RU2792201C2 - Linker for antibody-drug conjugates and their use - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a linker having a structure represented by formula I, in which each of M₁', M₂' is selected independently from

, M₁', M₂' are the same or different; L₁, L₂ are chosen, each independently, from C₁-C₉-alkyl; Q chosen from

, N; L₃' chosen from

C (=O) R⁸C (=O),

_NH2,

OH, and any combination thereof; X is chosen from Br; Y represents

,

; Z is selected from C; R¹ is selected from H, C₁-C₆-alkyl, R², R⁸, R¹⁰ are each independently selected from H, C₁-C₆-alkyl. The invention also relates to antibody-drug conjugates obtained using said linker, as well as to the use of antibody-drug conjugates in a preparation for the treatment of cancer associated with the HER2 antigen.

EFFECT: new linker is obtained, which is capable of simultaneously connecting with 2, 3 or 4 thiol groups of an antibody, which leads to a homogeneous and structurally stable coupling product.

19 cl, 14 dwg, 3 tbl, 30 ex

Description

The field of technology to which the invention belongs

The present invention relates to a linker for antibody-drug conjugates and antibody-drug conjugates made with the linker, as well as to the use of antibody-drug conjugates in the treatment of neoplastic and other diseases.

State of the art

Antibody-drug conjugates (ADCs) are a class of targeted therapeutic agents that are used primarily in the treatment of cancer, autoimmune diseases, and the like. Their structure usually consists of three components: an antibody or antibody-like ligand, a drug component, and a linker that connects the antibody or antibody-like ligand and the drug.

A conventional ADC preparation is prepared by connecting the drug via a linker to a lysine residue of an antibody or a cysteine residue obtained by reduction of an interchain disulfide bond of an antibody. When a lysine residue is used as the junction site, since the antibody contains about 40 or more lysine residues and the coupling reaction is not selective, the number and site of junctions have considerable uncertainty, and the product is very poor quality due to heterogeneity. When a cysteine residue is used as the junction, although the antibody (IgG1) has only 4 pairs of interchain disulfide bonds, and the DAR (drug to antibody ratio) of the drug ADC formed by the cysteine residue obtained by reduction of the interchain disulfide bond is relatively uniform , this method of connection destroys the disulfide bond of the antibody, affecting the stability of the antibody. And due to the poor selectivity of existing reducing agents (DTT, TCEP) for interchain disulfide bonds, the homogeneity of the final product is also poor. Therefore, it is necessary to provide drug ADCs with a well controlled drug to antibody ratio.

In recent years, some researchers have used genetic engineering technology to modify antibodies to achieve a targeted combination of antibodies and drugs, and the resulting ADCs also have very uniform DSR values. However, since the technology of genetic recombination requires a lot of work and many projects to find a suitable place for drug attachment or polyethylene glycol modification, it is very laborious and extensive in development.

Given the above disadvantages in the preparation of antibody-drug conjugates in the prior art, there is an urgent need in the art to develop a new linker to achieve targeted coupling of toxic drugs with antibodies in order to obtain ADC drugs with higher product uniformity and greater stability.

The essence of the invention

In order to solve the above problems, the present invention provides a linker for the preparation of antibody-drug conjugates, and antibody-drug conjugates obtained using a linker, as well as the use of antibody-drug conjugates in a tumor treatment preparation. The linker is capable of simultaneously connecting to the thiol(s) group(s) or amino group(s) of an antibody or a functional fragment of an antibody, in particular, it is capable of connecting to 2, 3 or 4 thiol groups of an antibody or a functional fragment of an antibody. The combined product is homogeneous and structurally stable.

In a first aspect, the present invention relates to a linker which has the structure of formula I:

wherein

,

,

,

,

,

,

, или он отсутствует, M₁', M₂' являются одинаковыми или различными, но не отсутствуют в одно и то же время; each of M ₁ ', M ₂ ' is chosen independently from

,

,

,

,

,

,

, or it is absent, M ₁ ', M ₂ ' are the same or different, but not absent at the same time;

each of X, Y, R ¹ , L ₁ , L ₂ , L ₃ ', Q is an arbitrary group.

In addition, the above X, Y, R ¹ , L ₁ , L ₂ , L ₃ ', Q may be as follows:

NR⁴

,

C (=O)

,

C (=O) O

,

C (=O) NR¹¹

,

C=NR³

,

C (=S) O

,

C (=S) NR¹²

,

C (=S) S

,

NR¹³ (C=O)

,

NR¹⁴ (C=S) NR⁵

,

O (C=O) NR¹⁵

,

S (=O) ₂

и их любой комбинации; L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₉ -alkyl, C ₂ -C ₉ -alkenyl, C ₂ -C ₉ -alkynyl, aryl, heteroaryl, C ₃ -C ₉ -cycloalkyl, C ₃ - C ₉ -heterocyclyl, polyethylene glycol, O, S,

NR ⁴

,

C (=O)

,

C(=O)O

,

C (=O) NR ¹¹

,

C=NR ³

,

C (=S)O

,

C (=S) NR ¹²

,

C (=S)S

,

NR ¹³ (C=O)

,

NR ¹⁴ (C=S) NR ⁵

,

O(C=O)NR ¹⁵

,

S(=O) ₂

and any combination of them;

,

, N, B, P, арила, гетероарила, циклоалкила, (гетероциклил)алкила; Q choose from

,

, N, B, P, aryl, heteroaryl, cycloalkyl, (heterocyclyl)alkyl;

R⁸ (C=O),

C (=O) R⁸C (=O), полиэтиленгликоля,

,

NH₂,

OH,

SH,

X и их любой комбинации;L ₃ ' choose from

R ⁸ (C=O),

C (=O) R ⁸ C (=O), polyethylene glycol,

,

_NH2 ,

Oh,

SH,

X and any combination thereof;

X is selected from F, Cl, Br, I;

,

,

; (в вышеуказанных двух структурах замещение цикла R² может иметь место в любом положении);Y represents

,

,

; (in the above two structures, the substitution of the R ² ring can take place at any position);

Z is selected from C, N;

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ are each independently selected from H, C ₁ -C ₆ -alkyl , C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, aryl, heteroaryl, C ₃ -C ₉ -cycloalkyl, C ₃ -C ₉ -heterocyclyl.

In addition, the above X, Y, R ¹ , L ₁ , L ₂ , L ₃ ', Q may be as follows:

NR⁴

,

C (=O)

,

C (=O) O

,

C (=O) NR¹¹

,

O (C=O) NR¹⁵

;L ₁ , L ₂ are each independently selected from C ₁ -C ₄ -alkyl, C ₆ -C ₈ -aryl, C ₆ -C ₈ -heteroaryl, C ₅ -C ₆ -cycloalkyl, C ₅ -C ₆ -heterocyclyl , C ₂ -C ₁₂ - polyethylene glycol, O, S,

NR ⁴

,

C (=O)

,

C(=O)O

,

C (=O) NR ¹¹

,

O(C=O)NR ¹⁵

;

, N, C₆-C₈-арила, C₆-C₈-гетероарила, C₃-C₈-циклоалкила, C₃-C₈-гетероциклила;Q choose from

, N, C ₆ -C ₈ -aryl, C ₆ -C ₈ -heteroaryl, C ₃ -C ₈ -cycloalkyl, C ₃ -C ₈ -heterocyclyl;

R⁸ (C=O),

C (=O) R⁸C (=O), полиэтиленгликоля,

,

NH₂,

OH,

SH,

X и их любой комбинации;L ₃ ' choose from

R ⁸ (C=O),

C (=O) R ⁸ C (=O), polyethylene glycol,

,

_NH2 ,

Oh,

SH,

X and any combination thereof;

X is Br;

;Y represents

;

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ^{8 , R 10 ,} R ¹¹ , R ¹⁵ are each independently selected from H, C ₁ -C ₄ -alkyl, C ₆ -C ₈ -aryl, C ₆ -C ₈ -heteroaryl, C ₅ -C ₆ -cycloalkyl, C ₅ -C ₆ -heterocyclyl.

In addition, the linker has a structure represented by the formulas

or

or

or

.

.

Preferably the linker may have the structure

A'-1 A'-2 A'-4

A'-5 A'-7 A'-8

A'-9 A'-10 A'-11

A'-12 A'-13 A'-14

A'-15 A'-16 A'-19

A'-20 A'-21 A'-22

A'-24 A'-26 A'-27

A'-28 A'-29 A'-31

A'-33 A'-34 .

The present invention also relates to the use of the above linker in the preparation of antibody-drug conjugates.

In another aspect, the present invention relates to an antibody-drug conjugate, wherein the antibody-drug conjugate has the structure represented by formula II:

,

,

wherein

Ab is any antibody or functional antibody fragment. In addition, an antibody includes a derivative or analogue of an antibody; a "functional" fragment is an antibody, fragment, derivative, or analog thereof capable of recognizing the same antigen, and capable of recognizing fragments, derivatives, or analogs derived from the antigen, which include, for example, but not limited to, F(ab')2 fragments , Fab, Fab', Fv and antibody heavy and light chain dimers, or any of the smaller fragments such as Fv or single chain antibody (single chain antibody fragment/single chain scFv variable fragment). In addition, the antibody may be an antibody fusion protein. An antibody may also include modified or unmodified (ie, covalently linked through any molecule) analogs and derivatives, as long as such covalent bonding allows the antibody to retain its antigen-binding immunological specificity. Included, for example, but not limited to, are antibody analogs and derivatives, including additional modifications such as glycosylation, acetylation, pegelation, phosphorylation, amidation, derivatization through known protecting/blocking groups, protease cleavage, association with cellular antibody units or other proteins, and the like. . Any large number of chemical modifications can be achieved using known methods, including, but not limited to, specific chemical cleavage, acylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. In addition, analogues or derivatives may include one or more unnatural amino acids . In some examples, antibodies may have modifications (eg, substitution, deletion, or addition) at certain amino acid residues that interact with the Fc receptor. And furthermore, the antibody is selected from murine antibodies, mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies.

The linker moiety includes a first linker moiety A and a second linker moiety L; the first linker moiety A includes a group covalently attached to a thiol group or an amino group Ab.

D is a drug moiety, where the drug includes, but is not limited to, cytotoxic drugs, cell differentiation factors, stem cell nutritional factors, steroid drugs, drugs for the treatment of autoimmune diseases, anti-inflammatory drugs, or drugs for the treatment of infectious diseases . In addition, drug moiety D includes, but is not limited to, tubulin inhibitors or DNA damaging agents. Tubulin inhibitors include, but are not limited to, dolastamine, auristatins, maytansines; DNA damaging agents include, but are not limited to, calicheamycins, duocamycins, anthramycin derivative PBD (pyrrolobenzadiazepine), camptothecin derivative SN-38, topoisomerase I inhibitors. Auristatin drugs include, but are not limited to, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and auristatin F (AF) or derivatives thereof. Maytansine drugs include, but are not limited to, DM1, DM3, DM4 or derivatives thereof (Research Progress on Warhead Molecules of antibody-drug conjugate, Hu Xinyue et al., Chinese Medicinal Biotechnology, Dec., 2017, Vol. 12, No. 6) (Research advance of maytansinoid class antibody drug conjugates, Zhou Lei et al., Chinese Journal of New Drugs, 2016, Vol. 25, No. 22, pages 2521-2530). The drug moiety D can also be amanithins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combrestatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate, nettropsins, rhizoxins, taxanes, tubulizins or vinca alkaloids. The drug moiety D may also be a vitamin A precursor, folic acid, and the like. The drug formulation D is not limited to the above categories, but also includes all drugs that can be used in the case of ADC.

In the above formula II

n is 1, 2, 3, or 4.

The linker moiety is characterized in that the first linker moiety A has the structure represented by formula III:

,

,

wherein

,

,

,

,

,

,

,

,

,

,или отсутствуют, M₁ и M₂ являются одинаковыми или различными, но не могут отсутствовать одновременно;M ₁ , M ₂ covalently linked to the thiol group or amino group of the antibody or component of the functional fragment of the antibody (which is represented by Ab), which are each independently selected from

,

,

,

,

,

,

,

,

,

, or absent, M ₁ and M ₂ are the same or different, but cannot be absent at the same time;

NR⁴

,

C (=O)

,

C (=O) O

,

C (=O) NR¹¹

,

C=NR³

,

C (=S) O

,

C (=S) NR¹²

,

C (=S) S

,

NR¹³ (C=O)

,

NR¹⁴ (C=S) NR⁵

,

O (C=O) NR¹⁵

,

S (=O) ₂

и их любой комбинации; L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₉ -alkyl, C ₂ -C ₉ -alkenyl, C ₂ -C ₉ -alkynyl, aryl, heteroaryl, C ₃ -C ₉ -cycloalkyl, C ₃ - C ₉ -heterocylyl, polyethylene glycol, O, S,

NR ⁴

,

C (=O)

,

C(=O)O

,

C (=O) NR ¹¹

,

C=NR ³

,

C (=S) O

,

C (=S) NR ¹²

,

C (=S)S

,

NR ¹³ (C=O)

,

NR ¹⁴ (C=S) NR ⁵

,

O(C=O)NR ¹⁵

,

S(=O) ₂

and any combination of them;

,

, N, B, P, арила, гетероарила, циклоалкила, (гетероциклил)алкила; Q choose from

,

, N, B, P, aryl, heteroaryl, cycloalkyl, (heterocyclyl)alkyl;

R² (C=O)

,

C (=O) R²C (=O)

, полиэтиленгликоля,

,

NR⁹

, O, S и их любой комбинации, или отсутствует; L ₃ is covalently linked to the second linker moiety L and is selected from

R ² (C=O)

,

C (=O) R ² C (=O)

, polyethylene glycol,

,

NR ⁹

, O, S and any combination thereof, or missing;

R ¹ is an arbitrary group or absent, R ² , R ³ , R ⁴ , R ⁵ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ are each independently selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, aryl, heteroaryl, C ₃ -C ₉ cycloalkyl, C ₃ -C ₉ heterocyclyl;

the second linker moiety, L, is either a cleavable linker or a non-cleavable linker, or is absent, when L is absent, L ₃ is directly covalently bound to the drug moiety D.

