WO2022001864A1 - Antibody-drug conjugate and preparation method therefor and use thereof - Google Patents

Abstract

An antibody-drug conjugate and a preparation method therefor and the use thereof. The antibody-drug conjugate is an antibody-drug conjugate as represented by formula (V) formed by means of connecting the compound represented by formula (I) to an antibody via a linker represented by formula (II) and a thioether bond formed via a disulfide bond part which is present in the hinge part of the antibody. The antibody-drug conjugate has a faster onset time, a longer half-life of the drug, a more moderate stability, a good biocompatibility, a low immunogenicity, and safety, and can prevent aggregation, such that the antibody-drug conjugate has excellent anti-tumor effects.

Description

An antibody-drug conjugate, its preparation method and application technical field

The present invention relates to the field of biomedicine, in particular, the present invention relates to a new type of linker structure, a drug-linker compound including the linker structure, and an antibody-drug conjugate including the drug-linker compound, the above-mentioned drugs - Preparation methods and applications of linker compounds and antibody-drug conjugates.

Background technique

Antibody-drug conjugates (hereinafter referred to as "ADCs" or "conjugates") have shown unique advantages over pure antibody drugs, by combining them with tumor cell surface antigen binding specificity. The monoclonal antibody is linked to a biologically active cytotoxin, thereby combining the tumor recognition targeting of the antibody with the high-efficiency killing effect of the cytotoxin. big flaw. Compared with traditional anti-tumor drugs, ADC can accurately target tumor cells while reducing the impact on normal cells, greatly improving the effectiveness and safety of anti-tumor drugs.

ADC generally consists of three parts: antibody, linker and toxin. Antibodies are targeted functional macromolecules of ADCs, which play the role of enriching toxins near the tumor tissue site to improve the killing efficiency of toxins. At present, the development of antibodies against major popular targets such as HER-2, Trop-2, PDL-1, CD30, etc. is in full swing, and it also drives the development of ADCs against these targets.

ADC linkers are divided into two types: cleavable and non-cleavable. The ideal linker should meet the requirements of "good stability and high release efficiency", that is, the ADC remains stable in the blood circulation and can be quickly released after reaching the tumor cells. toxins, killing tumor cells. The linker is crucial for the ADC to function. An unstable linker will lead to off-target ADC and increase the safety risk, while an overly stable linker will affect the release rate of the toxin, thereby affecting the efficacy of the drug. In addition, how to select a linker structure suitable for a specific ADC according to the physicochemical properties, spatial structure, and target cell physiological environment of specific antibodies and specific toxins is a hot issue in the current ADC research and development, and there is still an urgent need for research and development.

The toxin part of ADC is a small drug molecule that plays a killing role, and generally kills tumor cells by inhibiting DNA or protein synthesis, inhibiting cell mitosis, and the like. Toxins currently used for ADC development mainly include microtubule inhibitors maytansinoids (see EP0425235, US5208020, US5416064, US7276497) and auristatin (MMAE/MMAF, see US2016304621A). The representative drugs currently on the market are T-DM1 developed by Genetech. T-DM1 is a compound formed by a stable thioether linker MCC (4-[N-maleimidomethyl]cyclohexane-1 - carboxylate) conjugated to an ADC consisting of trastuzumab conjugated to the maytansinoid toxoid DM1 (US8337856). Other classes of cytotoxins include Calicheamicin (see US5606040), benzodipyrrole derivatives (duocarmycin, see US7129261), pyrrobenzodiazepines (PBDs, see WO2005/040170) and Derivatives of tree alkaloids. The camptothecin derivatives include SN-38, CPT-11, ixatecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like. IMMU-132 developed by Immunotherapy Company and DS-8201 developed by Daiichi Sankyo Co., Ltd. are ADCs with outstanding clinical effects. They both use camptothecin derivatives as the toxin part of ADC. Among them, IMMU-132 uses a moderately toxic drug SN-38, and DS-8201 uses a highly cytotoxic ixatecan.

However, whether it is T-DM1, IMMU-132, or DS-8201, there are still the following shortcomings:

As far as T-DM1 is concerned, first of all, the efficacy of T-DM1 is insufficient, one is because its DAR is low, only 3-4, and the other is because it uses the linker of SMCC to connect with DM-1, and SMCC is non-degradable. linker, which reduces the efficacy of T-DM1; secondly, T-DM1 uses DM-1 as a toxin, which is a microtubule inhibitor, and the permeability of cell membranes is weak; thirdly, the presence of T-DM1 reduces white blood cells Serious side effects.

As for IMMU-132, first, since SN-38 is moderately toxic, each antibody of IMMU-132 needs to link more toxins (about 7 SN-38 per antibody) to achieve a better effect. However, high drug loading will lead to an increase in the composition of drug multimers, resulting in decreased drug stability, increased toxicity, and increased immunogenicity. Secondly, IMMU-132 uses a moderately stable linker with only one cleavage site. In human serum, the half-life of SN-38 released from IMMU-132 is about 24h, the release time is longer, and the onset is slow. The elimination rate of -132 in humans and mice is relatively fast, the elimination half-life is about 11h, and the average retention time is about 15.4h, which means that the elimination rate of IMMU-132 in vivo is too fast and the drug half-life is too short, which will make The frequency of medication in clinical treatment is relatively high.

As far as DS-8201 is concerned, although ixatecan is 10 times more toxic than SN-38, it cannot be used as a single drug due to its strong cell-killing activity. There is also only one enzymatic cleavage site, which also prolongs the onset time of ADC in cells to some extent. In addition, ixatecan has a short half-life in the blood, which reduces the toxicity and side effects, but also faces the risk of a short half-life of the drug.

It can be seen that there is still a need to develop more effective and safe camptothecin-based ADCs in this field. The preparation has a faster onset time, longer drug half-life, and at the same time, it has the advantages of stability, hydrophilicity and hydrophobicity, and anti-aggregation. Camptothecin ADCs with superior safety indicators are imminent.

SUMMARY OF THE INVENTION

In order to solve the above problems, the inventors designed a linker structure suitable for camptothecin derivatives, and used it as a linking structure between camptothecin derivatives and antibodies, so as to form a linker structure with faster onset time, longer drug half-life, and better drug resistance. Moderate stability and good safety, anti-aggregation antibody-drug conjugate, this ADC has excellent anti-tumor effect.

A first aspect of the present invention provides an antibody-drug conjugate represented by formula (V), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof A solvate of a struct or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is a compound represented by the following formula (I) and an antibody via a linker represented by the following formula (II) through the presence of It is formed by connecting the thioether bond formed by the disulfide bond part of the hinge part of the antibody;

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

Wherein, the antibody is connected to ^{the end of L 1} , and the compound represented by formula (I) is connected to the right -C(=O)- moiety in the above formula (II) using the oxygen in the hydroxyl group at the 19th position as a connecting site,

In formula (I),

R ₁ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, the substituted ₇ R ₈ NR C ₁ -C ₆ alkyl, -SiMe ₃ Substituted C ₁ -C ₆ alkyl, or -CH=NO(C ₁ -C ₆ alkyl);

R ₂ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NR ₇ R ₈ or C ₁ -C ₆ substituted alkyl;

R ₃ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or NR ₇ R ₈ C(O)O- group;

R ₄ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy;

Or R ₁ and R ₂ may be joined together to form an optionally substituted 5-6 R ₉ membered ring with the parent moiety;

Or R ₃ and R ₄ may be joined together to form an optionally substituted ₉ membered oxygen-containing heterocyclic R 5-6 to the parent moiety;

Each occurrence of R ₇ and R ₈ is independently selected from hydrogen, C ₁ -C ₆ alkyl; or R ₇ and R ₈ can be taken together with the N atom to which they are attached to form a 5-6 membered _{optionally substituted with R 9} nitrogen-containing heterocycle;

Each occurrence of R ₉ is independently selected from halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, piper optionally substituted with C ₁ -C _{6 alkyl} pyridyl;

In formula (II),

L ¹ represents -(succinimide-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimide-3-yl-N)-(CH ₂ )n ^{1 '-C} ₃ -C ₆ cycloalkyl, -C (= O) -, n 1, n 1' each independently represents an integer of 1 to 8,

Preferably, L ¹ represents -(succinimid-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimid-3-yl-N)-(CH _{^{2) n 1 '-C 3 -C}} 6 cycloalkyl, -C (= O) -, n 1 represents an integer of 1 to 5, n ^1' represents an integer of 1 to 3;

More preferably, L ¹ represents -(succinimid-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimid-3-yl-N)-( CH ₂ )n ^1' -cyclohexyl-C(=O)-, n ¹ represents 2 or 5, n ^1' represents 1;

Most preferably, L ¹ represents

L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ CH(CH ₃ )-O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -5-membered nitrogen-containing heteroaryl-(CH ₂ CH ₂ -O)n ^2' -(CH ₂ )n ⁷ -NH -C(=O)-(CH ₂ )n ⁸ -O-(CH ₂ )n ⁸ -C(=O)- or single bond, n ² represents an integer of 1 to 6, and n ^2' represents an integer of 1 to 12 Integer, n ³ represents an integer from 1 to 4, and n ⁶ , n ⁷ , and n ⁸ each independently represent an integer from 1 to 5;

Preferably, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -1H-[1,2 ,3] Triazolyl-(CH ₂ CH ₂ -O)n ^2' -(CH ₂ )n ⁷ -NH-C(=O)-(CH ₂ )n ⁸ -O-(CH ₂ )n ⁸ - C(=O)- or single bond, n ² represents an integer from 1 to 3, n ^2' represents an integer from 6 to 10, n ³ represents an integer from 1 to 3, and n ⁶ , n ⁷ , and n ⁸ represent each independently an integer from 1 to 3;

More preferably, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -1H-[1, 2,3] triazol-yl _{- (CH 2 CH 2 -O)} n 2 '- (CH 2) n 7 -NH-C (= O) - (CH 2) n 8 -O- (CH 2) n 8 -C(=O)- or single bond, n ² means 2, n ^2' means 8, n ³ means 2, n ⁶ means 1, n ⁷ means 2, n ⁸ means 1;

或单键； Most preferably, L ² represents

or single key;

L ^P represents a peptide residue composed of 1 to 7 amino acids, and

L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen, optionally _{C 1} -C ₆ alkyl substituted with 1 or 2 hydroxy groups; Aryl represents a C ₆ -C ₁₀ aryl group _{optionally substituted by R 9} ^{, n 4} represents an integer of 1 to 4, and n ⁵ represents an integer of 1 to 4 ;

Preferably, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, and _{each occurrence of R 10} is independently selected from hydrogen, C ₁ -C ₄ alkyl; Aryl represents phenyl, n ⁴ represents an integer of 1-3, and n ⁵ represents an integer of 1-3;

More preferably, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen , methyl; Aryl represents phenyl, n ⁴ represents 2, n ⁵ represents 1;

Most preferably, L ^a represents

-(Succinimide-3-yl-N)- is of the formula:

The represented structure is linked to the antibody at the 3-position of the structure, and at the nitrogen atom of the 1-position to the methylene group in the linking group comprising the structure;

In formula (V), AB represents an antibody;

in:

The structure after the compound represented by the formula (I) is connected to the linker represented by the formula (II) is not

取代的C ₁-C ₄烷基、被-SiMe ₃取代的C ₁-C ₄烷基或-CH＝NO(C ₃-C ₆烷基)； In some embodiments, R ₁ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyl substituted with -NH(C 1 -C 4 alkyl), C ₁ -C ₄ alkyl substituted by

Substituted C ₁ -C ₄ alkyl, -SiMe ₃ substituted C ₁ -C ₄ alkyl or -CH = NO (C ₃ -C ₆ alkyl);

被

取代的甲基、

或-CH＝NO-叔丁基。 Preferably, R ₁ represents hydrogen, ethyl,

quilt

substituted methyl,

or -CH=NO-tert-butyl.

In some embodiments, R ₂ represents hydrogen, nitro, amino, or -N (C ₁ -C ₄ alkyl) ₂ substituted C ₁ -C ₄ alkyl;

Preferably, R ₂ represents hydrogen, nitro, amino or

In some embodiments, R ₃ represents hydrogen, halogen, hydroxy, C ₁ -C ₄ alkyl or

Preferably, R ₃ represents hydrogen, F, hydroxyl, methyl or

In some embodiments, R ₄ represents hydrogen or halogen;

Preferably, R ₄ represents hydrogen or F.

其中

部分表示连接于母体基团的键； In some embodiments, R ₁ and R _{2 are} joined together to form the group shown below

in

moiety represents a bond to the parent group;

其中

部分表示连接于母体基团的键。 Preferably, R ₁ and R ₂ are linked together to form a group shown below

in

A moiety represents a bond to the parent group.

其中

部分表示连接于母体基团的键。 In some embodiments, R ₃ and R _{4 are} joined together to form a group shown below

in

A moiety represents a bond to the parent group.

In some embodiments, the compound represented by formula (I) is a compound selected from the group consisting of:

Preferably, the compound represented by formula (I) is gimatecan:

In some embodiments, L ^P by peptide residue selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid amino acids formed peptide residues.

In some embodiments, L ^P by 1-5 amino acid peptide consisting of residues.

In some embodiments, L ^P is a peptide residue selected from the following:

-K-

-ggfg-;

-vc-;

-evc-;

-DVC;

-EGGFG-;

-DGGFG-;

Preferably, L ^P is a peptide residue selected from the following: -K -, - GGFG -, - VC -, - EVC-.

In some embodiments, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, n ² represents an integer from 1 to 4, and n ³ represents 2 an integer of ~4.

In some embodiments, L ² represents a single bond.

In some embodiments, L ^a represents -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, wherein R ₁₀ represents hydrogen or C ₁ -C ₄ alkyl; n ⁵ represents an integer from 1 to 2, and Aryl represents phenyl ring group. In some embodiments, the -NR ₁₀ - group and the -(CH ₂ )n ⁵ - group are located in the para position of the benzene ring.

In some embodiments, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - and _{each occurrence of R 10} is independently selected from hydrogen, C ₁ -C _{4 optionally substituted with 1 hydroxy} In the alkyl group, n ⁴ represents an integer of 2-4.

In some embodiments, the linker represented by formula (II) is a group selected from the group consisting of:

In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8 (eg, 2.7, 5.3, 6.3, 7.2, 7.3, 7.4, 7.5, 7.6), preferably 4-8 , and more preferably 6 to 8 pieces.

