RU2800137C1 - Antibody-drug conjugate, intermediate for its production, method of its production and its use - Google Patents

Abstract

FIELD: biotechnology and medicine.

SUBSTANCE: antibody-drug conjugate (ADC), an intermediate for its preparation, a method of its preparation and its use are disclosed. The invention relates to an antibody-drug conjugate (ADC), which is Ab-(L₃-L₂-L₁-D)_m, where Ab is an antibody; the antibody is an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof, or an anti-claudin 18.2 IMAB362 antibody or a variant thereof, an intermediate for its production, a method of its production and its use. ADC has better biological activity, stability and homogeneity, is characterized by reduced toxic and side effects, as well as a faster release rate of the cleavage enzyme in tumor cells.

EFFECT: use of the new ADC may provide a wide use of a cytotoxic drug, in particular camptothecin, in the field of ADC in the treatment of patients with tumors resistant to ADC affecting microtubules.

17 cl, 13 tbl

Description

[1] The present application claims priority under Chinese Patent Application CN2019105779096, filed June 28, 2019. The entire disclosure of the above Chinese patent application is incorporated herein by reference in its entirety.

Technical field

[2] The present invention relates to the field of biotechnology and medicine, in particular relates to an antibody-drug conjugate, an intermediate for its production, a method for its production and its use.

State of the art

[3] Antibody drug conjugate (ADC) has been a hot topic in the pharmaceutical industry in recent years. Due to the poor clinical performance of many antibody-based drugs, many industry giants are increasingly turning their attention to ADC-based drugs. To date, seven ADC-based drugs have been approved for sale abroad. On May 17, 2000, Pfizer's Gemtuzumabaozogamicin (trade name Mylotarg) was approved by the Food and Drug Administration (FDA) for the treatment of newly relapsed acute myeloid leukemia (AML) patients over 60 years of age with CD33+ expression who are unsuitable for cytotoxic chemotherapy. Gemtuzumabozogamicin was withdrawn from the market in 2010 but was reintroduced in 2017. In the same year, Pfizer's inotuzumabozogamicin (trade name Besponsa) was also approved by the FDA for the treatment of relapsed and refractory B-cell ALL in adults. (Adcetris)), developed by SeattleGenetics, for the treatment of CD30-positive Hodgkin's lymphoma (HL) and the rare disease systemic anaplastic large cell lymphoma (sALCL). On February 22, 2013, ado-trastuzumabemtansine (T-DM1, trade name Kadcyla), developed by Genentech, was approved for sale by the FDA and is primarily used for the treatment of Her2-positive advanced (metastatic) breast cancer. In 2019 alone, polatuzumabvedotin (trade name Polivy), enfortumabvedotin (trade name Padcev) and fam-trastuzumabderukstecan (trade name Enhertu) were successively approved for marketing. In addition, over 100 ADC-based drugs are in clinical and preclinical development around the world.

[4] The basic functional elements of an antibody-drug conjugate include an antibody, a linker, and an effector molecule. The antibody is used to transport the effector molecule to the tumor for enrichment, resulting in the killing of the tumor cells. Conventional effector molecules in most cases are highly active inhibitors of tubulin, which are usually characterized by relatively strong toxic and side effects that limit the use of ADC. Recently, Immunomedics has invented a new type of ADC-based drug - IMMU-132 (ZL200980156218) - containing a camptothecin compound as an effector molecule, showing a good antitumor effect. DaiichiSankyo has invented another ADC-based drug, DS-8201a (ZL201380053256), containing camptothecin compound as an effector molecule, also showing good antitumor effects. In the current ADC technology, the linker used to connect the camptothecin compound and the antibody is rarely studied. In general, an ideal linker in an ADC should meet the following requirements: firstly, to ensure that the small molecule drug does not separate from the antibody in the blood plasma, after entering the cell, the linker is degraded under suitable conditions to quickly release the active small molecule drug; secondly, the linker must have good physical and chemical properties so that it can join the antibody to form a conjugate; and the linker should be easy to obtain in order to lay the foundation for large-scale production of ADC. IMMU-132 uses a pH sensitive linker which is characterized by poor stability. DS-8201a uses a tetrapeptide structure containing glycine-glycine-phenylalanine-glycine compared to the common cathepsin B substrate sequence (such as valine-citrulline), while the enzymatic cleavage reaction is slow, poor physical and chemical properties, and difficult to synthesize.

The essence of the invention

[5] The technical problem to be solved in the present invention is to overcome the disadvantage of one type of the existing antibody-drug conjugate and provide a new antibody-drug conjugate, an intermediate for its production, a method for its production and its use. The antibody-drug conjugate can provide widespread use of cytotoxic drugs in the field of ADC and treatment of patients with tumors that are resistant to ADCs that attack microtubules.

[6] The present invention provides antibody-drug conjugates with a number of different specific structural linkers, wherein the antibody-drug conjugates inhibit the growth of tumors in mammals and can be used to treat various types of cancer. Antibody-drug conjugates have better biological activity, stability and homogeneity, are characterized by reduced toxic and side effects, as well as a faster rate of release of the cleavage enzyme in tumor cells.

[7] The present invention solves the above technical problems by the following technical solutions.

[8] The present invention provides an antibody-drug conjugate, wherein the general structural formula of the antibody-drug conjugate is Ab-(L ₃ -L ₂ -L ₁ -D) _m ;

[9] where Ab is an antibody;

[10] D is a cytotoxic drug;

[11] m is 2-8;

[12] the structure of L ₁ is as shown in formula I, II, III or IV, the a-terminus of L ₁ is connected to a cytotoxic drug, and the e-terminus of L ₁ is connected to the c-terminus of L ₂ ;

, , , ;

[13] where L is independently a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, or a valine residue; p is 2-4;

[14] R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl, C ₃ -C ₁₀ cycloalkyl, C ₆ -C ₁₄ aryl, or 5-14 membered heteroaryl; wherein the heteroatoms in the 5-14-membered heteroaryl are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4;

[15] R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₆ alkyl;

[16] L ₂ is , , , , , , , , , , , , , , , or where n is independently 1-12, the c-terminus of L ₂ is connected to the e-terminus of L ₁ , the f-terminus of L ₂ is connected to the d-terminus of L ₃ ;

[17] L ₃ is or , where the b-terminus of L ₃ is connected to Ab, the d-terminus of L ₃ is connected to the f-terminus of L ₂ ;

[18] if the structure of L ₁ is as shown in formula I, L ₃ is ; L ₂ is not .

[19] In a preferred embodiment of the present invention, in antibody-drug conjugates, some groups have the following definitions, and the definitions of unspecified groups are as described in any of the above explanations (the content of this paragraph is referred to in the text as "in a preferred embodiment of the present invention"):

[20] The antibody may be a conventional anti-tumor ADC antibody, preferably an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof, an anti-claudin 18.2 IMAB362 antibody or a variant thereof, or an anti-Trop2 RS7 antibody or variant, more preferably an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or variant thereof or a claudin 18.2 antibody IMAB362 or a variant thereof, even more preferably an anti-HER2 antibody trastuzumab or a variant thereof or an anti-claudin 18.2 IMAB362 antibody or a variant thereof, and most preferably an anti-HER2 antibody trastuzumab or an anti-claudin 18.2 IMAB362 antibody. The light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing. The light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing. The light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing. The light chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 3 in the sequence listing, and the heavy chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 4 in the sequence listing. The HER2 antibody variant trastuzumab has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology compared to the HER2 antibody trastuzumab. An anti-B7-H3 P2E5 variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology compared to an anti-B7-H3 P2E5 antibody. The Trop2 RS7 antibody variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology compared to the Trop2 RS7 antibody. The anti-claudin antibody 18.2 IMAB362 variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology compared to the anti-claudin antibody 18.2 IMAB362.

[21] In a preferred embodiment of the present invention, the b-terminus of L ₃ is preferably connected to the sulfhydryl in the antibody via a thioether bond. Taking as an example, a contact form with a cysteine residue in the antibody is .

[22] In a preferred embodiment of the present invention, the cytotoxic drug may be a conventional cytotoxic drug in the field of ADC, in particular, preferably a topoisomerase inhibitor containing a hydroxyl group, and more preferably a topoisomerase I inhibitor containing a hydroxyl group, even more preferably camptothecin or its derivatives, and most preferably .

[23] L ₁ is preferably connected to the hydroxyl group in the cytotoxic drug via an ether bond. After attaching L ₁ to the cytotoxic drug moiety remaining in the antibody-drug conjugate is preferably or .