In addition, the above L ₁ , L ₂ , L ₃ , Q, R ¹ can also be respectively as follows:

NR⁴

,

C (=O)

,

C (=O) O

,

C (=O) NR¹¹

,

O (C=O) NR¹⁵

;L ₁ , L ₂ are each independently selected from C ₁ -C ₄ -alkyl, C ₆ -C ₈ -aryl, C ₆ -C ₈ -heteroaryl, C ₅ -C ₆ -cycloalkyl, C ₅ -C ₆ -heterocyclyl , C ₂ -C ₁₂ - polyethylene glycol, O, S,

NR ⁴

,

C (=O)

,

C(=O)O

,

C (=O) NR ¹¹

,

O(C=O)NR ¹⁵

;

, N, C₆-C₈-арила, C₆-C₈-гетероарила, C₃-C₈-циклоалкила, C₃-C₈-гетероциклила;Q choose from

, N, C ₆ -C ₈ -aryl, C ₆ -C ₈ -heteroaryl, C ₃ -C ₈ -cycloalkyl, C ₃ -C ₈ -heterocyclyl;

R² (C=O)

,

C(=O)R²C(=O)

, полиэтиленгликоля,

,

NR⁹

, O, S и их любой комбинации или отсутствует;L ₃ choose from

R ² (C=O)

,

C(=O)R ² C(=O)

, polyethylene glycol,

,

NR ⁹

, O, S and any combination thereof or missing;

R ¹ is selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, aryl, heteroaryl, C ₃ -C ₉ cycloalkyl, C ₃ -C ₉ heterocyclyl.

In addition, the first linker component has the structure

(A-1')

(A-1')

(A-1'')

(A-1''')

(A-2')

(A-2'')

(A-2''')

(A-2'''')

(A-3')

(A-3'')

(A-3''')

(A-3'''')

(A-4')

(A-4'')

(A-4''')

(A-5')

(A-5'')

(A-5''')

(A-5'''')

(A-6')

(A-6'')

(A-6''')

(A-7')

(A-7'')

(A-8')

(A-8'')

Additionally, the antibody or functional antibody fragment specifically binds to cell surface receptors or tumor-associated antigens.

In addition, the first linker moiety A also includes a group that is covalently bonded to the thiol group or amino group of Ab.

In addition, even the first linker component A can have the structure

A-1 A-2 A-4

A-5 A-6 A-7

A-10 A-11 A-12

A-13 A-15 A-16

A-17 A-18 A-19

A-20 A-21 A-23

A-24 A-25 A-28

A-29 A-31 A-33

A-34 .

, O, S,

NR⁶

,

C (=O)

,

C (=O) O

,

C (=O) NR⁶

,

C=NR⁶

,

C (=S) O

,

C (=S) NR⁶

,

C (=S) S

,

NR⁶ (C=O)

,

NR⁶ (C=S) NR⁷

,

O (C=O) NR⁶

,

S (=O) ₂

, Val-Val-PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac)-PAB, Phe-Lys-PAB, Phe-Lys (Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB и их любую комбинацию, или отсутствует, причем R⁶, R⁷ выбирают, каждый независимо, из H, C₁-C₆-алкила, C₂-C₆-алкенила, C₂-C₆-алкинила, арила, гетероарила, C₃-C₉-циклоалкила, C₃-C₉-гетероциклила, e равен 0, 1, 2, 3, 4, 5, 6, 7 или 8.In addition, another second linker moiety may be C ₁ -C ₉ alkyl, C ₂ -C ₉ alkenyl, C ₂ -C ₉ alkynyl, aryl, heteroaryl, C ₃ -C ₉ cycloalkyl, C ₃ -C ₉ -heterocyclyl,

, O, S,

NR ⁶

,

C (=O)

,

C(=O)O

,

C (=O) NR ⁶

,

C= ^NR6

,

C (=S) O

,

C (=S) NR ⁶

,

C (=S)S

,

NR ⁶ (C=O)

,

NR ⁶ (C=S) NR ⁷

,

O (C=O) NR ⁶

,

S(=O) ₂

, Val-Val-PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac)-PAB, Phe-Lys-PAB, Phe-Lys (Ac)-PAB, D-Val-Leu-Lys , Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, and any combination thereof, or absent, wherein R ⁶ , R ⁷ are each independently selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, aryl, heteroaryl, C ₃ -C ₉ -cycloalkyl, C ₃ -C ₉ -heterocyclyl, e is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

Preferably, the second linker moiety L may be

In addition, another drug moiety is selected from cytotoxic drugs, cell differentiation factors, stem cell nutritional factors, steroid drugs, drugs for the treatment of autoimmune diseases, anti-inflammatory drugs or drugs for the treatment of infectious diseases.

In addition, another drug constituent includes, but is not limited to, tubulin inhibitors or DNA damaging agents.

In addition, another tubulin inhibitor includes, but is not limited to, dolastatins, auristatins, maytansins; the DNA damaging agent includes, but is not limited to, calicheamycins, duocamycins, anthramycin derivative PBD, camptothecin derivatives (preferably camptothecin derivative SN-38), topoisomerase I inhibitors (i.e., Dxd).

In addition, another component of the drug includes, but without limitation, amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combrestatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins, eleutherobins, epothilones, estramustines, lexytropsins, maytansins, methotrexate , netropsins, puromycins, rhizoxins, taxanes, tubulisins or vinca alkaloids, vitamin A precursors, folic acid, camptothecin derivatives SN-38, topoisomerase I inhibitors (i.e., Dxd).

Preferably, drug moiety D may be

In some preferred examples, the antibody drug conjugate has the structure

ADC1

ADC1

ADC2

ADC3

ADC4

ADC5

ADC6

ADC7

ADC8

ADC9

ADC10

ADC11

ADC12

ADC13

ADC14

ADC15

ADC16,

wherein

the antibody or functional fragment of an Ab antibody is an antibody against cell surface receptors and tumor-associated antigens;

n ¹ , n ² , n ³ are each independently selected from 0, 1, 2, 3, 4; n ¹ , n ² , n ³ are not equal to 0 at the same time, and n ¹ +n ² +n ³ ≤4;

n ⁴ , n ⁵ , n ⁶ , n ⁷ are each independently selected from 0, 1, 2, 3, 4; n ⁴ , n ⁵ , n ⁶ , n ⁷ are not equal to 0 at the same time, and n ⁴ +n ⁵ +n ⁶ +n ⁷ ≤4;

n ⁸ , n ⁹ are each independently selected from 0, 1, 2, 3, 4; n ⁸ , n ⁹ are not equal to 0 at the same time, and n ⁸ +n ⁹ ≤4;

m ¹ , m ² , m ³ are chosen, each independently, from 0, 1, 2; at least one of m ¹ , m ² , m ³ is 0, but m ¹ , m ² , m ³ are not 0 at the same time, and m ¹ +m ² +m ³ ≤2;

m ⁴ , m ⁵ are each independently selected from 0, 1, 2; m ⁴ , m ⁵ are not equal to 0 at the same time, and m ⁴ +m ⁵ ≤2.

In addition, the antibody-drug conjugate has the structure

ADC17

ADC17

ADC18

ADC19

ADC20

ADC21

ADC22

ADC23

ADC24

ADC25

ADC26

ADC27

ADC28

ADC29

ADC30

ADC31

ADC32

wherein

Ab is an antibody against cell surface receptors and tumor-associated antigens;

n ¹⁰ is 0, 1, 2, 3 or 4;

m ⁶ is 0, 1 or 2.

In addition, the antibody-drug conjugate has the structure

ADC33

ADC33

ADC34

ADC35

ADC36

ADC37

ADC38

ADC39

ADC40

ADC41

ADC42

ADC43

ADC44

ADC45

ADC46

ADC47

ADC48

wherein

Ab is an antibody against cell surface receptors and tumor-associated antigens;

n ¹¹ is 0, 1, 2, 3 or 4;

m ⁷ is 0, 1 or 2.

The present invention also relates to a pharmaceutical composition that includes any antibody-drug conjugate described in the present invention and a pharmaceutically acceptable carrier.

The present invention also relates to the use of any linker described in the present invention in the preparation of an antibody-drug conjugate.

The present invention also relates to the use of any antibody-drug conjugate described in the present invention, or any pharmaceutical composition described in the present invention, in the preparation of a drug for the treatment of cancer, autoimmune diseases, inflammatory diseases or infectious diseases.

Description of figures

Figure 1A shows a chromatogram of the HIC-HPLC antibody-drug conjugate Her2-A'-7-Val-Cit-PAB-MMAE (the molar ratio of drug to thiol group of the antibody added during coupling is 5:1).

Figure 1B shows a chromatogram of the HIC-HPLC antibody-drug conjugate Her2-A'-11-Val-Cit-PAB-MMAE (the molar ratio of drug to thiol group of the antibody added during coupling is 5:1).

Figure 1C shows a chromatogram of the HIC-HPLC antibody-drug conjugate Her2-A'-10-Val-Cit-PAB-MMAE (the molar ratio of drug to thiol group of the antibody added during coupling is 5:1).

Figure 1D shows a chromatogram of the HIC-HPLC antibody-drug conjugate Her2-575DZ-Val-Cit-PAB-MMAE (the molar ratio of drug to thiol group of the antibody added during coupling is 5:1).

Figure 2A shows SDS-PAGE patterns of Her2-A'-11-Val-Cit-PAB-MMAE (coupling buffers respectively pH 8.5, 9.0, 7.4), Her2-A'-7- Val-Cit-PAB-MMAE (coupling buffers respectively pH 8.5, 9.0, 7.4), Her2-A'-10-Val-Cit-PAB-MMAE (coupling buffers respectively pH 8, 5, 9.0, 7.4) (coupling buffer has a pH of 9.0).

Figure 2B shows SDS-PAGE electrophoresis patterns of Her2-A'-7-Val-Cit-PAB-MMAE, Her2-575DZ-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE (coupling buffer has a pH of 9.0).

Figure 3 shows antigen affinity curves for antibody-drug conjugates (wherein the antibody-drug conjugates are Her2-A'-7-Val-Cit-PAB-MMAE and Her2-A'-10-Val-Cit-PAB-MMAE; wherein the Her2 antibody and Her2-Mc-Val-Cit-PAB-MMAE are negative and positive controls) determined by ELISA, where the abscissa shows concentration, and the ordinate shows optical density, absorbance.

Figure 4A shows the degree of inhibition of proliferation of human breast cancer cells SK-BR-3 by antibody-drug conjugates (Her2-A'-11-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB -MMAE).

Figure 4B shows the degree of inhibition of proliferation of human breast cancer cells SK-BR-3 by antibody-drug conjugates (Her2-A'-8-Val-Cit-PAB-MMAE, Her2-A'-7-Val-Cit-PAB -MMAE).

Figure 5 shows the results of coupling the conjugates (Ab-A) of the antibody (Ab) and the first linker moiety (A), with Nos. 1,2, 3 referring to Her2-A'-7, the coupling buffers respectively having a pH of 8.5, 9.0, 7.4; No. No. 4, 5, 6 refer to Her2-A'-8, coupling buffers respectively have pH 8.5, 7.4, 9.0; Nos. 7, 8, 9 refer to Her2-A'-10, coupling buffers respectively have pH 8.5, 9.0, 7.4; Nos. 10, 11, 12 refer to Her2-A'-11, coupling buffers respectively have pH 9.0, 7.4, 9.5; Nos. 13, 14, 15 refer to Her2-A'-12, coupling buffers, respectively, have a pH of 7.4, 9.0, 8.5; Nos. 16, 17, 18 refer to Her2-A'-20, coupling buffers, respectively, have a pH of 8.5, 9.0, 7.4; Nos. 19, 20, 21 refer to Her2-A'-28, coupling buffers, respectively, have a pH of 9.0, 8.5, 7.4; Nos. 22, 23, 24 refer to Her2-A'-31, the coupling buffers respectively have a pH of 7.4, 8.5, 9.0.

Figure 6A shows the LC-MS spectrum of a naked antibody (anti-claudin 18.2).

Figure 6B shows the LC-MS spectrum of claudin 18.2-A'-7-VC-PAB-MMAE .

Figure 7A shows a map of the distribution of isoelectric points of a naked antibody (anti-claudin 18.2).

Figure 7B shows a map of the placement of isoelectric points of claudins 18.2-A'-7-VC-PAB-MMAE .

Embodiments of the invention

Embodiments of the present invention are described in detail below with the help of specific examples, but in any case they should not be construed as limiting the present invention.