In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof;

Preferably, the antibody is selected from anti-Trop-2 antibody, Her2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody; preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably, the humanized antibody is a fully human antibody;

Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv and dsFv;

More preferably, the antibody is an anti-Trop-2 antibody, and the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody includes CDR1 consisting of the amino acid sequence of KASQDVSIAVA, and CDR2 consisting of the amino acid sequence of SASYRYT. , and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, the anti-Trop The amino acid sequences of the light chain and heavy chain of the -2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; preferably, the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody As shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

In other embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof;

Preferably, the antibody is selected from anti-Her2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody; preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably, the humanized antibody is a fully human antibody;

Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv and dsFv;

More preferably, the antibody is an anti-Her2 antibody, and the complementarity determining regions (CDRs) of the light chain variable region of the anti-Her2 antibody include CDR1 consisting of the amino acid sequence of RASQDVNTAVA, CDR2 consisting of the amino acid sequence of SASFLYS, and CDR2 consisting of the amino acid sequence of QQHYTTPPT. CDR3 composed of amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; Preferably, the light chain of the anti-Her2 antibody and the amino acid sequences of the heavy chain are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

A second aspect of the present invention provides a linker-drug intermediate compound represented by formula (IV), wherein the compound represented by the following formula (I) and the linker structure represented by the following formula (III) are represented by formula (I) The oxygen in the 19-position hydroxyl group in the compound is connected as a connecting site;

QL ² -L ^P -L ^a -C(=O)- (III)

Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described in the description of the present invention;

L ^2, L ^P, L ^a are as defined in the description of the present invention;

Q represents (maleimide-N)-

in:

The linker-drug intermediate compound represented by formula (IV) is not

In some embodiments, the compound represented by the formula (I) is the aforementioned compound; preferably, the linker-drug intermediate compound is a compound selected from the group consisting of:

The third aspect of the present invention provides the linker structure shown in general formula (II):

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

^{Wherein, L 1, L 2, L} P, L a of the present invention as defined in the specification.

The fourth aspect of the present invention provides a method for preparing the antibody-drug conjugate of the first aspect of the present invention, the method comprising:

The linker-drug intermediate compound represented by the formula (IV) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (IV) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;

Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described in the description of the present invention;

^{L 1, L 2, L P} , L a are as defined in the description of the present invention;

Q represents (maleimide-N)-

AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.

The fifth aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the second aspect of the present invention, the method comprising:

The compound represented by the formula (VI) is reacted with the compound represented by the formula (VII) to obtain the compound represented by the formula (VIII);

The compound shown in formula (VIII) and the compound shown in formula (I) are reacted in the presence of alkoxycarbonylating reagents (alkoxycarbonylating agents) to obtain the linker-drug intermediate compound shown in (IV); the described The alkoxycarbonylation reagents include but are not limited to triphosgene, bis(2-pyridyl)carbonate (di(2-pyridyl)carbonate), N,N'-disuccinimidyl carbonate (N,N'- Disuccinimidyl carbonate) and 4-nitrophenyl chloroformate.

_{Wherein, R 1, R 2, R} 3, R 4, Q, L 2, L P, L a are as defined in the description of the present invention; G represents a leaving group, preferably halogen, hydroxy, C ₁ -C ₆ alkoxy group, succinimidyl group; N-terminal peptide residue represented by ^P L is connected to the group represented by L ^2, C-terminus to the group represented by L ^a.

The sixth aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate A solvate of the conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

The seventh aspect of the present invention provides a pharmaceutical preparation, which comprises the antibody-drug conjugate of the first aspect of the present invention, a stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody- Solvates of drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof.

The eighth aspect of the present invention provides the antibody-drug conjugate of the first aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer A solvate of an isomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect or the pharmaceutical preparation of the seventh aspect, for use in preventing and/or treating tumors or cancers.

or,

Provide the antibody-drug conjugate of the first aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt Use of a solvate of an acceptable salt, the pharmaceutical composition of the sixth aspect or the pharmaceutical preparation of the seventh aspect in the manufacture of a medicament for preventing and/or treating tumors or cancer.

The ninth aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate of the first aspect of the present invention, its stereoisomer antibody or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect or the seventh The pharmaceutical formulation of the aspect.

The tenth aspect of the present invention provides the antibody-drug conjugate of the first invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer Use of a solvate of a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect or the pharmaceutical preparation of the seventh aspect for the preparation of an agent for inhibiting cancer cell growth, proliferation or migrate.

The eleventh aspect of the present invention provides the antibody-drug conjugate of the first invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer A solvate of the body or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect or the pharmaceutical preparation of the seventh aspect, which are used for inhibiting the growth, proliferation or migration of cancer cells.

The twelfth aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug conjugate of the first aspect of the present invention, its stereoisomer antibody or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect or the seventh The pharmaceutical formulation of the aspect.

The thirteenth aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate described in the first aspect of the present invention, its stereoisomer or its pharmaceutically acceptable The accepted salt, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the sixth aspect, or the pharmaceutical formulation of the seventh aspect .

In addition, the present invention also provides the following technical solutions:

One aspect of the present invention provides the antibody-drug conjugate represented by formula (V), its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its A solvate of a pharmaceutically acceptable salt, the antibody-drug conjugate is a compound represented by the following formula (I) and an antibody via a linker represented by the following formula (II), through a bond existing in the hinge portion of the antibody. The sulfide bond formed by the disulfide bond part is connected;

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

Wherein, the antibody is connected to ^{the end of L 1} , and the compound represented by formula (I) is connected to the right side -C(=O)- in the linker represented by the above formula (II) using the oxygen in the hydroxyl group at position 19 as a linking site. part,

In formula (I),

R ₁ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, the substituted ₇ R ₈ NR C ₁ -C ₆ alkyl, -SiMe ₃ Substituted C ₁ -C ₆ alkyl, or -CH=NO(C ₁ -C ₆ alkyl);

R ₂ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NR ₇ R ₈ or C ₁ -C ₆ substituted alkyl;

R ₃ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or NR ₇ R ₈ C(O)O- group;

R ₄ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy;

Or R ₁ and R ₂ may be joined together to form an optionally substituted 5-6 R ₉ membered ring with the parent moiety;

Or R ₃ and R ₄ may be joined together to form an optionally substituted ₉ membered oxygen-containing heterocyclic R 5-6 to the parent moiety;

Each occurrence of R ₇ and R ₈ is independently selected from hydrogen, C ₁ -C ₆ alkyl; or R ₇ and R ₈ can be taken together with the N atom to which they are attached to form a 5-6 membered _{optionally substituted with R 9} nitrogen-containing heterocycle;

Each occurrence of R ₉ is independently selected from halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, piper optionally substituted with C ₁ -C _{6 alkyl} pyridyl;

In formula (II),

L ¹ represents -(succinimide-3-yl-N)-(CH ₂ )n ¹ -C(=O)-, n ¹ represents an integer of 1 to 8,

L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ CH(CH ₃ )-O)n ² -(CH ₂ ) n ³ -C(=O)- or single bond, n ² represents an integer of 1-6, n ³ represents an integer of 1-4,

L ^P represents a peptide residue composed of 2 to 7 amino acids and,

L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen, optionally _{C 1} -C ₆ alkyl substituted with 1 or 2 hydroxy groups; Aryl represents a C ₆ -C ₁₀ aryl group _{optionally substituted by R 9} ^{, n 4} represents an integer of 1 to 4, and n ⁵ represents an integer of 1 to 4 ;

-(Succinimide-3-yl-N)- is of the formula:

The represented structure is linked to the antibody at the 3-position of the structure and to the methylene group in the linker comprising the structure at the nitrogen atom at the 1-position;

In formula (V), AB represents an antibody.

取代的C ₁-C ₄烷基、被-SiMe ₃取代的C ₁-C ₄烷基或-CH＝NO(C ₃-C ₆烷基)。 In some embodiments, R ₁ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyl substituted with -NH(C 1 -C 4 alkyl), C ₁ -C ₄ alkyl substituted by

Substituted C ₁ -C ₄ alkyl, -SiMe ₃ substituted C ₁ -C ₄ alkyl or -CH = NO (C ₃ -C ₆ alkyl).

In some embodiments, R ₂ represents hydrogen, nitro, amino, or -N (C ₁ -C ₄ alkyl) ₂ substituted C ₁ -C ₄ alkyl.

In some embodiments, R ₃ represents hydrogen, halogen, hydroxyl, or

In some embodiments, R ₄ represents hydrogen or halogen.

其中

部分表示连接于母体基团的键。 In some embodiments, R ₁ and R _{2 are} joined together to form the group shown below

in

A moiety represents a bond to the parent group.

其中

部分表示连接于母体基团的键。 In some embodiments, R ₃ and R _{4 are} joined together to form a group shown below

in

A moiety represents a bond to the parent group.

In some embodiments, the compound represented by formula (I) is a compound selected from the group consisting of:

Preferably, the compound represented by formula (I) is gimatecan:

In some embodiments, L ^P by peptide residue selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid amino acids formed peptide residues.

In some embodiments, L ^P by 2-5 amino acid residues constituting the peptide.

In some embodiments, L ^P is a peptide residue selected from the following:

-ggfg-;

-vc-;

-evc-;

-DVC;

-EGGFG-;

-DGGFG-.

In some embodiments, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, n ² represents an integer from 1 to 4, and n ³ represents 2 an integer of ~4.

In some embodiments, L ² represents a single bond.

In some embodiments, L ^a represents -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, wherein R ₁₀ represents hydrogen or C ₁ -C ₄ alkyl; n ⁵ represents an integer from 1 to 2, and Aryl represents phenyl ring group. In some embodiments, the -NR ₁₀ - group and the -(CH ₂ )n ⁵ - group are located in the para position of the benzene ring.

In some embodiments, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - and _{each occurrence of R 10} is independently selected from hydrogen, C ₁ -C _{4 optionally substituted with 1 hydroxy} In the alkyl group, n ⁴ represents an integer of 2-4.

In some embodiments, the linker represented by formula (II) is a group selected from the group consisting of:

In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8.

In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof;

Preferably, the antibody is selected from anti-Trop-2 antibody, Her2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody; preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably, the humanized antibody is a fully human antibody;

Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv and dsFv;

More preferably, the antibody is an anti-Trop-2 antibody, and the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody includes CDR1 consisting of the amino acid sequence of KASQDVSIAVA, and CDR2 consisting of the amino acid sequence of SASYRYT. , and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, the anti-Trop The amino acid sequences of the light chain and heavy chain of the -2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; preferably, the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody As shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

In other embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof;

Preferably, the antibody is selected from anti-Her2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody; preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably, the humanized antibody is a fully human antibody;

Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv and dsFv;

More preferably, the antibody is an anti-Her2 antibody, and the complementarity determining regions (CDRs) of the light chain variable region of the anti-Her2 antibody include CDR1 consisting of the amino acid sequence of RASQDVNTAVA, CDR2 consisting of the amino acid sequence of SASFLYS, and CDR2 consisting of the amino acid sequence of QQHYTTPPT. CDR3 composed of amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; Preferably, the light chain of the anti-Her2 antibody and the amino acid sequences of the heavy chain are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Another aspect of the present invention provides a linker-drug intermediate compound represented by formula (IV), which is a compound represented by formula (I) in which a compound represented by formula (I) below and a linker structure represented by formula (III) below are represented by formula (I). The oxygen in the 19-position hydroxyl group is connected as a connecting site;

QL ² -L ^P -L ^a -C(=O)- (III)

Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described above;

L ^2, L ^P, L ^a is defined above;

Q represents (maleimide-N)-

In some embodiments, the compound represented by the formula (I) is the aforementioned compound; preferably, the linker-drug intermediate compound is a compound selected from the group consisting of:

Another aspect of the present invention provides the aforementioned antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A process for the preparation of a solvate of an accepted salt, the process comprising:

The linker-drug intermediate compound represented by the formula (IV) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (IV) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;

Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described above;

^{L 1, L 2, L P} , L a is defined above;

Q represents (maleimide-N)-

AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.

Another aspect of the present invention provides a method for preparing the aforementioned linker-drug intermediate compound, the method comprising:

The compound represented by the formula (VI) is reacted with the compound represented by the formula (VII) to obtain the compound represented by the formula (VIII);

The compound represented by the formula (VIII) is reacted with the compound represented by the formula (I) in the presence of an alkoxycarbonylation reagent to obtain the linker-drug intermediate compound represented by (IV); the alkoxycarbonylation reagent It is preferably at least one of triphosgene, bis(2-pyridine) carbonate, N,N'-disuccinimidyl carbonate and 4-nitrophenyl chloroformate;

_{Wherein, R 1, R 2, R} 3, 4, Q, L 2, L P, L a is as previously defined R; G represents a leaving group, preferably halogen, hydroxy, C ₁ -C ₆ alkyl oxy or succinimidyloxy; the N-terminus of the peptide residue represented by ^{L P is linked to the group represented by L 2} and the C-terminus is linked to the group represented by ^{L a.}

Another aspect of the present invention provides a linker, characterized in that it is represented by the following formula (II)

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

^{Wherein, L 1, L 2, L} P, L a is defined above.

Another aspect of the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer A solvate of an isomer or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a pharmaceutical preparation comprising the aforementioned antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer A solvate of a isomer or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides the aforementioned antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Use of a solvate of the accepted salt, the aforementioned pharmaceutical composition and/or the aforementioned pharmaceutical formulation for the prevention and/or treatment of tumors or cancer.

In some embodiments, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic carcinoma.

Another aspect of the present invention provides a method of preventing or treating cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the aforementioned antibody-drug conjugate, a stereoisomer thereof, or a pharmacy thereof An acceptable salt of the above, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the aforementioned pharmaceutical composition and/or the aforementioned pharmaceutical formulation.

Another aspect of the present invention provides the aforementioned antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Use of a solvate of the accepted salt, the aforementioned pharmaceutical composition and/or the aforementioned pharmaceutical formulation for the manufacture of an agent for inhibiting cancer cell growth, proliferation or migration.

Another aspect of the present invention provides the aforementioned antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of accepted salts, the aforementioned pharmaceutical compositions and/or the aforementioned pharmaceutical formulations for use in inhibiting the growth, proliferation or migration of cancer cells.

Another aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the aforementioned antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof , or a solvate of the antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, the aforementioned pharmaceutical composition and/or the aforementioned pharmaceutical preparation.

Another aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the aforementioned antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody - a drug conjugate, a solvate of a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the aforementioned pharmaceutical composition and/or the aforementioned pharmaceutical preparation.

Description of drawings

Figure 1 shows the SEC-HPLC results of ADC-1 and the antibody is Herceptin antibody (ie ADC-1-b).

Figure 2 shows the SEC-HPLC results of ADC-2 and the antibody is the hRS7 antibody prepared in Example 1 (ie ADC-2-a).