Taking And

as examples, -L ₁ -D may be or .

[24] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl or tert -butyl, most preferably ethyl. Each of R ^1-1 and R ^1-2 is independently preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl or tert -butyl, most preferably methyl.

[25] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or tert -butyl, most preferably ethyl. R ^1-3 is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl or tert -butyl, most preferably methyl.

[26] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably methyl or ethyl.

[27] In a preferred embodiment of the present invention, m is preferably 4-8, more preferably 7-8 (eg, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0).

[28] In a preferred embodiment of the present invention, L is preferably a valine residue or an alanine residue, and p is preferably 2. (L)p is more preferably where the amino end of (L)p is connected to the carbonyl end in formula III.

[29] In a preferred embodiment of the present invention, n is preferably 8-12 (eg, 8 and 12).

[30] In a preferred embodiment of the present invention, R ^1-1 , R ^1-2 and R ^1-3 are independently preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably methyl.

[31] In a preferred embodiment of the present invention, R ¹ is preferably C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C _{1 -C 6 alkyl substituted with R 1-3 S(O) 2 -, or C 1 -C 6 alkyl , more} preferably C ₁ -C ₆ alkyl substituted with ^{-NR 1-1} R ^1-2 , or C ₁ -C ₆ alkyl substituted R ^1-3 S(O) ₂ -, most preferably C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -. If R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably methyl or ethyl. C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - is preferably . C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 is preferably .

[32] In a preferred embodiment of the present invention preferably is , or .

[33] In a preferred embodiment of the present invention, L ₃ is preferably .

[34] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula I, L ₂ is preferably , , , , , , or , L ₃ is preferably .

[35] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula II, L ₂ is preferably , or ; L ₃ is preferably .

[36] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₂ is preferably , , , , or ; L ₃ is preferably .

[37] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula IV, L ₂ is preferably or ; L ₃ is preferably .

[38] In a preferred embodiment of the present invention, the structure of L ₁ is preferably as shown in formula I or III.

[39] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula I, L ₂ is preferably , , , , or .

[40] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula I, L ₂ is preferably .

[41] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₂ is preferably , , , or .

[42] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₂ is preferably , , or .

[43] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₂ is .

[44] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₁ is preferably .

[45] In a preferred embodiment of the present invention, in the antibody-drug conjugate Ab is an anti-HER2 antibody trastuzumab, an anti-B7-H3 P2E5 antibody or variant thereof, or an anti-claudin 18.2 IMAB362 antibody or variant thereof; D is a cytotoxic drug; m is 2-8;

[46] the structure of L ₁ is as shown in formula I, II, III or IV,

, , , ;

[47] L ₂ is , , , , , , , , , , , , , , , or , n is independently 8-12;

[48] L ₃ is ;

[49] L is independently a valine residue or an alanine residue; p is from 2 to 4;

[50] R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₆ alkyl;

[51] each of R ^1-1 , R ^1-2 and R ^1-3 independently represents C ₁ -C ₆ alkyl;

[52] wherein the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably represented under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing.

[53] In a preferred embodiment of the present invention, in the antibody-drug conjugate Ab is an anti-HER2 antibody trastuzumab, an anti-B7-H3 P2E5 antibody or variant thereof, or an anti-claudin 18.2 IMAB362 antibody or variant thereof; D represents ; m is 7-8;

[54] the structure of L ₁ is as shown in formula I or III,

, ;

[55] if the structure of L ₁ is as shown in formula I, L ₂ is , , , , or ; n is independently 8-12;

[56] if the structure of L ₁ is as shown in formula III, L ₂ is , , , or ; n is independently 8-12;

[57] L ₃ is ;

[58] L is independently a valine residue or an alanine residue; p is from 2 to 4;

[59] R ¹ is C ₁ -C ₄ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₄ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₄ alkyl; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₄ alkyl;

[60] The light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing.

[61] In a preferred embodiment of the present invention, where Ab is an antibody; D represents ;

[62] L ₁ is ; where L is a valine residue or an alanine residue, p is 2, (L)p is preferably ; R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R 1-3 S(O) ₂ -, or C ₁ -C ₆ alkyl, preferably C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , or C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, more preferably C ₁ -C ₆ alkyl substituted with ^{R 1-3 S} (O) ₂ -; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₄ alkyl, preferably methyl; C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 is preferably ; C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - is preferably ;

[63] L ₂ is , , or , where n is preferably equal to 8; L ₂ is preferably ; L ₃ is .

[64] In a preferred embodiment of the present invention, L ₂ is .

[65] In a preferred embodiment of the present invention, L ₂ is .

[66] In a preferred embodiment of the present invention, L ₂ is .

[67] In a preferred embodiment of the present invention, L ₂ is .

[68] In a preferred embodiment of the present invention, L ₂ is .

[69] In a preferred embodiment of the present invention, L ₂ is .

[70] In a preferred embodiment of the present invention, L ₂ is .

[71] In a preferred embodiment of the present invention, L ₂ is .

[72] In a preferred embodiment of the present invention, L ₂ is .

[73] In a preferred embodiment of the present invention, L ₂ is .

[74] In a preferred embodiment of the present invention, L ₂ is .

[75] In a preferred embodiment of the present invention, L ₂ is .

[76] In a preferred embodiment of the present invention, L ₂ is .

[77] In a preferred embodiment of the present invention, L ₂ is .

[78] In a preferred embodiment of the present invention, L ₂ is .

[79] In a preferred embodiment of the present invention, L ₂ is .

[80] In a preferred embodiment of the present invention, L ₂ is .

[81] In a preferred embodiment of the present invention, the structure of L ₁ is as shown in formula III, L ₂ is .

[82] In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any of the compounds below:

, , , , , , , , , , , , , , , , , , , , , , or ,

[83] where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0;

[84] Ab is anti-HER2 trastuzumab, anti-B7-H3 P2E5, or anti-claudin 18.2 IMAB362; the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 2 in the sequence listing.

[85] In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any of the compounds below:

, , , , , , , , , , , or ;

[86] where Ab is an anti-HER2 antibody trastuzumab; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing; where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0.

[87] In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any of the compounds below:

[88] where Ab is an anti-HER2 antibody trastuzumab; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing.

[89] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

[90] where Ab is an anti-HER2 antibody trastuzumab; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing.

[91] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

, , , , , , , , , or ,

[92] where Ab is an antibody to B7-H3 P2E5; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 8 in the sequence listing where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0.

[93] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

[94] where Ab is an antibody to B7-H3 P2E5; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 8 in the sequence listing.

[95] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

, , , , , , , , or ,

[96] where Ab is an antibody to claudin 18.2 IMAB362; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 2 in the sequence listing; where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0.

[97] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

[98] Ab is an antibody to claudin 18.2 IMAB362; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 2 in the sequence listing.

[99] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

, , , ,

[100] where Ab, m and R ¹ are as defined above.

[101] The present invention also provides a linker-drug conjugate, wherein the general structural formula of the linker-drug conjugate is L ₄ -L ₂ -L ₁ -D; where L ₄ is or ; L ₂ , L ₁ and D are as defined above, the f-terminus of L ₂ is connected to the d-terminus of L ₄ ; if L ₄ is if L ₁ is , L ₂ is not .

[102] In a preferred embodiment of the present invention, the linker-drug conjugate is preferably any of the compounds below:

, , , ,

[103] where R ¹ is as defined above.

[104] In a preferred embodiment of the present invention, the linker-drug conjugate is preferably any of the compounds below:

, , , , , , , , , , , , , , , , , , , , , , or .

[105] The present invention also provides the following connection:

,

[106] where R ¹ is as defined above;

[107] R ² is -N ₃ , -NH ₂ , or .

[108] The present invention also provides the following compounds:

, , , , , , or .

[109] The present invention provides an antibody-drug conjugate, wherein the general structural formula of the antibody-drug conjugate is Ab-(L ₃ -L ₂ -L ₁ -D) _m ;

[110] where Ab is an antibody;

[111] D is a cytotoxic drug;

[112] m is 2-8;

[113] the structure of L ₁ is as shown in formula I, II, III or IV, the a-terminus of L ₁ is connected to a cytotoxic drug, and the e-terminus of L ₁ is connected to the c-terminus of L ₂ ;

, , , ;

[114] L ₂ is , , , , , , , , , , , , , , , or where n is independently 1-12, the c-terminus of L ₂ is connected to the e-terminus of L ₁ , the f-terminus of L ₂ is connected to the d-terminus of L ₃ ;

[115] L ₃ is or , where the b-terminus of L ₃ is connected to Ab, the d-terminus of L ₃ is connected to the f-terminus of L ₂ ;

[116] where L is independently a phenylalanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, or a valine residue; p is 2-4;

[117] R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl, C ₃ -C ₁₀ cycloalkyl, C ₆ -C ₁₄ aryl, or 5-14 membered heteroaryl; wherein the heteroatoms in the 5-14-membered heteroaryl are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4;

[118] R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₆ alkyl;

[119] if the structure of L ₁ is as shown in formula I, L ₃ is , L ₂ is not .