[Definitions]

Unless otherwise defined, the scientific and technological terms used in the present description have the same meanings that are usually given to them by specialists in this field of technology.

While numerical ranges and approximate parameter values are shown within the broad scope of the present invention, the numerical values shown in specific examples are recorded as accurately as possible. However, any numerical value inherently inevitably contains a certain error, which is caused by the standard deviation in the measurement. In addition, all intervals disclosed in the present description, should be understood as overlapping any and all sub-intervals contained therein. For example, the recorded interval "1-10" should be understood to span any and all sub-intervals contained between a minimum value of 1 and a maximum value of 10 (including endpoints), i.e., all sub-intervals starting with a minimum value of 1 or greater, e.g. , 1-6.1, and all sub-intervals ending with a maximum value of 10 or less, such as 5.5-10. In addition, any reference indicated as "included in the present description" should be understood as fully included.

», используемый в настоящем изобретении, означает, что группа, содержащая «

», соединена в этом месте с другими группами через химическую связь.Symbol "

", used in the present invention, means that the group containing "

", is connected at this point with other groups through a chemical bond.

The term "antibody" is used in the present invention in its broadest sense and covers various antibody structures, including, but not limited to, monoclonal antibody, polyclonal antibody, multispecific antibody (such as a bispecific antibody), and antibody fragments. In particular, "antibody" as used herein refers to a protein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (VH for short) and a heavy chain constant region. The heavy chain constant region includes three domains CH1, CH2 and CH3. Each light chain includes a light chain variable region (VL for short) and a light chain constant region. The light chain constant region includes one CL domain. The VL and VH regions can further subdivide into several regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs in the following order from amino to carboxy: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These heavy chain and light chain variable regions contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells (such as effector cells) of the immune system and the first component (Clq) of the classical complement system. Chimeric or humanized antibodies are also included in the antibodies of the present invention.

The term "humanized antibody" refers to an antibody that contains a CDR region derived from a non-human antibody and the other portion of the antibody molecule is derived from one or more human antibodies. In addition, to maintain binding affinity, it is possible to modify certain framework residues (called FRs) (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). A humanized antibody or fragments thereof according to the present invention can be obtained by methods known to those skilled in the art (for example, methods described in Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet Eng Rev. 10:1-142, 1992; or Bebbington et al., Bio/Technology 10:169-175, 1992).

The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species. For example, the variable region sequence is from a mouse antibody, and the constant region sequence is from a human antibody. Chimeric antibody or fragments thereof can be obtained using genetic recombinant technology. For example, a chimeric antibody can be obtained by cloning recombinant DNA, and the recombinant DNA includes a promoter and a sequence encoding the variable regions of a non-human, in particular murine, antibody of the present invention and a sequence encoding the constant regions of a human antibody. The chimeric antibody of the present invention encoded by this recombinant gene would be, for example, a mouse-human chimera. The specificity of this antibody is determined by a variable region derived from mouse DNA and the isotype is determined by a constant region derived from human DNA. As a method for producing a chimeric antibody, for example, see Verhoeyn et al. (BioEssays, 8:74, 1988).

The term "monoclonal antibody" refers to products derived from antibody molecules of a single molecular composition. The composition of a monoclonal antibody shows a single binding specificity and affinity for a specific epitope.

The term "functional fragment" in the present invention means that the antibody fragment consists of, or includes, a partial sequence of the heavy or light variable chain of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity as the antibody from which it is derived and of sufficient affinity, preferably at least 1/100 of the affinity of the antibody from which it is derived, and more preferably at least 1/10. This functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 20, 50 and 100 consecutive amino acids from the antibody sequence from which it is derived.

The term "DAR", also referred to as the ratio of drug to antibody, in the present invention is a unique attribute of the quality of the antibody-drug conjugate. A high DAR value can affect the safety of ADC, and its therapeutic window is narrow. A low DAR may reduce the effectiveness of ADC, but its safety is significant and its therapeutic window is wide. The best average DAR range is 2-4.

The term "linker" in the present invention refers to a compound that can act as a "bridge" for the appropriate interaction of the antibody/functional fragment and drug and connect them. The "linker" included in the present invention includes a first linker moiety and a second linker moiety. The term "first liquor component" in the present invention includes two connecting groups that are the same or different. Linking groups are capable of simultaneously covalently bonding to the interchain(s) thiol(s) group(s) or amino(s) of an antibody/functional antibody fragment, and linking groups are any functional groups capable of covalently bonding to the thiol(s) group(s) or amino group(s). The term "second liquor moiety" is used in the present invention for combination with a drug and is any cleavable or non-cleavable linker. The cleavable linker releases toxins depending on intracellular processes such as intracytoplasmic repair, exposure to acidic conditions in the lysosome, or cleavage by an intracellular specific protease. The non-cleavable linker can release the drug only after degradation of the protein component of the ADC antibody.

The term "drug" in the present invention usually refers to any compound that has the desired biological activity and has a reactive functional group to obtain a conjugate of the present invention. The desired biological activity includes diagnosing, curing, alleviating, curing, preventing disease in a human or other animal. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs of the present invention. Specifically, drugs include, but are not limited to, cytotoxic drugs, cell differentiation factors, steroid drugs, drugs for the treatment of autoimmune diseases, anti-inflammatory drugs, or drugs for the treatment of infectious diseases. More specifically, drugs include, but are not limited to, tubulin inhibitors or DNA/RNA damaging agents.

In some embodiments of the present invention, the drug is selected from maytansine compounds, V-ATPase inhibitors, proapoptotic agents, Be 12 inhibitors, McL1 inhibitors, HSP90 inhibitors, IAP inhibitor, mTOr inhibitors, microtubulin stabilizers, microtubulin destabilizers, auristatin, dolastatin, MetAP (methionine aminopeptidase) , CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators , an agent that binds DNA minor grooves, DHFR inhibitors, as well as dolastatin peptide, vitamin A precursors, folic acid, camptothecin derivatives.

In some preferred embodiments of the present invention, the drug is a cytotoxic drug (such as antimetabolites, antitumor antibiotics, alkaloids), an immunostimulant, or a radioisotope. Preferably, the drug can be selected from amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatins, cryptophycins, disoderomolides, docetaxel, doxorubicin, echinomycins, eleutherobins, epothilones, estramustines, texitropsins, maytansins, methotrexate, netropycins, puromoxins, taxanes, tubulizins or vinca alkaloids. More preferably, the "drug" is selected from MMAD (Mnomethylauristatin D) and its derivatives, MMAE (Mnomethylauristatin E) and its derivatives, MMAF (Mnomethylauristatin F) and its derivatives, DM1 maytansin derivatives (M1 mertansin derivative), DM4 maytansin derivatives (M4 mertansin derivative). ), duocarmycin and its derivatives, calicheamicin and its derivatives, PBDA (pyrrolobenzodiazepines), doxorubicin, vinca alkaloids, methotrexate, vinblastine, daunorubicin and its derivatives, tubulizins and their derivatives, camptothecin derivative SN-38, topoisomerase I inhibitors (i.e. ., Dxd).

In some specific embodiments, the "drug" may be the substances listed below and their derivatives.

Maytansin:

Maytansine - Maytansine

Maytansine derivatives:

Ansamitocin derivatives:

A is C=O, (C=O)NR ⁴ , and C=O(O) - A is C=O, (C=O)NR ⁴ and C=O(O)

Y is a substituent group - Y is a substituent group

Ansamitocin derivatives - Ansamitocin derivatives

Alaninylmaytansinol:

Alaninylmaytansinol - Alaninylmaytansinol:

MMAE:

MMAD:
MMAD

MMAF:

Tubulisin D:

Tubulysin D

Calicheamicin:

Calicheamicin - Calicheamicin

Doxorubicin:

Doxorubicin - Doxorubicin

Pyrrolobenzodiazepine derivatives:

Vitamin A precursor:

Folic acid:

Topoisomerase I inhibitor (i.e., Dxd):

The term "antibody-drug conjugate" in the present invention refers to a compound obtained by combining an antibody/antibody functional fragment, a linker (first linker moiety, second linker moiety) and a drug moiety through a chemical reaction. Although the antibody-drug conjugate of the present invention is still a mixture, compared with the conventionally prepared conjugate, its DAR distribution range is very narrow, and the best antibody-drug conjugate has an average DAR range of 2-4. With respect to the preparation of an antibody-drug conjugate of the present invention, when the conjugate (A-L-D) of the first linker moiety (A) and the second linker moiety-drug conjugate (L-D) are combined with an antibody (such as the anti-claudin 18.2 antibody shown in the example) , it can be hydrolyzed under mild hydrolysis conditions, and the hydrolysis site is at the maleimide group of the first linker. Taking Ab-A'-7-VC-PAB-MMAE as an example (and in the case where one antibody is combined with 4 drugs), when the Ab-A'-7-VC-PAB-MMAE conjugate undergoes 4 hydrolysis, the image structure has the form

or

.

.

When the Ab-A'-7-VC-PAB-MMAE conjugate undergoes 7 hydrolysis, the structure image is

When the Ab-A'-7-VC-PAB-MMAE conjugate undergoes 8 hydrolysis, the structure image is

The term "pharmaceutical composition" in the present invention refers to a combination of at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient, which are combined to achieve a specific goal. In some embodiments, the pharmaceutical composition includes a combination of ingredients that are separated in time and/or space, as long as they work together to achieve the goal of the present invention. For example, the ingredients contained in a pharmaceutical composition may be administered to a subject as a whole or separately. When the ingredients contained in a pharmaceutical composition are administered to a subject separately, the ingredients may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline such as PBS (phosphate buffer), glucose solution, mannitol, dextroglucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid , ethanol or a polyalkylene glycol such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends in particular on whether the composition of the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may contain, as additives, wetting agents, emulsifiers or buffering agents.

The pharmaceutical composition, vaccine or pharmaceutical preparation of the present invention may be administered by any suitable route such as oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.

The term "alkyl" in the present invention refers to a linear or branched saturated hydrocarbon (ie, free from double or triple bonds). An alkyl group may have 1-9 carbon atoms (when used in the present invention, the numerical range "1-9" refers to any integer within this range, e.g. "1-9 carbon atoms" means that an alkyl group may contain 1 carbon atom , 2 carbons, 3 carbons, ... up to 9 carbons; however, the definition of "alkyl" also includes alkyl groups without a specified chain length). The alkyl group may be a medium sized alkyl group containing 1-9 carbon atoms. An exemplary alkyl group includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, and the like.

The term "alkenyl" in the present invention refers to a linear or branched hydrocarbon containing one or more double bonds. The alkenyl group may have 2-9 carbon atoms and also includes alkenyl groups with no specified chain length. The alkenyl group may be a medium sized alkenyl group containing 2-9 carbon atoms. The alkenyl group may also be a small alkenyl group containing 2-4 carbon atoms. An alkenyl group may be depicted as "C2-4-alkenyl" or similar representations. For example, "C2-4-alkenyl" means that 2-4 carbon atoms are present in the alkenyl chain, i.e., the alkenyl chain can be selected from ethenyl, propen-1-yl, propen-3-yl, buten-1- yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methylpropen-1-yl, 2-methylpropen-1-yl, 1-ethylethen-1-yl, 2-methylpropen-3- yl, buta-1,2-dienyl, buta-1,2-dien-4-yl. Exemplary alkenyl includes, but is not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

The term "alkynyl" in the present invention refers to a linear or branched hydrocarbon containing one or more triple bonds. The alkynyl group may have 2-9 carbon atoms and also includes alkynyl groups of no specified chain length. The alkynyl group may be a medium sized alkynyl group containing 2-9 carbon atoms. The alkynyl group may also be a lower alkynyl group containing 2-4 carbon atoms. An alkynyl group may be depicted as "C2-4-alkynyl" or similar representations. For example, "C2-4-alkynyl" means that 2-4 carbon atoms are present in the alkynyl chain, i.e., the alkynyl chain can be selected from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1- yl, butyn-2-yl, butyn-3-yl and 2-butynyl. Exemplary alkynyl includes, but is not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

The term "aryl" in the present invention refers to a cyclic system with a conjugated π-electron system and includes carbocyclic aryl (such as phenyl) and heterocyclic aryl (such as pyridine). The term includes groups with a single ring or multiple fused rings (ie, rings that have a common pair of adjacent atoms) and the entire ring system is aromatic.

The term "heteroaryl" in the present invention refers to an aromatic ring or ring system (ie, two or more fused rings sharing a pair of adjacent atoms) containing one or more heteroatoms. In other words, in addition to carbon, the cyclic framework includes, but is not limited to, nitrogen, oxygen, sulfur, and other elements. When the heteroaryl is a ring system, each ring in the system is aromatic. A heteroaryl may have 5-18 ring members (ie, the number of atoms constituting the cyclic framework, including carbon atoms and heteroatoms). The current definition also includes heteroaryl groups without a given ring size. Examples of heteroaryl include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, benzothienyl.