3 is the SEC-HPLC result of ADC-4 and the antibody is hRS7 antibody prepared in Example 1 (ie ADC-4-a).

4 is the SEC-HPLC result of ADC-5 and the antibody is hRS7 antibody prepared in Example 1 (ie ADC-5-a).

5 is the SEC-HPLC result of ADC-6 and the antibody is hRS7 antibody prepared in Example 1 (ie ADC-6-a).

Figure 6 shows the inhibition results of five ADCs on the activity of MDA-MB-468 cells.

Figure 7 shows the results of inhibition of KPL-4 cell activity by five ADCs.

_{Figure 8 is the IC 50} dose-response curve of the test drug on BxPC-3.

9 is a measured IC ₅₀ dose-response curves of the drug on COLO 205.

_{Figure 10 is the IC 50} dose-response curve of the test drug on Calu-3.

_{Figure 11 is the IC 50} dose-response curve of the test drug on Calu-6.

_{Figure 12 is the IC 50} dose-response curve of the test drug on NCI-N87.

Figure 13 shows the antitumor activity of the test drugs in the BxPC-3 tumor model.

Figure 14 shows the effect of the test drug on the body weight of the BxPC-3 model.

Figure 15 shows the antitumor activity of the tested drugs in the COLO 205 tumor model.

Figure 16 shows the effect of the test drug on the body weight of the COLO 205 model.

Figure 17 shows the antitumor activity of the test drugs in the BxPC-3 tumor model.

Figure 18 shows the effect of the test drug on the body weight of the BxPC-3 model.

Figure 19 shows the antitumor activity of the test drugs in the Calu-3 tumor model.

Figure 20 shows the effect of the drug to be tested on the body weight of the Calu-3 model.

Figure 21 shows the antitumor activity of the tested drugs in the Capan-1 tumor model.

Figure 22 shows the effect of the drug to be tested on the body weight of the Capan-1 model.

detailed description

definition

For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, and γ chains, respectively. , alpha chains, and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.

The antibody light chain of the present invention may further comprise a light chain constant region comprising human or murine κ, λ chains or variants thereof.

The antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3).

Antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

In the present invention, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable region of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any fragment of an antibody, which is inserted into the antibody framework (e.g., by replacing the corresponding region) binding (i.e., or exhibits at least 10 ⁷ -10 ⁸ M ^-1 Ka of at least about) obtained immunization antigens specifically . A "functional fragment" or "analog of an anti-Trop-2 or Her2 antibody" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, functional fragments generally have the same meaning as "antibody fragments" and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') _2, and so on. Dimer (V _H -V _L dimer) "Fv" fragments consisting of the variable domain of a heavy chain and a light chain variable domain in noncovalent association formed by way of composition. In this configuration, the three CDRs of each variable domain interact to define _{the target binding site on the surface of the VH- VL} dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.

In the present invention, the term "Bispecific antibody" (BsAb) refers to an antibody and/or an antigen-binding molecule that can specifically bind to two different antigenic determinants. Generally, a bispecific antibody and/or an antigen-binding molecule contains Two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

In the present invention, "monoclonal antibody" or "monoclonal antibody" refers to a population of the same antibody, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods. For example, monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

In the present invention, a full-length antibody has two full-length heavy chains (eg VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region antibodies, such as those naturally produced by antibody-secreting B cells and those produced synthetically with the same domains.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.

"Humanized" antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.

In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody . For example, PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations within a CDR. Accordingly, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 two amino acid mutations within the CDRs.

In the present invention, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the passage of an antibody or antigen-binding fragment between the antibody and antigen's antibody binding sites The ability of non-covalent interactions to form one or more non-covalent bonds with alloantigens. The antigen may be an isolated antigen or present in tumor cells. Typically, immunospecifically bind (or specifically binds) an antibody or antigen is from about 1 × 10 ⁷ M ^-1 or 1x10 ⁸ M ^-1 or greater affinity constant Ka (or 1x10 ^-7 M or 1 × A dissociation constant (Kd) of 10 ^{−8 M or lower binds the antigen.} Affinity constants can be determined by standard kinetic methods of antibody response, eg, immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for detecting and monitoring binding rates in real time are known and commercially available.

In the present invention, the terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

In the present invention, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

In the present invention, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

In the present invention, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.

In the present invention, a "host cell" is a cell for receiving, maintaining, replicating and amplifying a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

In the present invention, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell. References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

In the present invention, the vector also includes "viral vector" or "viral vector". A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.

In the present invention, an "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.

The "drug (drug compound)" in the present invention, that is, "toxin", refers to a cytotoxic drug, that is, a compound represented by formula (I) (anti-tumor compound), which can strongly disrupt the normal growth of tumor cells. chemical molecule. In principle, cytotoxic drugs can kill tumor cells at a high enough concentration, but due to the lack of specificity, they can also lead to normal cell apoptosis while killing tumor cells. The term includes toxins, such as small molecule toxins or enzymatically active toxins of fungal, bacterial, plant or animal origin, radioisotopes (eg I ¹³¹ , Y ⁹⁰ , Re ¹⁸⁶ , I ¹²⁵ ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic agents Enzymes, preferably toxic drugs, more preferably camptothecin derivatives, more preferably gimatecan.

In the present invention, "C _a -C _b " (a and b represent an integer of 1 or more, a<b) includes any specific case of a to b carbons, for example, C ₁ -C ₆ includes C ₁ and C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , also including any one range of a to b, for example, C ₁ -C ₆ includes C ₁ -C ₃ , C ₁ -C ₄ , C ₁ -C ₅ , C _2- C ₅ , C ₂ -C ₄ , C ₃ -C _{6 ,} etc.; similarly, "ab-membered ring" (a and b represent an integer of 1 or more, a<b) represents that the number of ring atoms is a to b The ring structure, for example, 3-6 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, and also includes any range from a to b, for example, 3-6 membered ring includes 3-4 membered ring , 3-5-membered ring, 4-6-membered ring, 4-5-membered ring, etc.

In the present invention, "halogen" refers to fluorine, chlorine, bromine and iodine.

In the present invention, "C ₁ -C ₆ alkyl" refers to a straight-chain or branched alkyl group derived from an alkane moiety containing 1-6 carbon atoms by removing one hydrogen atom, specifically, C _1-6 Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl , neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3- Dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl Dimethylbutyl, 2-ethylbutyl, 1-methyl-2-methylpropyl, etc.; the "C _1-4 alkyl" refers to the moiety removed from an alkane containing 1-4 carbon atoms A linear or branched alkyl group derived from a hydrogen atom, specifically, C _1-4 alkyl groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , sec-butyl, tert-butyl.

In the present invention, the term "cycloalkyl" refers to a cyclized alkyl group which does not contain unsaturated bonds such as double bonds, such as C _3- C ₈ cycloalkyl, C _3- C ₇ cycloalkyl or C _3- C ₆ cycloalkyl. C _3- C ₆ cycloalkyl is meant to include C _3, C _4, C ₅ and C ₆ cycloalkyl. Example C _3- C ₆ cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

In the present invention, "C _1-6 alkoxy" refers to a "C _1-6 alkyl" group attached via an oxygen atom to the remainder of the molecule as defined above, i.e., "C _1-6 alkyl -O -" groups, specifically, include, but are not limited to, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy group, neopentyloxy, n-hexyloxy, etc.; the "C _1-4 alkoxy" refers to a group in which the above-defined "C _1-4 alkyl" is connected to the rest of the molecule through an oxygen atom , namely "C _1-4 alkyl-O-" group, specifically, including but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy group, sec-butoxy, tert-butoxy.

In the present invention, "5-6 membered ring" refers to a non-aromatic cyclic structure with 5-6 ring atoms, and the ring atoms can be all carbon atoms, thereby forming a carbocyclic ring; it can also contain 1- 3 ring heteroatoms each independently selected from N, O, or S, thereby forming a heterocycle (eg, oxygen-containing heterocycle, nitrogen-containing heterocycle, sulfur-containing heterocycle); the 5-6 membered ring may be saturated The structure can also be an unsaturated structure containing 1 or 2 carbon-carbon double bonds or carbon-carbon triple bonds.

In the present invention, "C ₆ -C ₁₀ aryl group" refers to an aromatic cyclic hydrocarbon group having 6-10 ring-forming carbon atoms, which can be a monovalent group or a divalent or higher group as required, including Monocyclic aryl group and fused-ring aryl group, "fused-ring aryl group" refers to an aryl group containing multiple rings (eg, containing 2) in which each ring in the group shares an adjacent pair of ring carbon atoms with other rings. . In the present invention, the "C ₆ -C ₁₀ -membered aryl group" specifically includes a phenyl group and a naphthyl group.

In the present invention, the term "5-membered nitrogen-containing heteroaryl group" refers to an aromatic 5-membered monocyclic group having at least one nitrogen heteroatom. Exemplary 5-membered nitrogen-containing heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, 1H-[1,2,3]triazolyl, etc., preferably 1H-[1 ,2,3]triazolyl.

表示作为该部分或取代基与核心或骨架结构的连接点的键。 In the present invention, in the structural formula

Represents the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

The "linker", "linker structure" or "linker" or "linking unit" in the present invention refers to a chemical structural fragment or bond that is connected to an antibody at one end and a drug (drug compound) at the other end. Other linkers can also be connected. It is then linked to the drug compound. The linker structure of the present invention can be synthesized by methods known in the art, or can be synthesized using the methods described in the present invention.

The "antibody-drug conjugate" of the present invention, namely ADC, refers to a ligand linked to a biologically active drug through a stable linking unit. In the present invention, it refers to linking the monoclonal antibody or fragment with the biologically active toxic drug through the linker structure.

In the present invention, "pharmaceutically acceptable salts" refer to relatively nontoxic acid addition salts or base addition salts of the conjugates of the present invention. The acid addition salts are salts formed by the conjugates of the present invention with suitable inorganic or organic acids, and these salts can be prepared by subjecting the conjugates of the present invention with suitable organic or inorganic acids in a suitable solvent reaction to prepare. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearic acid Salt, laurosilicate, borate, benzoate, lactate, nitrate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleic acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalene sulfonate, tartrate, benzoate, mesylate, p-toluene Sulfonate, gluconate, lactobionate and lauryl sulfonate, etc. The base addition salts are the salts formed by the conjugates of the present invention and suitable inorganic or organic bases, and these salts can be carried out by making the conjugates of the present invention and suitable inorganic or organic bases in a suitable solvent. reaction to prepare. Representative base addition salts include, for example, salts formed with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium salts, etc.; amine salts, including salts formed with ammonia (NH ₃ ), primary, secondary or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, and the like.

The conjugates of the present invention may exist in specific geometric or stereoisomeric forms. In the conjugates of the present invention, the chiral center may exist in the antitumor compound (the compound represented by formula (I)), or may exist in the The linker structure (the linker represented by formula (II)) may also exist in antibodies and derivatives thereof. In the present invention, all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, are included in the present invention within the range.

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of the conjugates of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide Pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).

In the present invention, solvates (eg, hydrates) of the conjugates of the present invention are also within the scope of the present invention. Specific examples of suitable solvates include solvates of the conjugate of the present invention with acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, and the like. Hydrates or ethanolates can also be cited.

In the present invention, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease. Treatment also includes any pharmaceutical use of the provided ADCs as well as the pharmaceutical compositions, pharmaceutical formulations provided herein.

In the present invention, "therapeutic effect" means the effect resulting from the treatment of an individual, which alters, generally ameliorates or ameliorates the symptoms of a disease or disease condition, or cures a disease or disease condition.

In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.

In the present invention, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have a desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

Documents mentioned herein are incorporated by reference in their entirety.

[Anti-tumor compound]

Hereinafter, the antitumor compounds linked to the antibody-drug conjugates of the present invention will be described. The antitumor compound is not particularly limited as long as it is a compound having an antitumor effect or a compound having a substituent capable of being linked to a linker structure. For antitumor compounds, a part or the whole of the linker is cleaved in tumor cells to free the antitumor compound part, thereby exhibiting an antitumor effect. When the linker is cleaved with the linking part of the drug, the antitumor compound is released in its original structure, and its original antitumor effect is exerted.

The antitumor compound in the present invention is a compound represented by the following formula (I).

In formula (I),

R ₁ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, the substituted ₇ R ₈ NR C ₁ -C ₆ alkyl, -SiMe ₃ Substituted C ₁ -C ₆ alkyl, or -CH=NO(C ₁ -C ₆ alkyl);

R ₂ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NR ₇ R ₈ or C ₁ -C ₆ substituted alkyl;

R ₃ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or NR ₇ R ₈ C(O)O- group;

R ₄ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy;

Or R ₁ and R ₂ may be joined together to form an optionally substituted 5-6 R ₉ membered ring with the parent moiety;

Or R ₃ and R ₄ may be joined together to form an optionally substituted ₉ membered oxygen-containing heterocyclic R 5-6 to the parent moiety;

Each occurrence of R ₇ and R ₈ is independently selected from hydrogen, C ₁ -C ₆ alkyl; or R ₇ and R ₈ can be taken together with the N atom to which they are attached to form a 5-6 membered _{optionally substituted with R 9} nitrogen-containing heterocycle;

Each occurrence of R ₉ is independently selected from halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, piper optionally substituted with C ₁ -C _{6 alkyl} pyridyl.

取代的C ₁-C ₄烷基、被-SiMe ₃取代的C ₁-C ₄烷基或-CH＝NO(C ₃-C ₆烷基)。 In certain preferred embodiments, R ₁ represents hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyl substituted with -NH(C 1 -C 4 alkyl), C ₁ -C ₄ alkyl substituted by

Substituted C ₁ -C ₄ alkyl, -SiMe ₃ substituted C ₁ -C ₄ alkyl or -CH = NO (C ₃ -C ₆ alkyl).

In certain preferred embodiments, R ₂ represents hydrogen, nitro, amino, or -N (C ₁ -C ₄ alkyl) ₂ substituted C ₁ -C ₄ alkyl.

In certain preferred embodiments, R ₃ represents hydrogen, halogen, hydroxyl, or

In certain preferred embodiments, R ₄ represents hydrogen or halogen.

其中

部分表示连接于母体基团的键。 In certain preferred embodiments, R ₁ and R ₂ are linked together to form a group shown below

in

A moiety represents a bond to the parent group.

其中

部分表示连接于母体基团的键。 In certain preferred embodiments, R ₃ and R _{4 are} joined together to form a group shown below

in

A moiety represents a bond to the parent group.