[120] In a preferred embodiment of the present invention, in antibody-drug conjugates, some groups have the following definitions, and the definitions of unspecified groups are as described in any of the above explanations (the content of this paragraph is referred to in the text as "in a preferred embodiment of the present invention"):

[121] The antibody may be a conventional anticancer ADC antibody, preferably an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof, an anti-claudin 18.2 IMAB362 antibody or a variant thereof, or an anti-Trop2 RS7 antibody or variant, more preferably an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof IMAB362 variant or anti-claudin 18.2 antibody or variant thereof, even more preferably trastuzumab anti-HER2 antibody or variant thereof or IMAB362 anti-claudin 18.2 antibody or variant thereof, and most preferably trastuzumab anti-HER2 antibody or IMAB362 anti-claudin 18.2 antibody. The light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing. The light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing. The light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing. The light chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 3 in the sequence listing, and the heavy chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 4 in the sequence listing.

[122] In a preferred embodiment of the present invention, the b-terminus of L ₃ is preferably connected to the sulfhydryl in the antibody via a thioether bond. Taking as an example, a contact form with a cysteine residue in the antibody is .

[123] In a preferred embodiment of the present invention, the cytotoxic drug may be a conventional cytotoxic drug in the field of ADC, in particular, preferably a topoisomerase inhibitor containing a hydroxyl group, and more preferably a topoisomerase I inhibitor containing a hydroxyl group, even more preferably camptothecin or its derivatives, and most preferably .

[124] L ₁ is preferably connected to the hydroxyl group in the cytotoxic drug via an ether bond. After attaching L ₁ to the cytotoxic drug moiety remaining in the antibody-drug conjugate is preferably or . Taking And as examples, -L ₁ -D may be or .

[125] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably ethyl. Each of R ^1-1 and R ^1-2 is independently preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably methyl.

[126] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or tert -butyl, most preferably ethyl. R ^1-3 is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or tert -butyl, most preferably methyl.

[127] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably methyl.

[128] In a preferred embodiment of the present invention, m is preferably 4-8, more preferably 7-8 (eg, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8).

[129] In a preferred embodiment of the present invention, L is preferably a valine residue or an alanine residue, and p is preferably 2. More preferably, (L)p is where the amino end of (L)p is connected to the carbonyl end in formula III.

[130] In a preferred embodiment of the present invention, n is preferably 8-12 (eg, 8 and 12).

[131] In a preferred embodiment of the present invention, R ^1-1 , R ^1-2 and R ^1-3 are independently preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl or t -butyl, most preferably methyl.

[132] In a preferred embodiment of the present invention preferably is or .

[133] In a preferred embodiment of the present invention, L ₃ is preferably .

[134] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula I, L ₂ is preferably , , , , , , or .

[135] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula II, L ₂ is preferably , or ; L ₃ is preferably .

[136] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula III, L ₂ is preferably , , , , or ; L ₃ is preferably .

[137] In a preferred embodiment of the present invention, if the structure of L ₁ is as shown in formula IV, L ₂ is preferably or ; L ₃ is preferably .

[138] In a preferred embodiment of the present invention, R ¹ is preferably C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₆ alkyl.

[139] In a preferred embodiment of the present invention, in the antibody drug conjugate Ab is an anti-HER2 antibody trastuzumab, an anti-B7-H3 P2E5 antibody or variant thereof, an anti-claudin 18.2 IMAB362 antibody or variant thereof; D is a cytotoxic drug; m is 2-8;

[140] the structure of L ₁ is as shown in formula I, II, III or IV,

, , , ;

[141] L ₂ is , , , , , , , , , , , , , , , or , n is independently 8-12;

[142] L ₃ is ;

[143] L is independently a valine residue or an alanine residue; p is 2-4;

[144] R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₆ alkyl;

[145] R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₆ alkyl;

[146] wherein the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably represented under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing.

[147] In a preferred embodiment of the present invention, in the antibody drug conjugate Ab is an anti-HER2 antibody trastuzumab, an anti-B7-H3 P2E5 antibody or variant thereof, an anti-claudin18.2 IMAB362 antibody or variant thereof; D represents ; m is 7-8;

[148] if the structure of L ₁ is as shown in formula I, L ₂ is , , , , , , or ; n is independently 8-12;

[149] if the structure of L ₁ is as shown in formula II, L ₂ is , or ; n is 8-12;

[150] if the structure of L ₁ is as shown in formula III, L ₂ is , , , , or ; n is 8-12;

[151] if the structure of L ₁ is as shown in formula IV, L ₂ is or ;

[152] L ₃ is ;

[153] L is independently a valine residue or an alanine residue; p is from 2 to 4;

[154] R ¹ is C ₁ -C ₄ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₄ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₄ alkyl; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₄ alkyl;

[155] The light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing.

[156] In a preferred embodiment of the present invention, the antibody drug conjugate is preferably any of the compounds below:

, , , , , , , , , , , , , , , , , , , , , , or where Ab is anti-HER2 trastuzumab, anti-B7-H3 P2E5, or anti-claudin 18.2 IMAB362, m is 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8; the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing.

[157] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

Ab is an anti-HER2 antibody trastuzumab; the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing.

[158] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

where Ab is an antibody to HER2 trastuzumab; the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing.

[159] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

7.5

7.6

7.6
where Ab is an antibody to B7-H3 P2E5; the light chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 P2E5 antibody is preferably shown under SEQ ID No. 8 in the sequence listing.

[160] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably any of the compounds below:

where Ab is an antibody to claudin 18.2 IMAB362; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 2 in the sequence listing.

[161] The present invention also provides a linker-drug conjugate, wherein the general structural formula of the linker-drug conjugate is L ₄ -L ₂ -L ₁ -D; where L ₄ represents or ; L ₂ , L ₁ and D are as defined above, the f-terminus of L ₂ is connected to the d-terminus of L ₄ ; if L ₄ is , L ₁ is ; L ₂ is not .

[162] In a preferred embodiment of the present invention, the linker-drug conjugate is preferably any of the compounds below:

, , , , , , , , , , , , , , , , , , , , , , or .

[163] The present invention also provides the following compounds:

, .

[164] The present invention provides a method for preparing an antibody-drug conjugate comprising the steps of: coupling the linker-drug conjugate to an antibody.

[165] In the present invention, the combination conditions and actions for conducting the combination may be conventional conditions and actions for conducting the combination in the prior art.

[166] The present invention also provides a pharmaceutical composition comprising an antibody-drug conjugate and a pharmaceutically acceptable carrier.

[167] The present invention also provides for the use of an antibody-drug conjugate or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of cancer. The cancer is preferably gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer, or pancreatic cancer.

[168] The present invention also provides a method for preventing and/or treating cancer, comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutical composition. The cancer is preferably gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer, or pancreatic cancer.

[169] In the present invention, m is the molar ratio of the cytotoxic drug molecule and Ab (also known as DAR, that is, the ratio of the drug and the antibody when they are combined), and m can be an integer or a decimal number, and preferably it should be understood as the average value of the molar ratio of the drug molecule and the monoclonal antibody molecule in the antibody-drug conjugate obtained by combining one monoclonal antibody molecule with a cytotoxic drug means, and in general it can be measured using hydrophobic interaction chromatography (HIC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, electrophoresis), liquid chromatography-mass spectrometry (LC-MS) and other methods.

[170] In the present invention, the term "C ₁ -C ₆ alkyl" alone or in combination means a saturated linear or branched alkyl group containing from 1 to 6, in particular from 1 to 4 carbon atoms, such as methyl and ethyl, n- propyl, isopropyl, n- butyl, isobutyl or tert -butyl, preferably "C ₁ -C ₆ alkyl" means methyl or ethyl.

[171] The antibody of the present invention can be obtained using techniques well known in the art, such as hybridoma technologies, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations of such techniques, or other techniques known in the art.