The term "cycloalkyl" in the present invention refers to a fully saturated carbocyclic or cyclic system. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The term "(heterocyclyl)alkyl" in the present invention refers to heterocyclyl as a substituent connected to other groups via alkylene. Examples include, but are not limited to, imidazolinylmethyl and indolinylethyl. The term "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in its backbone. The heterocyclyl may be connected in the form of fused rings, bridged rings or spirocycles. At least one ring in the heterocyclyl system is non-aromatic and may have any degree of saturation. The heteroatom may be located in the non-aromatic or aromatic ring of the ring system. The heterocyclyl may have 3-20 ring atoms (ie, the number of atoms constituting the ring backbone, including the number of carbon atoms and heteroatoms). The current definition also includes heterocyclyl groups with no specified range of ring members. The heterocyclyl group may also be a medium sized heterocyclyl group containing 3-10 ring atoms. The heterocyclyl group may also be a small size heterocyclyl group containing 3-6 ring atoms. Examples of the heterocyclyl group include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thietanyl, piperidinyl, piperazinyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl , 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathianyl, 1,4-oxathianyl, 2H-1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithiolanyl , isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, oxazolidinone, thiazolidinyl, 1,3-oxathiolyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrochinoylbenzofuranyl, and tetrahydrobenzofuranyl.

[Examples]

General procedure A: general method for the synthesis of a bromomaleimide linker

Amines and their salts or Boc protected derivatives - Amines and their salts or Boc-protected derivatives

Bromomaleic anhydride - Bromomaleic anhydride

Amines and their salts or Boc-protected derivative (1 eq.) and anhydrous sodium acetate (2.5 eq.) are dissolved in acetic acid (1 mmol/2 ml) and heated to 50-100 ° C, then bromomaleic anhydride is quickly added (2.5 eq.), and carry out the reaction at 50-100°C during the night. The reaction solution was cooled to 45° C., concentrated under reduced pressure, ethyl acetate was added to the residue, then washed, dried and concentrated to give a crude reaction product. The crude product is purified by normal phase column chromatography or reverse phase preparative liquid chromatography to give the desired product.

General Procedure B: General Method for the Synthesis of a Thiophenol Maleimide Linker

DMF - DMF, Sodium thiophenolate _ Sodium thiophenolate

The bromomaleimide linker (1.0 eq.) was dissolved in anhydrous DMF (1 mmol/5 ml), pyridine (3.0 eq.) was added to the solution, and then stirred at room temperature for 5-10 minutes. Dissolve sodium thiophenolate (2.1 eq.) in anhydrous DMF (1 mmol/1 ml) and add dropwise to the reaction solution. After that, stirring is continued at room temperature for 0.5-5 hours. After completion of the reaction, 5 times the volume of distilled water was added to the mixture, the solids precipitated, and then the crude reaction product was obtained by filtration, washing, drying and concentration. The crude product is purified by normal phase column chromatography or reverse phase preparative liquid chromatography to give the desired product.

Example 1 Synthesis of A'-1

Bromomaleic anhydride - Bromomaleic anhydride

According to general procedure A, 300 mg of compound 1 (ie, 3,5-diaminobenzoic acid) are added while adding other materials in molar ratios at the same time. The reaction is carried out at 70° C. overnight. The reaction solution is purified by preparative liquid chromatography to give the target compound A'-1. LC-MS (ESI ⁺ ) 468.9 [M+H] ⁺ .

Example 2 Synthesis of A'-4

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 100 mg of compound A'-1 are added while other materials are added in molar ratios. The reaction solution is purified by preparative liquid chromatography to give the target compound A'-4. LC-MS (ESI ⁺ ) 529.0 [M+H] ⁺ .

Example 3 Synthesis of A'-2

Bromomaleic anhydride - Bromomaleic anhydride, DMF - DMF

Compound 2 (1 mol) was dissolved in a mixture of ethanol and water, stirred, reduced iron powder (5 mol), ammonium chloride (3 mol) were added to the solution in one step, and then the reaction was carried out at 80°C for 1 hour. After completion of the reaction, the reaction solution was cooled to room temperature, filtered to remove solids, and distilled under reduced pressure to remove the solvent, and compound 3 was obtained. LC-MS m/z (ES ⁺ ) 154.05 (M+H) ⁺ .

According to general procedure A, compound 3 (1 mol) is added while adding other materials in molar ratios at the same time. The reaction is carried out at 80° C. overnight. The reaction solution is purified by preparative liquid chromatography to give the target compound A'-2. LC-MS m/z (ES ⁺ ) 471.85 (M+H) ⁺ .

Example 4 Synthesis of A'-7

Bromomaleic anhydride - Bromomaleic anhydride

Compound 5 (Boc-imidazole, 2.37 g, 14.1 mmol) was dissolved in 30 ml of toluene, and compound 4 (diethylenetriamine, 0.66 g, 6.41 mmol) was added to the solution at room temperature, followed by stirring with heating at 60°C for 6 hours. After completion of the reaction, the reaction solution was distilled under reduced pressure to give an oily crude reaction product. The crude product was dissolved in 40 ml dichloromethane, washed with water (40 ml×3), the solution was dried, filtered and concentrated to give 1.84 g of compound 6 in 95% yield. LC-MS (ESI ⁺ ) 304.2 [M+H] ⁺ .

Compound 6 (3.3 g, 10.89 mmol) was dissolved in 30 ml of anhydrous toluene, and compound 7 (succinic anhydride, 1.3 g, 13 mmol) was added to the solution, followed by stirring with heating at 60° C. overnight . After completion of the reaction, the reaction solution was distilled under reduced pressure to remove toluene, the residue was dissolved in 20 ml of dichloromethane, washed with water (20 ml × 2), the solution was washed with saturated NaCl solution (20 ml × 1), dried, filtered, and distilled under reduced pressure, and get 3.5 g of compound 8 with a yield of 80%. LC-MS (ESI ⁺ ) 404.3 [M+H] ⁺ .

According to general procedure A, 100 mg of compound 8 are added while adding other materials in molar ratios at the same time. The reaction is carried out at 70° C. overnight. The reaction solution was purified by preparative liquid chromatography to give 25.8 g of the target compound A'-7 in 20% yield. LC-MS (ESI ⁺ ) 519.9 [M+H] ⁺ .

Example 5 Synthesis of A'-8

THF - THF, Bromomaleic anhydride - Bromomaleic anhydride

Compound 9 (1,3-diaminopropanol, 5.0 g, 55.47 mmol) was dissolved in 50 ml of THF and 20 ml of water with stirring. A solution of Boc ₂ O (25.4 g, 116.5 mmol, 2.1 eq.) in 30 ml THF is added dropwise at room temperature. Thereafter, the reaction solution was stirred at room temperature overnight. After completion of the reaction, the solution is concentrated under reduced pressure. The residue, to which 30 ml of petroleum ether was added, was stirred at room temperature for 20 minutes, a white solid precipitated, which was filtered off and dried to give 14.7 g of white compound 10 with a yield of 91%. LC-MS (ESI ⁺ ) 291.2 [M+H] ⁺ .

Compound 10 (14.7 g, 50.64 mmol) was dissolved with stirring in 100 ml of anhydrous THF, followed by cooling in an ice bath. NaH (4.8 g, 202.55 mmol, 4 eq.) was then added in portions, followed by stirring for 10 minutes. A solution of compound 11 (tert-butyl bromoacetate, 24.7 g, 126.6 mmol, 2.5 eq.) in 50 ml THF was added dropwise. Thereafter, the reaction solution was heated at 40° C. and stirred overnight. After completion of the reaction, the reaction solution was cooled to room temperature, diluted with 250 ml of ethyl acetate, an ice-water mixture was gradually added thereto until a large amount of bubbles formed, and then the liquid was separated. The aqueous phase is extracted with ethyl acetate, the organic phases are combined, washed, dried and concentrated. The residue, to which 50 ml of petroleum ether was added, was stirred at room temperature for 20 minutes, and a white solid precipitated, which was filtered off and dried to give 12.1 g of white compound 12 with a yield of 60%. LC-MS (ESI ⁺ ) 404.1 [M+H] ⁺ .

Compound 12 (12.1 g, 30.0 mmol) was dissolved with stirring in 120 ml of anhydrous DCM, then a solution (4M, 45 ml, 180 mmol) of hydrogen chloride in 1,4-dioxane was added to the solution, followed by stirring at room temperature in during the night. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residue is dispersed by ultrasound in 30 ml of methyl tert-butyl ether, centrifuged to remove the supernatant and then dispersed in 30 ml of petroleum ether to remove the supernatant. The solid is kept under reduced pressure to remove the solvent, and get 7.7 g of white compound 13 with a yield of 100%. LC-MS (ESI ⁺ ) 148.1 [M+H] ⁺ .

According to the general procedure A, add 1.2 g of compound 13, while adding other materials at the same time in molar ratios. The reaction is carried out at 85°C for 5 hours. The reaction solution was purified by preparative liquid chromatography to give 890 mg of the target compound A'-8 in 35% yield. LC-MS (ESI ⁺ ) 464.9 [M+H] ⁺ .

Example 6 Synthesis of A'-9

Compound - Compound, Bromomaleic anhydride - Bromomaleic anhydride

According to general procedure A, 500 mg of compound 14 are added while adding other materials in molar ratios at the same time. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 240 mg of the title compound A'-9 in 23% yield. LC-MS (ESI ⁺ ) 420.9 [M+H] ⁺ .

Example 7 Synthesis of A'-10

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 104 mg of compound A'-7 are added while adding other materials in molar ratios at the same time. The reaction is carried out at room temperature for 2.5 hours. The reaction solution was purified by preparative liquid chromatography to give 27.7 mg of the title compound A'-10 in 24% yield. LC-MS (ESI ⁺ ) 596.1 [M+H] ⁺ .

Example 8 Synthesis of A'-11

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 600 mg of compound A'-8 are added while adding other materials in molar ratios at the same time. The reaction is carried out at room temperature for 2.5 hours. The reaction solution was purified by preparative liquid chromatography to give 203 mg of the target compound A'-11 in 30% yield. LC-MS (ESI ⁺ ) 525.1 [M+H] ⁺ .

Example 9 Synthesis of A'-12

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 120 mg of compound A'-9 are added, while other materials are added in molar ratios. The reaction is carried out at room temperature for 0.7 hours. The reaction solution was purified by preparative liquid chromatography to give 30 mg of the target compound A'-12 in 22% yield. LC-MS (ESI ⁺ ) 481.0 [M+H] ⁺ .

Example 10 Synthesis of A'-13

Compound - Compound, Bromomaleic anhydride - Bromomaleic anhydride

According to the general procedure A, add 500 mg of compound 15 (L-lysine), while adding other materials at the same time in molar ratios. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 410 mg of the title compound A'-13 in 26% yield. LC-MS (ESI ⁺ ) 462.9 [M+H] ⁺ .

Example 11 Synthesis of A'-14

Bromomaleic anhydride - Bromomaleic anhydride

Compound 16 (500 mg, 2.03 mmol) was dissolved with stirring by adding 1 ml of THF and 1 ml of an aqueous NaHCO ₃ solution, followed by stirring in an ice bath for 5 minutes. Then, compound 17 (724.4 g, 4.67 mmol) was added gradually in portions, followed by stirring in an ice bath for 40 minutes, and then stirring at room temperature for 2.5 hours. After completion of the reaction, the reaction solution was subjected to rotary evaporation to remove THF, diluted with 1 ml of methanol and separated by preparative liquid chromatography, and 245 mg of compound 18 was obtained with a yield of 37%. LC-MS (ESI ⁺ ) 327.1 [M+H] ⁺ .

According to the general procedure A, add 245 mg of compound 18, while adding at the same time other materials are added in molar ratios. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 67 mg of the title compound A'-14 in 23% yield. LC-MS (ESI ⁺ ) 462.9 [M+H] ⁺ .

Example 12 Synthesis of A'-15

Compound - Compound, Bromomaleic anhydride - Bromomaleic anhydride

According to general procedure A, 56.1 mg of compound 19 (Boc-4-amino-L-phenylalanine) is added while adding other materials in molar ratios at the same time. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 17.9 mg of the title compound A'-15 in 18% yield. LC-MS (ESI ⁺ ) 496.9 [M+H] ⁺ .

Example 13 Synthesis of A'-16

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 218 mg of compound A'-13 are added while adding other materials in molar ratios at the same time. The reaction is carried out at room temperature for 0.7 hours. The reaction solution was purified by preparative liquid chromatography to give 65 mg of the title compound A'-16 in 26% yield. LC-MS (ESI ⁺ ) 523.1 [M+H] ⁺ .

Example 14 Synthesis of A'-19

Bromomaleic anhydride - Bromomaleic anhydride

Compound 20 (140 mg, 0.5 mmol) was dissolved in 3 ml of dichloromethane, and triethylamine (210 μl, 1.5 mmol) was added, followed by stirring in an ice bath. Compound 21 (62.5 μl, 0.75 mmol) was dissolved in 2 ml of DCM, and the solution was added dropwise to the reaction solution, followed by stirring in an ice bath for 0.5 hour, and stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated. The residue was dissolved in acetonitrile and separated by preparative liquid chromatography to give 93.5 mg of compound 22 in 56.1% yield. LC-MS (ESI ⁺ ) 335.2 [M+H] ⁺ .

According to general procedure A, 33.4 mg of compound 22 are added while other materials are added at the same time in molar ratios. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to obtain 7.5 mg of the target compound A'-19 with a yield of 19.1%. LC-MS (ESI ⁺ ) 393.0 [M+H] ⁺ .