In certain preferred embodiments, the compound represented by formula (I) is a compound selected from the group consisting of:

In certain preferred embodiments, the compound represented by formula (I) is gimatecan:

In the case where the compound represented by the formula (I) is gimatecan, when L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ -, the antitumor compound of gimatecan and the formula (II) The linker structure shown by ) is connected with the structure of carbomate (-NC(=O)O-), which is more stable in blood and has less toxic and side effects.

In the present invention, in the antibody-drug conjugate, the number of linker-drug linkages (drug loading (DAR, drug load ratio)) connected to one molecule of the antibody affects the effectiveness and safety of the conjugate. The production of antibody-drug conjugates can be carried out by specifying reaction conditions such as the amount of raw materials and reagents used for the reaction in order to make the number of linker-drug linkages constant, but it is different from the chemical reaction of low-molecular-weight compounds. , usually obtained as a mixture of linked different numbers of drugs. Therefore, in the present invention, the number of linker-drug linkages attached to each molecule of the antibody is expressed as an average value, that is, the average number of drug linkages. In the present invention, unless otherwise specified, in principle, except for the case where antibody-drug conjugates with a specific number of drug linkages contained in antibody-drug conjugate mixtures with different numbers of drug linkages, the The number of connections refers to the average. The number of antitumor compounds linked to the antibody molecule can be controlled, and as the average number of drug linkages per antibody, about 1 to 10 antitumor compounds can be linked, preferably 2 to 8, more preferably 4 to 8, More preferably, it is 6-8. It should be noted that those skilled in the art can design the reaction of linking the necessary number of drugs on the antibody according to the description of the examples of the present application, and can obtain the antibody with the number of links of the anti-tumor compound controlled. In the examples of this application, the number of free sulfhydryl groups attached to each antibody molecule is not actually measured. By controlling the molar ratio of reactants and reaction conditions, it can be expected that the average number m of sulfhydryl groups attached to each antibody molecule is 6- 8.

[Joint structure]

In the antibody-drug conjugate of the present invention, the linker structure for connecting the antitumor compound and the antibody will be described.

The linker has the structure shown in the following formula (II):

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

In formula (II), L ¹ represents -(succinimide-3-yl-N)-(CH ₂ )n ¹ -C(=O)-, and n ¹ represents an integer of 1 to 8,

L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ CH(CH ₃ )-O)n ² -(CH ₂ ) n ³ -C(=O)- or single bond, n ² represents an integer of 1-6, n ³ represents an integer of 1-4,

L ^P represents a peptide residue composed of 2 to 7 amino acids and,

L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen, optionally _{C 1} -C ₆ alkyl substituted with 1 or 2 hydroxy groups; Aryl represents a C ₆ -C ₁₀ aryl group _{optionally substituted by R 9} ^{, n 4} represents an integer of 1 to 4, and n ⁵ represents an integer of 1 to 4 ;

-(Succinimide-3-yl-N)- is of the formula:

The structure shown is connected to the antibody at the 3-position of the structure, and is connected to the methylene group in the linker including the structure at the nitrogen atom at the 1-position.

[L ¹ part]

L ¹ represents -(succinimide-3-yl-N)-(CH ₂ )n ¹ -C(=O)-, and n ¹ represents an integer of 1 to 8.

Wherein, -(succinimide-3-yl-N)- is the following formula:

The indicated structure is linked to the antibody at the 3-position of the structure.

In certain embodiments of the invention, the linkage to the antibody at position 3 is characterized by the formation of a thioether for linkage. On the other hand, the nitrogen atom at the 1-position of the moiety is connected to the carbon atom of the methylene group present in the linker including the structure. That is, -(succinimidyl-3-yl-N)-(CH ₂ )n ¹ -C(=O)- is connected to the antibody through a thioether bond, and has the structure shown in the following formula (here, ""AB" denotes an antibody, and AB-S- is a structure derived from an antibody).

In certain embodiments of the present invention, n ¹ represents 2, 3, 4, 5 or 6.

[Part ^{L 2]}

L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ CH(CH ₃ )-O)n ² -(CH ₂ ) n ³ -C(=O)- or a single bond, n ² represents an integer of 1-6, and n ³ represents an integer of 1-4.

In certain embodiments of the invention, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-. The solubility of the drug is enhanced by the polyethylene glycol moiety.

In certain embodiments of the present invention, L ² represents a single bond.

In certain embodiments of the present invention, n ² represents an integer from 1 to 4, preferably 2, 3 or 4.

In certain embodiments of the present invention, n ³ represents an integer from 1 to 4, preferably 2 or 3.

[L ^P section]

L ^P represents a peptide residue composed of 2 to 7 amino acids. That is, it consists of oligopeptide residues in which 2-7 amino acids are linked by peptide bonds. Is not particularly limited in the amino acids L ^P, for example, L- or D- amino acids, preferably L- amino acids. In addition to α-amino acids, amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may be used, and, for example, non-natural amino acids such as N-methylated amino acids may be used. type of amino acid.

^{The amino acid sequence of the LP} moiety is not particularly limited, and the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), and glycine (Gly). ; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit; C), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D) and the like. Among these, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid are preferably used. The pattern of drug release can be controlled according to the type of amino acid. The number of amino acids can be 2-7.

In certain embodiments of the invention, the peptide residues represented by ^P L ² to L N-terminus portion, connecting the L ^a portion of the C-terminus.

In certain embodiments of the present invention, L ^P by 2-5 amino acid residues constituting the peptide.

In certain embodiments of the present invention, L ^P is a peptide residue selected from the following:

-ggfg-;

-vc-;

-evc-;

-dvc-;

-EGGFG-;

-DGGFG-.

[L ^a part]

L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen, optionally _{C 1} -C ₆ alkyl substituted with 1 or 2 hydroxy groups; Aryl represents a C ₆ -C ₁₀ aryl group _{optionally substituted by R 9} ^{, n 4} represents an integer of 1 to 4, and n ⁵ represents an integer of 1 to 4 .

In certain embodiments of the invention, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ -, wherein n ⁴ represents 2, 3 or 4; each occurrence of _{R 10 independently represents hydrogen} , Methyl, ethyl, propyl, isopropyl optionally substituted by one hydroxyl group.

In certain embodiments of the present invention, L ^a represents -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, wherein R ₁₀ represents hydrogen, methyl, ethyl, propyl; n ⁵ represents 1-2 An integer of , Aryl represents a benzene ring group; preferably, the -NR ₁₀ - group and the -(CH ₂ )n ⁵ - group are located in the para position of the benzene ring

In certain embodiments of the present invention, L ^a represents a structure represented by 4-aminobenzyl alcohol derived.

In certain embodiments of the present invention, the C-terminal peptide residue represented by L ^P is connected to the group represented by L ^a and, more specifically, to the C-terminus end group represented by L ^a in amino.

In certain embodiments of the present invention, the linker represented by formula (II) is a group selected from the group consisting of:

[Linker-drug intermediate compound]

In the linker-drug intermediate compound of the present invention, the compound represented by the following formula (I) is linked to the linker structure represented by the following formula (III) using the oxygen in the 19-position hydroxyl group in the compound represented by the formula (I) as a linking site The linker-drug intermediate compound represented by formula (IV);

QL ² -L ^P -L ^a -C(=O)- (III)

Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described in the description of the present invention;

L ^2, L ^P, L ^a are as defined in the description of the present invention;

Q represents (maleimide-N)-

In certain embodiments of the invention, the linker-drug intermediate compound is a compound selected from the group consisting of,

[Antibody-Drug Conjugates]

In the antibody-drug conjugate of the present invention, a compound represented by the following formula (I) and an antibody are linked via a linker represented by the following formula (II) through a thioether bond formed by a disulfide bond moiety present in the hinge portion of the antibody The antibody-drug conjugate represented by formula (V) is obtained.

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II)

_{R 1, R 2, R 3} , R ₄ are as defined in the description of the present ^{invention; L 1, L 2, L} P, L a are as defined in the description of the present invention; AB represents the antibody.

[Manufacturing method of antibody-drug conjugate]

Next, a representative production method of the antibody-drug conjugate of the present invention or its production intermediate will be described.

Specifically, in the present invention, an antibody-drug conjugate in which an antibody and a linker structure are linked via a thioether can be produced, for example, by the following method.

That is, the linker-drug intermediate compound represented by the formula (IV) is reacted with AB-SH to connect the linker-drug represented by the formula (IV) through the thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The intermediate compound is linked with the antibody; the antibody-drug conjugate represented by formula (V) is prepared. _{Wherein, R 1, R 2, R} 3, R 4, L 1, L 2, L P, L a are as defined in the description of the present invention.

In the formula, AB-SH represents an antibody carrying a sulfhydryl group, AB represents an antibody, the compound represented by the formula (IV) is the above-mentioned linker-drug intermediate compound of the present invention, and the compound represented by the formula (V) as the product is Antibody-drug conjugates of the present invention.

For convenience of explanation, the compound represented by the formula (V) is described in the form of a structure in which one structural moiety from the drug to the linker terminal is linked to one antibody, but in fact, it is relative to one antibody molecule. In general, there are many cases where a plurality of such structural parts are connected. For example, as described above, 2 to 8, preferably 4 to 8, more preferably 6 to 8 linker-drug intermediate compounds are linked to one antibody molecule. This is also the same in the following description of the manufacturing method. In fact, as described above, in the present invention, the average number of linker-drugs linked to each molecule of antibody is expressed as the average number of drug linkages.

That is, as described above, the antibody-drug conjugate represented by formula (V) can be produced by reacting the above-mentioned linker-drug intermediate compound of the present invention with the antibody AB-SH having a thiol group.

Antibodies having thiol groups can be obtained by methods known to those skilled in the art (Hermanson, G.T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, the following methods are exemplified: allowing Traut to react with the amino group of the reagent and the antibody; and allowing N-succinimidyl S-acetylthioalkanoate to react with the amino group of the antibody After reacting with hydroxylamine; after reacting N-succinimidyl 3-(pyridyldithio)propionate, reacting reducing agent; reacting dithiothreitol, 2-mercaptoethanol, tri( A reducing agent such as 2-carboxyethyl) phosphine hydrochloride (TCEP) acts on the antibody to reduce the disulfide bond in the hinge portion of the antibody to generate a sulfhydryl group; etc., but not limited to these methods.

Specifically, using 0.3 to 3 molar equivalents of TCEP per disulfide bond in the hinge portion of the antibody as a reducing agent, and reacting it with the antibody in a buffer containing a chelating agent, partial or A sulfhydryl group-carrying antibody (AB-SH) obtained by completely reducing the disulfide in the hinge part of the antibody. As a chelating agent, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), etc. are mentioned, for example. They can be used in concentrations of 1 mM to 20 mM. As the buffer solution, sodium phosphate, sodium borate, sodium acetate solution or the like can be used. In a specific example, by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours, an antibody AB-SH partially or completely reduced to have a thiol group can be obtained.

With respect to each antibody AB-SH having a thiol group, 2 to 20 molar equivalents of the compound represented by the formula (IV) can be used to produce an antibody-drug conjugate in which 2 to 8 drugs are linked to one antibody ( V). Specifically, a solution in which the compound represented by the formula (IV) is dissolved is added to a buffer solution containing the antibody AB-SH having a thiol group and allowed to react. Here, as the buffer solution, sodium acetate solution, sodium phosphate, sodium borate, or the like can be used. The pH during the reaction is 5 to 9, and the reaction is more preferably near pH 7. As a solvent for dissolving compound (2), dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP) can be used and other organic solvents. The organic solvent solution in which the compound represented by formula (IV) is dissolved at 1 to 20% v/v can be added to the buffer solution containing the antibody AB-SH having a thiol group, and the reaction can be carried out. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by inactivating the reactivity of the unreacted compound of formula (IV) with a thiol-containing reagent. Thiol-containing reagents are, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, 1 to 2 molar equivalents of NAC are added to the compound represented by the formula (IV) to be used, and the reaction is completed by incubating at room temperature for 10 to 30 minutes.

For the produced antibody-drug conjugate (V), operations such as concentration, buffer exchange, and purification can be performed by the following common operations.

Common Procedure A: Concentration of Aqueous Antibody or Antibody-Drug Conjugates

In the container, put the antibody or antibody-drug conjugate solution, use a centrifuge for centrifugation (for example, centrifuge at 2000G-3800G for 5-20 minutes), and concentrate the antibody or antibody-drug conjugate solution.

Common operation B: Determination of antibody concentration

Antibody concentration was measured using a UV analyzer according to the manufacturer's instructions.

At this time, the absorbance at 280 nm (1.3 mLmg ^-1 cm ^-1 to 1.8 mL mg ^-1 cm ^-1 ) which differs depending on the antibody was used.

Common operation C-1: Buffer exchange of antibodies

Phosphate buffer (eg, 10 mM, pH 6.0) containing sodium chloride (eg, 137 mM) and ethylenediaminetetraacetic acid (EDTA, eg, 5 mM) (also referred to in this specification as PBS6.0/EDTA). The NAP-25 column using Sephadex G-25 vector was equilibrated. One of the NAP-25 columns was packed with 2.5 mL of aqueous antibody solution, and then the fraction (3.5 mL) eluted with PBS6.0/EDTA 3.5 mL was separated. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

Common operation C-2: Buffer exchange of antibodies

Phosphate buffer (eg, 50 mM, pH 6.5) containing sodium chloride (eg, 50 mM) and EDTA (eg, 2 mM) (also referred to in this specification as PBS6.5/EDTA) according to the manufacturer's protocol . The NAP-25 column using Sephadex G-25 vector was equilibrated. One of the NAP-25 columns was packed with 2.5 mL of aqueous antibody solution, and then a fraction (3.5 mL) eluted with PBS6.5/EDTA 3.5 mL was obtained by separation. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 20 mg/mL using PBS6.5/EDTA.

Common Operation D-1: Purification of Antibody-Drug Conjugates

Use commercially available phosphate buffer (eg, PBS7.4), sodium phosphate buffer (eg, 10 mM, pH 6.0; also referred to as PBS6.0 in this specification) containing sodium chloride (eg, 137 mM), or The NAP-25 column is equilibrated with any of the acetic acid buffers (eg, 10 mM, pH 5.5; also referred to herein as ABS) containing sorbitol (eg, 5%). An antibody-drug conjugate reaction aqueous solution (for example, about 1.5 mL) is packed in the NAP-25 column, and the antibody fraction is obtained by separation by eluting with an amount of buffer specified by the manufacturer. The fractions obtained by this separation were re-packed on a NAP-25 column, eluted with a buffer, and subjected to a gel filtration purification operation. The operation was repeated 2 to 3 times, whereby unconnected drug linkers were removed. Antibody-drug conjugates of molecular compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide).