[172] Variants refer to amino acid sequence mutants of the antibody and covalent derivatives of natural polypeptides, provided that biological activity equivalent to that of natural polypeptides is maintained. The difference between mutant amino acid sequences and natural amino acid sequences is usually that one or more amino acids in the natural amino acid sequence are changed or one or more amino acids are removed from and/or inserted into the polypeptide sequence. Deletion mutants include native polypeptide fragments and N-terminal and/or C-terminal truncation mutants. Typically, mutant amino acid sequences have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology relative to the natural sequence.

[173] The term "treatment" or equivalent expressions in relation to, for example, cancer refers to a procedure or process used to reduce or eliminate a certain number of cancer cells in a patient's body or alleviate the symptoms of cancer. "Treatment" of cancer or other proliferative disorders does not necessarily mean that the cancer cells or other disorders will actually be eliminated, the number of cells or disorders will actually be reduced, or the symptoms of the cancer or other disorders will actually be alleviated. Usually, even with a low success rate, a cancer treatment will be used, but given the patient's medical history and expected survival expectations, it will still be considered to provide an overall beneficial effect.

[174] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that can deliver an effective amount of the active agent of the present invention, does not adversely affect the biological activity of the active agent, and does not produce toxic side effects on the host or patient. Exemplary carriers include water, vegetable and mineral oils, cream base, lotion base, ointment base, and the like. Such bases include suspending agents, tackifiers, penetration enhancers, and the like. Their formulations are well known to those skilled in the art of cosmetics or topical preparations.

[175] Based on the avoidance of violation of well-known knowledge in the art, the preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[176] All reagents and starting materials used in the present invention are commercially available.

[177] The positive and progressive effect of the present invention is as follows: the antibody-drug conjugate of the present invention has better biological activity, stability and uniformity, is characterized by reduced toxic and side effects, as well as a faster release rate of the cleavage enzyme in tumor cells. Through the use of this new type of antibody-drug conjugate, a wide application of cytotoxic drugs, in particular camptothecin compounds, in the field of ADC and the treatment of patients with tumors that are resistant to microtubule-acting ADCs can be achieved.

Detailed Description of the Preferred Embodiment

[178] Table 1. Description of abbreviations

[179] The following embodiments further illustrate the present invention, but the present invention is not limited to them. In the following embodiments, the experimental methods without specific conditions are selected in accordance with generally accepted methods and conditions or in accordance with the product specification.

[180] Embodiment 1. Synthesis of LE 01

[181] Commercially available SMCC (1 mmol, 0.32 g) and compound A (0.5 mmol, 0.42 g) were dissolved in 10 ml of DMF, the mixture was stirred at room temperature for 3 h and the solvent was removed by distillation under reduced pressure, the crude product was purified by silica gel column chromatography [chloroform - chloroform:methanol = 9:1 (v/v .)] to give the title compound (0.5 g, 0.47 mmol) as a pale yellow solid, yield: 94%, ESI-MS mass/charge: 1060.3 (M+H); where compound A can be synthesized according to the known method described in WO2015146132A1.

[182] The GGFG-Dxd compound (structure given below) was also synthesized according to the known method described in WO2015146132A1, ESI-MS mass/charge: 1034.5 (M+H),¹H-NMR (400 MHz, DMSO-d ₆ ) δ 8.61 (t,J= 6.4 Hz, 1H), 8.50 (d,J= 8.5 Hz, 1H), 8.28 (t,J= 5.1 Hz, 1H), 8.11 (d,J= 7.5 Hz, 1H), 8.05 (t,J= 5.7 Hz, 1H), 7.99 (t,J= 5.9 Hz, 1H), 7.77 (d,J= 11.0 Hz, 1H), 7.31 (s, 1H), 7.25 - 7.16 (m, 5H), 6.98 (s, 2H), 6.51 (s, 1H), 5.59 (dt,J= 7.4, 4.1 Hz, 1H), 5.41 (s, 2H), 5.20 (s, 2H), 4.64 (d,J= 6.1 Hz, 2H), 4.53 - 4.40 (m, 1H), 4.02 (s, 2H), 3.74 - 3.37 (m, 8H), 3.18 - 3.00 (m, 2H), 3.04 - 2.97 (m, 1H), 2.77 (dd,J= 13.5, 9.4 Hz, 1H), 2.38 (s, 3H), 2.19 (dd,J= 14.9, 8.5 Hz, 2H), 2.11 - 2.05 (m, 2H), 1.86 (dd,J= 14.0, 6.7 Hz, 2H), 1.45 (s, 4H), 1.20 - 1.14 (m, 2H), 0.87 (t,J= 7.1 Hz, 3H).

[183] Embodiment 2: Synthesis of LE02-LE06 and LE08

, , , , , .

[184] Referring to Embodiment 1, compounds were prepared by carrying out a condensation reaction (LE02, LE03 and LE06 must be acidified after the condensation reaction) between the appropriate maleamide moieties and Compound A. The structures of the specific maleamide moieties used are shown in Table 2. Compound LE02: pale yellow solid, ESI-MS mass/charge: 1114.2 (M+H); compound LE03: pale yellow solid, ESI-MS mass/charge: 1007.2 (M+H); compound LE04: slightly yellow solid, ESI-MS mass/charge: 1344.5 (M+H); compound LE05: yellow solid, ESI-MS mass/charge: 1112.3 (M+H); compound LE06: yellow solid, ESI-MS mass/charge: 1162.5 (M+H); compound LE08: pale yellow oil, ESI-MS mass/charge: 1719.1 (M+H).

[185] Table 2. Structures of the maleamide fragments used in the synthesis of LE02-LE06 and LE08

[186] Embodiment 3: Synthesis of LE07

[187] Commercially available LE07-S (1 mmol, 0.34 g) and compound B (0.5 mmol, 0.38 g) were dissolved in 10 ml of DMF; HATU (0.5 mmol, 0.19 g), 0.5 ml of triethylamine was added and the mixture was stirred at room temperature for 3 hours, 0.5 ml of trifluoroacetic acid was added, then the mixture was stirred at room temperature for 10 minutes, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform-chloroform:methanol= 9:1 (v/v)] to give the trifluoroacetic acid salt and the title compound (0.33 g, 0.3 mmol) as a pale yellow solid, yield: 60%, ESI-MS w/charge: 1105.3 (M+H). Compound B can be synthesized according to the known method described in WO2015146132A1.

[188] Embodiment 4: Synthesis of LE09-LE11

, , .

[189] Referring to the method in Embodiment 3, compounds were obtained by performing a condensation reaction between Compound B and suitable carboxylic acid moieties (commercially available) using a condensing agent. The structures of the specific carboxylic acid moieties used are shown in Table 3. Compound LE09: pale yellow to light yellow oil, ESI-MS mass/charge: 1705.9 (M+H). Соединение LE10: ESI-MS: масса/заряд: 1115,9 (M+H), ¹ H-ЯМР (400 MГц, DMSO- d ₆ ) δ 8,66 (t, J = 6,7 Гц, 1H), 8,53 (d, J = 9,0 Гц, 1H), 8,47 (t, J = 5,2 Гц, 1H), 8,37 (t, J = 5,8 Гц, 1H), 8,30 (d, J = 8,4 Гц, 2H), 7,81 (s, 1H), 7,78 (d, J = 11,1 Гц, 1H), 7,31 (s, 1H), 7,20 (dd, J = 21,9, 7,3 Гц, 5H), 7,00 (d, J = 5,3 Гц, 2H), 6,54 (s, 1H), 5,60 (dd, J = 13,7, 6,8 Гц, 1H), 5,42 (s, 2H), 5,20 (s, 2H), 5,10 (s, 2H), 4,64 (d, J = 6,3 Гц, 2H), 4,55 - 4,45 (m, 1H), 4,26 (d, J = 5,3 Гц, 2H), 4,02 (s, 2H), 3,74 (ddd, J = 31,4, 16,7, 5,6 Гц, 6H), 3,17 (dd, J = 14,3, 8,5 Гц, 2H), 3,02 (dd, J = 14,1, 4,3 Гц, 1H), 2,74 (dd, J = 13,4, 10,0 Гц, 1H), 2,38 (s, 3H), 2,23 - 2,15 (m, 2H), 2,05 ( t, J = 7,4 Гц, 2H), 1,90 - 1,80 (m, 2H), 1,46 (dd, J = 14,7, 7,3 Гц, 4H), 1,19 - 1,14 (m, 2H), 0,87 (t, J = 7,2 Гц, 3H). Compound LE11: pale yellow solid, ESI-MS mass/charge: 1141.5 (M+H).