Example 15 Synthesis of A'-20

Compound 23 (230 mg, 1 mmol) was dissolved in 3 ml of THF, and bromomaleic anhydride (354 mg, 2 mmol) was added, followed by stirring at room temperature for 3 hours. After completion of the reaction, the reaction solution was subjected to rotary evaporation to remove THF, and the crude reaction product compound 24 was obtained. Compound 24 was dissolved in 3 ml of acetic anhydride, and sodium acetate (41 mg, 0.5 mmol) was added, followed by stirring at 70° C within 8 hours. After completion of the reaction, the reaction solution is subjected to rotary evaporation to remove the solvent. The residue was dissolved in 4 ml of acetonitrile/water (1:1), purified by preparative liquid chromatography to give 70 mg of compound 25 in 18% yield. LC-MS (ESI ⁺ ) 389.0 [M+H] ⁺ .

To compound 25 (99.2 mg, 0.32 mmol) was added 2 ml of a solution (4M, 8 mmol) of hydrogen chloride in 1,4-dioxane, followed by stirring in an ice bath for 2 hours. After completion of the reaction, the reaction solution is subjected to rotary evaporation to remove the solvent. The residue was washed with diethyl ether (5 ml x 3) and dried to give 64.1 mg of compound 26 in 95.3% yield. LC-MS (ESI ⁺ ) 289.0 [M+H] ⁺ .

Compound 26 (21 mg, 0.1 mmol) was dissolved in 1 ml of dichloromethane, and triethylamine (42 μl, 0.3 mmol) was added, followed by stirring in an ice bath. Compound 21 (12.5 μl, 0.15 mmol) was dissolved in 1 ml of DCM, and the solution was added dropwise to the reaction solution, followed by stirring in an ice bath for 0.5 hour and stirring at room temperature for 2 hours. After completion of the reaction, the reaction solution was concentrated. The residue was dissolved in acetonitrile and separated by preparative liquid chromatography to give 18.2 mg of A'-20 in 69.1% yield. LC-MS (ESI ⁺ ) 343.0 [M+H] ⁺ .

Example 16 Synthesis of A'-26

THF - THF, DMF - DMF, dioxane - dioxane

Bromomaleic anhydride - Bromomaleic anhydride

Compound 27 (150 mg, 0.63 mmol, 1.0 eq.) was dissolved in 5 ml anhydrous THF and then cooled to -5° C. under Ar protection, and BH ₃ -THF (1N, 2.5 mmol, 4.0 eq.) followed by stirring for 10 minutes and then stirring for 10 minutes at room temperature. Thereafter, the solution is gradually heated to 65° C. and the reaction is carried out overnight. After completion of the reaction, the reaction solution was cooled to 0°C, and 5 ml of methanol was gradually added dropwise to the reaction solution, followed by stirring for 30 minutes and then stirring for 10 minutes at room temperature. Thereafter, the reaction solution was heated to 60° C., stirred for 3 hours, and concentrated under reduced pressure to give a crude reaction product of 140 mg of compound 28 as an oily liquid, which was passed directly to the next step without purification. LC-MS (ESI ⁺ ) 167.1 [M+H] ⁺ .

The crude compound 28 (140.0 mg, 0.58 mmol, 1.0 eq.) was dissolved in 5 ml of THF, 3 ml of water was added and then a solution of BOC ₂ O (255.5 g, 1.17 mmol) was added dropwise. , 2.0 eq.) in 2.5 ml THF, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residue, to which 30 ml of EA was added, was washed, dried, concentrated and purified to give 185 mg of compound 29 as a white solid in 72% yield. LC-MS (ESI ⁺ ) 367.1 [M+H] ⁺ .

Compound 29 (94.0 mg, 0.25 mmol, 1.0 eq.) is dissolved in 3 ml of DMF, K ₂ CO ₃ (71.0 mg, 0.51 mmol, 2.0 eq.) is added to the solution and then a solution of compound 11 (75.0 mg, 0.38 mmol, 1.5 eq.) in 1 ml DMF was added dropwise, followed by heating to 55° C. and stirring overnight. After completion of the reaction, 20 ml of EA was added to the reaction solution. The pH of the reaction solution was adjusted with 30% citric acid to pH=3, the solution was extracted, washed, dried and concentrated to give 108 mg of compound 30 as an off-white solid in 88% yield. LC-MS (ESI ⁺ ) 481.1 [M+H] ⁺ .

Compound 30 (108 mg, 0.22 mmol, 1.0 eq.) was dissolved in 5 ml anhydrous DCM, then a solution of (4M, 1.5 ml, 6.0 mmol, 27.0 eq.) HCl in 1,4-dioxane followed by stirring at room temperature for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residue is dispersed by ultrasound in 10 ml of methyl tert-butyl ether, centrifuged to remove the supernatant, and then dispersed in 10 ml of petroleum ether and centrifuged to remove the supernatant. The solid is kept under reduced pressure to remove the solvent, and receive 66.6 mg of compound 31 as a white solid with a yield of 100%. LC-MS (ESI ⁺ ) 225.1.1 [M+H] ⁺ .

According to general procedure A, 66.6 mg of compound 31 are added while other materials are added at the same time in molar ratios. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 40 mg of the title compound A'-26 in 33% yield. LC-MS (ESI ⁺ ) 540.9 [M+H] ⁺ .

Example 17 Synthesis of A'-27

Sodium thiophenolate _ Sodium thiophenolate

Compound A'-26 (40.0 mg, 0.074 mmol, 1.0 eq.) was dissolved in 1.0 ml of anhydrous THF, and TEA solution (22.5 mg, 0.22 mmol, 3.0 eq. .) in 500 µl THF followed by stirring at room temperature for 5 min. Compound 32 (20.5 mg, 0.16 mmol, 2.2 eq.) was dissolved in 500 µl of DMF, and the solution was added dropwise to the reaction solution, followed by stirring at room temperature overnight. After completion of the reaction, 15 ml of ethyl acetate was added to the reaction solution. The reaction solution was washed with 15% citric acid solution, washed with water, washed with saturated NaCl solution, dried, concentrated and purified to give 12 mg of A'-27 as a white solid in 26% yield. LC-MS (ESI ⁺ ) 629.1 [M+H] ⁺ .

Example 18 Synthesis of A'-28

Dioxane - dioxane, DMF - DMF

Bromomaleic anhydride - Bromomaleic anhydride

Compound 33 (150 mg, 2 mmol, 1.0 eq.) is dissolved in 5 ml of dioxane, 3 ml of saturated aqueous sodium bicarbonate solution is added and then a solution of BOC ₂ O (523 mg, 2.40 mmol, 1.2 equiv.) in 2.5 ml of dioxane, followed by stirring at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure. The residue, to which 30 ml of EA was added, was adjusted to pH=3 with 30% citric acid, extracted, washed, dried, concentrated, dispersed in 15 ml of PE, filtered and dried, and 344 mg of compound 34 was obtained with a yield of 62%. LC-MS (ESI ⁺ ) 176.1 [M+H] ⁺ .

Compound 34 (350 mg, 2.0 mmol, 1.0 equiv.) is dissolved in 4 ml of DMF and cooled to -15°C in an Ar atmosphere, TEA solution (606.0 mg, 6.00 mmol, 3 0 eq.) in 1 ml DMF, followed by stirring for 5 min, and then a solution of T3P (700.0 mg, 2.20 mmol, 1.1 eq.) in 1 ml DMF was added, followed by stirring for 1, 5 o'clock A solution of compound 35 (135.0 mg, 1.00 mmol, 0.5 eq.) in 1 ml of DMF was added dropwise to the reaction solution, followed by stirring overnight. The pH of the reaction solution, to which 30 ml of EA was added, was adjusted to pH=3 with 30% citric acid, extracted, washed, dried, and concentrated to give 350 mg of compound 36 as an oily liquid, which was passed directly to the next step without purification. LC-MS (ESI ⁺ ) 449.2 [M+H] ⁺ .

Compound 36 (350 mg, 0.78 mmol, 1.0 eq.) was dissolved with stirring in 5 ml of anhydrous DCM, and then a solution (4M, 2.0 ml, 8.0 mmol, 10.2 eq. ) HCl in 1,4-dioxane, followed by stirring at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure. The residue is dispersed by ultrasound in 20 ml of methyl tert-butyl ether, centrifuged to remove the supernatant and then dispersed in 20 ml of petroleum ether and centrifuged to remove the supernatant. The solid is kept under reduced pressure to remove the solvent, and receive 254 mg of compound 37 with a yield of 100%. LC-MS (ESI ⁺ ) 249.1.1 [M+H] ⁺ .

According to the general procedure A, add 195 mg of compound 37, while adding other materials at the same time in molar ratios. The reaction is carried out at 70° C. overnight. The reaction solution was purified by normal phase column chromatography to give 58 mg of the title compound A'-28 as a light yellow solid in 17% yield. LC-MS (ESI ⁺ ) 564.9 [M+H] ⁺ .

Example 19 Synthesis of A'-29

Sodium thiophenolate _ Sodium thiophenolate

According to general procedure B, 58 mg of compound 38 are added while other materials are added at the same time in molar ratios. The reaction is carried out at room temperature for 0.7 hours. The reaction solution was purified by normal phase column chromatography to give 14.1 mg of the title compound A'-29 in 22% yield. LC-MS (ESI ⁺ ) 625.1 [M+H] ⁺ .

Example 20 Synthesis of A'-31

Bromomaleic anhydride - Bromomaleic anhydride

Compound 43 (342 mg, 3.00 mmol, 1.0 eq.) was dissolved in 15 ml of anhydrous THF, and bromomaleic anhydride (1110.0 mg, 6.30 mmol, 2.1 eq.) was added dropwise, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution is filtered. The filter cake was washed with DCM and dried to give 1160 mg of compound 44 as a white solid in 83% yield. LC-MS (ESI ⁺ ) 466.9 [M+H] ⁺ .

To compound 45 (300 mg, 0.64 mmol, 1.0 eq.) and anhydrous sodium acetate (158.0 mg, 1.93 mmol, 3.0 eq.) was added 6 ml of acetic acid, followed by stirring at 85° With and response during the night. The reaction solution is cooled to 45°C and concentrated under reduced pressure. The residue, to which 20 ml of acetonitrile was added, was filtered. The filtrate was purified by preparative liquid chromatography to give 15 mg of a light yellow solid (ie Compound A'-31) in 5% yield. LC-MS (ESI ⁺ ) 430.9 [M+H] ⁺ .

Example 21 Synthesis of A'-33

According to general procedure A, compound 48 (95 mg, 0.5 mmol) and compound 8 (80.6 mg, 0.2 mmol) are added to give 10.9 mg of compound A'-33 in 10.1% yield. LC-MS (ESI ⁺ ) 548.0 [M+H] ⁺ .

Example 22 Synthesis of A'-34

Bromomaleic anhydride - Bromomaleic anhydride

According to general procedure A, compound 49 (2-amino-6-chloroisonicotinic acid, 85 mg, 0.5 mmol) was added to give 21.3 mg of compound A'-34 in 13% yield. LC-MS (ESI ⁺ ) 330.9 [M+H] ⁺ .

Example 23 Synthesis of the conjugate (Ab-A) of the antibody (Ab) and the first linker moiety (A)

For coupling with an antibody, the antibody-drug conjugate primarily uses the linker (ie, the first linker moiety A) of the present invention. In order to confirm the ability to combine a linker of the present invention with an antibody, the linkers synthesized in the above examples were individually combined with an antibody. The combination method used is described below.

1) Obtaining mother solutions of the reducing agent and the protective agent. Stock solutions of reducing agent and protective agent - 1~20 mM TCEP (tris-2-carboxyethylphosphine) (reductant) and 1~20 mm DTPA (diethylenetriaminepentaacetic acid) (protective agent) - are prepared respectively with purified water.

2) Antibody recovery reaction. Stock solutions of DTPA and TCEP are added to the monoclonal antibody solution (eg 5-30 mg/ml), respectively. The DTPA stock solution is mixed with a monoclonal antibody solution (eg 5-30 mg/ml) according to a certain volume ratio (1~5:10), and the molar ratio of TCEP to monoclonal antibody is 0.5~6.0:1. After adding the reducing agent and protective agent, the reaction is carried out at 25° C. with stirring for 1 hour.

3) Obtaining a solution of the first linker component (A). The first linker moiety (A) is dissolved in 25% DMSO (dimethyl sulfoxide) to form a 5 mM solution of the first linker moiety (A).

4) Coupling reaction between the first linker moiety (A) and the antibody. The solution of the first linker component (A) obtained in step 3) is gradually added to the solution of the reduced monoclonal antibody obtained in step 2), and according to the actual need, the molar ratio of the first linker component (A) to the thio group of the antibody is adjusted in the range of 0 ,3~5:1, the reaction is carried out at 25°C with stirring for 4 hours.

5) Stages of purification and detection. After completion of the reaction in step 4), the reaction solution is centrifuged and ultrafiltered 3 times with PBS buffer to purify and remove remaining unreacted chemicals and free small molecules such as DMSO, and the coupling result is determined by SDS-PAGE electrophoresis.

The result of the coupling of the antibody-linker conjugate is analyzed by electrophoresis in SDS-PAGE. The following method is used: for detection in SDS-PAGE, a NUPAGE 4-12% gel plate is used, 1.5 µl of sample is placed in a 1.5 ml EP tube, 2.5 µl of buffer is added, then 1.5 µl of reducing agent is added, and add water to obtain a volume of 10 μl. The mixture was stirred on an oscillator and heated in a microwave oven for 1 min. After sample loading, gel electrophoresis is performed. Upon completion of gel electrophoresis, the gel is removed and placed in a fresh storage case, to which 50 ml of fast dye is added, heated in a microwave oven for 5 minutes, and then dried on a shaker for 5 minutes. The dye is drained off and then 100 ml of purified water is added, heated on an induction cooker for 5 minutes and decolorized on a shaker for 1 hour. This operation is repeated 3 times.

