, halogen, alkyl, haloalkyl, cyano, NR g R h and OR i is substituted by one or more substituents;

[0042] R 9 is a hydroxyl group or an alkoxy group;

[0043] R 10 is selected from the group consisting of a hydrogen atom, a hydroxyl group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, and an alkoxy group;

[0044] Ring A is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0045] R 11 Selected from hydrogen atom, halogen, alkyl, haloalkyl, OR j 、C(O)R k 、C(O)OR k , cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

[0046] R a and R b are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, cyano, amino and hydroxyalkyl;

[0047] R c 、R e 、R f 、R g and R h are the same or different and are each independently selected from a hydrogen atom, an alkyl group and a haloalkyl group;

[0048] R d 、R i and R j are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from an oxo group, a halogen group, an alkyl group, a haloalkyl group, a cyano group, an amino group, a hydroxyl group, an alkoxy group and a hydroxyalkyl group;

[0049] R k are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyl group, and a hydroxyalkyl group;

[0050] s is 1, 2, 3, 4, 5, or 6;

[0051] t is 1, 2, 3, 4, or 5.

[0052] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Y and Z are both oxygen atoms.

[0053] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 1 A hydrogen atom.

[0054] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 2 A hydrogen atom.

[0055] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ring A is a 6- to 10-membered aryl group; preferably, Ring A is a phenyl group.

[0056] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein D is represented by formula (D2), formula (D2-S) or formula (D2-R):

[0057] Among them, X, R 3 to R 11 and t are as defined in general formula (I).

[0058] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein X is (CR a R b ) s , R a and R b are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, hydroxyl and C 1-6 Hydroxyalkyl; preferably, X is (CH2) s ; where s is 1, 2, 3, 4, 5, or 6.

[0059] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R3 and R 4 Both OR d , R d Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, 3 to 6 membered cycloalkyl and 3 to 6 membered heterocyclic group, wherein the 3 to 6 membered cycloalkyl or 3 to 6 membered heterocyclic group is independently selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, cyano, amino, hydroxyl, C 1-6 Alkoxy and C 1-6 Preferably, R 3 and R 4 Both OR d , and R d C 1-6 alkyl or 3 to 6 membered cycloalkyl; more preferably, R 3 C 1-6 Alkoxy, and R 4 is a 3- to 6-membered cycloalkyloxy group; most preferably, R 3 is a methoxy group, and R 4 It is cyclopropyloxy.

[0060] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 5 A hydrogen atom.

[0061] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 6 A hydrogen atom.

[0062] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 7 and R 8 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl and C 1-6 haloalkyl; or R 7 and R 8 Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group are each independently optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 is substituted by one or more substituents selected from haloalkyl, hydroxyl and amino groups; preferably, R 7 and R 8Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl group, wherein the 3 to 6-membered cycloalkyl group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxy, C 1-6 More preferably, R 7 and R 8 Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group; most preferably, R 7 and R 8 Together with the carbon atom to which they are attached they form a cyclopropyl group.

[0063] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 9 It is a hydroxyl group.

[0064] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 10 Selected from hydrogen atoms, hydroxyl groups, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl and C 1-6 Alkoxy; preferably, R 10 A hydrogen atom.

[0065] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 11 Selected from hydrogen atoms, halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxyl and C 1-6 Alkoxy; preferably, R 11 A hydrogen atom or C 1-6 Alkoxy; more preferably, R 11 It is a methoxy group.

[0066] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein D is selected from the following structures:

[0067] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is -L a -L b -L c -L d -,

[0068] L a Selected from: The asterisk * indicates that b Combined, wavy lines Indicates binding to the sugar chain or reconstructed sugar chain of Ab;

[0069] L b Selected from -C(O)-(CR m R n -CR p R q ) t1 -C(O)-、-C(O)-(CR m R n -CR p R q ) t1 -C(O)-NR s -(CR m R n -CR p R q ) t2 -C(O)-, -C(O)-(CR m R n -CR p R q )t1-C(O)-NR s -(CR m R n -CR p R q -O) t2 -CR u R v -C(O)-, -C(O)-(CR m R n -CR p R q ) t1 -NR s -C(O)-(CR m R n -CR p R q -O) t2 -(CR m R n -CR p R q ) t3 -C(O)- and -(CR u R v ) t4 -OC(O)-;

[0070] L c is a peptide residue consisting of 2 to 7 amino acids;

[0071] L d -NR w -W-CR x Ry OC(O)-、-NR w -CR x R y OR z -C(O)- or chemical bond;

[0072] R m 、R n 、R p 、R q 、R u 、R v 、R x and R y are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino;

[0073] or R m and R n Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R p and R q Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R u and R v Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R x and R y Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0074] or R m and R p Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0075] R s 、R w and R z are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from an oxo group, a halogen group, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxyl group, a cyano group and an amino group;

[0076] W is an aryl or heteroaryl group, and the aryl or heteroaryl group is independently substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, haloalkyl and alkoxy; preferably, W is a phenyl group or a 5- or 6-membered heteroaryl group, and the phenyl group or the 5- or 6-membered heteroaryl group is independently substituted by one or more substituents selected from halogen, oxo, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 substituted by one or more substituents in the alkoxy group;

[0077] t1, t2, t3 and t4 are the same or different and are each independently 1, 2, 3, 4, 5 or 6.

[0078] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L b -C(O)-(CR m R n -CR p R q ) t1 -C(O)-, and R m 、R n 、R p and R q are the same or different and are each independently a hydrogen atom or a C 1-6 Alkyl, t1 is 1, 2, 3, 4, 5 or 6; preferably, L b It is -C(O)-CH2-CH2-C(O)-.

[0079] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L c Peptide residues formed by amino acids selected from phenylalanine (F), alanine (A), proline (P), isoleucine (I), leucine (L), glycine (G), valine (V), lysine (K), citrulline (Cit), serine (S), glutamic acid (E) and aspartic acid (D), wherein each of the peptide residues is independently optionally selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 alkoxy, 3 to 6 membered cycloalkyl, 3 to 6 membered heterocyclyl, 6 to 10 membered aryl and 5 to 10 membered heteroaryl; preferably, L cis selected from -GGVA-(SEQ ID NO: 45), -GG-(D-)VA-(SEQ ID NO: 46), -VA-, -GGFG-(SEQ ID NO: 44), -GGPI-(SEQ ID NO: 47), -GGVCit-(SEQ ID NO: 48), -GGVK-(SEQ ID NO: 49), -GG-(D-)PI-(SEQ ID NO: 50) and -GGPL-(SEQ ID NO: 51), wherein (D-)V represents D-valine, (D-)P represents D-proline; more preferably, L c is -GGVA- (SEQ ID NO: 45).

[0080] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L d -NR w -W-CR x R y OC(O)-,R w 、R x and R y The same or different, and each independently a hydrogen atom or a C 1-6 alkyl, and W is selected from 1,4-phenyl, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidinyl and 2,5-thienyl; preferably, L d is -NH-W-CH2-OC(O)-, and W is 1,4-phenyl.

[0081] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is

[0082] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is directly bound to L from its amino acid residue at position 297, or Ab is bound to L from its sugar chain or remodeled sugar chain at position 297; preferably, Ab is bound to L from its remodeled sugar chain at Asn297.

[0083] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the remodeled sugar chain structure is:

[0084] In the formula, the wavy line Indicates binding to Asn 297 of the Ab heavy chain;

[0085] P1 and P2 are the same or different and are each independently selected from hydroxyl, *-(CRp1 R q1 -CR s1 R t1 -O)s 1 - and *-(CR p1 R q1 -CR s1 R t1 -O)s 2 -(CR p1 R q1 -CR s1 R t1 -CR x1 R y1 -O)s 3 -(CR p1 R q1 -CR s1 R t1 -O)s 4 -, where R p1 、R q1 、R s1 、R t1 、R x1 and R y1 are the same or different and are each independently selected from hydrogen atom, halogen, C 1- 6 alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, hydroxyl, cyano, amino, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl, wherein the 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl are each independently selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 It is substituted by one or more substituents selected from hydroxyalkyl, hydroxyl, cyano and amino groups; the asterisk * indicates that it is bound to the linker L;

[0086] s 1 1-10, preferably 1-5; s 2 0-10, preferably 1-5; s 3 1-10, preferably 1-5; s 4 0-10, preferably 1-5;

[0087] The condition is that P1 and P2 are not hydroxyl or *-(CH2CH2O)s at the same time 1 -.

[0088] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the remodeled sugar chain structure is:

[0089] In the formula, the wavy line Indicates binding to Asn 297 of the Ab heavy chain; P1 is hydroxyl, P2 is *-(CR p1 R q1 -CR s1 R t1 -O) s2 -(CR p1 R q1 -CR s1 R t1 -CR x1 R y1 -O) s3 -(CR p1 R q1 -CR s1 R t1 -O) s4 -, where R p1 、R q1 、R s1 、R t1 、R x1 and R y1 are the same or different and are each independently selected from hydrogen atom, halogen, C 1- 6 alkyl and C 1-6 Halogenated alkyl, s2 is 1, s3 is 1, and s4 is 1.

[0090] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the remodeled sugar chain structure is:

[0091] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items is represented by the general formula (II):

[0092] in:

[0093] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0094] y is 1 to 10; preferably, y is 1 to 4; more preferably, y is 1 to 2; most preferably, y is 2.

[0095] In another aspect, the present disclosure relates to a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as represented by formula (II), comprising the following steps:

[0096] The compound represented by general formula (IIa) or its salt reacts with the compound represented by general formula (Ib) or its salt to obtain the antibody-drug conjugate represented by general formula (II) or its pharmaceutically acceptable salt;

[0097] in:

[0098] L' is L aa -L b -L c -L d -,

[0099] L aa Selected from: The asterisk * indicates that b Combine;

[0100] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0101] y is 1 to 10; preferably, y is 1 to 4; more preferably, y is 1 to 2; most preferably, y is 2;

[0102] L b , L c , L d The definition of is as defined in general formula (I);

[0103] R is as defined in formula (II);

[0104] D is as defined in the general formula (I).

[0105] In some embodiments, the present disclosure relates to a method for preparing an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0106] The compound represented by general formula (IIa) or a salt thereof reacts with compound LD-11 or a salt thereof to obtain an antibody-drug conjugate represented by general formula (II) or a pharmaceutically acceptable salt thereof;

[0107] in:

[0108] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0109] y is 1 to 10; preferably, y is 1 to 4; more preferably, y is 1 to 2; most preferably, y is 2;

[0110] R is as defined in the general formula (II).

[0111] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the average of any two values; preferably, y is 1-10; more preferably, y is 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 3-8, or 3-7, or 3-6, or 4-8, or 4-7, or 4-6, or the average of 4-5; most preferably, y is 2.

[0112] In another aspect, the present disclosure relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is represented by the general formula (IM):

[0113] wherein Ab is an anti-CLDN6 antibody as described in any of the preceding items;

[0114] M is selected from -O-(CR 12a R 12b ) m1 -CR 13a R 13b -C(O)-, -O-CR 13a R 13b -(CR 12a R 12b ) m1 -、-O-CR 13a R 13b -、-NH-(CR 12a R 12b ) m1 -CR 13a R 13b -C(O)- and -S-(CR 12a R 12b ) m1 -CR 13a R 13b -C(O)-;

[0115] R 12a and R 12b are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

[0116] Or, R 12a and R 12b Together with the carbon atom to which it is attached, it forms a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino;

[0117] R 13a is selected from the group consisting of halogen, alkyl, haloalkyl, hydroxy, hydroxyalkyl, alkoxy, cyano, amino, carboxyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl, wherein the cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0118] R 13b is selected from hydrogen, halogen, alkyl, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, cyano, amino, -(CR 19a R 19b ) m2 -NR 20a R 20b 、-(CR 19a R 19b ) m2 -COOH, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl, wherein the cycloalkyl, heterocyclyl, aryl and heteroaryl groups are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0119] Or, R 13a and R 13b Together with the carbon atoms to which they are attached, they form a cycloalkyl, heterocyclyl, aryl and heteroaryl group, wherein the cycloalkyl, heterocyclyl, aryl and heteroaryl group are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino groups;

[0120] Or, R 12a and R 13a Together with the carbon atom to which it is attached, it forms a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino;

[0121] R 19a and R 19b are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

[0122] Or, R 19a and R 19bTogether with the carbon atom to which it is attached, it forms a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino;

[0123] R 20a and R 20b are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from an oxo group, a halogen group, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxyl group, a cyano group and an amino group;

[0124] or R 20a and R 20b Together with the nitrogen atom to which they are attached, they form a heterocyclic group, wherein the heterocyclic group is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0125] m1 is 0, 1, 2, 3, or 4;

[0126] m2 is 0, 1, 2, 3, 4, 5 or 6;

[0127] n is 1 to 10;

[0128] L" is the joint unit.