[190] Table 3. Structures of carboxylic acid fragments used in the synthesis of LE09-LE11

[191] Embodiment 5: Synthesis of LE23-LE24

[192] Commercially available compound LE23-S or LE24-S (2 equivalents) and compound C (1 equivalent) were dissolved in an appropriate amount of DMF, the mixture was stirred at room temperature for 3 hours, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform-chloroform:methanol=10:1 (v/v)] to give the title compound as a pale yellow solid. Here, compound C can be synthesized according to the known method described in WO2015146132A1. Compound LE23: yellow solid, ESI-MS mass/charge: 1090.2 (M+H); compound LE24: yellow solid, ESI-MS mass/charge: 1122.1 (M+H).

[193] Embodiment 6: Synthesis of LE12

[194] Synthesis of intermediate 2

[195] (S) -2-Azidopropanic acid (10 g, 86.9 mmol) and 4-aminobenzyl alcohol (21.40 g, 173.8 mmol) was dissolved in 300 ml of a mixed solvent consisting of dichloromethane and methanol (volumetric ratio: 2: 1), 2: 1) was added 2: 1), added 2: 1) (21.49 g, 86.9 mmol), ensured the reaction in the reaction medium at room temperature for 5 hours, the solvent was evaporated at reduced pressure and then the resulting residue was cleaned with the help of column chromatography on silicahel [dichloromethane: ethyl acetate = 1: 1 (vol. SI-MS mass/charge: 221 (m+h).

[196] Synthesis of intermediate 3

[197] Intermediate 2 (15 g, 68.2 mmol) and bis( p -nitrophenyl) carbonate (22.82 g, 75.02 mmol) were mixed and dissolved in 200 ml of anhydrous N,N-dimethylformamide, and 25 ml of triethylamine was added, allowing the mixture to react at room temperature for 2 hours. After completion of the reaction, the starting materials were monitored by liquid chromatography-mass spectrometry, methylamine hydrochloride (6.91 g, 102.3 mmol) was added and the reaction was allowed to continue for 1 hour. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure, and then 200 ml of water and 200 ml of ethyl acetate were added. After phase separation, the organic phase was collected, dried and concentrated. The resulting crude product was purified by silica gel column chromatography [dichloromethane:ethyl acetate = 10:1 (v/v)] to give intermediate 3 (18.9 g, yield: 100%), ESI-MS w/charge: 278 (M+H).

[198] Synthesis of intermediate 5

[199] Intermediate 3 (10 g, 36.1 mmol) and paraformaldehyde (1.63 g, 54.2 mmol) were dissolved in 150 ml of anhydrous dichloromethane, trimethylchlorosilane (6.28 g, 57.76 mmol) was added slowly and the mixture was allowed to react at room temperature for 2 hours to obtain a solution of the crude product, which was intermediate 4. After selection and a sample reaction mixture was quenched by adding methanol and the reaction was monitored by LC/MS. After completion of the reaction, the reaction solution was filtered, and then tert -butyl glycolate (9.54 g, 72.2 mmol) and triethylamine (10 ml, 72.2 mmol) were added to the filtrate, and the mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [petroleum ether:ethyl acetate = 3:1 (v/v)] to give intermediate 5 (11.2 g, yield: 74%), ESI-MS mass/charge: 422 (M+H).

[200] Synthesis of intermediate 6

[201] Intermediate 5 (10 g, 23.8 mmol) was dissolved in 80 ml of anhydrous tetrahydrofuran, 80 ml of water was added, then tris(2-carboxyethyl)phosphine hydrochloride (13.6 g, 47.6 mmol) was added and the mixture was allowed to react at room temperature for 4 hours. After completion of the reaction, tetrahydrofuran was removed by distillation under reduced pressure, and then the residue was extracted with ethyl acetate. After drying the resulting organic phase, the solvent was evaporated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give intermediate 6 (8.1 g, yield: 86%), ESI-MS mass/charge: 396 (M+H).

[202] Synthesis of intermediate 8

[203] Intermediate 6 (5 g, 12.7 mmol) was dissolved in 60 ml of a mixed solvent of dichloromethane and methanol (v/v = 2:1), 3 ml of trifluoroacetic acid was added slowly, and the mixture was allowed to react at room temperature for 30 minutes. After completion of the reaction, equal volumes of water and ethyl acetate were added, the organic phase was dried and then concentrated, and the resulting crude product was directly used in the next step.

[204] The crude product obtained in the previous step was dissolved in 50 ml of anhydrous N,N-dimethylformamide, Fmoc-valine hydroxysuccinimide ester (8.3 g, 19.1 mmol), triethylamine (5 ml) was added, the mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give intermediate 8 (5.4 g, yield: 64%), ESI-MS mass/charge: 661 (M+H).

[205] Synthesis of intermediate 9

[206] Intermediate 8 (1 g, 1.5 mmol) and exatecanemethanesulfonate (0.568 g, 1 mmol) were mixed in 30 ml of anhydrous N,N-dimethylformamide and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.14 g, 3.0 mmol) was added, 2 ml of triethylamine, allowed the mixture to react at room temperature for 2 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform:methanol = 10:1 (v/v)] to give intermediate 9 (0.94 g, 87% yield), ESI-MS mass/charge: 1078 (M+H ).

[207] Synthesis of compound LE12

[208] Intermediate 9 (1 g, 0.929 mmol) was dissolved in 20 ml of anhydrous DMF, 0.5 ml of 1,8-diazabicycloundec-7-ene was added and the mixture was allowed to react at room temperature for 1 hour. After completion of the reaction of the starting materials, 6-(maleimido)hexanoic acid succinimidyl ester (428.5 mg, 1.39 mmol) was added directly, and the mixture was stirred at room temperature for 1 hour. The solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform:methanol = 8:1 (v/v)] to give the title compound (0.7 g, yield: 73%), ESI-MS mass/charge: 1035 (M+H).

[209] Embodiment 7: Synthesis of LE13-LE20

[210] Intermediate VI can be prepared by using Fmoc-L-valine-L-alanine as a starting material, referring to steps 6a and 6b in the method for the synthesis of intermediate 3 in embodiment 6, where the methylamine hydrochloride in step 6b is replaced by the corresponding commercially available amino compound. The subsequent steps started from intermediate VI according to the same method as steps 6c, 6d, 6f and 6h in Embodiment 6 to give an intermediate IX similar to Intermediate 9, and then according to the same steps as Steps 6i and 6j in Embodiment 6 to work up, deprotect the amino group, and then the residue was condensed with various commercially available maleimides to give final product. The amine compounds used and the maleimide structures are shown in Table 4. Compound LE13: pale yellow solid, ESI-MS mass/charge: 1106.5 (M+H); compound LE14: pale yellow solid, ESI-MS mass/charge: 1141.4 (M+H); compound LE15: off-white solid, ESI-MS mass/charge: 1121.2 (M+H); compound LE16: pale yellow solid, ESI-MS mass/charge: 1167.1 (M+H); compound LE17: yellow solid, ESI-MS mass/charge: 1132.3 (M+H); compound LE18: pale yellow solid, ESI-MS mass/charge: 1305.4 (M+H); compound LE19: pale yellow solid, ESI-MS mass/charge: 1307.4 (M+H); compound LE20: pale yellow solid, ESI-MS mass/charge: 1337.6 (M+H).

[211] Table 4. Intermediates used in the synthesis of LE13-LE20

, , , , , , , .

[212] Embodiment 8: Synthesis of LE21-LE22

R' represents or .

[213] Synthesis of compound DXD-1

[214] Commercially available exatecanemethanesulfonate (0.568 g, 1 mmol) and 2-( tert -butyldimethylsiloxy)acetic acid (CAS: 105459-05-0, 0.38 g, 2 mmol) were dissolved in 20 ml of anhydrous dichloromethane, condensing agent HATU (0.76 g, 2 mmol) was added and 1 ml pyridine and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the solvent was evaporated to dryness under reduced pressure, and the resulting crude product was purified by column chromatography [dichloromethane:methanol = 50:1 (v/v)] to give the title compound DXD-1 (0.55 g, yield: 90%), ESI-MS mass/charge: 608.1 (M+H). ¹ H ЯМР (400 MГц, CDCl ₃ ) δ 7,73 (d, J = 10,5 Гц, 1H), 7,64 (s, 1H), 7,05 (d, J = 9,2 Гц, 1H), 5,80 - 5,62 (m, 2H), 5,41 - 5,14 (m, 4H), 4,29 - 4,15 (m, 2H), 4,08 - 4,03 (m, 1H), 3,27 - 3,07 (m, 2H), 2,45 (s, 3H), 2,38 - 2,28 (m, 2H), 1,96 - 1,81 (m, 2H), 1,04 (t, J = 7,4 Гц, 3H), 0,80 (s, 9H), 0,11 (s, 3H), 0,03 (s, 3H).