[Production method of linker-drug intermediate compound]

Next, a typical production method of the linker-drug intermediate compound of the present invention will be described.

Wherein, in the compound represented by formula (VI), ^{the definitions of Q and L 2} are as described in the present invention, and G represents a leaving group commonly used in the art, such as halogen, hydroxyl, C ₁ -C ₆ alkoxy , succinimidyloxy.

A compound represented by formula (VII), the L ^P as defined in the present invention, L ^a of the present invention as defined; C-terminal peptide residue represented by L ^P L ^a is connected to a group represented by . More specifically, the C-terminal end of L ^a shown connected to the amino group.

The compound represented by the formula (VI) and the compound represented by the formula (VII) can be reacted under conditions conventional in the art. For example, the reaction can be carried out in an organic solvent in the presence of a base.

Make the compound shown in formula (VIII) and the compound shown in formula (I) react in the presence of alkoxycarbonylating reagents (alkoxycarbonylating agents) to obtain the linker-drug intermediate compound shown in (IV); the described Alkoxycarbonylation reagents include, but are not limited to, triphosgene, di(2-pyridyl)carbonate; N,N'-disuccinimidyl carbonate carbonate); 4-nitrophenyl chloroformate. The compound represented by the formula (VIII) and the compound represented by the formula (I) can be reacted under conditions conventional in the art. For example, the reaction can be carried out in an organic solvent in the presence of a base.

Examples of the base include carbonates and alkali metal alkoxides of alkali metals or alkaline earth metals such as sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride, and potassium hydride. , alkali metal hydroxides or hydrides, or organometallic bases represented by alkyl lithiums such as n-butyllithium, and dialkyl lithium amides such as lithium diisopropylamide; bis(trimethylmethane) Silyl) Organometallic bases of bissilylamines such as lithium amide; or pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methyl Organic bases such as morpholine, diisopropylethylamine, diazabicyclo[5.4.0]undec-7-ene (DBU), and the like. Examples of the inert solvent usable in this reaction include halogenated hydrocarbon-based solvents such as dichloromethane, chloroform, and carbon tetrachloride; tetrahydrofuran, 1,2-dimethoxyethane, dioxane, and the like. Ether-based solvents; aromatic hydrocarbon-based solvents such as benzene and toluene; amide-based solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidin-2-one. In addition to the above-mentioned inert solvents, sulfoxide-based solvents such as dimethyl sulfoxide and sulfolane; ketone-based solvents such as acetone and methyl ethyl ketone, etc. can be used in some cases.

In the reaction step of producing the antibody-drug conjugate or the linker-drug intermediate compound of the present invention, groups that need to be protected, such as amino groups, can be protected with a protecting group as needed. For example, suitable amino protecting groups include, but are not limited to: tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), benzyl (Bn), acetyl and trifluoroacetyl. More specifically, the Boc protecting group can be removed by treatment with an acid such as trifluoroacetic acid or hydrochloric acid. Cbz and Bn protecting groups can be removed by catalytic hydrogenation, and acetyl and trifluoroacetyl protecting groups can be removed by a variety of conditions including the use of sodium, potassium or lithium hydroxide in aqueous organic or alcoholic solvents. Methods of using protecting groups and suitable amino protecting groups are described in Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1999).

In the antibody-drug conjugate of the present invention, the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof. In certain embodiments of the invention, the antibody is selected from the group consisting of anti-Trop-2 antibody, Her2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20, CD30 Antibodies, CD19 antibodies, CD33 antibodies.

In some embodiments, preferred antibodies of the invention are Trop-2 antibodies or antigen-binding fragments thereof, including bispecific antibodies and functional derivatives of antibodies. The full name of Trop-2 antibody is antibody human trophoblast cell surface antigen 2 (Trop-2), which is a 40kDa transmembrane glycoprotein encoded by the TACSTD2 gene. Trop-2 was first identified in a human trophoblastic choriocarcinoma cell line, and its intracellular tail plays an important role in controlling many signaling pathways that regulate cellular functions, such as cell-cell adhesion, cell proliferation, and cell migration. Trop-2 protein is found in many human tumors (such as breast, colorectal, lung, pancreatic, ovarian, prostate, cervical, renal, urethral, glioblastoma, melanoma, liver, bladder, Gastric cancer, esophageal cancer) are expressed on the cell surface, but only limited in normal human tissues. Trop-2 has the functions of regulating tumor cell growth and promoting tumor cell invasion and metastasis. Examples of Trop-2 antibodies that can be used in the present invention include, but are not limited to: m7E6, h7E6, h7E6_SVG, h7E6_SVG1, h7E6_SVG2, h7E6_SVG3, h7E6_SVG4, h7E6_SVG5, h7E6_SVG6, h7E6_SVG7, h7E6_SVG8, h7E6_SVG9, h7E6_SVG5, h7E6_SVG6, h7E6_SVG7, h7E6_SVG8, h7E6_SVG1 h7E6_SVG12, h7E6_SVG13, h7E6_SVG14, h7E6_SVG15, h7E6_SVG16, h7E6_SVG17, h7E6_SVG18, h7E6_SVG19, h7E6_SVG20, h7E6_SVG21, h7E6_SVG22, h7E6_SVG23, h7E6_SVG24, h7E6_SVG25, h7E6_SVG26, h7E6_SVG27, h7E6_SVG28, h7E6_SVG29, h7E6_SVG30, h7E6_SVG31, h7E6_SVG32, h7E6_SVGL, h7E6_SVGL1, h7E6_SVGL2, h7E6_SVGL3, h7E6_SVGL4, h7E6_SVGL5, h7E6_SVGN, m6G11, h6G11, h6G11_FKG_SF; AR47A6.4.2 described in US Patent No. 7,420,041; BR110 described in US Patent No. 5,850,854; RS7 described in US Patent No. 6,653,104; RS7 in Patent No. 7,517,964; and hRS7 described in US2012/0237518. The anti-Trop-2 antibody that can be used in the present invention can also be obtained by screening the method of vector design, construction and construction of an antibody library for displaying antibodies disclosed in CN103476941A, or it can also be obtained from the library of Sorrento Therapeutics, Inc. filter to obtain.

The Trop-2 of the natural sequence in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or a combination thereof.

The antibody used in the present invention is preferably an anti-human Trop-2 antibody.

In certain preferred embodiments, the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human Trop-2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.

In certain preferred embodiments, the anti-human Trop-2 antibody may be a humanized antibody or a fully human antibody.

In certain preferred embodiments, the antibody is the RS7 antibody of CN100360567C, wherein the complementarity determining region (CDR) of the light chain variable region of the humanized or chimeric RS7 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIAVA; by CDR2 consisting of the SASYRYT amino acid sequence; and CDR3 consisting of the QQHYITPLT amino acid sequence, and wherein the CDRs of the heavy chain variable region of the humanized or chimeric RS7MAb include CDR1 consisting of the NYGMN amino acid sequence; CDR2 consisting of the WINTYTGEPTYTDDFKG amino acid sequence; and CDR3 consisting of the amino acid sequence of GGFGSSYWYFDV. The light chain sequence and heavy chain sequence of RS7 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Also included are those antibodies that retain Trop-2 binding activity after conservative amino acid substitutions to the above-mentioned antibodies.

In other embodiments, preferred antibodies of the invention are Her2 antibodies or antigen-binding fragments thereof, including bispecific antibodies and antibody functional derivatives. Her2 is also known as human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2), or receptor tyrosine protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent animal) or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans.

Her2 overexpression has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. Her2 overexpression occurs in approximately 15-30% of breast cancers. In recent years, this protein has become an important biomarker and therapeutic target in about 30% of breast cancer patients. Her2 overexpression also occurs in ovarian cancer, intestinal gastric cancer, and invasive forms of uterine cancer such as serous endometrial cancer.

Examples of Her2 antibodies that can be used in the present invention include, but are not limited to: trastuzumab described in US5821337, pertuzumab described in CN101981056B, antibodies that can be used in the present invention can also be obtained by CN103476941A The methods for designing, constructing and constructing an antibody library for displaying antibodies disclosed in the vector can be obtained by screening, and can also be obtained by screening the library of Sorrento Therapeutics, Inc.

The natural sequence Her2 in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or their combination.

The antibody used in the present invention is preferably an anti-human Her2 antibody.

In certain preferred embodiments, the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human Her2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.

In certain preferred embodiments, the anti-human Her2 antibody may be a humanized antibody or a fully human antibody.

In certain preferred embodiments, the Her2 antibody is the trastuzumab antibody described in US5821337, and the complementarity determining region (CDR) of its light chain variable region comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA; CDR2 consisting of the amino acid sequence of SASFLYS and CDR3 consisting of the QQHYTTPPT amino acid sequence, and the CDRs of its heavy chain variable region include CDR1 consisting of the DTYIH amino acid sequence; CDR2 consisting of the RIYPTNGYTRY amino acid sequence; and CDR3 consisting of the WGGDGFYAMDY amino acid sequence. The light chain sequence and heavy chain sequence of the trastuzumab antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Also included are those antibodies that retain Her2-binding activity after conservative amino acid substitutions to the above-mentioned antibodies.

The antibody-drug conjugates of the present invention can be preferably administered to mammals, more preferably humans.

The substance to be used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention can be appropriately selected from formulation additives or others commonly used in the art in consideration of the dose and concentration to be administered.

In certain embodiments of the present invention, the antibody-drug conjugates of the present invention may be administered in the form of a pharmaceutical composition or pharmaceutical formulation containing one or more pharmaceutically suitable ingredients. For example, the above-mentioned pharmaceutical compositions or pharmaceutical preparations may typically contain one or more pharmaceutically acceptable carriers (such as sterile liquids (such as water and oils (including petroleum, animal, vegetable, or synthetic origin) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case of intravenous administration of the above-mentioned pharmaceutical composition, water is a more representative carrier. In addition, saline solution, as well as aqueous dextrose and glycerol solutions are also Can be used as liquid carrier, especially can be used for injection solution.Appropriate pharmaceutical excipients are known in this field.As required, the above-mentioned composition can also contain a trace amount of wetting agent or emulsifying agent, or pH buffering agent Examples of suitable pharmaceutical carriers are described in "Examples of W. Martin Carriers armaceutical Sciences" by EW Martin. The prescription corresponds to the mode of administration.

Various delivery systems are known and can be used for administration of the antibody-drug conjugates of the invention. The introduction method includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration, for example, can be by infusion or bolus injection. In certain preferred embodiments, the administration of the antibody-drug conjugates described above is performed by infusion. Parenteral administration is the preferred route of administration.

In a representative embodiment, the above-mentioned pharmaceutical composition is formulated into a pharmaceutical composition for intravenous administration to humans, and is formulated according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The above-mentioned pharmaceutical composition may further contain a solubilizer and a local anesthetic (eg, lidocaine) for relieving pain at the injection site, as required. Generally, the above ingredients may be supplied either as a dry freeze-dried powder or anhydrous concentrate in a sealed container (eg, an ampule or sachet, etc., which indicates the amount of active agent), respectively, Or mixed together in unit dosage form. When the above-mentioned medicine is intended to be administered by infusion, for example, the above-mentioned medicine may be put into an infusion bottle containing sterilized pharmaceutical-grade water or saline. When the above-mentioned drugs are administered by injection, ampoules of sterile water for injection or saline may be provided so that, for example, the above-mentioned components are mixed before administration.

The pharmaceutical composition or pharmaceutical preparation of the present invention may be a pharmaceutical composition or pharmaceutical preparation containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition or pharmaceutical preparation containing the antibody-drug conjugate and at least one other cancer therapeutic agent pharmaceutical composition. The antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, whereby the anticancer effect can be enhanced. Other anticancer agents used for this purpose may be administered to the individual simultaneously, separately or sequentially with the antibody-drug conjugate, or may be administered at varying intervals. Examples of such cancer therapeutic agents include albumin-bound paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, or international publications. The agents described in the pamphlet No. WO2003/038043, as well as LH-RH analogs (leuprolide, goserelin, etc.), estradiol phosphate mustard, estrogen antagonists (tamoxifen, ranloxacin, etc.) Xifen, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane), etc., but they are not limited as long as they have antitumor activity.

Such pharmaceutical compositions can be formulated in the form of freeze-dried preparations or liquid preparations as preparations having a selected composition and necessary purity. When a preparation is formed as a freeze-dried preparation, it may be a preparation containing appropriate preparation additives available in the art. In addition, also in the case of a liquid preparation, a preparation can be formed as a liquid preparation containing various preparation additives usable in the art.

The composition and concentration of the pharmaceutical composition vary depending on the administration method, but the affinity of the antibody-drug conjugate contained in the pharmaceutical composition of the present invention for the antigen of the antibody-drug conjugate, that is, the affinity for the antigen, varies. Considering the dissociation constant (Kd value), when the affinity is higher (the Kd value is lower), the drug effect can be exhibited even with a small dose. Therefore, when determining the administration amount of the antibody-drug conjugate, the administration amount can also be set based on the state of the affinity of the antibody-drug conjugate with the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, it may be administered once at about 0.001 to 100 mg/kg, or may be administered multiple times at intervals of 1 to 180 days.

The antibody-drug conjugates, pharmaceutical compositions, and pharmaceutical preparations of the present invention can be used to prevent and/or treat tumors or cancers. The tumor or cancer targeted for prevention and/or treatment may be any cancer cell that expresses a protein recognized by the antibody in the antibody-drug conjugate. In certain embodiments of the invention, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma tumor, liver cancer, bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic carcinoma.

In certain embodiments of the invention, a prophylactically or therapeutically effective amount of an antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the invention is administered to a subject in need thereof for inhibiting the growth, proliferation or migrate.

In certain embodiments of the present invention, there is provided a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the present invention.

Technical effect of the present invention:

The antibody-drug conjugate of the present invention has fast and efficient tumor cell killing activity, and at the same time has good biocompatibility, low immunogenicity, biological safety and stability.

The linker structure of the present invention such as formula (II) has the following advantages: (1) the linker structure of the present invention has suitable molecular weight and hydrophobicity, and most of the prepared products have higher drug loading (DAR, dug to antibody ratio)> 7; (2) The linker structure of the present invention can improve the anti-aggregation ability of the linker-drug compound; help to improve the hydrophilicity of gimatecan in the antitumor compound, and increase the biological safety of ADC; (3) the present invention The release of the linker structure is especially suitable for the cytotoxicity, pharmacokinetics, and tumor inhibition properties of the preferred toxin gimatecan. The self-cleavage of the linker is faster, which is conducive to the rapid release of gimatecan, greatly Enhanced drug efficacy; since the linker of the present invention is rapidly cleaved in the cell, the intracellular effect is rapid; (4) the size, physicochemical properties and coupling site of the linker designed by the present invention will not affect the physiological activity of the antibody; (5) The synthesis method of the linker compound of the present invention is simple and suitable for industrial production.