[0129] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein M is -O-(CR 12a R 12b ) m1 -CR 13a R 13b -C(O)-;

[0130] Among them, R 12a and R 12b are the same or different and are each independently selected from hydrogen, halogen and C 1-6 alkyl;

[0131] R 13a is a 3- to 6-membered cycloalkyl-C 1-6 Alkyl or 3- to 6-membered cycloalkyl;

[0132] R 13b Selected from hydrogen atoms, C 1-6 Haloalkyl and 3- to 6-membered cycloalkyl;

[0133] Or, R 13a and R 13bTogether with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group;

[0134] m1 is 0, 1, 2, 3 or 4.

[0135] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein M is -O-(CR 12a R 12b ) m1 -CR 13a R 13b -C(O)-;

[0136] Among them, R 12a and R 12b are the same or different and are each independently a hydrogen atom or a C 1-6 alkyl;

[0137] R 13a is a 3- to 6-membered cycloalkyl group;

[0138] R 13b Selected from hydrogen atoms, C 1-6 Haloalkyl and 3- to 6-membered cycloalkyl;

[0139] Or, R 13a and R 13b Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group;

[0140] m1 is 0, 1, 2, 3 or 4.

[0141] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -L"- is -L 1 -L 2 -L 3 -L 4 -,

[0142] Where: L 1 Selected from -(succinimidyl-3-yl-N)-W 1 -C(O)-, -CH2-C(O)-NR 14 -W 1 -C(O)- and -C(O)-W 1 -C(O)-,W 1 Selected from C 1-6 Alkylene and C 1-6 Alkylene-3 to 6-membered cycloalkyl, wherein the C 1-6 Alkylene or C 1-6 Alkylene-3 to 6 membered cycloalkyl groups are each independently optionally selected from halogen, hydroxy, cyano, amino, alkyl, C 1-6 Halogenated alkyl, C 1-6substituted by one or more substituents selected from alkoxy and 3- to 6-membered cycloalkyl;

[0143] L 2 Selected from-NR 15 (CH2CH2O) p1 CH2CH2C(O)-、-NR 15 (CH2CH2O) p1 CH2C(O)-、-S(CH2) p1 C(O)- and chemical bonds, wherein p1 is an integer from 1 to 20;

[0144] L 3 The peptide residue is composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from the group consisting of phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and is optionally selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted by one or more substituents selected from alkoxy and 3- to 6-membered cycloalkyl;

[0145] L 4 Selected from-NR 16 (CR 17 R 18 ) q -、-C(O)NR 16 -、-C(O)NR 16 (CH2) q - and chemical bonds, wherein q is 1, 2, 3, 4, 5 or 6;

[0146] R 14 、R 15 and R 16 are the same or different and are each independently selected from hydrogen atom, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 hydroxyalkyl;

[0147] R 17 and R 18 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxy, C 1-6 Alkoxy, C 1-6 Haloalkoxy and C 1-6 Hydroxyalkyl.

[0148] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L 1 for where s 1 is 1, 2, 3, 4, 5 or 6; preferably, L 1 for where s 1 is 5.

[0149] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L 4 -NR 16 (CR 17 R 18 ) q -, where R 16 、R 17 and R 18 The same or different, and each independently a hydrogen atom or a C 1-6 alkyl, and q is 1 or 2; preferably, L 4 -NR 16 (CR 17 R 18 ) q -, where R 16 、R 17 and R 18 are all hydrogen atoms, and q is 1.

[0150] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -L"- is -L 1 -L 2 -L 3 -L 4 -,

[0151] L 1 for where s 1 is 1, 2, 3, 4, 5, or 6;

[0152] L 2 is a chemical bond;

[0153] L 3 is a tetrapeptide residue; preferably, L 3 is the tetrapeptide residue represented by GGFG (SEQ ID NO: 44);

[0154] L 4 -NR 16 (CR 17 R 18 ) q -, where R 16 、R 17 and R 18 The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl, q is 1 or 2;

[0155] wherein the -L"-L 1 The end is connected to Ab, L 4 Connect the Y end.

[0156] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 12a and R 12b are the same or different and are each independently a hydrogen atom or a C 1-6 Alkyl; preferably, R 12a and R 12b All are hydrogen atoms.

[0157] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by the general formula (IIM):

[0158] in,

[0159] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0160] m1 is 0, 1, 2, 3, or 4;

[0161] n is 1 to 10;

[0162] R 13a is a 3- to 6-membered cycloalkyl-C 1-6 Alkyl or 3- to 6-membered cycloalkyl;

[0163] R 13b Selected from hydrogen atoms, C 1-6 Haloalkyl and 3- to 6-membered cycloalkyl;

[0164] Or, R 13a and R 13b Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group;

[0165] W 1 Selected from C 1-6 Alkylene and C 1-6 Alkylene-3 to 6-membered cycloalkyl, wherein the C 1-6 Alkylene and C 1-6 Alkylene-3 to 6 membered cycloalkyl groups are each independently optionally selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted by one or more substituents of alkoxy and 3- to 6-membered cycloalkyl;

[0166] L 2 Selected from-NR15 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 15 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 From 1 to 20;

[0167] L 3 The peptide residue is composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from the group consisting of phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and is optionally selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1- 6 haloalkyl, C 1-6 substituted by one or more substituents selected from alkoxy and 3- to 6-membered cycloalkyl;

[0168] R 15 and R 16 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 hydroxyalkyl;

[0169] R 17 and R 18 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 Hydroxyalkyl.

[0170] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein R 13a is a 3- to 6-membered cycloalkyl group; R 13b is a hydrogen atom; or, R 13a and R 13b Together with the carbon atom to which it is attached, it forms a 3- to 6-membered cycloalkyl group.

[0171] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein m1 is 0 or 1; preferably, m1 is 0.

[0172] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein M is selected from: Preferably, M is (include ); wherein the O-end of M is connected to L".

[0173] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L 2 For chemical bonds.

[0174] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L 3 is a tetrapeptide residue; preferably, L 3 It is a tetrapeptide residue represented by GGFG (SEQ ID NO: 44).

[0175] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items is represented by Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S):

[0176] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0177] n is 1 to 10; preferably, n is 2 to 8; more preferably, n is 4 to 8; most preferably, n is 8.

[0178] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items is represented by the general formula (IIIM-R):

[0179] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0180] n is 1 to 10; preferably, n is 2 to 8; more preferably, n is 4 to 8; most preferably, n is 8.

[0181] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the average of any two values; preferably, n is 1-10; more preferably, n is 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 3-8, or 3-7, or 3-6, or 4-8, or 4-7, or 4-6, or the average of 4-5; most preferably, n is 8.

[0182] In another aspect, the present disclosure relates to a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as represented by the general formula (IIM), comprising the following steps:

[0183] After Ab is reduced, a coupling reaction is carried out with the compound represented by the general formula (IIMa) or a salt thereof to obtain an antibody-drug conjugate represented by the general formula (IIM) or a pharmaceutically acceptable salt thereof;

[0184] in:

[0185] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0186] W 1 , L 2 , L 3 、R 13a 、R 13b 、R 16 to R 18 , m1 and n are as defined in the general formula (IIM).

[0187] In some embodiments, the present disclosure relates to a method for preparing an antibody-drug conjugate of formula (IIIM-R) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0188] After Ab is reduced, a coupling reaction is carried out with compound 9-A or a salt thereof to obtain an antibody-drug conjugate represented by the general formula (IIIM-R) or a pharmaceutically acceptable salt thereof.

[0189] In some embodiments, the preparation method of the antibody-drug conjugate represented by the general formula (IIIM) or the general formula (IIIM-S) or a pharmaceutically acceptable salt thereof disclosed herein can refer to the preparation method of the antibody-drug conjugate represented by the general formula (IIIM-R) or a pharmaceutically acceptable salt thereof, and the corresponding chiral raw material is used to replace compound 9-A.

[0190] On the other hand, the present disclosure relates to a pharmaceutical composition comprising an anti-CLDN6 antibody as described in any of the preceding items, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0191] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising the anti-CLDN6 antibody as described in any of the preceding items and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0192] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0193] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of an anti-CLDN6 antibody, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof, based on the total weight of the pharmaceutical composition.

[0194] In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the anti-CLDN6 antibody as described in any of the preceding items, or the antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof.

[0195] In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of an anti-CLDN6 antibody, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof.

[0196] In some embodiments, the pharmaceutical composition contains 1%-99% of an anti-CLDN6 antibody, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof.

[0197] In some embodiments, the pharmaceutical composition contains 2%-98% of an anti-CLDN6 antibody, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof.

[0198] In some embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable diluent or excipient based on the total weight of the pharmaceutical composition.

[0199] In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable diluent or excipient.

[0200] In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable excipient.

[0201] In some embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable diluent or excipient.

[0202] In some embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable diluent or excipient.

[0203] In another aspect, the present disclosure relates to an isolated nucleic acid encoding an anti-CLDN6 antibody as described in any of the preceding items.

[0204] In another aspect, the disclosure relates to a host cell comprising the isolated nucleic acid as described above.

[0205] On the other hand, the present disclosure relates to a method for preventing or treating tumors, comprising administering to a subject an anti-CLDN6 antibody as described in any of the preceding items, an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R), or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

[0206] In some embodiments, the present disclosure relates to a use in preparing a medicament for preventing or treating tumors, comprising administering to a subject the anti-CLDN6 antibody or pharmaceutical composition as described in any of the preceding items.

[0207] In some embodiments, the present disclosure relates to a use in preparing a medicament for preventing or treating tumors, comprising administering to a subject an antibody-drug conjugate represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

[0208] In another aspect, the present disclosure relates to an anti-CLDN6 antibody as described in any of the preceding items, an antibody-drug conjugate as represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R), or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, for use as a medicament. In some embodiments, the medicament is used to prevent or treat tumors.

[0209] In some embodiments, the present disclosure relates to an anti-CLDN6 antibody or a pharmaceutical composition thereof for use as a medicament. In some embodiments, the medicament is used to prevent or treat tumors.

[0210] In some embodiments, the present disclosure relates to an antibody-drug conjugate as represented by Formula (I), Formula (II), Formula (IM), Formula (IIM), Formula (IIIM), Formula (IIIM-R), or Formula (IIIM-S), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, for use as a medicament. In some embodiments, the medicament is used to prevent or treat tumors.

[0211] The tumor described in the present disclosure is selected from ovarian cancer, lung cancer, endometrial cancer, gastric cancer, cervical cancer, testicular cancer, placental choriocarcinoma, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer and esophageal cancer; preferably, the tumor is selected from ovarian cancer, lung cancer, endometrial cancer and testicular cancer; more preferably, the tumor is ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0212] Figures 1 to 7 show the binding of each antibody to CLDN3 and CLDN4; Figure A shows the binding to CLDN3, and Figure B shows the binding to CLDN4.

[0213] FIG8A and FIG8B show the efficacy evaluation of ADC in the PA-1 in vivo model.

[0214] FIG8C shows the efficacy evaluation of ADC in the OVCAR3 in vivo model.

[0215] FIG8D shows the efficacy evaluation of ADC in the OV90 in vivo model. DETAILED DESCRIPTION

[0216] the term

[0217] In order to make the present disclosure more easily understood, certain technical and scientific terms are described below. Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0218] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0219] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0220] The term "and / or" is intended to include both "and" and "or". For example, the phrase "A, B and / or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0221] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0222] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0223] The term "amino acid mutation" includes amino acid substitutions (also known as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 82aR means that the amino acid residue at position 82a is R. S82aR means that the amino acid residue at position 82a (also known as 82A) has mutated from S to R.