[215] Preparation of Intermediate V

[216] Intermediate V can be obtained by referring to the method of obtaining compound 4 in embodiment 6, where the methylamine hydrochloride in step 6b is replaced by the corresponding commercially available amino compound.

[217] Synthesis of LE21-LE22

[218] Intermediate V was reacted with DXD-1, then the residue was treated with a 10% trifluoroacetic acid/dichloromethane solution to give intermediate X, and then intermediate X was reacted according to subsequent steps 6e, 6g, 6i and 6j for compound 5 in Embodiment 6: intermediate X was subjected to a reduction reaction to obtain an amino compound, the resulting amino compound was subjected to a condensation reaction with complex Fmoc-valine hydroxysuccinimide ester and then the amino group Fmoc protecting group in the obtained product was removed, and the resulting amino product was reacted with 6-(maleimido)hexanoic acid succinimidyl ester to obtain the final product. Compound LE21: yellow solid, ESI-MS mass/charge: 1141.2 (M+H); compound LE22: yellow solid, ESI-MS mass/charge: 1106.6 (M+H).

[219] Embodiment 9. Synthesis of Compound LE13

[220] Connection LE13 can be obtained in accordance with the following synthetic route:

[221] The specific preparation steps were as follows.

[222] Synthesis of intermediate 14

[223] Commercially available intermediate 12 (267 mg, 0.8 mmol) and paraformaldehyde (50 mg, 1.6 mmol) were dissolved in 20 ml of anhydrous dichloromethane and trimethylchlorosilane (0.3 ml, 3.4 mmol) was added slowly, allowing the mixture to react at room temperature for 2 hours after addition was complete. The reaction mixture was then sampled and quenched by adding methanol while monitoring the reaction by liquid chromatography-mass spectrometry. After completion of the reaction, the reaction solution was filtered, and then tert -butyl glycolate (211 mg, 1.6 mmol) and 0.5 ml of pempidine were added to the filtrate, and the mixture was allowed to react at room temperature for about 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography [dichloromethane:methanol = 20:1 (v/v)] to give Intermediate 14 (260 mg, yield: 68%), ESI-MS mass/charge: 479 (M+H).

[224] Synthesis of intermediate 15

[225] Intermediate 14 (238 mg, 0.50 mmol) was dissolved in 6 ml of a mixed solvent of dichloromethane and methanol (v/v = 2:1), 0.3 ml of trifluoroacetic acid was added slowly, and the mixture was allowed to react at room temperature for 30 minutes. After completion of the reaction, equal volumes of water and ethyl acetate were added, the organic phase was dried and concentrated, and the resulting crude product was used directly in the next step.

[226] Synthesis of intermediate 16

[227] The crude product obtained in the previous step and exatecanemethanesulfonate (170 mg, 0.30 mmol) were mixed in 5 ml of anhydrous N,N-dimethylformamide and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (341 mg, 0.90 mmol), 0.60 ml was added triethylamine allowed the reaction to proceed in the mixture at room temperature for 2 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform:methanol = 10:1 (v/v)] to give intermediate 16 (210 mg, 83%), ESI-MS mass/charge: 840 (M+H).

[228] Synthesis of intermediate 17

[229] Intermediate 16 (100 mg, 0.12 mmol) was dissolved in 15 ml of anhydrous tetrahydrofuran, 3 ml of water was added, then 0.3 ml of an aqueous solution of triethylphosphine, 1 mol/l was added, and the mixture was allowed to react at room temperature for 4 hours. The reaction was followed to completion, the reaction solution was distilled under reduced pressure to remove tetrahydrofuran, sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added to extract. The resulting organic phase was dried and the solvent was evaporated under reduced pressure, the crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give intermediate 17 (69 mg, yield: 71%), ESI-MS mass/charge: 814 (M+H).

[230] Synthesis of LE13

[231] Intermediate 17 (120 mg, 0.15 mmol) obtained in the previous step and commercially available starting material MC-V (102 mg, 0.33 mmol) were mixed in 40 ml of dichloromethane, the condensing agent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) was added and the mixture was allowed to react at room temperature. temperature during the night. After completion of the reaction, the solvent was evaporated to dryness under reduced pressure. The obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give compound LE13 (116 mg, yield: 70%), ESI-MS mass/charge: 1106.5 (M+H).

[232] Embodiment 10 Synthesis of Compound LE14

[233] Connection LE14 can be obtained in accordance with the following synthetic route:

[234] The specific preparation steps were as follows.

[235] Synthesis of intermediate 19

[236] Commercially available intermediate 18 (300 mg, 0.8 mmol) and paraformaldehyde (50 mg, 1.6 mmol) were dissolved in 20 ml of anhydrous dichloromethane, and trimethylchlorosilane (0.3 ml, 3.4 mmol) was added slowly, allowing the mixture to react at room temperature for 2 hours. The reaction mixture was then sampled and quenched by adding methanol while monitoring the reaction by liquid chromatography-mass spectrometry. After completion of the reaction, the reaction solution was filtered, and then tert -butyl glycolate (211 mg, 1.6 mmol) and triethylamine (0.22 mg, 1.6 mmol) were added to the filtrate, and the mixture was allowed to react at room temperature for about 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure. Полученный неочищенный продукт очищали с помощью колоночной хроматографии на силикагеле [дихлорметан:метанол = 20:1 (об./об.)] с получением промежуточного соединения 19 (349 мг, выход 85%), ESI-MS масса/заряд: 514 (M+H), ¹ H ЯМР (400 MГц, CDCl ₃ ) δ 8,13 (s, 1H), 7,56 (d, J = 7,5 Гц, 2H), 7,35 (s, 2H), 5,14 (s, 2H), 4,91 (s, 2H), 4,25 (q, J = 7,1 Гц, 1H), 3,99 (d, J = 42,5 Гц, 2H), 3,85 (t, J = 6,2 Гц, 2H), 3,40 (dd, J = 18,5, 7,6 Гц, 2H), 2,89 (d, J = 48,6 Гц, 3H), 1,65 (d, J = 6,8 Гц, 3H), 1,46 (s, 9H).

[237] Synthesis of intermediate 20

[238] Intermediate 19 (257 mg, 0.50 mmol) was dissolved in 6 ml of a mixed solvent of dichloromethane and methanol (v/v = 2:1), 0.3 ml of trifluoroacetic acid was added slowly, and the mixture was allowed to react at room temperature for 30 minutes. After completion of the reaction, equal volumes of water and ethyl acetate were added, the organic phase was dried and concentrated, and the resulting crude product was used directly in the next step.

[239] The obtained crude product and exatecanemethanesulfonate (170 mg, 0.30 mmol) were mixed in 5 ml of anhydrous N,N-dimethylformamide and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (341 mg, 0.90 mmol), 0.60 ml of triethylamine, the mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [chloroform:methanol = 20:1 (v/v)] to give intermediate 20 (212 mg, yield: 81%), ESI-MS mass/charge: 875 (M+H). ¹ H ЯМР (400 MГц, CDCl ₃ ) δ 8,27 (d, J = 34,7 Гц, 1H), 7,63 - 7,35 (m, 5H), 7,21 - 7,10 (m, 1H), 5,71 - 5,48 (m, 2H), 5,24 - 4,95 (m, 3H), 4,95 - 4,72 (m, 4H), 4,45 (s, 1H), 4,33 - 3,97 (m, 3H), 3,75 (s, 2H), 3,39 - 2,99 (m, 4H), 2,76 (d, J = 15,3 Гц, 3H), 2,43 - 2,15 (m, 5H), 2,04 (s, 1H), 1,94 - 1,75 (m, 2H), 1,62 (d, J = 6,6 Гц, 3H), 1,11 - 0,89 (m, 3H).