The antibody used is the monoclonal antibody Her2 (for information about the sequence of the antibody, please refer to paragraphs 41, 42, 43 of the specification in Chinese Patent Application Publication No. CN105008398B), and the linkers are

A'-7 A'-8 A'-10

A'-11 A'-12 A'-20

A'-28 A'-31 .

The results of the coupling are shown in Figure 5. In Figure 5, Nos. 1, 2, 3 refer to Her2-A'-7, the coupling buffers respectively have a pH of 8.5, 9.0, 7.4; Nos. 4, 5, 6 refer to Her2-A'-8, coupling buffers, respectively, have a pH of 8.5, 7.4, 9.0; Nos. 7, 8, 9 refer to Her2-A'-10, coupling buffers respectively have pH 8.5, 9.0, 7.4; Nos. 10, 11, 12 refer to Her2-A'-11, coupling buffers respectively have pH 9.0, 7.4, 9.5; Nos. 13, 14, 15 refer to Her2-A'-12, coupling buffers, respectively, have a pH of 7.4, 9.0, 8.5; Nos. 16, 17, 18 refer to Her2-A'-20, coupling buffers, respectively, have a pH of 8.5, 9.0, 7.4; Nos. 19, 20, 21 refer to Her2-A'-28, coupling buffers, respectively, have a pH of 9.0, 8.5, 7.4; Nos. 22, 23, 24 refer to Her2-A'-31, the coupling buffers respectively have a pH of 7.4, 8.5, 9.0.

Due to the use of reductive electrophoresis, all thio groups in the antibodies are exposed. Therefore, in Figure 5, 25 kDa represent the molecular weight of one antibody light chain, 50 kDa represent the molecular weight of one heavy chain, and 75 kDa represent the molecular weight of one light chain and one heavy chain (i.e., one linker covalently binds to a thio group between one heavy chain and one light chain), 100 kDa represent the molecular weight of two heavy chains (i.e., one linker covalently binds to a thio group between two heavy chains), 125 kDa represent the molecular weight of one light chain and two heavy chains (i.e. ., one linker covalently bonds to thio groups between two heavy chains and one light chain), 150 kDa represents the molecular weight of two heavy chains and two light chains (i.e., one linker covalently bonds to thio groups between two heavy chains and two light chains ).

The results show that the antibody-linker conjugates described above each have linkers that simultaneously covalently bind to thio groups respectively between one heavy chain and one light chain, between two heavy chains, between two heavy chains and one light chain, or between two heavy chains and two light chains. And in most of the antibody-linker conjugates, there are 150-75 kDa bands, which confirm that the 4 pairs of disulfide bonds in the antibody fully or partially form bridges, and the bridging effect is better. From figure 5 it can also be seen that the coupling result in a buffer medium with pH=9.0 is better than in media with pH=8.5 and 7.4.

Considering the better effect of bridging the linker of the present invention with an antibody, A'-7, A'-8, A'-10, A'-11, A'-28 are selected for preparing antibody-drug conjugates. The antibody used is Her2 monoclonal antibody (same as above), and the drugs used are MMAE, MMAF, MMAD, CBI, DM1, DM4.

With respect to MMAE and MMAD, the preparation of the antibody-drug conjugate included in the present invention is generally divided into three steps: the first step is to prepare the L-D conjugate of the second linker L and the drug D; the second stage is to obtain a conjugate A-L-D of the first linker A and L-D; and the third step is to prepare an Ab-A-L-D antibody conjugate, i.e., an Ab-A-L-D antibody-drug conjugate.

With respect to MMAF, CBI, DM1, DM4, the preparation of the antibody-drug conjugate included in the present invention is generally divided into two steps: the first step is to prepare the first linker A-drug D conjugate A-D; and the second step is to prepare an Ab-A-D antibody conjugate, i.e., an Ab-A-D antibody-drug conjugate.

A specific production method is shown in Examples 26-29.

Example 24 Synthesis of the conjugate (L-D) of the second linker moiety (L) and drug (D)

General synthesis method (L-D). The second linker moiety L was dissolved in an appropriate amount of DMF, drug (D), HOBt, DIPEA and pyridine were added to the resulting solution under the protection of nitrogen gas in appropriate amounts, and then the mixture was stirred at room temperature for 25 hours using TLC to monitor reaction development. After completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography, the resulting solution was lyophilized to obtain a conjugate (L-D) of the second linker moiety and a drug.

Val-Cit-PAB-MMAE is prepared by the general method described above and the synthesis route is shown below.

Compound 53 (i.e., Fomc-Val-Cit-PAB-PNP, 995.4 mg, 1.3 mmol) and HOBt (67.6 mg, 0.65 mmol) are placed in a reaction flask and dissolved in 5 ml of anhydrous DMF, and DIPEA (258.5 mg, 2 mmol) was added to the resulting solution, followed by stirring at room temperature for 10 minutes. Compound 54 (ie, MMAE, 717 mg, 1 mmol) was dissolved in 5 ml of anhydrous DMF and added dropwise to the reaction solution, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution is filtered. The organic phase is concentrated. The residue was separated by preparative liquid chromatography to give 636.2 mg of compound 55 (i.e., Fmoc-Val-Cit-PAB-MMAE) as a white solid in 47.3% yield, LC-MS (ESI ⁺ ) 1345 ,8 [M+H] ⁺ .

Compound 55 (270 mg, 0.2 mmol) was added to 3 ml of a 20% solution of piperidine in acetonitrile (v/v), followed by stirring at room temperature for 2.5 hours to gradually dissolve the solid. After completion of the reaction, the reaction solution was distilled under reduced pressure to remove the solvent. The residue was separated by preparative liquid chromatography to give 214 mg of compound 62 (ie, Val-Cit-PAB-MMAE) in 95% yield, LC-MS (ESI ⁺ ) 1123.7 [M+H] ⁺ .

Synthesis using a linker moiety (L) and a drug (D) is a common method for the synthesis of other L-D compounds such as Val-Cit-PAB-MMAD.

Example 25 Synthesis of the first linker moiety (A) conjugate (A-L-D) and the second linker moiety-drug conjugate (L-D)

General method for the synthesis of A-L-D. The first linker moiety, EDCI, HOBt, was dissolved in appropriate amounts in an appropriate amount of anhydrous DMF and stirred at room temperature for 2 hours. An appropriate amount of the above-obtained L-D was dissolved in an appropriate amount of anhydrous DMF and gradually added dropwise to the reaction solution, followed by stirring at room temperature for 4 hours. After completion of the reaction, the reaction solution was diluted with acetonitrile and then separated by preparative liquid chromatography. The eluent is lyophilized to give a conjugate of the first linker moiety (A) and a second linker moiety-drug conjugate (L-D).

Conjugate A'-7 and compound 56 (Val-Cit-PAB-MMAE) was synthesized using the general method described above. The synthesis path is shown below.

DMF - DMF

Compound A'-7 (27.8 mg, 0.053 mmol), EDCI (11.2 mg, 0.058 mmol), HOBt (1.4 mg, 0.01 mmol) was dissolved in 0.3 ml anhydrous DMF and stirred at room temperature. temperature for 2 hours. Compound 56 (ie, Val-Cit-PAB-MMAE, 40 mg, 0.035 mmol) was dissolved in 0.2 ml of anhydrous DMF and added dropwise to the reaction solution, followed by stirring at room temperature for 4 hours. After completion of the reaction, the reaction solution was diluted with 2 ml of acetonitrile and then separated by preparative liquid chromatography. The eluent was lyophilized to give 8.6 mg of compound 57 (i.e., A'-7-Val-Cit-PAB-MMAE) as a light yellow solid in 15% yield. LC-MS (ESI ⁺ ) 1624.6 [M+H] ⁺ .

Using the general synthesis method described above, other conjugates of the first linker moiety and the L-D compound are synthesized, such as A'-8-Val-Cit-PAB-MMAE, A'-10-Val-Cit-PAB-MMAE, A'-11- Val-Cit-PAB-MMAE, A'-11-Val-Cit-PAB-MMAD, A'-28-Val-Cit-PAB-MMAE and the like.

For comparison, the present description also describes the preparation of the 575DZ-Val-Cit-PAB-MMAE conjugate of compound 6 (this compound is defined in the present description as 575DZ) disclosed on page 7 of the claims of Chinese Patent No. CN103933575A, with a second linker moiety (Val- Cit-PAB) and drug constituent (MMAE).

Example 26 Synthesis of the conjugate (A-D) of the first linker moiety (A) and drug (D)

(1) Synthesis of A'-7-MMAF

Compound A'-7 (51.8 mg, 0.1 mmol), EDCI (21.1 mg, 0.11 mmol), HOBt (2.7 mg, 0.01 mmol) are dissolved in 2 ml anhydrous DMF and stirred at room temperature for 2 hours. Compound 58 (ie, MMAF, 48.8 mg, 0.066 mmol) was dissolved in 0.8 ml of anhydrous DMF and added dropwise to the reaction solution, followed by stirring at room temperature for 4 hours. After completion of the reaction, the reaction solution was diluted with 4 ml of acetonitrile and then separated by preparative liquid chromatography. The eluent was lyophilized to give 25.9 mg of A'-7-MMAF as a light yellow solid in 21% yield. LC-MS (ESI ⁺ ) 1233.4 [M+H] ⁺ .

(2) Synthesis of A'-8-CBI

The method of synthesis is the same as the synthesis of A'-7-MMAF. A'-8 (22.0 mg, 0.047 mmol, 1.0 eq.) and compound 59 (i.e., CBI, 14.0 mg, 0.020 mmol, 0.7 eq.) are added to give 6, 0 mg white solid A'-8-CBI in 21% yield. LC-MS (ESI ⁺ ) 868.0 [M+H] ⁺ .

(3) Synthesis of A'-8-DM1

Compound 60 (45.0 mg, 0.21 mmol, 1.0 eq.) was dissolved in 2 ml anhydrous THF, 1 ml sat. an aqueous solution of NaHCO ₃ , and a solution of DM1 (61, 150.0 mg, 0.21 mmol, 1.0 eq.) in 1 ml of DMF was added dropwise, followed by stirring at room temperature for 2 hours. The reaction solution was diluted with 10 ml of water and extracted with EA. The pH of the aqueous phase was adjusted to pH=3 with 15% citric acid solution, extracted with 15 ml of EA, the extract was washed with saturated NaCl solution, dried and concentrated to give 165.7 mg of compound 62 as a white solid in 85% yield. LC-MS (ESI ⁺ ) 949.3 [M+H] ⁺ .

A'-8 (150.0 mg, 0.33 mmol, 1.0 eq.) was dissolved in 5 ml DMF, cooled to -15° C. under Ar, and TEA solution (98.0 mg, 0.99 mmol, 3.0 eq) in 1 ml DMF followed by stirring for 5 min, and then a solution of T3P (154.0 mg, 0.48 mmol, 1.5 eq) in 1 ml DMF with followed by stirring for 1.5 hours. A solution of compound 63 (46.0 mg, 0.29 mmol, 0.9 eq.) in 1 ml of DMF was added dropwise to the reaction solution, followed by stirring overnight. After completion of the reaction, 30 ml of EA was added, the reaction solution was adjusted to pH=3 with 30% citric acid, extracted, washed, dried, concentrated and purified by normal phase column chromatography to give 90 mg of compound 64 as a white solid with a yield of 52 %. LC-MS (ESI ⁺ ) 607.1 [M+H] ⁺ .

Compound 64 (90 mg, 0.15 mmol, 1.0 eq.) is dissolved in 3 ml of anhydrous DCM with stirring, and then a solution of (4 M, 1.0 ml, 4 mmol, 26.7 eq.) HCl in 1,4-dioxane followed by stirring at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure. The residue is dispersed by ultrasound in 20 ml of methyl tert-butyl ether, centrifuged to remove the supernatant and then dispersed in 20 ml of petroleum ether and centrifuged to remove the supernatant. The solid is kept under reduced pressure to remove the solvent, and receive 82 mg of compound 65 as a white solid with a yield of 100%. LC-MS (ESI ⁺ ) 506.9 [M+H] ⁺ .

Compound 62 (30.0 mg, 0.03 mmol, 1.0 eq.) was dissolved in 2 ml DMF, cooled to -15° C. under Ar, and TEA solution (11.0 mg, 0.095 mmol , 4.0 eq.) in 500 μl DMF followed by stirring for 5 min, and then a solution of T3P (15.0 mg, 0.047 mmol, 1.5 eq.) in 500 μl DMF was added followed by stirring for 1, 5 o'clock A solution of compound 65 (19.0 mg, 0.035 mmol, 1.1 eq.) in 500 µl of DMF was added dropwise to the reaction solution, followed by stirring overnight. After completion of the reaction, the reaction solution was diluted with 2 ml of acetonitrile and purified by preparative liquid chromatography to obtain 9 mg of product A'-8-DM1 in 20% yield. LC-MS (ESI ⁺ ) 1437.3 [M+H] ⁺ .