[0224] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity.

[0225] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bispecific antibodies with different valencies, such as bispecific antibodies with different valencies. Based on whether the structure is symmetrical, they can be divided into bispecific antibodies with different symmetric structures and bispecific antibodies with different asymmetric structures. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight. The Fc fragment helps purify the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn to increase the antibody serum half-life.

[0226] "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by a heavy chain constant region, and the natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL). The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody structure or having a heavy chain with an Fc region as defined herein. A natural complete antibody light chain comprises a light chain variable region VL and a constant region CL, wherein VL is located at the amino terminus of the light chain, and the light chain constant region comprises a kappa chain and a lambda chain; a heavy chain comprises a variable region VH and constant regions (CH1, CH2, and CH3), wherein VH is located at the amino terminus of the heavy chain, and the constant region is at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0227] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain involved in antigen binding. Herein, the heavy chain variable region (VH) and light chain variable region (VL) of an antibody each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The term "complementarity determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. The VH comprises three CDR regions: HCDR1, HCDR2, and HCDR3; the VL comprises three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus), in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0228] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. Exemplary, as shown in Table 1 below.

[0229] Table 1. Relationships between CDR numbering systems

[0230] Unless otherwise specified, the variable regions and CDRs in this disclosure are numbered using the Kabat numbering convention.

[0231] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0232] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and reconstructed Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable Fc regions for antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, such as by deleting the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or by deleting the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise indicated, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0233] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0234] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework region portion of the variable region).

[0235] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably to refer to antibodies whose variable and constant regions are human sequences. The term encompasses antibodies that are derived from human genes but have altered sequences, for example, to reduce potential immunogenicity, increase affinity, or eliminate cysteines or glycosylation sites that may cause undesirable folding. The term encompasses antibodies that are recombinantly produced in non-human cells (which may confer glycosylation that is not characteristic of human cells). The term also encompasses antibodies that have been bred in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0236] The term "affinity" refers to the overall strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0237] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. For example, a biosensor system such as a system measuring surface plasmon resonance (e.g., Biacore) is used, or affinity in a solution is measured by solution equilibrium titration (SET).

[0238] The term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0239] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0240] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for the natural mutations that may be present in small amounts. In contrast, polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" represents the characteristic of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of an antibody by any ad hoc method. In some embodiments, the antibody provided by the present disclosure is a monoclonal antibody.

[0241] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody) and that can additionally be used in an animal to produce antibodies that can bind to the antigen. An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0242] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope can be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Conformational epitopes differ from linear epitopes in that antibody binding to a conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0243] The terms "capable of specific binding", "specific binding" or "binding" refer to an antibody that is able to bind to an antigen or an epitope of the antigen with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, for example, by However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0244] The term "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc gamma receptors (Fc gamma R) expressed on effector cells. For example, NK cells express Fc gamma RIIIa, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIIIa. The ADCC activity of the antibodies provided herein can be assessed using in vitro assays using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from the lysed cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins).

[0245] The term "antibody-dependent cellular phagocytosis (ADCP)" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.

[0246] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of a target-bound antibody binds to and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0247] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0248] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions when two sequences are optimally aligned, introducing gaps when necessary, to obtain maximum sequence identity percentage, and not considering any conservative substitutions as part of sequence identity. For measuring sequence identity percentage, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applicable to measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0249] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and contains a nucleic acid sequence for the expression of one or more heterologous coding regions that are operably connected thereto, including guidance and / or control (together with the host cell). Expression constructs can include but are not limited to affecting or controlling transcription, translation and affecting the sequence of RNA splicing of the coding region operably connected thereto when there are introns.

[0250] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia truncatula, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha, polymorpha), Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa.

[0251] "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cell are included.

[0252] The term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., C 1-20 The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 alkyl), more preferably an alkyl group having 1 to 6 carbon atoms (i.e., C 1-6 Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 ,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. The alkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0253] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups may be substituted or unsubstituted. When substituted, they may be substituted at any available point of attachment, with the substituent preferably being selected from one or more of a D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0254] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic all-carbon ring (i.e., a monocyclic cycloalkyl) or a polycyclic ring system (i.e., a polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered cycloalkyl). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3- to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3- to 8-membered cycloalkyl group), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered cycloalkyl group).

[0255] Non-limiting examples of the monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl and cyclooctyl.

[0256] The polycyclic cycloalkyl group includes: spirocycloalkyl group, fused cycloalkyl group and bridged cycloalkyl group.

[0257] The term "spiroalkyl" refers to a polycyclic ring system having a common carbon atom (called a spiro atom) between the rings, which may contain one or more double bonds within the ring, or one or more heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized to form nitrogen oxides; the sulfur may be optionally oxoed to form sulfoxides or sulfones, but does not include -OO-, -OS- or -SS-), provided that it contains at least one all-carbon ring and the point of attachment is on the all-carbon ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroalkyl). The spiroalkyl preferably has 6 to 14 ring atoms (i.e., a 6- to 14-membered spiroalkyl), and more preferably has 7 to 10 ring atoms (i.e., a 7- to 10-membered spiroalkyl). The spirocycloalkyl group includes a monospirocycloalkyl group and a polyspirocycloalkyl group (such as a bispirocycloalkyl group, etc.), preferably a monospirocycloalkyl group or a bispirocycloalkyl group, more preferably a 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered monospirocycloalkyl group. Non-limiting examples include:

[0258] Its connection point can be at any position;

[0259] wait.

[0260] The term "fused cycloalkyl" refers to a polycyclic ring system in which two adjacent carbon atoms are shared between the rings, which is a monocyclic cycloalkyl fused to one or more monocyclic cycloalkyls, or a monocyclic cycloalkyl fused to one or more heterocyclyls, aryls, or heteroaryls, wherein the point of attachment is on the monocyclic cycloalkyl, which may contain one or more double bonds within the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered fused cycloalkyl). The fused cycloalkyl is preferably a fused cycloalkyl having 6 to 14 ring atoms (i.e., a 6- to 14-membered fused cycloalkyl), more preferably a fused cycloalkyl having 7 to 10 ring atoms (i.e., a 7- to 10-membered fused cycloalkyl). The fused cycloalkyl group includes bicyclic fused cycloalkyl groups and polycyclic fused cycloalkyl groups (such as tricyclic fused cycloalkyl groups, tetracyclic fused cycloalkyl groups, etc.), preferably bicyclic fused cycloalkyl groups or tricyclic fused cycloalkyl groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered bicyclic fused cycloalkyl groups. Non-limiting examples include:

[0261] Its connection point can be at any position;

[0262] wait.

[0263] The term "bridged cycloalkyl" refers to a full carbon polycyclic ring system that shares two carbon atoms that are not directly connected between the rings, which may contain one or more double bonds within the ring and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., a 5 to 20-membered bridged cycloalkyl). The bridged cycloalkyl preferably has a bridged cycloalkyl of 6 to 14 carbon atoms (i.e., a 6 to 14-membered bridged cycloalkyl), more preferably a bridged cycloalkyl of 7 to 10 carbon atoms (i.e., a 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic bridged cycloalkyl or tricyclic bridged cycloalkyl. Non-limiting examples include:

[0264] Its connection point can be at any position.

[0265] The cycloalkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0266] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl) containing at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxoed, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-) in the ring, and having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered heterocyclyl). The heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclic group), for example, a 4- to 12-membered heterocyclic group containing at least one nitrogen atom; further preferably, a heterocyclic group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclic group); more preferably, a heterocyclic group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclic group); and most preferably, a heterocyclic group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclic group).

[0267] Non-limiting examples of the monocyclic heterocyclic group include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and homopiperazinyl.

[0268] The polycyclic heterocyclic group includes a spiro heterocyclic group, a fused heterocyclic group and a bridged heterocyclic group.

[0269] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic ring system in which the rings share one atom (called a spiro atom), which may contain one or more double bonds in the ring and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS- or -SS-), provided that it contains at least one monocyclic heterocyclic group and the point of attachment is on the monocyclic heterocyclic group, which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroheterocyclyl). The spiro heterocyclic radical preferably has a spiro heterocyclic radical (i.e., a 6 to 14 yuan spiro heterocyclic radical) of 6 to 14 ring atoms, more preferably a spiro heterocyclic radical (i.e., a 7 to 10 yuan spiro heterocyclic radical) with 7 to 10 ring atoms. The spiro heterocyclic radical includes monospiro heterocyclic radical and polyspiro heterocyclic radical (such as dispiro heterocyclic radical etc.), preferably monospiro heterocyclic radical or dispiro heterocyclic radical, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan monospiro heterocyclic radical. Non-limiting examples include:

[0270] wait.

[0271] The term "fused heterocyclyl" refers to a polycyclic heterocyclic ring system that shares two adjacent atoms between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), which is a monocyclic heterocyclyl fused to one or more monocyclic heterocyclyls, or a monocyclic heterocyclyl fused to one or more cycloalkyl, aryl, or heteroaryl groups, wherein the point of attachment is on the monocyclic heterocyclyl, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered fused heterocyclyl). The fused heterocyclic radical preferably has a fused heterocyclic radical of 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic radical), more preferably a fused heterocyclic radical of 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic radical). The fused heterocyclic radical includes bicyclic and polycyclic fused heterocyclic radicals (such as tricyclic fused heterocyclic radicals, tetracyclic fused heterocyclic radicals, etc.), preferably bicyclic fused heterocyclic radicals or tricyclic fused heterocyclic radicals, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan bicyclic fused heterocyclic radicals. Non-limiting examples include:

[0272] wait.

[0273] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic ring system that shares two atoms that are not directly connected between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered bridged heterocyclic group). The bridged heterocyclic group is preferably a bridged heterocyclic group having 6 to 14 ring atoms (i.e., a 6- to 14-membered bridged heterocyclic group), and more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e., a 7- to 10-membered bridged heterocyclic group). According to the number of constituent rings, heterocyclic groups can be divided into bicyclic bridged heterocyclic groups and polycyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), preferably bicyclic bridged heterocyclic groups or tricyclic bridged heterocyclic groups. Non-limiting examples include:

[0274] wait.

[0275] The heterocyclic group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclic group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group.

[0276] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., a polycyclic aromatic group) having a conjugated π electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 6- to 14-membered aromatic group). The aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aromatic group). The monocyclic aromatic group is, for example, a phenyl group. Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthrenyl, etc. The polycyclic aromatic group also includes a phenyl group fused with one or more heterocyclic groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclic groups or cycloalkyl groups, wherein the connection point is on the phenyl group or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples include:

[0277] wait.

[0278] The aryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0279] The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated π electron system, which contains at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS- or -SS-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl group), more preferably a monocyclic heteroaryl group having 5 or 6 ring atoms (i.e., a 5- or 6-membered monocyclic heteroaryl group) or a bicyclic heteroaryl group having 8 to 10 ring atoms (i.e., an 8- to 10-membered bicyclic heteroaryl group), and most preferably a 5- or 6-membered monocyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, or an 8- to 10-membered bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring.

[0280] The monocyclic heteroaryl groups include, but are not limited to, furyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidinyl, pyridonyl, N-alkylpyridone (e.g. etc.), pyrazinyl, pyridazinyl, etc.

[0281] The polycyclic heteroaryl groups include, but are not limited to, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophenyl, quinazolinyl, benzothiazolyl, carbazolyl, and the like. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more aromatic groups, wherein the point of attachment is on the aromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more cycloalkyl or heterocyclic groups, wherein the point of attachment is on the monocyclic heteroaromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. Non-limiting examples include:

[0282] wait.

[0283] The heteroaryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0284] The above-mentioned cycloalkyl, heterocyclyl, aryl and heteroaryl groups include residues derived from a parent ring atom by removing one hydrogen atom, or residues derived from the same parent ring atom or two different ring atoms by removing two hydrogen atoms, i.e., "divalent cycloalkyl", "divalent heterocyclyl", "arylene" and "heteroarylene".

[0285] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations.