Preferably, the anti-tumor compound of the present invention selects gimatecan, whose toxicity is about 10 times that of SN-38, and is comparable to ixatecan, but its safety is much better than ixatecan, and it can be used alone as Oral preparation is made into medicine, and the present invention selects this molecule as the toxin of ADC, which greatly increases the biological safety of ADC. In addition, gimatecan has a strong ability to penetrate cell membranes, allowing them to kill nearby cancer cells after killing the cancer cells that have engulfed the ADC.

Example

The solution of the present invention will be explained below with reference to specific embodiments. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention. If no specific technique or condition is indicated in the following examples, the technique or condition described in the literature in this field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Trastuzumab antibody (Herceptin antibody) was purchased from Genentech Inc.

Example 1: Preparation and detection of hRS7 antibody

1. Gene synthesis, transfection and antibody preparation

hRS7 antibody was produced in CHO cells. The expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods, and the nucleotide sequences of the light chain and heavy chain of the hRS7 antibody were shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Insert the above two sequences into the same expression vector, extract and prepare transfection plasmids in large quantities, and transfect them into CHO-K1 cells (ATCC CCL-61). The specific transfection and antibody preparation processes are as follows:

(1) Cell culture: CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium at 37°C, 7% CO ₂ , 140 rpm, and 90% relative humidity;

(2) Transfection: After entering the logarithmic growth phase, centrifuge the cells, resuspend them in fresh ActiPro medium, count and adjust the cell density to 1.35×10 ⁷ cells/ml, and transfer 500 μl of the cell suspension into the electroporation cup , then add 40 μg of the constructed plasmid, mix the cells with the plasmid, and then introduce the plasmid by electroporation (Bio-rad electroporator);

(3) Subcloning: The electroporated cells were resuspended in ActiPro medium at 37°C, and 100 μl per well was dispensed into a 96-well plate. Cell supernatants were assayed to determine antibody expression levels. The clones with higher expression levels were transferred from the 96-well plate to the 24-well plate for culture, and then transferred to the 6-well plate for culture. The antibody production and yield of the cells were determined, and the 3 clones with the highest expression levels were selected for subcloning, and then transferred to into a shaker flask and placed in an incubator to continue culturing.

2 Purification of antibodies

The highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity (GE, Mab Select SuRe) and ion exchange (GE, Capto S). The molecular weight and purity of the purified antibodies were analyzed by SDS-PAGE and SEC-HPLC. The results of SDS-PAGE showed that the molecular weight of the prepared hRS7 was in line with expectations, and the purity of the antibody was 99.1% measured by SEC-HPLC.

Example 2: Preparation of conjugate ADC-1

Preparation of I-1 Intermediate A

I-1.1 Dissolve A1 (170 mg, 0.4 mmol) and VC-PABOH (150 mg, 0.4 mmol) in DMF (5 mL), react at room temperature overnight, HPLC shows that a new peak is generated, and A2 is obtained after Pre-HPLC purification Pure about 120 mg, about 43.5% yield. LCMS: [M+1] ⁺ = 690.6.

I-1.2. Gimatecan (44.7 mg, 0.1 mmol), DMAP (36.6 mg, 0.3 mmol) and triphosgene (14.8 mg, 0.05 mmol) were weighed and added to a round bottom flask. After adding DCM (2 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then A2 (68.9 mg, 0.1 mmol) was added, and stirring was continued for about 4 minutes, the reaction solution was passed through a short silica gel column, and DCM:MeOH was used. After eluting with the eluent of =10:1, concentrating, and purifying by Pre-HPLC to obtain about 10 mg of product A, the yield: 8.6%. LCMS: [M+1] ⁺ =1164.3.

Synthesis of I-2 Coupling Crude Product ADC-1

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound A dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the coupled crude product ADC-1-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold substance amount of Compound A dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-1-b.

Detection of I-3 Coupling Crude Product ADC-1

The coupling reaction crude product is detected by SEC, and the SEC chromatographic conditions are as follows:

Column model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm

Detector wavelength: 280nm/363nm

Column temperature: 30℃

Flow rate: 1mL/min

Elution method: isocratic elution

Injection volume: 10 μL

Running time: 55min

Through the detection of SEC, 280nm and 363nm were compared, and the absorption at 363nm at the peak position of the protein was significantly enhanced, indicating that the small molecule has been coupled to the protein.

I-4 coupling reaction product purification:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-1 is shown in Figure 1. The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of I-5 DAR

The absorbance values of conjugate and naked antibody at 280nm and 363nm were measured by UV spectrophotometer. The concentration of Gimatecan in the conjugate was calculated from the absorbance at 363 nm according to the standard curve. The concentration of antibody in the conjugate was calculated by subtracting the absorbance of gimatecan at 280 from the absorbance at 280 nm. The results are shown in Figure 1 and below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-1-a was calculated from the ratio of these two concentrations to be 2.7, ie, n was 2.7.

When the antibody is Herceptin antibody:

The DAR value of ADC-1-b was calculated from the ratio of these two concentrations to be 7.3, ie, n was 7.3.

Example 3 Preparation of conjugate ADC-2

Preparation of II-1 Intermediate B

II-1.1. A1 (85 mg, 0.2 mmol) and L-glutamic acid-5-tert-butyl ester (40.6 mg, 0.2 mmol) were dissolved in DMF (3 mL), stirred at room temperature overnight, HPLC showed a new peak Generated and purified by Pre-HPLC to obtain about 60 mg of the target product B2, yield: 58.8%. LCMS: [M-1] ^- =512.5.

II-1.2 B2 (51.3 mg, 0.1 mmol), VC-PABOH (40 mg, 0.11 mmol) and EEDQ (37.0 mg, 0.15 mmol) were dissolved in DCM/MeOH (3 mL/3 mL) and reacted at room temperature overnight, HPLC detects that a new peak is generated, and the raw material B2 has disappeared. After adding an appropriate amount of ether to the reaction solution for sedimentation, a solid is precipitated. After 5 times of centrifugation, the solid can be directly used in the next reaction. The solid was added to the solution of TFA (2mL). , after 2 hours of reaction, a new peak was detected by HPLC. After Pre-HPLC purification, about 40 mg of the target product B3 was obtained, and the yield was 48.9%. LCMS: [M-1] ^- = 817.6.

II-1.3 Gimatecan (22.3 mg, 0.05 mmol), DMAP (18.3 mg, 0.15 mmol) and triphosgene (7.4 mg, 0.025 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then B3 (38 mg, 0.046 mmol) was added, and stirring was continued for about 4 minutes, and the reaction solution was passed through a short silica gel column, using DCM:MeOH= After eluting with 10:1 eluent, concentration, and Pre-HPLC purification, about 5 mg of product B was obtained, yield: 7.8%. LCMS: [M-1] ^- = 1291.2.

Synthesis of II-2 Coupling Crude Product ADC-2

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound B dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the coupled crude product ADC-2-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold amount of Compound B dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-2-b.

Detection of II-3 Coupling Crude Product ADC-2

The detection method is as described in step I-3 of Example 2.

II-4 Coupling reaction product purification:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-2 was similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of II-5 DAR

DAR was determined as described in Example 2, steps 1-5. The results are shown in Figure 2 and below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-2 of ADC-2-a is 7.6, ie, n is 7.6.

When the antibody is Herceptin antibody:

The DAR value of ADC-2 of ADC-2-b is 7.5, ie, n is 7.5.

Example 4 Preparation of conjugate ADC-3

Preparation of III-1 Intermediate C

III-1.1 Dissolve C1 (87.2 mg, 0.2 mmol), PABOH (25 mg, 0.2 mmol) and EEDQ (74.1 mg, 0.3 mmol) in DCM/MeOH (5 mL/5 mL), stir at room temperature overnight, and detect by HPLC A new peak is generated, and the raw material C1 has disappeared. After adding an appropriate amount of ether to the reaction solution for sedimentation, a solid is precipitated. After 5 times of centrifugation, a white solid crude product of about 100 mg is obtained, and the solid is added to TFA/DCM=1/1 (2mL) After 3 hours of reaction, a new peak was detected by HPLC. After Pre-HPLC purification, about 48 mg of the target product C2 was obtained, and the yield was 54.4%. LCMS: [M+1] ⁺ = 442.6.

III-1.2 C2 (44.1 mg, 0.1 mmol) and 6-(maleimido)hexanoic acid succinimidyl ester (31 mg, 0.1) were dissolved in DMF (3 mL) and reacted at room temperature overnight, HPLC of the reaction solution showed that a new peak was formed, and after Pre-HPLC purification, about 38 mg of C3 was obtained, and the yield was about 59.9%. LCMS: [M+1] ⁺ =635.5;

III-1.3 Gimatecan (22.3 mg, 0.05 mmol), DMAP (18.3 mg, 0.15 mmol) and triphosgene (7.4 mg, 0.025 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then C3 (32 mg, 0.05 mmol) was added, and stirring was continued for about 4 minutes, and the reaction solution was passed through a short silica gel column, using DCM:MeOH= After elution with 10:1 eluent, concentration, and Pre-HPLC purification, about 5 mg of product C was obtained, yield: 9.0%. LCMS: [M+1] ⁺ = 1109.3.

Synthesis of III-2 Coupling Crude Product ADC-3

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, compound C dissolved in DMF (final DMF concentration 15%) was added to the antibody solution in an 8-fold amount of the substance. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the coupled crude product ADC-3-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold amount of Compound C dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-3-b.

Detection of III-3-conjugated crude product ADC-3

The detection method is as described in step I-3 of Example 2.

Purification of III-4 Coupling Reaction Products:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-3 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of III-5 DAR

DAR was determined as described in Example 2, steps 1-5. The results are shown below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-3-a was 7.2, ie, n was 7.2.

When the antibody is Herceptin antibody:

The DAR value of ADC-3-b was 7.2, ie, n was 7.2.

Example 5 Preparation of conjugate ADC-4

Preparation of IV-1 Intermediate D

IV-1.1. Compound D1 (109 mg, 0.25 mmol) was dissolved in a solution of TFA/DCM (1 mL/1 mL). After 2 hours at room temperature, HPLC detected that D1 had completely disappeared. TFA/DCM was distilled off under reduced pressure. Add an appropriate amount of TEA and DMF (3mL) to adjust the pH value to about 8, add 6-(maleimido)hexanoic acid (N-hydroxysuccinimide) ester (77mg, 0.25mmol) to the above liquid ) and TEA (25mg, 0.25mmol) continued to react overnight, the reaction solution was detected by HPLC, a new peak was formed, and the product was obtained after Pre-HPLC purification to obtain the target product D2 about 55mg; Yield: 41.6%; LCMS: [M- 1] ^- = 528.7.

IV-1.2. Combine D2 (53 mg, 0.1 mmol), methyl tert-butyl[2-(methylamino)ethyl]carbamic acid (28 mg, 0.15 mmol), DIEA (39 mg, 0.3 mmol) with HATU (57 mg, 0.15 mmol) mmol) was dissolved in DMF (3 mL), and after stirring for 4 hours at room temperature, a new peak was generated in the reaction solution by HPLC. After adding an appropriate amount of water, a solid was precipitated. After suction filtration, the solid crude product was obtained after drying, and then TFA/DCM was added. (1mL/1mL), after standing at room temperature for 1 hour, HPLC showed that a peak was formed, and after pre-HPLC purification, about 35 mg of product D3 was obtained, yield: 58.33%. LCMS: [M+1] ⁺ =600.5.

IV-1.3. Gimatecan (22.3 mg, 0.05 mmol), DMAP (18.3 mg, 0.15 mmol) and triphosgene (7.4 mg, 0.025 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then D3 (30 mg, 0.05 mmol) was added, and stirring was continued for about 4 minutes, the reaction solution was passed through a short silica gel column, and DCM:MeOH= After eluting with 10:1 eluent, concentration, and Pre-HPLC purification to obtain about 6 mg of product D, yield: 11.17%. LCMS: [M+1] ⁺ = 1074.2.

Synthesis of IV-2 Coupling Crude Product ADC-4

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound D dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-4-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold amount of Compound D dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-4-b.

Detection of IV-3 conjugated crude product ADC-4

The detection method is as described in Step 2, Step 1-3 of Step Example 2.

Purification of IV-4 coupling reaction products:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-4 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of IV-5 DAR

DAR was determined as described in Example 2, steps 1-5. The results are shown in Figure 3 and below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-4-a was 5.3, ie, n was 5.3.

When the antibody is Herceptin antibody:

The DAR value of ADC-4-b was 7.4, ie, n was 7.4.

Example 6 Preparation of conjugate ADC-5

Preparation of V-1 Intermediate A

V-1.1. Combine D2 (53 mg, 0.1 mmol), tert-butyl N-(2-aminoethyl)-N-methylcarbamate (26 mg, 0.15 mmol), DIEA (39 mg, 0.3 mmol) with HATU (57 mg , 0.15mmol) was dissolved in DMF (3mL), after stirring for 4 hours at room temperature, the reaction solution HPLC detected a new peak formation, after adding an appropriate amount of water, there was solid precipitation, suction filtration, after drying to obtain a solid crude product, then add TFA /DCM (1mL/1mL), after standing at room temperature for 1 hour, HPLC showed that a peak was generated, and the target product E1 was obtained after pre-HPLC purification, about 32 mg, yield: 54.70%. LCMS: [M+1] ⁺ =586.5.

V-1.2. Gimatecan (22.3 mg, 0.05 mmol), DMAP (18.3 mg, 0.15 mmol) and triphosgene (7.4 mg, 0.025 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then E1 (29 mg, 0.05 mmol) was added, and stirring was continued for about 4 minutes, the reaction solution was passed through a short silica gel column, and DCM:MeOH= After eluting with 10:1 eluent, concentration and Pre-HPLC purification, about 7 mg of product E was obtained, yield: 13.21%. LCMS: [M+1] ⁺ = 1060.2.

Synthesis of V-2 Coupling Crude Product ADC-5

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound E dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-5-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold amount of Compound E dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled product ADC-5-b.

Detection of V-3 conjugated crude product ADC-5

The detection method is as described in step I-3 of Example 2.

Product purification of V-4 coupling reaction:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-5 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of V-5 DAR

DAR was determined as described in Example 2, steps 1-5. The results are shown in Figure 4 and below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-5-a was 6.3, ie, n was 6.3.