[240] Synthesis of intermediate 21

[241] Intermediate 20 (77 mg, 0.09 mmol) was dissolved in 12 ml of anhydrous tetrahydrofuran, 3 ml of water was added, and then 0.3 ml of an aqueous solution of triethylphosphine, 1 mol/l was added, and the mixture was allowed to react at room temperature for 4 hours. After completion of the reaction, tetrahydrofuran was removed by distillation under reduced pressure, sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added to extract. The resulting organic phase was dried and the solvent was evaporated under reduced pressure, the resulting crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give intermediate 21 (53 mg, yield: 69%), ESI-MS mass/charge: 849 (M+H). ¹ H ЯМР (400 MГц, DMSO) δ 8,52 (s, 1H), 7,79 (d, J = 10,8 Гц, 1H), 7,67 - 7,55 (m, 2H), 7,47 - 7,21 (m, 3H), 6,51 (s, 1H), 5,60 (s, 1H), 5,52 - 5,32 (m, 2H), 5,30 - 5,11 (m, 2H), 5,11 - 4,94 (m, 2H), 4,94 - 4,74 (m, 2H), 4,02 (s, 2H), 3,81 - 3,66 (m, 2H), 3,60 - 3,35 (m, 4H), 3,24 - 3,08 (m, 2H), 2,94 (d, J = 30,8 Гц, 3H), 2,39 (s, 3H), 2,28 - 2,04 (m,2H), 2,00 - 1,73 (m, 2H), 1,22 (d, J = 6,6 Гц, 3H), 0,96 - 0,70 (m, 3H).

[242] Synthesis of compound LE14

[243] Intermediate 21 (134 mg, 0.16 mmol) and commercially available starting material MC-V (102 mg, 0.33 mmol) were mixed in 40 mL of dichloromethane, the condensing agent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) was added and the mixture was allowed to react at room temperature overnight. After completion of the reaction, the solvent was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give compound LE14 (137 mg, yield: 75%), ESI-MS mass/charge: 1141.4 (M+H). ¹ H ЯМР (400 MГц, DMSO) δ 9,97 (s, 1H), 8,52 (s, 1H), 8,27 - 8,09 (m, 1H), 7,88 - 7,70 (m, 2H), 7,63 - 7,51 (m, 2H), 7,28 (s, 3H), 6,99 (s, 2H), 6,51 (s, 1H), 5,59 (s, 1H), 5,50 - 5,32 (m, 2H), 5,17 (s, 2H), 4,98 (s, 2H), 4,85 (d, J = 17,3 Гц, 2H), 4,43 - 4,33 (m, 1H), 4,21 - 4,12 (m, 1H), 4,03 (s, 2H), 3,74 - 3,64 (m, 2H), 3,20 - 3,03 (m, 3H), 3,02 - 2,84 (m, 4H), 2,36 (s, 3H), 2,23 - 2,09 (m, 4H), 2,01 - 1,90 (m, 1H), 1,90 - 1,78 (m, 2H), 1,55 - 1,39 (m, 4H), 1,30 (d, J = 6,7 Гц, 3H), 1,23 - 1,11 (m, 2H), 0,93 - 0,77 (m, 9H).

[244] Embodiment 11 Synthesis of Compound LS13

[245] Referring to the synthesis method of LE14 in Embodiment 7, after reacting SN-38 (7-ethyl-10-hydroxycamptothecin) with Intermediate VII (R¹ is methylsulfonethyl) compound LS13 was obtained by deprotection, condensation reaction and other steps:¹H NMR (400 MHz, DMSO) δ 9.92 (d,J = 22.4Hz, 1H), 8.14(s, 1H), 8.08(d,J = 9.1 Hz, 1H), 7.81 (d,J = 8.0 Hz, 1H), 7.70 - 7.50 (m, 3H), 7.47 (d,J = 7.2 Hz, 1H), 7.34 (d,J = 7.2 Hz, 1H), 7.27 (s, 1H), 7.20 (s, 1H), 6.98 (s, 2H), 6.51 (s, 1H), 5.61 (s, 2H), 5.48 - 5.35 (m, 2H), 5.27 (s, 2H), 5.10 (d,J = 20.6 Hz, 2H), 4.36 (s, 1H), 4.21 - 4.07 (m, 1H), 3.84 (s, 2H), 3.48 (s, 2H), 3.21 - 2.92 (m, 6H), 2.25 - 2.04 (m, 2H), 2.04 - 1.78 (m, 3H), 1 .55 - 1.36 (m, 4H), 1.36 - 1.10 (m, 9H), 0.95 - 0.71 (m, 10H).

[246] Embodiment 12. General Method for Coupling Linker-Drug Conjugates with Antibodies

[247] Anti-B7-H3 antibody P2E5, anti-claudin antibody 18.2 IMAB362, and anti-HER2 antibody trastuzumab (concentration was 15 mg/mL) were replaced with a buffer of 50 mM PB/1.0 mM EDTA (pH 7.0) using a G25 desalting column in that order. 12 equivalents of TECP was added and the mixture was stirred at 37° C. for 2 hours to fully open the interchain disulfide bonds of the antibody. Phosphoric acid was then used to adjust the pH of the reconstituted antibody solution to 6.0, and the water bath temperature was lowered to 25° C. to carry out the coupling reaction. The linker-drug conjugate prepared in Embodiments 1-11 and GGFG-Dxd were appropriately dissolved in DMSO, 12 equivalents of the linker-drug conjugate was taken and added dropwise to the reconstituted antibody solution, DMSO was added until the final concentration in the solution was 10% (v/v), the mixture was stirred and allowed to react at 25°C within 0.5 hours. After completion of the reaction, the sample was filtered with a 0.22 µm membrane. Unbound small molecules were purified and removed using a tangential flow ultrafiltration system. The buffer was a solution of 50 mm PB/1.0 mm EDTA (pH 6.0). After purification, sucrose was added (final concentration was 6%) and the mixture was stored in a refrigeration unit at -20°C. A UV method was used to measure absorbance values at 280 nm and 370 nm, respectively, and the DAR value was calculated. The results are presented in table 5 below. The light chain amino acid sequence of the anti-HER2 antibody trastuzumab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is shown under SEQ ID No. 6 in the sequence listing. The light chain amino acid sequence of the anti-B7-H3 antibody P2E5 is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is shown under SEQ ID No. 8 in the sequence listing. The light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 1 in the sequence table, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 2 in the sequence listing.

[248] Table 5. DAR values for different antibody-drug conjugates (ADC) measured by UV method

[249] Efficient Embodiment 1. In vitro cell activity test

[250] HEK293 cells stably transfected with high expression of claudin 18.2, SK-BR-3 and NCI-N87 cells with high expression of HER2 were selected as cell lines for determining in vitro activity in this experiment. NCI-N87 cells also highly expressed B7-H3. A dose-dependent effect of different body-drug conjugates on cell killing was observed. Pre-selected seeding density tablets for each type of cells: 2×10 ³ cells/well, and tested for cytotoxic activity after 16-24 hours; secondly, the final concentration of the antibody-drug conjugate obtained in Embodiment 12 was tested after loading, and the initial concentration was set to 5000 nM. A series of solutions was prepared at 10 concentrations of 5000-0.006 nM (diluted 4-10 times), after 96 hours, changes in kill (or suppression) were observed, chemiluminescent staining was performed using the CellTiter-Glo® luminescent cell viability assay kit, after reading the fluorescence data, the IC ₅₀ was calculated. Based on the activity test results (see Table 6), all ADCs showed some anti-tumor activity, with some ADCs having better activity than ADC-8201.

[251] Table 6 Cytotoxic activity of various ADCs in vitro

[252] * These data were obtained in accordance with the same experimental operation as in this effective embodiment, based on the initial concentration of 30 μm.

[253] Efficient Embodiment 2. In Vitro Plasma Stability Test

[254] In this embodiment, the stability of the antibody-drug conjugate of embodiment 12 in human plasma is evaluated. Specifically, in this embodiment, a portion of the antibody-drug conjugates of Embodiment 12 was added to human plasma and placed in a 37°C water bath for 1, 3, 7, 14, 21, 28 days, an internal standard (Exatecan was used as an internal standard substance) was added, and the mixture was extracted, and then the amount of release was determined by high performance liquid chromatography. free-form drug. The results are presented in table 7.

[255] Table 7. Evaluation of the stability of different ADCs in human plasma

[256] The plasma stability results demonstrate that the stability of the ADCs produced with the novel solution is no worse than those of ADC-8201, with some being more stable. At the same time, the above potency test results also prove that the potency values of some of the first ADCs are better than ADC-8201.

[257] Efficient Embodiment 3 Evaluation of Different Linker-Drug Conjugates

[258] If the linker-drug conjugate has been bound to different antibodies, the versatility of the linker-drug conjugate is reflected in aggregate formation, release rate and whether precipitation occurs, etc. Although trastuzumab does not precipitate during the ADC-8201 (GGFG-Dxd) linker-drug coupling process, among the antibodies listed in the present invention, P2E5 and IMAB362 antibodies precipitate during the GGFG-Dxd coupling process. Therefore, P2E5, IMAB362, and trastuzumab were selected to assess the versatility of the conjugate-drug of the present invention in this experiment, coupling reactions were performed according to the method in Embodiment 12, samples were prepared according to the highest DAR value (i.e., overcoupling), and the results are presented in Table 8.