[0286] The compounds of the present disclosure include all suitable isotopic derivatives of the compounds thereof. The term "isotopic derivative" refers to a compound in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass. Examples of isotopes that can be introduced into the compounds of the present disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, 2 H (deuterium, D), 3 H (tritium, T), 11 C. 13 C. 14 C. 15 N. 17 O. 18 O. 32 p、 33 p、 33 S. 34 S. 35 S. 36 S. 18 F. 36 Cl, 82 Br, 123 I. 124 I. 125 I. 129 I and 131 I, etc., preferably deuterium.

[0287] Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are encompassed by the present disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom, where the deuterium replacement can be partial or complete. Partial deuterium replacement refers to the replacement of at least one hydrogen atom with at least one deuterium atom.

[0288] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0289] The term "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates described herein or pharmaceutically acceptable salts thereof and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0290] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0291] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. In certain embodiments, the individual or subject is a human.

[0292] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0293] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0294] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions that attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0295] "Effective amount" is generally enough to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the damage caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is enough to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. Complete treatment or prevention may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0296] "Pharmaceutically acceptable salts" refer to salts formed by the reaction of a pharmaceutically active ingredient (such as a cytotoxic agent) with an appropriate acid or base. These salts maintain the pharmacological activity of the pharmaceutically active ingredient while also having better physicochemical properties, such as improving the solubility, stability, and bioavailability of the drug. After being ingested by a subject, this salt can release the active pharmaceutical ingredient under physiological conditions and exert a therapeutic effect. Examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, hydrochlorides, sulfates, phosphates, acetates, citrates, tartrates, and maleates. "Reconstructed sugar chains" refer to sugar chains obtained by modifying or remodeling the original sugar chain structure on a biological molecule (such as an antibody). In vivo, antibodies undergo glycosylation modifications, and these sugar chain structures play a vital role in protein folding, stability, localization, activity, and interaction with other molecules. Reconstructed sugar chains are modified by chemical or biotechnological means to impart new properties to the original sugar chain structure.

[0297] Exemplary antibody-drug conjugates or pharmaceutically acceptable salts thereof

[0298] Embodiments of the present disclosure relate to an anti-CLDN6 antibody comprising a heavy chain variable region and a light chain variable region, wherein HCDR1 of the heavy chain variable region has an amino acid sequence of SEQ ID NO: 14, HCDR2 has an amino acid sequence of SEQ ID NO: 15, and HCDR3 has an amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region has an amino acid sequence of SEQ ID NO: 26, LCDR2 has an amino acid sequence of SEQ ID NO: 24, and LCDR3 has an amino acid sequence of SEQ ID NO: 19.

[0299] In some embodiments, the anti-CLDN6 antibody as described above, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32.

[0300] In some embodiments, the anti-CLDN6 antibody as described above, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 38.

[0301] On the other hand, embodiments of the present disclosure relate to an anti-CLDN6 antibody comprising a heavy chain variable region and a light chain variable region, wherein HCDR1 of the heavy chain variable region has an amino acid sequence of SEQ ID NO: 14, HCDR2 has an amino acid sequence of SEQ ID NO: 15, and HCDR3 has an amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region has an amino acid sequence of SEQ ID NO: 25, LCDR2 has an amino acid sequence of SEQ ID NO: 24, and LCDR3 has an amino acid sequence of SEQ ID NO: 19.

[0302] In some embodiments, the anti-CLDN6 antibody as described above, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 31.

[0303] In some embodiments, the anti-CLDN6 antibody as described above, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 37.

[0304] In another aspect, the present disclosure relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is represented by the general formula (II):

[0305] in:

[0306] Ab is the anti-CLDN6 antibody as described in any of the preceding items;

[0307] y is 1 to 2; preferably, y is 2.

[0308] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof is represented by the general formula (II):

[0309] in:

[0310] the HCDR1 of the heavy chain variable region of Ab has the amino acid sequence of SEQ ID NO: 14, the HCDR2 has the amino acid sequence of SEQ ID NO: 15, and the HCDR3 has the amino acid sequence of SEQ ID NO: 16, and the LCDR1 of the light chain variable region of Ab has the amino acid sequence of SEQ ID NO: 26, the LCDR2 has the amino acid sequence of SEQ ID NO: 24, and the LCDR3 has the amino acid sequence of SEQ ID NO: 19;

[0311] Preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of Ab is as shown in SEQ ID NO: 32;

[0312] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 38;

[0313] And y is 2.

[0314] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items is represented by the general formula (II):

[0315] in:

[0316] HCDR1 of the heavy chain variable region of Ab has the amino acid sequence of SEQ ID NO: 14, HCDR2 has the amino acid sequence of SEQ ID NO: 15, and HCDR3 has the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region of Ab has the amino acid sequence of SEQ ID NO: 25, LCDR2 has the amino acid sequence of SEQ ID NO: 24, and LCDR3 has the amino acid sequence of SEQ ID NO: 19;

[0317] Preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of Ab is as shown in SEQ ID NO: 31;

[0318] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 37;

[0319] And y is 2.

[0320] In another aspect, the present disclosure relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is represented by Formula (IIIM), Formula (IIIM-R) or Formula (IIIM-S):

[0321] wherein Ab is an anti-CLDN6 antibody as described in any of the preceding items;

[0322] n is 4 to 8; preferably, n is 8.

[0323] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above is represented by the general formula (IIIM-R):

[0324] wherein Ab is an anti-CLDN6 antibody as described in any of the preceding items;

[0325] n is 4 to 8; preferably, n is 8.

[0326] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above is represented by the general formula (IIIM-R):

[0327] in:

[0328] the HCDR1 of the heavy chain variable region of Ab has the amino acid sequence of SEQ ID NO: 14, the HCDR2 has the amino acid sequence of SEQ ID NO: 15, and the HCDR3 has the amino acid sequence of SEQ ID NO: 16, and the LCDR1 of the light chain variable region of Ab has the amino acid sequence of SEQ ID NO: 26, the LCDR2 has the amino acid sequence of SEQ ID NO: 24, and the LCDR3 has the amino acid sequence of SEQ ID NO: 19;

[0329] Preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of Ab is as shown in SEQ ID NO: 32;

[0330] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 38;

[0331] And n is 8.

[0332] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above is represented by the general formula (IIIM-R):

[0333] in:

[0334] HCDR1 of the heavy chain variable region of Ab has the amino acid sequence of SEQ ID NO: 14, HCDR2 has the amino acid sequence of SEQ ID NO: 15, and HCDR3 has the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region of Ab has the amino acid sequence of SEQ ID NO: 25, LCDR2 has the amino acid sequence of SEQ ID NO: 24, and LCDR3 has the amino acid sequence of SEQ ID NO: 19;

[0335] Preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of Ab is as shown in SEQ ID NO: 31;

[0336] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 37;

[0337] And n is 8.

[0338] Example

[0339] The present disclosure is further described below with reference to the following examples and test cases, but these examples and test cases are not intended to limit the scope of the present disclosure. Experimental methods not specifying specific conditions in the examples or test cases of this disclosure generally followed conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents and materials not specifying their sources were purchased commercially.

[0340] 1. Antibody Preparation

[0341] Example 1: Preparation of CLDN6 Stable Transgenic Cell Line

[0342] CLDN6 and CLDN9 genes from different species, as well as human CLDN3 and CLDN4 genes, were transfected into human embryonic kidney 293T cells to construct cell lines expressing CLDN6 and CLDN9 from different species, as well as human CLDN3 and CLDN4, for subsequent antibody screening and identification. The amino acid sequences of the relevant proteins are as follows:

[0343] >Human CLDN6 full-length protein (Uniprot: P56747):

[0344] >Cynomolgus monkey CLDN6 full-length protein (Uniprot:G7Q0B0):

[0345] >Human CLDN9 full-length protein (Uniprot:O95484):

[0346] >Cynomolgus monkey CLDN9 full-length protein (Uniprot:A0A2K5UCC0):

[0347] >Human CLDN6 full-length protein variant (Uniprot: P56747 Variant p. Ile143 Val):

[0348] > Mouse CLDN6 full-length protein (Uniprot:Q9Z262):

[0349] > Mouse CLDN9 full-length protein (Uniprot:Q9Z0S7):

[0350] >Rat CLDN6 full-length protein (Uniprot: B4F7F0):

[0351] >Rat CLDN9 full-length protein (Uniprot:Q5PPJ3):

[0352] >Human CLDN3 full-length protein (Uniprot:O15551):

[0353] >Human CLDN4 full-length protein (Uniprot:O14493):

[0354] Note: The underlined part is the extracellular domain of the protein (hereinafter referred to as "ECD").

[0355] Construction of cell lines overexpressing CLDN6, CLDN9, CLDN3, and CLDN4

[0356] The pCDH lentiviral expression vector plasmid containing SEQ ID NOs: 1-11 (synthesized by GENEWIZ) and the pCDH plasmid were transfected with pVSVG and pCMV lentiviral packaging vectors using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17). The virus-containing culture supernatant was collected, filtered, and subjected to ultracentrifugation. The supernatant was discarded and resuspended in 0.2 mL of sterile PBS. The concentrated virus was used to infect 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17), selected with puromycin for two to three weeks, and then single-cell sorted by FACS. The selected monoclonal cell lines were expanded and frozen.

[0357] Note: The ECD sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are identical, the ECD sequences of SEQ ID NO: 3 and SEQ ID NO: 4 are identical, and the ECD sequences of SEQ ID NO: 7 and SEQ ID NO: 9 are identical. Therefore, it suffices to construct any one of these sequences; that is, binding to human CLDN6 is assumed to also bind to cynomolgus monkey CLDN6, binding to human CLDN9 is assumed to also bind to cynomolgus monkey CLDN9, and binding to mouse CLDN9 is assumed to also bind to rat CLDN9.

[0358] Example 2: Preparation of anti-human CLDN6 monoclonal antibodies

[0359] The humanized heavy chain variable region sequence VH45 and light chain variable region sequence VL43 of CLDN6 were obtained from patent WO2021006328A1 and cloned into the pTT 5 vector plasmid containing the human IgG1 heavy chain constant region set forth in SEQ ID NO:20 and the kappa light chain constant region set forth in SEQ ID NO:21. HEK293 cells were then transfected to generate the anti-CLDN6 antibody Ab-1.

[0360] Table 2. CDR sequences of antibody Ab-1 Note: The amino acid residues of the CDRs of VH / VL are determined by the Kabat numbering system and are underlined, the same below.

[0361] >Human IgG1 (LALA) heavy chain constant region:

[0362] >Human kappa light chain constant region:

[0363] >Heavy chain of antibody Ab-1:

[0364] >Light chain of antibody Ab-1:

[0365] Example 3: Hotspot Removal and Modification of Anti-CLDN6 Monoclonal Antibodies

[0366] The hotspot was removed by performing point mutation on the CDR of VL43.

[0367] Table 3. CDR sequences after VL43 hotspot removal

[0368] The light chain variable region of the antibody was reconstructed using the CDRs in Table 3. The specific sequences are as follows:

[0369] Note: VL43+D56E represents the light chain variable region sequence obtained by mutating amino acid D at position 56 of VL43 to E; VL43+D56E+S31R represents the light chain variable region sequence obtained by mutating amino acid D at position 56 of VL43 to E and amino acid S at position 31 to R. The same applies to other sequences.

[0370] Single underlines indicate CDR regions, and double underlines indicate mutation sites.

[0371] SEQ ID NOs: 30-35 were cloned into the pTT 5 vector plasmid containing the kappa light chain constant region shown in SEQ ID NO: 21, and were cloned together with SEQ ID NO: 12 into the pTT 5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO: 20, and transfected together into HEK293 cells to obtain anti-CLDN6 antibodies Ab-2, Ab-3, Ab-4, Ab-5, Ab-6 and Ab-7.

[0372] >Heavy chain of antibodies Ab-2, Ab-3, Ab-4, Ab-5, Ab-6 and Ab-7: SEQ ID NO: 22, same as the heavy chain of antibody Ab-1.