When the antibody is Herceptin antibody:

The DAR value of ADC-5-b was 7.4, ie, n was 7.4.

Example 7 Preparation of conjugate ADC-6

Preparation of VI-1 Intermediate A

VI-1.1 Gimatecan (20 mg, 0.043 mmol), DMAP (24 mg, 0.2 mmol) and triphosgene (7 mg, 0.26 mmol) were weighed and added to a round-bottomed test tube. After adding DCM (0.5 mL), stirring for about 7 minutes, TLC showed that Boc-CMTC had reacted completely, then CL2-L, (45 mg, 0.43 mmol) was added, stirring was continued for about 10 minutes, 1d methanol was added to quench, and spin-dried Then, after Pre-HPLC purification, about 25 mg of target product F1 was obtained, LCMS: [M+1]+=1475.3.

VI-1.2 F1 (25mg) and F2 (5mg) were dissolved in DMSO/H2O (1mL/1mL), CuBr (0.3mg) was added at room temperature, the reaction solution was stirred at room temperature for 2 hours, HPLC showed new peaks Generated, LCMS showed that the target product was generated, and the product F3 was about 15 mg after Pre-HPLC purification. [M+1]+=1807.9.

VI-1.3 F3 (15mg) was added to the reaction solution of DCM/DCA/anisole. After half an hour of reaction, HPLC showed that a new peak was generated, and the target product F was purified by Pre-HPLC to obtain about 7mg, LCMS: [1/2M+1 ]+=768.8.

Synthesis of VI-2 Coupling Crude Product ADC-6

The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 7 times the amount of TCEP at 37°C for 2 hours. Next, a 12-fold amount of compound F dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 20 minutes to obtain the coupled crude product ADC-6-a.

or:

The Herceptin antibody was first reduced with 9.5 times the amount of TCEP in a 6 mg/mL solution in PBS pH 7.2 for 30 minutes at room temperature. Next, a 12-fold amount of Compound F dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled product ADC-6-b.

Detection of VI-3 conjugated crude product ADC-6

The detection method is as described in step I-3 of Example 2.

Purification of VI-4 coupling reaction product:

After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-6 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.

Determination of VI-5 DAR

DAR was determined as described in Example 2, steps 1-5. The results are shown in Figure 5 and below.

When the antibody is the hRS7 antibody prepared in Example 1:

The DAR value of ADC-6-a was 7.2, ie, n was 7.2.

Example 8 ADC (when the antibody is the hRS7 antibody prepared in Example 1) cell killing test

HTB-132 ^TM)细胞覆盖整个培养皿底面积的80％以上时，对细胞进行传代并计数，将细胞浓度调整到1×10 ⁵个/mL，取100μL加入到96孔板中继续培养。在37℃，CO ₂细胞培养箱中继续培养24h后取出。弃去原培养液后，改用含有1％FBS的培养液继续培养30min。 DMEM/F12 (Gibco, 11320033) medium and FBS (Sijiqing, 13011-8611) were mixed at a ratio of 9:1 to prepare a complete medium. When triple negative breast cancer MDA-MB-468 (

When HTB-132 ^TM ) cells covered more than 80% of the bottom area of the entire culture dish, the cells were passaged and counted, the cell concentration was adjusted to 1×10 ⁵ cells/mL, and 100 μL was added to a 96-well plate to continue culturing. Incubate for 24 h at 37 °C in a CO ₂ cell incubator. After discarding the original culture medium, the culture medium containing 1% FBS was used to continue culturing for 30 min.

Prepare test substances of different concentrations: ADC-1-a to ADC-6-a prepared in Example 2-7 and hSR7 mAb prepared in Example 1 were used as negative controls, and the culture medium of 1% FBS was used at 81 nmol The initial gradient of /L was diluted 3 times to obtain 8 concentrations of 81 nmol/L, 27 nmol/L, 9 nmol/L, 3 nmol/L, 1 nmol/L, 0.3 nmol/L, 0.1 nmol/L and 0.03 nmol/L point, three replicate wells per well, discard the original culture solution in the 96-well plate, add the above-prepared test substances of different concentrations into the 96-well plate, 100 μl per well, and put them into a CO ₂ incubator to continue. Cultivated for 72h. Add 10 μL of CCK8 reagent (Beyotime Biotechnology, C0040) to each well, and put it into a CO ₂ cell incubator for incubation for 2 h. Then, the SpectraMax M5 multifunctional microplate reader was used for reading at a wavelength of 450 nm, and the inhibitory effect on cell proliferation was measured by detecting the dehydrogenase activity in the mitochondria.

The results of the ADC in vitro inhibition experiment of the present invention are shown in Fig. 6, wherein the ordinate is specifically, after the administration of cells, the absorbance value will also decrease after the amount of cells is reduced, and then the ratio of the detected value to the blank control is used. drawing.

Compared with the blank control group, the six ADC test products all showed inhibitory effects on the activity of MDA-MB-468, and with the increase of the test product concentration, the cell activity decreased significantly, that is, in a dose-dependent manner. In addition, the inhibitory activities of 6 ADCs on NCI-N87, BxPC-3, Capan-1, H1975, MDA-MB-231 and HCC1806 cells were also tested respectively, and it was found that the ADC prepared by the present invention also inhibited the activities of these cells effect (Table 1).

Table 1

test article IC ₅₀ value (nmol/L) R ² ADC-1-a 2.77 0.983 ADC-2-a 1.19 0.967 ADC-3-a 1.53 0.971 ADC-4-a 1.12 0.966 ADC-5-a 1.34 0.978 ADC-6-a 1.11 0.969

Example 9 ADC (when the antibody is Herceptin antibody) cell killing experiment

RPMI-1640 (Gibco, 22400089) medium and FBS (Sijiqing, 13011-8611) were mixed at a ratio of 9:1 to prepare a complete medium. Cells were passaged when KPL-4 cells (Nanjing Kebai Biotechnology Co., Ltd.) covered more than 80% of the bottom area of the entire culture dish. Before the test, the cell concentration was adjusted to 1×10 ⁵ cells/mL, and after mixing, 100 μL/well was added to a 96-well plate (NEST, 701001) and cultured for 24 h. After that, the culture medium containing 1% FBS was used to continue the culture for 30 min.

Prepare test articles of different concentrations: use trastuzumab as a negative control. At the same time, seven test articles including trastuzumab, ADC-1-b, ADC-2-b, ADC-3-b, ADC-4-b, ADC-5-b and ADC-6-b were used The culture medium containing 1% FBS was diluted three times from the initial gradient of 11731.068pmol/L to obtain 3910.356pmol/L, 1303.452pmol/L, 434.484pmol/L, 144.828pmol/L, 48.276pmol/L L, 16.092pmol/L, 5.364pmol/L, 1.78800pmol/L, 0.5960pmol/L, a total of 10 concentration points, three replicate wells per well. The original culture solution was discarded and added to a 96-well plate, and then placed in a carbon dioxide incubator for 120 h. Add 10 μL of CCK8 reagent (Beyotime Biotechnology, C0040) to each well, and put it into a carbon dioxide cell incubator for 2h incubation. Then, the SpectraMax M5 multifunctional microplate reader was used for reading at a wavelength of 450 nm, and the inhibitory effect on cell proliferation was measured by detecting the dehydrogenase activity in the mitochondria (see FIG. 7 and Table 2).

Table 2

test article IC ₅₀ value (pmol/L) R ² ADC-1-b 22.8 0.981 ADC-2-b 21.5 0.981 ADC-3-b 32.43 0.980 ADC-4-b 31.38 0.956 ADC-5-b 15.75 0.973 ADC-6-b 12.13 0.979

Cell Killing Assay of Example 10 Samples

ADC sample 137 to be tested: Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0

The purpose of this experiment was to evaluate the effect of one drug on cell proliferation in five cell lines. The 50% inhibitory concentration was calculated by detecting cell viability after treatment with different drug concentrations. Specifically, the cell killing activity of one ADC sample 137, its corresponding small molecule sample SN-38 (XYD-CX1) and another small molecule sample gemitecan (XYD-CX4) was determined in 5 cell lines, And set a quality control reference control, a blank control and a vehicle control for each cell line. Each compound has 9 concentrations, 3 replicate wells, and the cell viability is detected after 72 hours, and IC _{50 is} calculated.

(1) Experimental materials

cell line

serial number cell line tissue source quality control reference processing time 1 BxPC-3 Pancreas Cisplatin 72h 2 Calu-3 Lung Cisplatin 72h 3 COLO 205 Large testine/Colorectum Cisplatin 72h 4 NCI-N87 Stomach Cisplatin 72h 5 Calu-6 Lung Cisplatin 72h

All cells were placed 37 ℃, cultured under 5% CO ₂ and 95% humidity. The brand of media used for cell culture is Hyclone/Gibco with 10-15% fetal bovine serum.

Reagents and Consumables

1. Cell culture medium and consumables

2. Fetal bovine serum FBS (ExCell Bio., Cat#FND500)

3. CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat#G7573)

4.96-well black-walled clear flat-bottom cell culture plate (Corning, Cat#3340)

Drug to be tested

Quality control reference drug

name molecular weight Package supplier concentration save Cisplatin 300.05 20mg Qilu Pharmaceutical 3.33mM RT

instrument

EnVision Multi-label Microplate Reader, PerkinElmer, 2104-0010A;

Cell counter, Inno-Alliance Biotech, Countstar;

CO ₂ incubator, Thermo Scientific, Model 3100Series;

Biological Safety Cabinet, Thermo Scientific, Model 1300Series A2;

Inverted microscope, Olympus, CKX41SF;

Refrigerator, SIEMENS, KK25E76TI.

(2) Experimental method

1. Harvest cells in logarithmic growth phase and dilute them with culture medium to form a cell suspension, transfer to a 96-well cell plate, _{and culture overnight at 37°C, 5% CO 2} and 95% humidity. Drug treatment begins when the density reaches 80%.

2. Dissolve the tested compound with the corresponding solvent to form a stock solution and perform gradient dilution to obtain a 10-fold working concentration solution; also prepare a 10-fold solution of the cisplatin positive drug.

3. Add 10 μL of drug solution to each well of the 96-well plate that has been seeded with cells, and set up three replicate wells for each cell concentration. The highest concentration of the tested compound was 10 μM, 9 concentrations, 3.16 times dilution.

4. The cells in the medicated 96-well plate were placed under the conditions of 37° C., 5% CO ₂ , and 95% humidity for further culturing for 72 hours.

5. After 72 hours, equilibrate the CellTiter-Glo reagent and drug-treated cell culture plates for 30 minutes at room temperature.

6. Add 50 μL of CellTiter-Glo reagent to each well.

7. Shake for 2 minutes on an orbital shaker to fully lyse the cells.

8. Allow the cell culture to equilibrate at room temperature for 10 minutes.

9. Read the chemiluminescence value with EnVision.

(3) Data processing

Using GraphPad Prism 5.0 data analysis software, using a nonlinear regression curve fit data S derived dose - response curves, _IC50 values calculated therefrom IC.

Cell viability (%)=(Lum _{test drug-} Lum _{culture solution control} )/(Lum _{cell control-} Lum _{culture solution control} )×100%.

Lum _{cell control -} Lum _{culture medium control is} set to 100%, and Lum _{Medium control} value is set to 0%.

Amplification fold = (the fifth day Lum _{None treated} -Lum _{Medium control} )/(the second day Lum _{None treated} -Lum _{Medium control} )

(3) Experimental results

The experimental results are shown in the following table and Figures 8-12.

Table 3-1. ₅₀ Compound IC 5 value in cell lines lines

Table 3-2. The maximum concentration inhibition rate of compounds in 5 cell lines

The results of the animal tumor inhibition experiment of the sample of Example 11

ADC sample 137 to be tested: Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0

ADC sample 138 to be tested: ADC-6-a prepared in the previous example (the antibody is the hRS7 antibody prepared in Example 1)

(1) Experimental method

cell culture

Human pancreatic adenocarcinoma cells BxPC-3 (ATCC CRL-1687), human colon cancer cells Colo205 (ATCC CCL-222), human lung adenocarcinoma cells Calu-3 (ATCC HTB-55) and human pancreatic adenocarcinoma cells Capan-1 (ATCC HTB-79) in vitro monolayer culture, the culture conditions are 1640 medium with 10% heat-inactivated fetal bovine serum and agar, and cultured at 37°C in an _{incubator containing 5% CO 2} air. Digestion and passage were performed twice a week with 0.25% trypsin. When cells are in exponential growth phase, cells are harvested, counted, and seeded.

Tumor cell inoculation and tumor mass passage

5.0×10 ⁶ tumor cells were suspended in 0.1 ml of a mixture of PBS and Matrigel (1:1), and inoculated into the right scapula of 5 nude mice (passage P1). When the tumor grows to 500-800mm ³ , the tumor-bearing mice are _{sacrificed with CO 2} anesthesia, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into 20-30 mm ³ small tumor pieces, Inoculated into a new batch of nude mice (P2 generation).

Tumor mass inoculation and group administration

In this experiment, the P6 generation tumor tissue was used to evaluate the antitumor activity of the test article. Generation P5 to be 500-800mm ³ tumor grew, the tumor-bearing mice were anesthetized with CO ₂ sacrificed and the tumor mass, the removal of necrotic tissue around the tumor mass state would preferably cut into small tumor of 20-30mm ³ The block was inoculated to the right scapula of the formal experimental mice, and a total of 70 mice were inoculated. When the average tumor volume reached about 135 mm ³ 13 days after tumor mass inoculation, mice with too small or too large tumor volume were eliminated, and the remaining 50 mice were randomly grouped according to tumor volume and started to be administered.

Experimental observation and data collection

After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were also monitored: the activity of experimental animals, food and water intake, body weight changes (body weight was measured twice a week), eyes, coat and other abnormal situation. The clinical symptoms observed during the experiment were all recorded in the raw data. The tumor volume was calculated as follows: tumor volume (mm ³ )=1/2×(a×b ² ) (where a represents the long diameter, and b represents the short diameter).

When the body weight of a single animal decreased by more than 15% (BWL>15%), the corresponding single animal was given drug withdrawal treatment, and the body weight decreased to less than 10%, and the drug was resumed. When individual mice lost >20% body weight, they were euthanized in accordance with animal welfare.

Efficacy Evaluation Criteria

The relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C%=T _RTV /C _RTV × 100% (T _RTV : the average RTV of the treatment group; C _RTV : the average RTV of the vehicle control group; RTV=V _t /V ₀ , V ₀ is the animal at the time of grouping the tumor volume, V _t is the tumor volume of the animals after treatment); or _{T / C% = T TW /} C TW × 100% (T TW: mean tumor weight of treatment group end experiment; C _TW: vehicle control experiments mean tumor weight at termination).