(4) Synthesis of A'-7-DM4

Compound 67 (737.5 mg, 4.03 mmol), EDCI (846.13 mg, 4.43 mmol), HOBt (108.9 mg, 0.806 mmol) was dissolved in 10 ml of DCM, followed by stirring at room temperature for 2 o'clock Compound 66 (1 g, 4.03 mmol) was dissolved in 5 ml of dichloromethane and slowly added dropwise to the reaction solution in 3 additions, followed by stirring at room temperature overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent. The residue was dissolved in 6 ml of acetonitrile/water (1:1), separated by preparative liquid chromatography and lyophilized to give 1.11 g of compound 68 as a colorless oil in 67% yield. LC-MS (ESI ⁺ ) 414.2 [M+H] ⁺ .

Compound 68 (1.11 g, 2.7 mmol) was dissolved in 1.1 ml DMA. The resulting solution, 0.1 ml, was dissolved in 1 ml DMA and DIPEA (41.3 mg, 0.32 mmol) was added, followed by stirring at room temperature for 2 min, and then DM4 (69, 202.5 mg, 0.26 mmol) followed by stirring at room temperature for 6 hours. The reaction solution was directly separated by preparative liquid chromatography and lyophilized to give 260 mg of compound 70 as a white solid in 84% yield. LC-MS (ESI ⁺ ) 1193.5 [M+H] ⁺ .

The method for the synthesis of compound 71 is the same as that for the synthesis of compound 65. 119.3 mg of compound 70 are added to give 106 mg of compound 71 as a white solid in 96% yield. LC-MS (ESI ⁺ ) 1093.5 [M+H] ⁺ .

The method for the synthesis of A'-7-DM4 is the same as the method for the synthesis of A'-8-DM1. 52 mg of A'-7 and 54.6 mg of compound 71 are added to give 15 mg of A'-8-DM1 as a white solid in 19% yield. LC-MS (ESI ⁺ ) 1594.4 [M+H] ⁺ .

Example 27 Antibody-Drug Conjugate Synthesis and Product Homogeneity Analysis

General Method for Synthesizing an Antibody-Drug Conjugate

1) Obtaining mother solutions of the reducing agent and the protective agent. Stock solutions of reducing agent and protective agent - 1~20 mM TCEP (tris-2-carboxyethylphosphine) (reductant) and 1~20 mm DTPA (diethylenetriaminepentaacetic acid) (protective agent) - are prepared respectively with purified water.

2) Antibody recovery reaction. Stock solutions of DTPA and TCEP are added to the monoclonal antibody solution (eg 5-30 mg/ml), respectively. The DTPA stock solution is mixed with a monoclonal antibody solution (eg 5-30 mg/ml) according to a certain volume ratio (1~5:10), and the molar ratio of TCEP to monoclonal antibody is 0.5~6.0:1. After adding the reducing agent and protective agent, the reaction is carried out at 25° C. with stirring for 1 hour.

3) Synthesis of A-L-D and obtaining a solution. The conjugate (A-L-D) first linker moiety-second linker moiety-drug moiety is synthesized, the synthesized A-L-D is dissolved in 25% DMSO (dimethyl sulfoxide), and a 5 mM A-L-D solution is obtained.

4) The reaction of the combination of the drug and antibodies. The A-L-D solution obtained in step 3) is slowly added to the reconstituted antibody solution obtained in step 1), and according to the actual need, and the molar ratio of A-L-D to the thio group of the antibody is adjusted in the range of 0.3~5:1, the reaction is carried out at 25° With stirring for 4 hours.

5) Stages of purification and detection. After completion of the reaction in step 4), the reaction solution is centrifuged and ultrafiltered 3 times with PBS buffer to purify and remove remaining unreacted chemicals and small free molecules such as DMSO, and the coupling result is determined by SDS-PAGE electrophoresis and hydrophobic high performance liquid chromatography (HIC-HPLC).

The result of the combination of the antibody-drug conjugate analyzed by electrophoresis in SDS-PAGE and HIC-HPLC. Use the method described below.

1) Polyacrylamide gel electrophoresis (SDS-PAGE)

For detection in SDS-PAGE, a NUPAGE 4-12% gel plate is used, 1.5 µl of sample is placed in a 1.5 ml EP tube, 2.5 µl of buffer is added, then 1.5 µl of reducing agent is added, and water is added until volume of 10 μl, followed by stirring the mixture on an oscillator and heating in a microwave oven for 1 min. After sample loading, gel electrophoresis is performed. Upon completion of gel electrophoresis, the gel is removed and placed in a fresh storage case, to which 50 ml of fast dye is added, heated in a microwave oven for 5 minutes, and then dried on a shaker for 5 minutes. The dye is drained off and then 100 ml of purified water is added, heated on an induction cooker for 5 minutes and decolorized on a shaker for 1 hour. This operation is repeated three times.

2) Hydrophobic High Performance Liquid Chromatography (HIC)

Test conditions. HIC-HPLC: Tosoh TSK Gel Butyl, 4.6 mm × 3.5 cm, 10 mm; buffer A: 20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0; buffer B: 20 mM sodium phosphate, 25% (v/v) isopropanol, pH 7.0; flow rate 1 ml/min; gradient: 10% buffer B to 100% buffer B in 15 minutes; 10 µl of sample.

Using the method described above, the following 4 antibody-drug conjugates were prepared: Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-11-Val-Cit-PAB-MMAE, Her2-A'-10 -Val-Cit-PAB-MMAE, Her2-575DZ-Val-Cit-PAB-MMAE, the antibody being Her2 monoclonal antibody (same as above). Coupling results of the above 4 antibody-drug conjugates analyzed by HIC-HPLC and SDS-PAGE electrophoresis are shown in Table 1, Figure 1A, Figure 1B, Figure 1C, Figure 1D and Figure 2A, Figure 2B.

In Table 1 and in Figure 1A, Figure 1B, Figure 1C, Figure 1D, D0% represents the proportion of antibody-drug conjugates with 0 drug coupled to the linker, i.e., the proportion of naked antibody, D1% represents the proportion of antibody-drug conjugates drug with 1 drug linked to a single antibody via a linker, D2% represents the proportion of antibody-drug conjugates to 2 drugs linked to a single antibody via a linker, D3% represents the proportion of antibody-drug conjugates to 3 drugs linked with one antibody via a linker, D4% represents the proportion of 4-drug antibody-drug conjugates linked to a single antibody via a linker, and D5% represents the proportion of 5-drug antibody-drug conjugates linked to a single antibody via a linker.

The results show that the average DAR value of Her2-A'-7-Val-Cit-PAB-MMAE is 4.10 (molar ratio of drug to antibody during coupling is 5:1); the average DAR value of Her2-A'-11-Val-Cit-PAB-MMAE is 2.12 (molar ratio of drug to antibody during coupling is 5:1); the average DAR value of Her2-A'-10-Val-Cit-PAB-MMAE is 2.26 (molar ratio of drug to antibody during coupling is 5:1); the average DAR value of Her2-575DZ-Val-Cit-PAB-MMAE is 4.10 (the molar ratio of drug to antibody during coupling is 5:1). The results show that Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-11-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE are all have a good coupling effect (average DAR is 2-4 under ideal conditions), with DAR2 Her2-A'-11-Val-Cit-PAB-MMAE and Her2-A'-10-Val-Cit-PAB-MMAE are 65.574% and 78.9% respectively, the D4% Her2-A'-7-Val-Cit-PAB-MMAE ratio is 59.45%, the DAR value distribution is very narrow, and the product uniformity is very good (all better than at Her2-575DZ-Val-Cit-PAB-MMAE).

Table 1. Results of analysis of conjugates by hydrophobic high performance liquid chromatography (HIC-HPLC) ADC D0% D1% D2% D3% D4% D5% DAR Her2-A'-7-Val-Cit-PAB-MMAE 0 0.07 1.93 12.16 59.45 26.40 4.10 Her2-A'-10-Val-Cit-PAB-MMAE 0 9.03 78.9 11.9 0 0 2.26 Her2-A'-11-Val-Cit-PAB-MMAE 0 11.41 65.57 23.02 0 0 2.12 Her2-575DZ-Val-Cit-PAB-MMAE 0 0 1.99 18.46 52.94 21.17 4.10

Figure 2A, Figure 2B show SDS-PAGE patterns of the above antibody-drug conjugates, with Figure 2A showing SDS-PAGE patterns of Her2-A'-11-Val-Cit-PAB-MMAE (coupling buffers respectively have pH 8.5, 9.0, 7.4), Her2-A'-7-Val-Cit-PAB-MMAE (coupling buffers respectively have pH 8.5, 9.0, 7.4), Her2-A '-10-Val-Cit-PAB-MMAE (coupling buffers respectively have pH 8.5, 9.0, 7.4) and Figure 2B shows electrophoresis patterns in SDS-PAGE, left to right, Her2-A'- 7-Val-Cit-PAB-MMAE, Her2-575DZ-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE (coupling buffer suitably has a pH of 9.0).

Due to the use of reductive electrophoresis, all thio groups in the antibodies are exposed. Therefore, 25 kDa represents the molecular weight of one antibody light chain, 50 kDa represents the molecular weight of one heavy chain, and 75 kDa represents the molecular weight of one light chain and one heavy chain (i.e., one linker covalently binds to a thio group between one heavy chain and one light chain), 100 kDa represents the molecular weight of two heavy chains (i.e., one linker covalently binds to a thio group between two heavy chains), 125 kDa represents the molecular weight of one light chain and two heavy chains (i.e., one linker covalently bonds to thio groups between two heavy chains and one light chain), 150 kDa represents the molecular weight of two heavy chains and two light chains (i.e., one linker covalently bonds to thio groups between two heavy chains and two light chains).

Figure 2A shows that the antibody-drug conjugates (Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE, Her2-A'-11-Val- Cit-PAB-MMAE) presented in the examples in the present description, each have linkers that simultaneously covalently bind to thio groups, respectively, between one heavy chain and one light chain (75 kDa), between two heavy chains (100 kDa), between two heavy chains and one light chain (125 kDa) or between two heavy chains and two light chains (150 kDa), which proves that 4 pairs of disulfide bonds in the antibody fully or partially form bridges, and the bridging effect is stronger.

Figure 2B shows parallel contrast electrophoresis patterns in SDS-PAGE Her2-A'-7-Val-Cit-PAB-MMAE, Her2-575DZ-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB -MMAE (coupling buffer has a pH of 9.0). It can be seen from this figure that the ratio of the 150 kDa and 125 kDa bands in the Her2-A'-7-Val-Cit-PAB-MMAE and Her2-A'-10-Val-Cit-PAB-MMAE conjugates is significantly higher than the ratio corresponding bands in the Her2-575DZ-Val-Cit-PAB-MMAE conjugate. At the same time, the ratio of the 50 kDa and 25 kDa bands is also significantly lower, proving that the first linker moiety of the present invention has greater coupling efficiency and can result in an antibody-drug conjugate that has a higher degree of bridging.

Example 28 Antibody Drug Conjugate Hydrolysis Assay

The result of hydrolysis of the antibody-drug conjugate (claudin 18.2-A'-7-VC-PAB-MMAE) is described by molecular weight analysis and protein isoelectric point analysis.

1) Molecular weight analysis (LC-MS)

Test conditions: instrument model Thermo Q Exactive Plus; mobile phase 25 mM ammonium acetate solution; flow rate 0.3 ml/min; injection volume 1 mg/ml, 20 µl, i.e., 20 µg; detection interval 5000-8000 Da; instrument performance interval 140000-160000 Yes.

2) Protein isoelectric point analysis

Instrument Details: Isoelectric Focusing Capillary Electrophoresis Imaging Instrument Model iCE 3.

Instrument setting: focus time 1: 1500 V for 1.00 min; focus time 2: 3000 V for 8.00 min.

Naked antibody (anti-claudin 18.2): vehicle amphoteric electrolyte: 1% Pharmalyte 5-8, 3% Pharmalyte 8-10.5; marker for lower isoelectric point 8.18; marker for the upper electrical point 9.77; protein concentration 0.25 mg/ml; tray temperature 15°C.

Claudine 18.2-A'-7-VC-PAB-MMAE: amphoteric electrolyte carrier: 3% Servalyt 5-9, 1% Servalyt 9-11; marker for lower isoelectric point 5.85; marker for the upper electrical point 9.46; protein concentration 0.50 mg/ml; tray temperature 15°C.

Figure 6A shows the result of the measurement of the molecular weight of the naked antibody, and figure 6B shows the result of the measurement of the molecular weight of claudin 18.2-A'-7-VC-PAB-MMAE. From the figures it can be seen that the molecular weight of the naked antibody is 145144 Da and the molecular weights of claudin 18.2-A'-7-VC-PAB-MMAE (when the ratio of A'-7-VC-PAB-MMAE to antibody is 4:1) are 152078 Yes, 152119 Yes, 152147 Yes. When one antibody is combined with 4A'-7-VC-PAB-MMAE without hydrolysis, its theoretical molecular weight is 151999 Da. The actual determined molecular weight is 152078 Da, 152119 Da, 152147 Da, which has a significant deviation from the theoretical molecular weight, exceeding the deviation interval (20 Da) of the instrument. When the claudin18.2-A'-7-VC-PAB-MMAE conjugate undergoes hydrolysis in which 4, 7, 8 maleimides are hydrolyzed, its theoretical molecular weight is 152071 Da, 152125 Da, 152143 Da. When compared with these theoretical values, the relative deviation of the actually determined values is in the range of instrument deviations. This suggests that the antibody-A'-7-VC-PAB-MMAE is partially hydrolyzed.