[0373] >Light chain of antibody Ab-2:

[0374] >Light chain of antibody Ab-3:

[0375] >Light chain of antibody Ab-4:

[0376] >Light chain of antibody Ab-5:

[0377] >Light chain of antibody Ab-6:

[0378] >Light chain of antibody Ab-7:

[0379] The VH / VL sequences of the negative control antibody Isotype used in this disclosure are from patent US6114143A, and the heavy chain constant region and light chain constant region sequences are SEQ ID NO: 20 and SEQ ID NO: 21, respectively. The full-length sequences are as follows:

[0380] Isotype heavy chain:

[0381] Isotype light chain:

[0382] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0383] 2. Preparation of ADC

[0384] Analysis of drug loading in ADC stock solution

[0385] ADCs are antibody-drug conjugates that treat diseases by relying on the antibody's targeted properties to deliver the drug into cells, thereby killing or inhibiting cell growth. The drug loading plays a decisive role in its efficacy.

[0386] This disclosure uses RP-HPLC to analyze drug loading, and the process is basically as follows:

[0387] Reagents and instruments:

[0388] Trifluoroacetic acid (TFA): produced by Sigma, 100 mL / bottle; acetonitrile: LC grade, 4 L / bottle, produced by Thermo Fisher; DTT: produced by Sigma, 1 g / bottle.

[0389] High performance liquid chromatograph: Agilent 1200.

[0390] Solution preparation:

[0391] 1) 0.25M DTT solution:

[0392] Preparation example: Take 5.78 mg of DTT and add 150 μL of purified water to fully dissolve it to prepare 0.25 M DTT solution. Store at -20°C.

[0393] 2) Mobile phase A (0.1% TFA aqueous solution):

[0394] Preparation example: Measure 1000 mL of purified water into a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0395] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0396] Preparation example: Measure 1000 mL of acetonitrile into a measuring cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0397] Naked antibody and test sample (concentration 1 mg / mL, about 200 μL) were reduced by adding 4 μL DTT and incubated in a 37°C water bath for 1 hour. After reduction, the samples were removed from the tube and used for injection.

[0398] Chromatographic conditions:

[0399] Chromatographic column: Agilent PLRP-S1000A 8μm 4.6*250mm; column temperature: 80℃;

[0400] DAD detector: detection wavelength 280 nm; sample chamber temperature: 4°C; flow rate: 1 mL / min;

[0401] Injection volume: 40 μL;

[0402] The chromatographic gradient is shown in Table 4:

[0403] Table 4

[0404] Data Analysis:

[0405] By comparing the spectra of the sample and naked antibody, the positions of the light and heavy chains are distinguished, and then the spectrum of the test sample is integrated to calculate the DAR value. The calculation formula is as follows:

[0406] Table 5

[0407] Sum of LC peak areas = LC peak area + LC+1 peak area;

[0408] The sum of HC peak areas = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area;

[0409] LC DAR = Σ(number of linked drugs * peak area percentage) / total LC peak area;

[0410] HC DAR = Σ(number of connected drugs * peak area percentage) / sum of HC peak areas;

[0411] DAR=LC DAR+HC DAR.

[0412] Example 4-1 Synthesis of Sugar Chain OLS-4

[0413] (2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-2-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-6-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,5-diol OLS-4

[0414] first step

[0415] 2-((4-methoxybenzyl)oxy)ethan-1-ol OLS-4b

[0416] Ethylene glycol OLS-4a (50.00 g, 805.57 mmol) was dissolved in anhydrous tetrahydrofuran (300 mL), and sodium hydroxide (4.93 g, 128.89 mmol, content 60%) and tetrabutylammonium iodide (5.37 g, 16.11 mmol) were added in sequence under nitrogen protection. The mixture was stirred at 0°C for 30 minutes, and then p-methoxybenzyl chloride (20.19 g, 128.89 mmol) was slowly added dropwise. The addition was completed in about 30 minutes, and the mixture was heated to 80°C and stirred for 1 minute. After 6 hours, the reaction solution was poured into ice water with stirring to quench, and ethyl acetate (500 mL) was added with stirring. The layers were separated, and the obtained aqueous phase was extracted with ethyl acetate (150 mL×3). The organic phases were combined, and the organic phases were washed with water (250 mL×3) and saturated sodium chloride solution (250 mL×2), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with eluent system C to give the title product OLS-4b (22.00 g, yield: 14.9%).

[0417] MS m / z(ESI):205.2[M+23].

[0418] Step 2

[0419] 2-((4-methoxybenzyl)oxy)ethyl 4-methylbenzenesulfonate OLS-4c

[0420] OLS-4b (22.00 g, 120.74 mmol) was dissolved in pyridine (98 mL). p-Toluenesulfonyl chloride (27.62 g, 144.88 mmol) was added at 0°C under nitrogen. The reaction was stirred at 0°C and slowly allowed to warm to room temperature for 16 hours. Ethyl acetate (300 mL) was added to dilute the reaction solution and washed with hydrochloric acid (10%, 150 mL × 3). The organic phase was washed sequentially with water (150 mL × 3) and saturated sodium chloride solution (250 mL × 2), dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4c (36.40 g, 89.6% yield).

[0421] 1H NMR(400MHz, CDCl3)δ7.81(d,2H),7.33(d,2H),7.21(d,2H),6.88(d,2H),4 .43(s,2H),4.23-4.17(m,2H),3.82(s,3H),3.68-3.61(m,2H),2.45(s,3H).

[0422] Step 3

[0423] 3-(2-((4-methoxybenzyl)oxy)ethoxy)propan-1-ol OLS-4d

[0424] OLS-4c (36.40 g, 108.20 mmol) and 1,3-propylene glycol (82.34 g, 1.08 mol) were dissolved in toluene (100 mL). Powdered potassium hydroxide (15.18 g, 270.51 mmol) was added at room temperature. The mixture was heated to 50°C under nitrogen and stirred for 72 hours. The reaction mixture was poured into ice water (200 mL). Concentrated hydrochloric acid was added dropwise until the pH reached 3-4. Ethyl acetate (300 mL) was added for extraction. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (100 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate for 30 minutes. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4d (15.60 g, 60.0% yield).

[0425] 1H NMR(400MHz, CDCl3)δ7.28(d,2H),6.89(d,2H),4.51(s,2H),3.81(s,3H),3.7 9(t,2H),3.68(t,2H),3.65-3.59(m,4H),2.5i1(br.s,1H),1.88-1.82(m,2H).

[0426] Step 4

[0427] 3-(2-((4-methoxybenzyl)oxy)ethoxy)propyl 4-methylbenzenesulfonate OLS-4e

[0428] OLS-4d (15.60 g, 64.92 mmol) was dissolved in a mixture of pyridine (52 mL) and dichloromethane (52 mL). p-Toluenesulfonyl chloride (18.56 g, 97.38 mmol) was added at 0°C. Under nitrogen, the reaction was stirred at 0°C and slowly returned to room temperature for 16 hours. Ethyl acetate (300 mL) was added to dilute the reaction solution, which was then washed sequentially with hydrochloric acid (10%, 150 mL × 3), water (150 mL × 3), and saturated sodium chloride solution (250 mL × 2). The solution was dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4e (18.00 g, 70.3% yield).

[0429] MS m / z(ESI):417.3[M+23].

[0430] 1H NMR(400MHz, CDCl3)δ7.80(d,2H),7.34(d,2H),7.27(d,2H),6.90(d,2H),4.48(s, 2H),4.16(t,2H),3.83(s,3H),3.53-3.49(m,6H),2.45(s,3H),1.96-1.91(m,2H).

[0431] Step 5

[0432] 14-Azido-1-(4-methoxyphenyl)-2,5,9,12-tetraoxatetradecane OLS-4f

[0433] OLS-4e (8.00 g, 20.28 mmol) and azide-diethylene glycol (3.72 g, 28.39 mol, AIMATE) were dissolved in toluene (33 mL). Powdered potassium hydroxide (2.84 g, 50.70 mmol) was added at room temperature. The mixture was heated to 60°C under nitrogen and stirred for 16 hours. The reaction mixture was poured into ice water (100 mL). Concentrated hydrochloric acid was added dropwise until the pH reached 3-4. Ethyl acetate (100 mL) was added for extraction. The mixture was separated and the aqueous phase was extracted with ethyl acetate (50 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate for 30 minutes. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4f (6.3 g, 87.9% yield).

[0434] MS m / z(ESI):376.3[M+23].

[0435] 1H NMR (400MHz, CDCl3) δ7.29(d,2H),6.89(d,2H),4.52(s,2H),3.82(s,3H),3.70–3.56(m,14H),3.40(t,2H),1.93-1.87(m,2H).

[0436] Step 6

[0437] 2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethan-1-ol OLS-4g

[0438] OLS-4f (6.30 g, 17.83 mmol) was dissolved in dichloromethane (31 mL) and water (3.1 mL). Dichlorodicyanobenzoquinone (4.45 g, 19.61 mmol, Aladdin) was added at room temperature under nitrogen and stirred at 25°C for 1.5 hours. The reaction solution was filtered through celite, and the filtrate was diluted with dichloromethane (150 mL) and washed sequentially with saturated sodium bicarbonate solution (100 mL × 2), saturated sodium bisulfite solution (100 mL × 2), water (100 mL), and saturated sodium chloride solution (100 mL × 2). The product was dried over anhydrous sodium sulfate for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4g (3.2 g, 76.9% yield).

[0439] Step 7

[0440] 2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethyl 4-methylbenzenesulfonate OLS-4h

[0441] OLS-4g (3.20 g, 13.72 mmol) was dissolved in pyridine (11 mL) and dichloromethane (11 mL), cooled to 0°C, and p-toluenesulfonyl chloride (3.14 g, 16.46 mmol) was added. The reaction was stirred at 0°C under nitrogen and slowly returned to room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (200 mL) and washed sequentially with hydrochloric acid (10%, 150 mL × 3), water (150 mL × 3), and saturated sodium chloride solution (250 mL × 2). The product was dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4h (4.80 g, 90.3% yield).

[0442] 1H NMR (400MHz, CDCl3) δ7.79(d,2H),7.34(d,2H),4.14(t,2H),3.67-3.57(m,8H),3.51-3.46(m,4H),3.38(t,2H),2.44(s,3H),1.81-1.75(m,2H).

[0443] Step 8

[0444] N-((2R,3R,4R,5S,6R)-5-(((2R,4aR,6S,7S,8S,8aR)-8-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-7-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxan-6-yl)oxy)-2,4-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4i

[0445] OLS-3c (340 mg, 0.41 mmol), N,N-dimethylformamide (4.8 mL), and 2 mL of a solution of OLS-4h (398 mg, 1.03 mmol) in N,N-dimethylformamide were added sequentially to a reaction flask and cooled to 0°C. Sodium hydride (86 mg, 2.15 mmol, 60% content) was added and the mixture was allowed to react at 0°C for approximately 0.5 hour, then warmed to 20-30°C and reacted for approximately 0.5 hour. The reaction solution was poured into a mixture of saturated ammonium chloride solution (10 mL) and water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed sequentially with water (10 mL) and saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent System C to obtain the title product, OLS-4i (362 mg, 84.6% yield).

[0446] MS m / z(ESI):1047.4[M+1].

[0447] 1H NMR(400MHz, CDCl3)δ7.40-7.16(m,25H),5.70(d,1H),5.44(s,1H),4.87(d,1H) ),4.84-4.78(m,3H),4.71-4.68(m,1H),4.57-4.50(m,4H),4.41-4.38(m,1H),4 .04-4.01(m,1H),3.97(t,1H),3.93-3.84(m,2H),3.76-3.68(m,3H),3.61-3.40 (m,16H),3.34-3.27(m,3H),3.11-3.05(m,2H),1.78-1.72(m,2H),1.51(s,3H).

[0448] Step 9

[0449] N-((2R,3R,4R,5S,6R)-5-(((2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-3-(benzyloxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,4-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4j

[0450] Compound OLS-4i (360 mg, 0.34 mmol) and dichloromethane (26 mL) were added sequentially to a reaction flask, cooled to -30 to -20°C under nitrogen, and trifluoroacetic acid (3.6 mL) was added dropwise. After the addition was complete, the temperature was raised to -5 to 5°C and stirred for approximately 1.5 hours. Methanol (5.1 mL) was added to quench the reaction, which was then diluted with dichloromethane (12.3 mL). The product was washed sequentially with saturated sodium bicarbonate solution (26 mL x 2) and saturated sodium chloride solution (13 mL), dried over anhydrous sodium sulfate, and filtered to remove the desiccant. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin-layer chromatography using developing solvent System C to afford the title product, OLS-4j (234 mg, 71.0% yield).