Statistical Analysis

In this experiment, one-way ANOVA was used to compare the mean tumor values among the groups. Homogeneity of variance analysis showed that the F value was significantly different, and Dunnet's T3 (unequal variance) method was used for multiple comparisons after ANOVA analysis. All data analyses were performed with SPSS 17.0. p<0.05 was considered a significant difference.

(2) Experimental results

The experimental results are shown in the following table and Figures 13-22.

Industrial Applicability

In the present invention, through the antibody-drug conjugate comprising a novel linker structure, high drug loading is achieved, and at the same time, faster onset time, longer drug half-life, excellent stability, good biocompatibility, and low immunity are obtained. Antibody-drug conjugates with good originality and safety. The antibody-drug conjugate of the present invention exhibits excellent antitumor effect.

Claims (20)

Antibody-drug conjugate represented by formula (V), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof The solvate is characterized in that the antibody-drug conjugate is a compound represented by the following formula (I) and an antibody via a linker represented by the following formula (II), through the disulfide present in the hinge part of the antibody. The thioether bond formed by the bond part is connected;

-L ¹ -L ² -L ^P -L ^a -C(=O)- (II) Wherein, the antibody is connected to ^{the end of L 1} , and the compound represented by formula (I) is connected to the right side -C(=O)- in the linker represented by the above formula (II) using the oxygen in the hydroxyl group at position 19 as a linking site. part, In formula (I), R ₁ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, the substituted ₇ R ₈ NR C ₁ -C ₆ alkyl, -SiMe ₃ Substituted C ₁ -C ₆ alkyl, or -CH=NO(C ₁ -C ₆ alkyl); R ₂ represents hydrogen, halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NR ₇ R ₈ or C ₁ -C ₆ substituted alkyl; R ₃ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or NR ₇ R ₈ C(O)O- group; R ₄ represents hydrogen, halogen, hydroxyl, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy; Or R ₁ and R ₂ may be joined together to form an optionally substituted 5-6 R ₉ membered ring with the parent moiety; Or R ₃ and R ₄ may be joined together to form an optionally substituted ₉ membered oxygen-containing heterocyclic R 5-6 to the parent moiety; Each occurrence of R ₇ and R ₈ is independently selected from hydrogen, C ₁ -C ₆ alkyl; or R ₇ and R ₈ can be taken together with the N atom to which they are attached to form a 5-6 membered _{optionally substituted with R 9} nitrogen-containing heterocycle; Each occurrence of R ₉ is independently selected from halogen, hydroxy, nitro, amino, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, piper optionally substituted with C ₁ -C _{6 alkyl} pyridyl; In formula (II), L ¹ represents -(succinimide-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimide-3-yl-N)-(CH ₂ )n ^{1 '-C} ₃ -C ₆ cycloalkyl, -C (= O) -, n 1, n 1' each independently represents an integer of 1 to 8, Preferably, L ¹ represents -(succinimid-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimid-3-yl-N)-(CH _{^{2) n 1 '-C 3 -C}} 6 cycloalkyl, -C (= O) -, n 1 represents an integer of 1 to 5, n ^1' represents an integer of 1 to 3; More preferably, L ¹ represents -(succinimid-3-yl-N)-(CH ₂ )n ¹ -C(=O)- or -(succinimid-3-yl-N)-( CH ₂ )n ^1' -cyclohexyl-C(=O)-, n ¹ represents 2 or 5, n ^1' represents 1; Most preferably, L ¹ represents

L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ CH(CH ₃ )-O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -5-membered nitrogen-containing heteroaryl-(CH ₂ CH ₂ -O)n ^2' -(CH ₂ )n ⁷ -NH -C(=O)-(CH ₂ )n ⁸ -O-(CH ₂ )n ⁸ -C(=O)- or single bond, n ² represents an integer of 1 to 6, and n ^2' represents an integer of 1 to 12 Integer, n ³ represents an integer from 1 to 4, and n ⁶ , n ⁷ , and n ⁸ each independently represent an integer from 1 to 5; Preferably, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -1H-[1,2 ,3] Triazolyl-(CH ₂ CH ₂ -O)n ^2' -(CH ₂ )n ⁷ -NH-C(=O)-(CH ₂ )n ⁸ -O-(CH ₂ )n ⁸ - C(=O)- or single bond, n ² represents an integer from 1 to 3, n ^2' represents an integer from 6 to 10, n ³ represents an integer from 1 to 3, and n ⁶ , n ⁷ , and n ⁸ represent each independently an integer from 1 to 3; More preferably, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, -NH-(CH ₂ )n ⁶ -1H-[1, 2,3] triazol-yl _{- (CH 2 CH 2 -O)} n 2 '- (CH 2) n 7 -NH-C (= O) - (CH 2) n 8 -O- (CH 2) n 8 -C(=O)- or single bond, n ² means 2, n ^2' means 8, n ³ means 2, n ⁶ means 1, n ⁷ means 2, n ⁸ means 1; Most preferably, L ² represents

or single key; L ^P represents a peptide residue composed of 1 to 7 amino acids, and L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen, optionally _{C 1} -C ₆ alkyl substituted with 1 or 2 hydroxy groups; Aryl represents a C ₆ -C ₁₀ aryl group _{optionally substituted by R 9} ^{, n 4} represents an integer of 1 to 4, and n ⁵ represents an integer of 1 to 4 ; Preferably, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, and _{each occurrence of R 10} is independently selected from hydrogen, C ₁ -C ₄ alkyl; Aryl represents phenyl, n ⁴ represents an integer of 1-3, and n ⁵ represents an integer of 1-3; More preferably, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ - or -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, _{each occurrence of R 10} is independently selected from hydrogen , methyl; Aryl represents phenyl, n ⁴ represents 2, n ⁵ represents 1; Most preferably, L ^a represents

-(Succinimide-3-yl-N)- is of the formula:

The represented structure is linked to the antibody at the 3-position of the structure and to the methylene group in the linker comprising the structure at the nitrogen atom at the 1-position; In formula (V), AB represents an antibody; in: The structure after the compound represented by the formula (I) is connected to the linker represented by the formula (II) is not

The antibody-drug conjugate according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof solvates of the salts, wherein, R ₁ represents hydrogen, C ₁ -C ₄ alkyl, (C ₁ -C ₄ alkyl group) -NH C ₁ -C ₄ alkyl,

Substituted C ₁ -C ₄ alkyl, -SiMe ₃ substituted C ₁ -C ₄ alkyl or -CH = NO (C ₃ -C ₆ alkyl); Preferably, R ₁ represents hydrogen, ethyl,

quilt

substituted methyl,

or -CH=NO-tert-butyl; Alternatively, R ₂ represents hydrogen, nitro, amino, or -N (C ₁ -C ₄ alkyl) ₂ substituted C ₁ -C ₄ alkyl; Preferably, R ₂ represents hydrogen, nitro, amino or

Alternatively, R ₃ represents hydrogen, halogen, hydroxy, C ₁ -C ₄ alkyl or

Preferably, R ₃ represents hydrogen, F, hydroxyl, methyl or

Alternatively, R ₄ represents hydrogen or halogen; Preferably, R ₄ represents hydrogen or F; Alternatively, R ₁ and R ₂ are linked together to form a group shown below

in

moiety represents a bond to the parent group; Preferably, R ₁ and R ₂ are linked together to form a group shown below

in

moiety represents a bond to the parent group; Alternatively, R ₃ and R _{4 are} joined together to form a group shown below

in

A moiety represents a bond to the parent group. The antibody-drug conjugate according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a salt, wherein the compound represented by the formula (I) is a compound selected from the group consisting of:

Preferably, the compound represented by formula (I) is gimatecan:

The antibody-drug conjugate according to any one of claims 1-3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof pharmaceutically acceptable solvates of the salts, wherein the peptide residues by L ^P selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and asparagine Peptide residues formed by amino acids in amino acids; Alternatively, L ^P by 1-5 amino acids of the peptide residue; Alternatively, L ^P is a peptide residue selected from the following: -K- -ggfg-; -vc-; -evc-; -DVC; -EGGFG-; -DGGFG-; Preferably, L ^P is a peptide residue selected from the following: -K -, - GGFG -, - VC -, - EVC-; Alternatively, L ² represents -NH-(CH ₂ CH ₂ -O)n ² -(CH ₂ )n ³ -C(=O)-, n ² represents an integer of 1 to 4, and n ³ represents an integer of 2 to 4 ; Alternatively, L ² represents a single bond; Or, L ^a represents -NR ₁₀ -Aryl-(CH ₂ )n ⁵ -O-, wherein R ₁₀ represents hydrogen or C ₁ -C ₄ alkyl; n ⁵ represents an integer from 1 to 2, and Aryl represents a benzene ring group ; Preferably, the -NR ₁₀ -group and the -(CH ₂ )n ⁵ -group are located in the para position of the benzene ring; Alternatively, L ^a represents -NR ₁₀ -(CH ₂ )n ⁴ -NR ₁₀ -, _{each occurrence of R 10} is independently selected from hydrogen, C ₁ -C ₄ alkyl optionally substituted with 1 hydroxy, n ⁴ represents an integer of 2 to 4. The antibody-drug conjugate according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a salt, wherein the linker represented by the formula (II) is a group selected from the group consisting of:

The antibody-drug conjugate according to any one of claims 1-5, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of pharmaceutically acceptable salts, wherein, for one antibody molecule, the average number of linker-drug linkages is 2-8 (eg 2.7, 5.3, 6.3, 7.2, 7.3, 7.4, 7.5, 7.6) , preferably 4 to 8, more preferably 6 to 8. The antibody-drug conjugate according to any one of claims 1-6, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof; Preferably, the antibody is selected from anti-Trop-2 antibody, Her2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody; preferably, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably, the humanized antibody is a fully human antibody; Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv and dsFv; More preferably, the antibody is an anti-Trop-2 antibody, and the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody includes CDR1 consisting of the amino acid sequence of KASQDVSIAVA, and CDR2 consisting of the amino acid sequence of SASYRYT. , and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, the anti-Trop The amino acid sequences of the light chain and heavy chain of the -2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; preferably, the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody As shown in SEQ ID NO:3 and SEQ ID NO:4 respectively; Or more preferably, the antibody is an anti-Her2 antibody, and the complementarity determining region (CDR) of the light chain variable region of the anti-Her2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, CDR2 consisting of the amino acid sequence of SASFLYS, and CDR2 consisting of the amino acid sequence of SASFLYS. CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; The amino acid sequences of the chain and heavy chain are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. The linker-drug intermediate compound represented by the formula (IV) is characterized in that the compound represented by the following formula (I) and the compound represented by the following formula (III) whose linker structure is represented by the formula (I) are at the 19th position The oxygen in the hydroxyl group is connected as a connecting site;

QL ² -L ^P -L ^a -C(=O)- (III) Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described in any one of claims 1-3; L ^2, L ^P, L ^a are as defined in any of claims 1, 4; Q represents (maleimide-N)-

in: The linker-drug intermediate compound represented by formula (IV) is not

The linker-drug intermediate compound of claim 8, wherein the compound represented by the formula (I) is the compound of claim 3; preferably, the linker-drug intermediate compound is selected from the following compound,

The antibody-drug conjugate of any one of claims 1-7, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its pharmacy A process for the preparation of a solvate of an acceptable salt of the above, comprising:

The linker-drug intermediate compound represented by the formula (IV) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (IV) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody; Wherein, _{the definitions of R 1} , R ₂ , R ₃ and R ₄ are as described in any one of claims 1-3; ^{L 1, L 2, L P} , L a is defined as claimed in any one of claims 1, 4; Q represents (maleimide-N)-

AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody. The preparation method of the described linker-drug intermediate compound of claim 8, the method comprises:

The compound represented by the formula (VI) is reacted with the compound represented by the formula (VII) to obtain the compound represented by the formula (VIII); The compound represented by the formula (VIII) is reacted with the compound represented by the formula (I) in the presence of an alkoxycarbonylation reagent to obtain the linker-drug intermediate compound represented by (IV); the alkoxycarbonylation reagent It is preferably at least one of triphosgene, bis(2-pyridine) carbonate, N,N'-disuccinimidyl carbonate and 4-nitrophenyl chloroformate; _{Wherein, R 1, R 2, R} 3, R 4, Q, L 2, L P, L a are as defined according to claim 8; G represents a leaving group, preferably halogen, hydroxy, C ₁ -C ₆ alkoxy group or a succinimidyl group; N-terminal peptide residue represented by ^P L is connected to the group represented by L ^2, C-terminus to the group represented by L ^a. A linker characterized in that it is represented by the following formula (II) -L ¹ -L ² -L ^P -L ^a -C(=O)- (II) ^{Wherein, L 1, L 2, L} P, L a is defined as claimed in any one of claims 1, 4; Preferably, the linker is selected from the group shown in claim 4 . A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, A solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier. A pharmaceutical preparation comprising the antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its A solvate of a stereoisomer or a pharmaceutically acceptable salt thereof. The antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof Use of a solvate of an acceptable salt of the above, a pharmaceutical composition according to claim 13 and/or a pharmaceutical preparation according to claim 14, for the prevention and/or treatment of tumors or cancer; or, The antibody-drug conjugate of any one of claims 1-7, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its pharmacy Use of a solvate of an acceptable salt above, the pharmaceutical composition of claim 13 and/or the pharmaceutical preparation of claim 14 in the preparation of a medicament for the prevention and/or treatment of tumor or cancer; Preferably, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer, bladder cancer , gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic carcinoma. A method of preventing or treating cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof or A pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 13 and/or claims The pharmaceutical preparation of 14. The antibody-drug conjugate of any one of claims 1-7, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its pharmacy Use of a solvate of an acceptable salt of the above, the pharmaceutical composition of claim 13 and/or the pharmaceutical formulation of claim 14 for the manufacture of an agent for inhibiting the growth, proliferation or migration of cancer cells . The antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof A solvate of an acceptable salt above, the pharmaceutical composition of claim 13 and/or the pharmaceutical formulation of claim 14 for use in inhibiting the growth, proliferation or migration of cancer cells. A method of inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof, or a pharmaceutically acceptable or the solvate of the antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, the pharmaceutical composition of claim 13 and/or the medicine of claim 14 preparation. A kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate of any one of claims 1-7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the The antibody-drug conjugate, a solvate of a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 13 and/or the pharmaceutical preparation of claim 14.

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