Next, the hydrolyzed component is investigated. Figure 7A shows the result of placing the isoelectric point of the naked antibody, and Figure 7B shows the result of placing the isoelectric point of claudin18.2-A'-7-VC-PAB-MMAE. From Figure 7A, it can be seen that the main peak of the naked antibody isoelectric point is at 8.91, and the ratio of the acid peak (isoelectric point <8.91) to the alkaline peak (isoelectric point >8.91) is relatively small. From figure 7B it can be seen that the isoelectric point of antibody-A'-7-VC-PAB-MMAE is located between 7.86 and 8.70. Compared to naked antibody, the isoelectric point of antibody-A'-7-VC-PAB-MMAE is significantly reduced, indicating that it has more acidic groups, such as carboxyl groups, on its surface. By combining with structural analysis, it can be seen that the position of hydrolysis is in the maleimide moiety of antibody-A'-7-VC-PAB-MMAE, and by adjusting the environment for synthesizing and hydrolyzing the antibody-drug conjugate, it is possible to control the degree of maleimide hydrolysis in the linker of the antibody conjugate. -drug, for example, to achieve complete hydrolysis or no hydrolysis. In addition, purification methods can also be used to separate products that hydrolyze to the same extent from the synthesized products.

Example 29 Determination of Affinity of Antibody-Drug Conjugates for Antigen (ELISA Method)

General procedure: Antigen is diluted with coating buffer to the desired concentration (such as 500 ng/ml, 250 ng/ml, 100 mg/ml, or adjusted as is according to the needs of the experiment) at 100 µl/well and left at 4°C overnight ; plate washing: the plate is washed 3 times with PBST wash solution at 350 μl/well and dried accordingly; blocking: the plate is blocked with 3% BSA blocking solution at 200 μl/well and left at 4° C. overnight; plate washing: the plate is washed twice with 300 μl/well PBST and the solution in the well is analyzed by blotting; sample loading: sample diluted with 1% BSA-PBST diluent, then add the diluted sample to a blocked cell culture plate at 100 µl/well in triplicate, and diluent as a control, and incubate in an incubator at 37°C for 2 hours ( the dilution scheme can be changed according to the actual situation); plate washing: the plate is washed twice with 200 μl/well PBST and the solution in the well is analyzed by blotting; test antibody loading: HRP conjugated to goat antA-Human IgG-Fc diluted 1:5000 with 1% BSA-PBST at 100 µl/well and incubated at 37°C for 1 hour (diluent can be changed according to actual situation); plate washing: the plate is washed 4 times with PBST at 200 μl/well, and the solution in the well is analyzed by blotting; color detection: add TMB substrate at 100 µl/well for color development effect within 2 min; completion: add 2 MH ₂ SO ₄ at 50 µl/well to complete the reaction; reading: the absorbance or absorbance at 450/655 nm is measured with a microplate reader, and the experimental results are analyzed with the prism analysis program, where the abscissa shows the concentration, and the absorbance shows the absorbance along the ordinate, EC ₅₀ values and the like calculated automatically by the program. The results are shown in table 2 and figure 3.

Table 2 shows the EC ₅₀ values of the antibody-drug conjugates (Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE) against the Her2 antigen, Her2 being naked antibody, with Her2-Mc-vc-PAB-MMAE as ADC control. The results show that Her2-A'-10-Val-Cit-PAB-MMAE and Her2-A'-7-Val-Cit-PAB-MMAE both have good antigen affinity and the results show that the method used binding does not change the original affinity of the antibody.

Table 2. Affinity of antibody-drug conjugates for antigen (EC ₅₀ value) ADC EC ₅₀ (ng/ml) ^R2 Her2 1.073 0.999 Her2-Mc-vc-PAB-MMAE 2.132 0.994 Her2-A'-10-Val-Cit-PAB-MMAE 1.725 0.999 Her2-A'-7-Val-Cit-PAB-MMAE 2.692 1,000

Example 30 In Vitro Efficacy Test

A culture dish containing SK-BR-3 human breast cancer cells is placed in an incubator, 37° C., 5% CO ₂ , for incubation. Cells in a good growth state are collected and the original culture medium is discarded, then the obtained cells are suspended with the culture medium and counted separately. The cell suspension is added to a 96-well plate (5000 cells per well) and incubated in an incubator, 37°C, 5% CO ₂ overnight. The drug, which is diluted, is then added to the cell suspension for incubation. After incubation for 72 hours, a Cell Counting Kit-8 (abbreviated as CCK-8 kit) was used to actively detect the color, and after color appeared, the 96-well plate was detected with a microplate reader and an OD value at 450 nm was obtained. The IC ₅₀ value is calculated from the OD value using the Prism program. Fit four parametric curves using the program, with the degree of inhibition as the y value and the drug concentration as the x value, and record the drug concentration value (which is set by default as the IC ₅₀ value in the program) corresponding to the degree of inhibition between the maximum degree of inhibition and minimum degree of inhibition. When the constructed curve is an "S-curve" and R ² ≥ 0.95, the IC ₅₀ value is applicable. IC ₅₀ values of antibody-drug conjugates Her2-A'-8-Val-Cit-PAB-MMAE, Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-11-Val-Cit-PAB -MMAE, Her2-A'-10-Val-Cit-PAB-MMAE against SK-BR-3 human breast cancer cells are shown in Table 3, and the degree of inhibition curves are shown in Figure 4A and Figure 4B.

Table 3 shows the IC ₅₀ degree of inhibition of the antibody-drug conjugates (Her2-A'-8-Val-Cit-PAB-MMAE, Her2-A'-7-Val-Cit-PAB-MMAE, Her2-A'-11- Val-Cit-PAB-MMAE, Her2-A'-10-Val-Cit-PAB-MMAE) against SK-BR-3 human breast cancer cells.

Table 3. In vitro efficacy of antibody-drug conjugates against human breast cancer cells SK-BR-3 ADC IC ₅₀ (ng/ml) Her2-A'-8-Val-Cit-PAB-MMAE 5.13 Her2-A'-7-Val-Cit-PAB-MMAE 1.57 Her2-A'-11-Val-Cit-PAB-MMAE 2.53 Her2-A'-10-Val-Cit-PAB-MMAE 2.11

The present invention is illustrated by examples of various specific embodiments. However, a person skilled in the art may understand that the present invention is not limited to any particular embodiment, and various changes or modifications may be made by those skilled in the art within the scope of the present invention, and various technical features referred to in the present description may be combined. with each other without departing from the spirit and scope of the present invention. Such changes and modifications are all within the scope of the present invention.

Claims (90)

1. Linker having the structure represented by formula I

(I) , wherein each of M ₁ ', M ₂ ' is chosen independently from

, M ₁ ', M ₂ ' are the same or different; L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₉ -alkyl; Q choose from

,N; L ₃ ' choose from

C (=O) R ⁸ C (=O),

_NH2 ,

OH, and any combination thereof; X is selected from Br; Y represents

,

; Z is selected from C; R ¹ is selected from H, C ₁ -C ₆ -alkyl, R ² , R ⁸ , R ¹⁰ are each independently selected from H, C ₁ -C ₆ -alkyl. 2. Linker according to claim 1, where each of M ₁ ', M ₂ ' is chosen independently from

; L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₄ -alkyl; Q is selected from N; L ₃ ' choose from

C (=O) R ⁸ C (=O),

OH, and any combination thereof; X is Br; Y represents

or

; R ¹ , R ⁸ are each independently selected from H, C ₁ -C ₄ alkyl. 3. Linker according to claim 1 or 2, which has the structure

(A'-1') . 4. A linker that has a structure

A'-1 A'-2 A'-4

A'-7 A'-8 A'-9

A'-10 A'-11 A'-12

A'-13 A'-14 A'-15

A'-16 A'-19 A'-20

A'-26 A'-27 A'-28

A'-29 A'-31 A'-33

A'-34 . 5. The use of a linker according to any one of paragraphs. 1-4 in the preparation of antibody-drug conjugates. 6. An antibody-drug conjugate which has the structure represented by formula II Ab-(A-L-D) n (II), wherein Ab is an antibody or a functional fragment of an antibody; the linker moiety includes a first linker moiety A and a second linker moiety L; D is a drug moiety; n is 1, 2, 3 or 4; Where the first linker moiety A includes a group covalently linked to the thiol group in Ab, which has the structure represented by formula III:

(III), in which M ₁ , M ₂ are chosen, each independently, from

,

,

,

, M ₁ and M ₂ are the same or different; L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₉ -alkyl; Q choose from

,N; L ₃ is covalently linked to the second linker moiety L, and L ₃ is selected from

R ² (C=O)

,

C (=O) R ² C (=O)

; R ¹ is selected from H, C ₁ -C ₆ -alkyl, R ² , R ¹⁰ are each independently selected from H, C ₁ -C ₆ -alkyl; the second linker component L is absent or selected from:

or

e is 0 or 1. 7. The antibody-drug conjugate according to claim 6, characterized in that L ₁ , L ₂ are chosen, each independently, from C ₁ -C ₄ -alkyl; Q choose from

,N; L ₃ choose from

R ² (C=O)

,

C(=O)R ² C(=O)

; R ¹ is selected from H, C ₁ -C ₆ -alkyl, R ² , R ¹⁰ are each independently selected from H, C ₁ -C ₆ -alkyl. 8. Conjugate antibody-drug according to any one of paragraphs. 6, 7, characterized in that the first linker component has the structure

(A-1')

(A-1'')

(A-1''')

(A-2')

(A-2'')

(A-2''')

(A-2'''')

(A-4')

(A-4'')

(A-4''')

(A-6'')

(A-6'''). 9. Conjugate antibody-drug according to any one of paragraphs. 6-8, characterized in that the antibody or antibody functional fragment specifically binds to cell surface receptors or tumor-associated antigens. 10. Conjugate antibody-drug according to any one of paragraphs. 6-9, characterized in that the first linker moiety A includes a group covalently linked to the thiol group in Ab. 11. An antibody-drug conjugate that has the structure represented by formula II: Ab-(A-L-D) n (II), wherein Ab is an antibody or a functional fragment of an antibody; the linker moiety includes a first linker moiety A and a second linker moiety L; D is a drug moiety; n is 1, 2, 3 or 4; Where the first linker moiety A includes a group covalently linked to the thiol group in Ab, which has the structure represented as:

A-4 A-5 A-16

A-17 A-18 A-19

A-29 the second linker component L is absent or selected from:

L-4 L-6

or

, L-7 e is 0 or 1. 12. Conjugate antibody-drug according to any one of paragraphs. 6-11, characterized in that drug moiety D is cytotoxic drugs, cell differentiation factors, stem cell nutrient factors, steroid drugs, drugs for the treatment of autoimmune diseases, anti-inflammatory drugs or drugs for the treatment of infectious diseases; drug moiety D is preferably tubulin inhibitors or DNA damaging agents; drug moiety D is also preferably dolastatins, auristatins, maytansins, calicheamycins, duocamycins, anthramycin derivative PBD, camptothecin derivative SN-38, topoisomerase I inhibitors, amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combrestatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansins, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulisins, vinca alkaloids, vitamin A precursors, folic acid. 13. Conjugate antibody-drug according to any one of paragraphs. 6-12, characterized in that the component of the drug is

MMAD

MMAE

MMAF

DM1

DM4

PBD

CBI

Dxd

SN-38 14. Conjugate antibody-drug according to any one of paragraphs. 6-13, characterized in that it has the structure

ADC1

ADC2

ADC10

ADC11, wherein Ab is an antibody against cell surface receptors and tumor-associated antigens; n ¹ , n ² , n ³ are each independently selected from 0, 1, 2, 3, 4; n ¹ , n ² , n ³ are not equal to 0 at the same time, and n ¹ +n ² +n ³ ≤4; m ¹ , m ² , m ³ are chosen, each independently, from 0, 1, 2; at least one of m ¹ , m ² , m ³ is 0, but m ¹ , m ² , m ³ are not 0 at the same time, and m ¹ +m ² +m ³ ≤2. 15. The antibody-drug conjugate according to claim 14, characterized in that it has the structure

ADC17

ADC18

ADC26

AD27, wherein Ab is an antibody against cell surface receptors and tumor-associated antigens; n ¹⁰ is 0, 1, 2, 3 or 4; m ⁶ is 0, 1 or 2. 16. The antibody-drug conjugate according to claim 14, characterized in that it has the structure

ADC33

ADC34

ADC42

ADC43, wherein Ab is an antibody against cell surface receptors and tumor-associated antigens; n ¹¹ is 0, 1, 2, 3 or 4; m ⁷ is 0, 1 or 2. 17. Conjugate antibody-drug according to any one of paragraphs. 6-16, characterized in that the antibody is selected from mouse antibodies, mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies. 18. Pharmaceutical composition for the treatment of cancer associated with the HER2 antigen, which includes an effective amount of an antibody-drug conjugate according to any one of paragraphs. 6-17 and a pharmaceutically acceptable carrier. 19. The use of an antibody-drug conjugate according to any one of paragraphs. 6-17 or the pharmaceutical composition according to claim 18 when receiving a drug for the treatment of cancer associated with the HER2 antigen.

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