[0451] MS m / z(ESI):959.7[M+1].

[0452] 1H NMR (400MHz, CDCl3) δ7.31-7.17(m,20H),5.64(d,1H),4.87(d,1H),4.84-4.79(m,2H),4.70(s,2H),4.58-4.50(m,3H),4.43-4.39(m,2H),4.0 5(t,1H),3.82(t,1H),3.74-3.36(m,23H),3.30(t,2H),3.10-3.07(m,1 H),3.00-2.97(m,1H),2.21(br.s,1H),1.81-1.75(m,2H),1.66(s,3H).

[0453] Step 10

[0454] N-((3R,4R,5S,6R)-5-(((2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4k (diastereoisomer mixture)

[0455] OLS-4j (150 mg, 158.72 μmol) was dissolved in tetrahydrofuran (8 mL) and water (2 mL), and palladium hydroxide (50.0 mg, 15% content), palladium on carbon (61 mg, 10% content), and hydrochloric acid (1 drop) were added. The atmosphere was replaced with hydrogen three times, and the reaction was stirred at room temperature for 12 hours. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in methanol (7 mL), and imidazole-1-sulfonyl azide hydrochloride (6 mg, 220.41 μmol), potassium carbonate (68 mg, 492.00 μmol), and copper sulfate (5.7 mg, 22.83 μmol) were added. The reaction was stirred at room temperature for 12 hours. Inorganic salts were removed by filtration, and the solid was rinsed with methanol (3 mL). The organic phases were combined and the filtrate was concentrated under reduced pressure to obtain the crude title product OLS-4k (161 mg). The product was directly used in the next reaction without purification.

[0456] MS m / z(ESI):599.3[M+1].

[0457] Step 11

[0458] (2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-2-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-6-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,5-diol OLS-4

[0459] Crude compound OLS-4k (161 mg, 148.01 μmol) was dissolved in pure water (8 mL) under ice, and 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride (557 mg, 3.07 mmol) and cesium carbonate (3.00 g, 9.20 mmol) were added. The mixture was stirred at 0°C for 12 hours. The reaction mixture was purified by HPLC (Phenomenex C18 column, 150 x 25 mm, 10 μm; mobile phase: water (0.01% ammonia) and acetonitrile, gradient: acetonitrile 0% to 30%, flow rate: 25 mL / min) and lyophilized from 5 mol% sodium hydroxide solution to afford the title product, OLS-4 (35 mg, 38.2% total yield for the tenth and eleventh steps).

[0460] MS m / z(ESI):581.3[M+1].

[0461] 1H NMR(400MHz, CDCl3)δ6.02(d,1H),4.65(s,2H),4.30(s,2H),4.09-3.93(m,5H),3.86 -3.77(m,4H),3.73-3.51(m,16H),3.40-3.28(m,5H),2.01(s,3H),1.88-1.81(m,2H).

[0462] Example 4-2 Synthesis of ADC-1, ADC-2, and ADC-3

[0463] The following reaction formula is applicable to the preparation of ADC-1, ADC-2, and ADC-3:

[0464] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains two structures shown in R.)

[0465] Among them, the linker-toxin in the third step reaction adopts compound LD-11 (prepared by the method disclosed in Example 3-11 of the patent application "WO2023051814A1, pages 142-146"), and the specific structure is shown below:

[0466] Example 4-2-1 ADC-1

[0467] first step

[0468] Endo S enzyme (19.16 mg / mL, 10.4 μL) was added to a PBS buffer solution of antibody Ab-3 (0.05 M PBS buffer solution, pH 6.3; 10 mg / mL, 2 mL) at 37°C. The mixture was shaken in a water bath at 37°C for 12 hours, after which the reaction was stopped. The reaction solution was purified using a Protein A purification column (elution phase: acetic acid buffer solution, pH 3.0) to obtain affinity eluate a-1. The solution was exchanged to 0.05 M PBS buffer solution, pH 6.3, and stored refrigerated at 4°C.

[0469] Step 2

[0470] At 37°C, Endo S enzyme (19.16 mg / mL, 10.4 μL) and OLS-4 (2.32 mg) were added to a-1 in PBS buffer (0.05 M PBS buffer at pH 6.3; 10.0 mg / mL, 2 mL). The mixture was placed in a water bath shaker and shaken at 37°C for 1 hour to stop the reaction. The reaction solution was purified using a Protein A protein purification column (elution phase: acetate buffer at pH 3.0) to obtain affinity eluate b-1. The solution was exchanged with PBS buffer at pH 7.4 and stored refrigerated at 4°C. y1 represents the average number of sugar chains remodeled to position N297 of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0471] Step 3

[0472] At 25°C, a PBS buffer solution of b-1 (pH = 7.4 PBS buffer; 8.0 mg / mL, 1.0 mL) was added, followed by the addition of 0.8 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of a solution of LD-11 (0.388 mg, 266 nmol, dissolved in a mixture of 100 μL of dimethyl sulfoxide and 100 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 15 mL of pH = 7.4 PBS buffer. The supernatant was then purified using a Protein A column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to a pH of approximately 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, ADC-1, which was stored refrigerated at 4°C.

[0473] The average number of drug binding per antibody molecule was calculated by MS: y = 1.7.

[0474] Example 4-2-2 ADC-2

[0475] first step

[0476] Endo S enzyme (19.16 mg / mL, 15.6 μL) was added to a PBS buffer solution of antibody Ab-4 (0.05 M PBS buffer solution, pH 6.3; 10 mg / mL, 3 mL) at 37°C. The mixture was shaken in a water bath at 37°C for 12 hours, after which the reaction was stopped. The reaction solution was purified using a Protein A purification column (elution phase: acetic acid buffer solution, pH 3.0) to obtain affinity eluate a-2. The solution was exchanged to 0.05 M PBS buffer solution, pH 6.3, and stored refrigerated at 4°C.

[0477] Step 2

[0478] At 37°C, Endo S enzyme (19.16 mg / mL, 10.4 μL) and OLS-4 (2.32 mg) were added to a-2 PBS buffer solution (0.05 M PBS buffer solution at pH 6.3; 10.0 mg / mL, 2 mL). The solution was placed in a water bath shaker and shaken at 37°C for 1 hour to stop the reaction. The reaction solution was purified using a Protein A protein purification column (elution phase: acetate buffer solution at pH 3.0) to obtain affinity eluate b-2. The solution was exchanged to PBS buffer solution at pH 7.4 and stored refrigerated at 4°C. y1 represents the average number of sugar chains remodeled to position N297 of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0479] Step 3

[0480] At 25°C, a PBS buffer solution of b-2 (pH = 7.4 PBS buffer; 8.0 mg / mL, 1.0 mL) was added, followed by the addition of 0.8 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of a solution of LD-11 (0.388 mg, 266 nmol, dissolved in a mixture of 100 μL of dimethyl sulfoxide and 100 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 15 mL of pH = 7.4 PBS buffer. The supernatant was then purified using a Protein A column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to approximately pH 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, ADC-2, which was stored refrigerated at 4°C.

[0481] The average number of drug binding per antibody molecule was calculated by MS: y = 1.7.

[0482] Example 4-2-3 ADC-3

[0483] first step

[0484] Endo S enzyme (19.16 mg / mL, 16.8 μL) was added to the antibody isotype PBS buffer solution (0.05 M PBS buffer solution, pH 6.3; 10 mg / mL, 3.23 mL) at 37°C. The mixture was shaken in a water bath at 37°C for 12 hours, after which the reaction was stopped. The reaction solution was purified using a Protein A purification column (elution phase: acetic acid buffer solution, pH 3.0) to obtain the a-3 affinity eluate. The solution was exchanged to 0.05 M PBS buffer solution, pH 6.3, and stored refrigerated at 4°C.

[0485] Step 2

[0486] At 37°C, Endo S enzyme (19.16 mg / mL, 15.6 μL) and OLS-4 (3.48 mg) were added to a-3 PBS buffer solution (0.05 M PBS buffer solution at pH 6.3; 10.0 mg / mL, 3.0 mL). The solution was placed in a water bath shaker and shaken at 37°C for 1 hour to stop the reaction. The reaction solution was purified using a Protein A protein purification column (elution phase: acetate buffer solution at pH 3.0) to obtain the affinity eluate b-3. The solution was exchanged to PBS buffer solution at pH 7.4 and stored refrigerated at 4°C. y1 represents the average number of sugar chains remodeled to position N297 of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0487] Step 3

[0488] At 25°C, a PBS buffer solution of b-3 (pH = 7.4 PBS buffer; 8.0 mg / mL, 3.5 mL) was added, followed by the addition of 2.8 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of a solution of LD-11 (1.36 mg, 931 nmol, dissolved in a mixture of 350 μL of dimethyl sulfoxide and 350 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 30 mL of pH = 7.4 PBS buffer. The supernatant was then purified using a Protein A column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to approximately pH 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, ADC-3, which was stored refrigerated at 4°C.

[0489] The average drug binding number per antibody molecule was calculated by MS: y = 1.71.

[0490] Example 4-3 Synthesis of ADC-4, ADC-5, and ADC-6

[0491] The following reaction formula is applicable to the preparation of ADC-4, ADC-5, and ADC-6:

[0492] Among them, compound 9-A was prepared using the method disclosed in Example 9 of patent application "WO2020063676A1, pages 58-61 of the specification".

[0493] Example 4-3-1 ADC-4

[0494] At 37°C, a solution of antibody Ab-3 in PBS buffer (0.05 M PBS buffer, pH 6.3; 10.0 mg / mL, 2.0 mL, 133.3 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 73.3 μL, 733 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C and then exchanged with 30 mM histidine-acetate buffer, pH 5.0, using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0495] Compound 9-A (2.15 mg, 2.0 μmol) was dissolved in 100 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed on a thermostatic oscillator and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.01 M His buffer solution at pH 5.5, containing 8% sucrose) to obtain the title product ADC-4 in His buffer solution, which was then refrigerated and stored at 4°C.

[0496] RP-HPLC calculated average value: n=7.44.

[0497] Example 4-3-2 ADC-5

[0498] At 37°C, a solution of antibody Ab-4 in PBS buffer (0.05 M PBS buffer, pH 6.3; 10.0 mg / mL, 10.0 mL, 666.7 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 366.7 μL, 3667 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C and then exchanged with 30 mM histidine-acetate buffer, pH 5.0, using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0499] Compound 9-A (10.74 mg, 10.0 μmol) was dissolved in 500 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed on a thermostatic oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.01 M His buffer solution at pH 5.5, containing 8% sucrose) to obtain the title product ADC-5 in His buffer solution, which was stored refrigerated at 4°C.

[0500] RP-HPLC calculated average value: n=7.56.

[0501] Example 4-3-3 ADC-6

[0502] To an antibody isotype solution in PBS buffer (pH 6.3, 0.05 M PBS buffer; 10.0 mg / mL, 2.2 mL, 146.7 nmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 81.8 μL, 818 nmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C.

[0503] Compound 9-A (2.39 mg, 2.23 μmol) was dissolved in 110 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed on a thermostatic oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.01 M His buffer solution at pH 5.5, containing 8% sucrose) to obtain the title product ADC-6 in His buffer solution, which was stored refrigerated at 4°C.

[0504] RP-HPLC calculated average value: n=7.6.

[0505] Test Case

[0506] Test Example 1: FACS detection of antibody binding at the cellular level

[0507] CLDN6-expressing tumor cells PA-1 (human ovarian teratoma cells, ATCC, catalog number CRL-1572), OVCAR3 (human ovarian cancer cells, ATCC, HTB-161), OV90 (human ovarian cancer cells, Shanghai Zhongqiao Xinzhou, ZQ0073), and 293T / human CLDN9 cells were prepared into 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the test antibody, 100μL / well. Incubate in a 4℃ refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add the working concentration of APC anti-human IgG Fc (BioLegend, 410712) and incubate in a 4℃ refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to these cells. 50 The results are shown in Table 6-1 and Table 6-2.

[0508] Table 6-1. Antibody binding activity to cells Note: / represents no combination.

[0509] Table 6-2. Antibody binding activity to cells Note: / represents no binding; NA represents not tested.

[0510] The results showed that the antibodies disclosed herein can specifically bind to CLDN6 expressed in cells, but not to CLDN9.

[0511] Test Example 2: FACS detection of antibody binding to 293T / human CLDN3 and CLDN4 cells

[0512] 293T / human CLDN3 and CLDN4 cells were prepared with FACS buffer (containing 1% BSA and pH 7.4 PBS) to prepare 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add 100ug / mL of the antibody to be tested, 100μL / well, and incubate in a 4°C refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add the working concentration of APC F(ab')2-goat anti-human IgG Fc (BioLegend, 410712) and incubate in a 4°C refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was measured on an Invitrogen flow cytometer. The results are shown in Figures 1 to 7.

[0513] The results showed that the antibody disclosed herein had the same isotype as the negative control antibody and did not bind to human CLDN3 and CLDN4.

[0514] Test Example 3: FACS detection of antibody binding to 293T / human / rat CLDN6 / 9 cells

[0515] 293T / mouse / rat CLDN6, 293T / human CLDN6-I143V and 293T / mouse CLDN9 cells were prepared with FACS buffer (1% BSA + pH 7.4 PBS) to a volume of 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the test antibody, 100μL / well. Incubate in a 4℃ refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add the working concentration of APC anti-human IgG Fc (BioLegend, 410712) and incubate in a 4℃ refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to these cells. 50 The results are shown in Tables 7-1 to 7-4.

[0516] Table 7-1 Antibody binding activity to cells

[0517] Table 7-2 Antibody binding activity to cells

[0518] Table 7-3 Antibody binding activity to cells

[0519] Table 7-4 Antibody binding activity to cells Note: / represents no combination.

[0520] The results showed that the disclosed antibodies had cross-species activity with both mouse and rat CLDN6, and did not bind to mouse CLDN9. Furthermore, the disclosed antibodies also had binding activity with the human CLDN6 variant I143V.

[0521] Test Example 4: Endocytosis Activity Detection of Anti-CLDN6 Antibodies

[0522] DT3C is a recombinantly expressed fusion protein, which is composed of the Fragment A of diphtheria toxin (toxin only) and the 3C fragment of group G streptococcus (IgG binding part). This protein has a high affinity with the Fc structure of antibodies and enters cells together with the antibodies during endocytosis. The activated diphtheria toxin (DT) kills cells, which indirectly reflects the endocytosis of antibodies. According to IC 50 and Imax to evaluate the in vitro endocytic activity of antibodies.

[0523] PA-1 cell suspension was prepared using fresh cell culture medium containing 20% ​​low IgG FBS (Bosheng Bio, BS-0007-500) at a cell density of 4 × 10 4 cells / mL, added into the cell culture plate at 50 μL / well, i.e., 2000 cells per well, and cultured at 37°C with 5% carbon dioxide for 16 hours.

[0524] Prepare 4× concentration of DT3C (2400nM or 600nM) in serum-free MEM medium and sterilize by filtering with a 0.22μm filter. Prepare 4× concentration of antibody (400nM or 100nM) in serum-free MEM medium, mix 75μL DT3C (about 70KD) and 75μL antibody (about 150KD) in a volume ratio of 1:1, incubate at room temperature for 30 minutes, then dilute the mixture 5-fold with MEM medium without FBS, for a total of 9 doses, with the 10th point being pure culture medium. The well with only culture medium is used as the zero point for calculating the killing rate. Take 50μL of diluted antibody and add it to 50μL of cells, incubate at 37°C, 5% CO2 for 1-3 days. Add 50μL CellTiter-Glo to each well. TM (CTG) (Promega, G7573), incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read on a multi-label detector VICTOR3 (PerkinElmer). The results are shown in Table 8-1 and Table 8-2. The results show that the antibodies disclosed herein have good endocytic activity.

[0525] Table 8-1 Endocytic activity of antibodies

[0526] Table 8-2 Endocytic activity of antibodies

[0527] Test Example 5: FACS detection of ADC binding at the cellular level

[0528] The CLDN6-expressing tumor cells PA-1 (ATCC, catalog number CRL-1572) were prepared with FACS buffer (1% BSA + pH 7.4 PBS) to a volume of 1×10 6 / mL cell suspension, 100 μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the ADC to be tested, 100 μL / well. Incubate in a 4°C refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add the working concentration of APC anti-human IgG Fc (BioLegend, 410712) and incubate in a 4°C refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to cells expressing CLDN6. 50 The results are shown in Table 9-1 and Table 9-2.

[0529] Table 9-1. Binding activity of ADC to cells

[0530] Table 9-2. Binding activity of ADC to cells Note: / represents no combination.

[0531] The results showed that ADC-1, ADC-2, ADC-4 and ADC-5 disclosed herein can all bind well to PA-1 cells.

[0532] Test Example 6: Cytotoxicity of ADC against cells with different CLDN6 expression levels

[0533] CLDN6-expressing tumor cells PA-1 (ATCC, catalog number CRL-1572), OVCAR3 (ATCC, HTB-161), and OV90 (Shanghai Zhongqiao Xinzhou, ZQ0073) were trypsinized, neutralized with fresh culture medium, centrifuged at 1000 rpm, and resuspended in culture medium. After counting, the cell suspension densities were adjusted to 3700 cells / mL for PA-1 cells, 7400 cells / mL for OVCAR3 cells, and 7400 cells / mL for OV90 cells. 135 μL of the cell suspension was added to 96-well cell culture plates. OV90 cells were seeded in round-bottom 96-well plates (CLS7007-24EA, Corning), while the other cells were seeded in flat-bottom 96-well plates (62096, Xinyou). That is, 500 PA-1 cells, 1000 OVCAR3 cells and 1000 OV90 cells were plated per well. No cells were plated in the peripheral wells of the 96-well plate, and only 135 μL of culture medium was added. The plates were cultured at 37° C. with 5% carbon dioxide for 16 hours.

[0534] The ADC sample was diluted with PBS to 5 μM (10× concentration), and this was used as the first concentration. PBS was used for five-fold continuous gradient dilution, for a total of 9 concentrations, and the 10th concentration point was used as the control well without drug addition. For OV90 cells dosed alone, the ADC sample was diluted with PBS to 10 μM (10× concentration), and this was used as the first concentration. PBS was used for three-fold continuous gradient dilution, for a total of 9 concentrations, and the 10th concentration point was used as the control well without drug addition. After drug dilution, 15 μL of 10× gradient dilution solution was added to each well, with the 10th well being culture medium. Each concentration point was repeated and cultured at 37°C with 5% carbon dioxide for 6 days.

[0535] After culture is complete, 75 μL of CTG (G7573, Promega) was added to each well of a 96-well plate containing PA-1 and OVCAR3 cells. The plates were incubated in the dark for 10 minutes at room temperature. A white bottom film was attached to the bottom of the cell culture plate, and the chemiluminescence was read on an ENVISION. For OV90 cells, 75 μL of 3D CTG (G9683, Promega) was added to each well. After shaking at room temperature for 20 minutes, 100 μL of the liquid was transferred using a dispenser to a 96-well plate with a white bottom film. The chemiluminescence was read on an ENVISION.

[0536] The results of in vitro cell killing activity are shown in Tables 10-1 to 10-3.

[0537] Table 10-1 In vitro cell killing activity of ADC

[0538] Table 10-2 In vitro cell killing activity of ADC

[0539] Table 10-3 In vitro cell killing activity of ADC

[0540] The results showed that ADC-1, ADC-2, ADC-4 and ADC-5 disclosed herein had good killing activity against PA-1, OVCAR3 and OV90 cells.

[0541] In vivo biological evaluation

[0542] Test Example 7-1: In vivo efficacy evaluation in the PA-1 cell CDX mouse model

[0543] Human ovarian cancer cell PA-1 cells (ATCC, catalog number CRL-1572) (4-5×10 6 200 μL / mouse of 50% Matrigel (#356234) was inoculated subcutaneously in the right flank of NOD SCID mice. 10-12 days after inoculation, when the tumor volume was 175 to 190 mm 3 After removing weight, excessive and small tumors, mice were randomly divided into groups according to tumor volume, with 8 mice per group, and drug administration began on the same day. ADC was injected through the tail vein at a dose of 0.3-0.1 or 0.3-0.15 mg / kg, and the drug was administered once. Tumor growth was observed. Tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for graphing, and two-way ANOVA or one-way ANOVA was used for statistical analysis of the data.

[0544] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2 .

[0545] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0546] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0547] The results are shown in Tables 11-1 and 11-2, and Figures 8A and 8B (vehicle refers to Isotype-ADC).

[0548] Table 11-1. Efficacy of ADC on PA-1 xenograft tumors in NOD SCID mice

[0549] Table 11-2. Efficacy of ADC on PA-1 xenograft tumors in NOD SCID mice Note: ns means not statistically significant.

[0550] The results showed that ADC-1 at a dose of 0.3 mpk could significantly inhibit the growth of PA-1 cell subcutaneous transplanted tumors. ADC-2 at doses of 0.3 mpk and 0.15 mpk could significantly inhibit the growth of PA-1 cell subcutaneous transplanted tumors.

[0551] Test Example 7-2: In vivo efficacy evaluation in the OVCAR3 cell CDX mouse model

[0552] Human ovarian cancer OVCAR3 cells (ATCC, HTB-161) (1×10 7 200 μL / mouse of NDG mice (containing 50% Matrigel / mouse, #356234) was inoculated subcutaneously on the right flank of the mice. 18 days after inoculation, when the tumor volume was ~185 mm 3 After weight loss and tumor size were eliminated, mice were randomly divided into groups of 8 per group according to tumor volume, and drug administration began on the same day. ADC was injected through the tail vein at a dose of 1-0.3 mg / kg for a total of 2 doses, and tumor growth was observed. Tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for graphing, and data were statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0553] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2 .

[0554] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0555] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0556] The results are shown in Table 11-3 and Figure 8C (vehicle refers to Isotype-ADC).

[0557] Table 11-3. Efficacy of ADC on OVCAR3 transplanted tumors in NDG mice

[0558] The results showed that ADC-2 at doses of 0.3 mpk and 1 mpk could significantly inhibit the growth of subcutaneously transplanted OVCAR3 cell tumors.

[0559] Test Example 7-3: In vivo efficacy evaluation in the OV90 cell CDX mouse model

[0560] OV90 cells (Shanghai Zhongqiao Xinzhou, ZQ0073) (3×10 6 200 μL / mouse of 50% Matrigel (#356234) was inoculated subcutaneously in the right rib of NOD SCID mice. After 7 days of inoculation, the tumor volume was about 185 mm. 3 After weight loss and tumor size were eliminated, mice were randomly divided into groups of 8 per group based on tumor volume, and dosing began on the same day. ADC was injected via the tail vein at a dose of 1-0.3 mg / kg for a total of 1 dose. Tumor volume was measured twice weekly, body weight was measured, and data were recorded. Data were recorded using Excel statistical software: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group). Graphs were generated using GraphPad Prism software, and data were statistically analyzed using two-way ANOVA or one-way ANOVA.

[0561] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2 .

[0562] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0563] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0564] The results are shown in Table 11-4 and Figure 8D (vehicle refers to Isotype-ADC).

[0565] Table 11-4. Efficacy of ADC on OV90 transplanted tumors in NOD SCID mice

[0566] The results showed that ADC-2 could significantly inhibit the growth of OV90 tumors at 1 mpk and 0.3 mpk.

Claims

1. An anti-CLDN6 antibody comprising a heavy chain variable region and a light chain variable region, wherein: The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 of the light chain variable region comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 32, 31, 30, 33, 34, or 35, respectively; Preferably, The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, 25, 17, 27, 28, or 29, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19; More preferably, The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26 or 25, LCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

19.

2. The anti-CLDN6 antibody according to claim 1, which is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; preferably a humanized antibody.

3. The anti-CLDN6 antibody according to claim 1 or 2, wherein: the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32, 31, 30, 33, 34, or 35; Preferably, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32 or 31.

4. The anti-CLDN6 antibody according to any one of claims 1 to 3, wherein the anti-CLDN6 antibody is an antibody fragment; preferably, the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv or dAb.

5. The anti-CLDN6 antibody according to any one of claims 1 to 3, wherein the anti-CLDN6 antibody comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain constant region is the light chain constant region of human κ, λ or a variant thereof; More preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

21.

6. The anti-CLDN6 antibody according to claim 5, wherein the anti-CLDN6 antibody comprises a heavy chain and a light chain, wherein: the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 38, 37, 36, 39, 40, or 41; Preferably, the heavy chain comprises the amino acid sequence of SEQ ID NO: 22, and the light chain comprises the amino acid sequence of SEQ ID NO: 38 or 37.

7. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is represented by the general formula (I): wherein Ab is an anti-CLDN6 antibody according to any one of claims 1 to 6; L is a linker connecting Ab and D; Ab binds to L directly from its amino acid, or Ab binds to L from its sugar chain or reconstructed sugar chain; preferably, Ab binds to L directly from its amino acid residue at position 297, or Ab binds to L from its sugar chain or reconstructed sugar chain at position 297; more preferably, Ab binds to L from its sugar chain reconstructed at Asn297; y is 1 to 10; D is represented by the general formula (D1), formula (D1-S) or formula (D1-R): in: X is (CR a R b ) s ; Y and Z are the same or different and are each independently selected from an oxygen atom, a sulfur atom and NR c ; R 1 is selected from hydrogen, halogen and alkyl; or R 1 Together with the carbon atom to which it is attached, it forms C=O; R 2 is a hydrogen atom or an alkyl group; or R 1 and R 2 Together with the carbon and nitrogen atoms to which they are attached, they form C=N; R 3 and R 4 The same or different, and each independently selected from hydrogen atom, alkyl, OR d , halogen, haloalkyl, hydroxyalkyl, SH, S-alkyl and NR e R f ; R 5 is selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, and a haloalkyl group; R 6 is selected from hydrogen, halogen, alkyl and haloalkyl; or R 6 Together with the carbon atom to which it is attached, it forms C=O; R 7 and R 8 are the same or different and are each independently selected from hydrogen, halogen, alkyl and haloalkyl; or R 7 and R 8 Together with the carbon atom to which it is attached, it forms C=O; or R 7 and R 8 Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently selected from oxo, halogen, alkyl, haloalkyl, cyano, NR g R h and OR i is substituted by one or more substituents; R 9 is a hydroxyl group or an alkoxy group; R 10 is selected from the group consisting of a hydrogen atom, a hydroxyl group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, and an alkoxy group; Ring A is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl; R 11 Selected from hydrogen atom, halogen, alkyl, haloalkyl, OR j 、C(O)R k 、C(O)OR k , cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; R a and R b are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, cyano, amino and hydroxyalkyl; R c 、R e 、R f 、R g and R h are the same or different and are each independently selected from a hydrogen atom, an alkyl group and a haloalkyl group; R d 、R i and R j are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from an oxo group, a halogen group, an alkyl group, a haloalkyl group, a cyano group, an amino group, a hydroxyl group, an alkoxy group and a hydroxyalkyl group; R k are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyl group, and a hydroxyalkyl group; s is 1, 2, 3, 4, 5, or 6; t is 1, 2, 3, 4, or 5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7 , wherein Y and Z are both oxygen atoms.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7 or 8, wherein R 1 and R 2 All are hydrogen atoms. 10 . The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7 , wherein ring A is a 6- to 10-membered aryl group; preferably, ring A is a phenyl group.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 10, wherein D is represented by general formula (D2), formula (D2-S) or formula (D2-R): Among them, X, R 3 to R 11 and t as defined in claim 7.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 11, wherein: X is (CR a R b ) s , R a and R b are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, hydroxyl and C 1-6 hydroxyalkyl; Preferably, X is (CH2) s ; wherein s is as defined in claim 7.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 12, wherein: R 3 and R 4 Both OR d , and R d Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, 3 to 6 membered cycloalkyl and 3 to 6 membered heterocyclic group, wherein the 3 to 6 membered cycloalkyl or 3 to 6 membered heterocyclic group is independently selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, cyano, amino, hydroxyl, C 1-6 Alkoxy and C 1-6 substituted by one or more substituents in the hydroxyalkyl group; Preferably, R 3 and R 4 Both OR d , and R d C 1-6 alkyl or 3 to 6 membered cycloalkyl; more preferably, R 3 C 1-6 Alkoxy, and R 4 is a 3- to 6-membered cycloalkyloxy group; most preferably, R 3 is a methoxy group, and R 4 It is cyclopropyloxy.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 13, wherein R 5 and R 6 All are hydrogen atoms.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 14, wherein R 7 and R 8 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl and C 1- 6 haloalkyl; or R 7 and R 8 Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group are each independently optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 is substituted by one or more substituents selected from haloalkyl, hydroxyl and amino groups; preferably, R 7 and R 8 Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl group, wherein the 3 to 6-membered cycloalkyl group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxy, C 1-6 More preferably, R 7 and R 8 Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group; most preferably, R 7 and R 8 Together with the carbon atom to which they are attached they form a cyclopropyl group.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 15, wherein R 9 It is a hydroxyl group.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 16, wherein R 10 Selected from hydrogen atoms, hydroxyl groups, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl and C 1-6 Alkoxy; preferably, R 10 A hydrogen atom.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 17, wherein R 11 Selected from hydrogen atoms, halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, hydroxyl and C 1-6 Alkoxy; preferably, R 11 A hydrogen atom or C 1-6 Alkoxy.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 18, wherein D is selected from the following structures:

20. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 19, wherein L is -L a -L b -L c -L d -, L a Selected from: The asterisk * indicates that b Combined, wavy lines Indicates binding to the sugar chain or reconstructed sugar chain of Ab; L b selected from -C(O)-(CR m R n -CR p R q ) t1 -C(O)-、-C(O)-(CR m R n -CR p R q ) t1 -C(O)-NR s -(CR m R n -CR p R q ) t2 -C(O)-、-C(O)-(CR m R n -CR p R q )t1-C(O)-NR s -(CR m R n -CR p R q -O) t2 -CR u R v -C(O)-、-C(O)-(CR m R n -CR p R q ) t1 -NR s -C(O)-(CR m R n -CR p R q -O) t2 -(CR m R n -CR p R q ) t3 -C(O)- and -(CR u R v ) t4 -O-C(O)-; L c is a peptide residue consisting of 2 to 7 amino acids; L d -NR w -W-CR x R y OC(O)-、-NR w -CR x R y OR z -C(O)- or chemical bond; R m 、R n 、R p 、R q 、R u 、R v 、R x and R y are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino; or R m and R n Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R p and R q Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R u and R v Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R x and R y Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; or R m and R p Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is independently optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; R s 、R w and R z are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, wherein the cycloalkyl group, the heterocyclic group, the aryl group and the heteroaryl group are each independently optionally substituted with one or more substituents selected from an oxo group, a halogen group, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxyl group, a cyano group and an amino group; W is an aryl or heteroaryl group, and the aryl or heteroaryl group is independently substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, haloalkyl and alkoxy; preferably, W is a phenyl group or a 5- or 6-membered heteroaryl group, and the phenyl group or the 5- or 6-membered heteroaryl group is independently substituted by one or more substituents selected from halogen, oxo, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 substituted by one or more substituents in the alkoxy group; t1, t2, t3 and t4 are the same or different and are each independently 1, 2, 3, 4, 5 or 6.

21. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein L b -C(O)-(CR m R n -CR p R q ) t1 -C(O)-, and R m 、R n 、R p and R q The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl, t1 is 1, 2, 3, 4, 5 or 6; preferably, L b It is -C(O)-CH2-CH2-C(O)-.

22. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 20 or 21, wherein L c Peptide residues formed by amino acids selected from phenylalanine (F), alanine (A), proline (P), isoleucine (I), leucine (L), glycine (G), valine (V), lysine (K), citrulline (Cit), serine (S), glutamic acid (E) and aspartic acid (D), wherein each of the peptide residues is independently optionally selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 alkoxy, 3 to 6 membered cycloalkyl, 3 to 6 membered heterocyclyl, 6 to 10 membered aryl and 5 to 10 membered heteroaryl; preferably, L c is selected from -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI- and -GGPL-; more preferably, L c It is -GGVA-.

23. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 20 to 22, wherein L d -NR w -W-CR x R y OC(O)-,R w 、R x and R y The same or different, and each independently a hydrogen atom or a C 1-6 alkyl, and W is selected from 1,4-phenyl, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidinyl and 2,5-thienyl; Preferably, L d is -NH-W-CH2-OC(O)-, and W is 1,4-phenyl.

24. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 20 to 23, wherein L is 25. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 20 to 24, wherein the remodeled sugar chain structure is: In the formula, the wavy line Indicates binding to Asn 297 of the Ab heavy chain; P1 and P2 are the same or different and are each independently selected from hydroxyl, *-(CR p1 R q1 -CR s1 R t1 -O)s 1 - and *-(CR p1 R q1 -CR s1 R t1 -O)s 2 -(CR p1 R q1 -CR s1 R t1 -CR x1 R y1 -O)s 3 -(CR p1 R q1 -CR s1 R t1 -O)s 4 -, where R p1 、R q1 、R s1 、R t1 、R x1 and R y1 are the same or different and are each independently selected from hydrogen atom, halogen, C 1- 6 alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, hydroxyl, cyano, amino, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl, wherein the 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl are each independently selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 It is substituted by one or more substituents selected from hydroxyalkyl, hydroxyl, cyano and amino groups; the asterisk * indicates that it is bound to the linker L; s 1 1-10, preferably 1-5; s 2 0-10, preferably 1-5; s 3 1-10, preferably 1-5; s 4 0-10, preferably 1-5; The condition is that P1 and P2 are not hydroxyl or *-(CH2CH2O)s at the same time 1 -; Preferably, P1 is hydroxyl, P2 is *-(CR p1 R q1 -CR s1 R t1 -O) s2 -(CR p1 R q1 -CR s1 R t1 -CR x1 R y1 -O) s3 -(CR p1 R q1 -CR s1 R t1 -O) s4 -, where R p1 、R q1 、R s1 、R t1 、R x1 and R y1 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl, s2 is 1, s3 is 1, s4 is 1; More preferably, the reconstructed sugar chain structure is:

26. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 25, which is represented by general formula (II): in: Ab is an anti-CLDN6 antibody according to any one of claims 1 to 6; y is 1 to 10; preferably, y is 1 to 4; more preferably, y is 1 to 2; most preferably, y is 2.

27. A method for preparing an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof, comprising the following steps: The compound represented by general formula (IIa) or its salt reacts with the compound represented by general formula (Ib) or its salt to obtain the antibody-drug conjugate represented by general formula (II) or its pharmaceutically acceptable salt; in: L' is L aa -L b -L c -L d -, L aa Selected from: The asterisk * indicates that b Combine; Ab is an anti-CLDN6 antibody according to any one of claims 1 to 6; y is 1 to 10; L b , L c , L d as defined in any one of claims 20 to 26; R is defined as in claim 26; D is as defined in any one of claims 7 to 19.

28. A pharmaceutical composition comprising: the anti-CLDN6 antibody of any one of claims 1 to 6, the antibody-drug conjugate of any one of claims 7 to 26, or a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable carriers, diluents, or excipients.

29. An isolated nucleic acid encoding the anti-CLDN6 antibody of any one of claims 1 to 6.

30. A host cell comprising the isolated nucleic acid of claim 29.

31. A method for preventing or treating tumors, comprising administering to a subject the anti-CLDN6 antibody of any one of claims 1 to 6, the antibody-drug conjugate of any one of claims 7 to 26 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 28; Preferably, the tumor is selected from ovarian cancer, lung cancer, endometrial cancer, gastric cancer, cervical cancer, testicular cancer, placental choriocarcinoma, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer and esophageal cancer; More preferably, the tumor is selected from ovarian cancer, lung cancer, endometrial cancer and testicular cancer; Most preferably, the tumor is ovarian cancer.

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