[259] Table 8. Circumstances regarding different linker-drug conjugates when combined with different antibodies

[260] "/" means that the recovery rate was not calculated.

[261] The current study also found that precipitation would occur if the ADC-8201 (GGFG-Dxd) drug conjugate was bound to other antibodies and the relative aggregate content would be high, characterizing it as non-universal. However, most of the linker-drug conjugates in this technical solution were coupled with different antibodies, and no precipitation occurred, while the relative content of aggregates was in the normal range, indicating that the linker-drug conjugates of the present invention are characterized by better physical and chemical properties.

[262] Efficient Embodiment 4. In Vitro Enzymatic Cleavage Experiment of Linker-Drug Conjugates

[263] Linker-drug conjugates (LE14 and GGFG-Dxd) and cathepsin B were incubated in buffers at three different pH values (5.0, 6.0, 7.0) and sampled at different time points for high performance liquid chromatography-mass spectrometry. Percent drug release was determined using an external standard method (with Dxd as external standard). Experimental results (shown in Table 9) show that GGFG-Dxd has a slower rate of enzymatic degradation over the applied pH range, while LE14 of the present invention provides fast degradation over the pH 5.0 to pH 7.0 range.

[264] Table 9. Enzymatic digestion of LE14 and GGFG-Dxd at different pH values in vitro

[265] Efficient Embodiment 5 In Vitro Enzymatic Cleavage Experiment of ADC030

[266] The NCI-N87 cell line was chosen as the experimental cell line. After incubation of the sample in a system based on cathepsin B (100 mM buffer containing sodium acetate and acetic acid, 4 mM dithiothreitol, pH 5.0) at 37°C for 4 hours, the obtained sample was diluted to different concentrations with culture medium, 8 concentrations (1.5-10 times diluted solutions) were prepared from 70 nM to 0.003 nM according to the concentration of SN-38, within 144 hours, the changes in the ability to kill (suppression) for the indicated cell line were observed and chemiluminescent staining was performed using the CellTiter-Glo® luminescent cell viability assay kit, after reading the fluorescence data, the IC ₅₀ value was calculated.

[267] An enzymatically digested sample obtained by incubation in a cathepsin B-based system at 37°C for 4 hours was exposed to an appropriate amount of ethanol to precipitate and remove the protein, the released small molecular weight compounds were determined using high performance liquid chromatography, and an equal amount of SN-38 was used as a reference. After 4 hours, the release rate was determined, the results showed that the release rate reached 99%.

[268] Experimental results (shown in Table 10) show that the cytotoxic activity of ADC030 after enzymatic cleavage was nearly the same as that of an equivalent amount of SN-38, and also show that ADC030 almost completely releases SN-38 and functions via cathepsin B. SN-38 rationing.

[269] Table 10. Changes in the activity of ADC030 in relation to the destruction of the NCI-N87 cell line before and after enzymatic cleavage by a system based on cathepsin B

[270] Efficient Embodiment 6. In Vivo Evaluation 1

[271] Balb/c female nude mice aged 6-8 weeks were subcutaneously injected into the back of the neck with 5×10 ⁶ human pancreatic cancer cells (Capan-1) diluted in 100 μl of PBS solution. When the average tumor volume reached approximately 160 mm ³ , nude mice were randomly divided into groups according to tumor size. 36 nude mice were randomly divided into 6 groups of 6 animals in each group, and the following groups were injected into the tail vein: 01 - blank control group and 02 - ADC-8201 (5 mg/kg), 03 - ADC-8201 (2 mg/kg), 04 - ADC-029 (5 mg/kg), 05 - ADC-029 (2 mg/kg), 06 - ADC-030 (5 mg/kg), while the introduction was made once. The body weight and tumor volume of the experimental animals were measured twice a week, while the vital status of the animals was monitored during the experiment. The experimental results (shown in Table 11) show that ADC029 has good antitumor activity in vivo . However, all experimental mice did not die or lose weight, indicating that ADC029 is safe.

[272] Table 11. Results of the experiment to evaluate the effectiveness of ADC in vivo

[273] Effect 7. In vivo evaluation 2

[274] Balb/c female nude mice aged 6-8 weeks were subcutaneously injected into the right side of the back of the neck with 1×10 ⁷ human gastric cancer cells (NCI-N87) diluted in 100 μl of PBS solution. When the average tumor volume was approximately 200 mm ³ , nude mice were randomly divided into groups according to tumor size. 42 nude mice were randomly divided into 7 groups of 6 animals in each group, and the following groups were injected into the tail vein: 01 - blank control group and 02 - ADC-8201 (2 mg/kg), 03 - ADC-8201 (1 mg /kg), 04 - ADC-029 (4 mg/kg), 05 - ADC-029 (2 mg/kg), 06 - ADC-029 (1 mg/kg), 07 - ADC-030 (4 mg/kg), and the introduction was made once. The body weight and tumor volume of the experimental animals were measured twice a week, while the vital status of the animals was monitored during the experiment. The results of the experiment (shown in Table 12) show that ADC029 has good antitumor activity in vivo . However, all experimental mice did not die or lose weight, indicating that ADC029 is safe.

[275] Table 12. Results of the experiment to evaluate the effectiveness of ADC in vivo

[276] Effective Embodiment 8: Safety Assessment

[277] Male and female ICR mice were divided proportionally into two groups. ADC-8201 and ADC029 were dosed at 300 mg/kg, respectively, with a body weight of 18.6-21.8 g at the time of administration. Mice were administered via tail vein injection. Within 14 days after administration, the body weight of the mice was measured at different time points. The results are summarized in the table below. Groups 01 and 02 were treated with ADC-8201, groups 03 and 04 were treated with ADC029, groups 01 and 03 were male mice, and groups 02 and 04 were female mice. The results of the test (shown in Table 13) show that the body weight of mice did not decrease significantly when the dose of ADC029 administered to mice reached 300 mg/kg, indicating that ADC has adequate safety.

[278] Table 13. In vivo safety assessment of ADC in mice

[279] Although specific embodiments of the present invention have been described above, one skilled in the art will appreciate that they are merely embodiments, and various changes or modifications can be made to these embodiments without departing from the spirit and spirit of the present invention. Therefore, the protection scope of the present invention is defined by the appended claims.

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SEQUENCE LIST

<110> Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd.

<120> Antibody drug conjugate, intermediate

to obtain it, the method of obtaining it and its application

<130> P21415844EN

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IMAB362 light chain sequence

<400> 1

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 2

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IMAB362 heavy chain sequence

<400> 2

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 3

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> RS7 light chain sequence

<400> 3

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain sequence RS7

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 5

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Trastuzumab light chain sequence

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Trastuzumab heavy chain sequence

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 7

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain sequence P2E5

<400> 7

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Phe Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Leu Leu Tyr Met Asp Ser

85 90 95

Gly Pro His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 8

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain sequence P2E5

<400> 8

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

Ala Ser Met Lys Pro Asp Gly Ser Val Lys His Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Ser Leu Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<---

Claims (108)

1. An antibody-drug conjugate, wherein the general structural formula of the antibody-drug conjugate is Ab-(L ₃ -L ₂ -L ₁ -D) _m , where Ab is an antibody; the antibody is an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof, or an anti-claudin 18.2 IMAB362 antibody or a variant thereof; the HER2 antibody variant trastuzumab has at least 90% homology compared to the HER2 antibody trastuzumab; the variant of the antibody to B7-H3 P2E5 has at least 90% homology compared to the antibody to B7-H3 P2E5; the anti-claudin antibody 18.2 IMAB362 variant has at least 90% homology compared to the anti-claudin antibody 18.2 IMAB362; D represents m is 2-8; the structure of L ₁ is as shown in formula I, II, III or IV, the a-terminus of L ₁ is connected to a cytotoxic drug and the e-terminus of L ₁ is connected to the c-terminus of L ₂ ; where L independently represents a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, or a valine residue; p is 2-4; R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - or C ₁ -C ₆ alkyl; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₆ alkyl; L ₂ is where n is independently 1-12, the c-terminus of L ₂ is connected to the e-terminus of L _1, the f-terminus of L ₂ is connected to the d-terminus of L ₃ ; L ₃ is where the b-terminus of L ₃ is connected to Ab, the d-terminus of L ₃ is connected to the f-terminus of L ₂ ; if the structure of L ₁ is as shown in formula I, L ₂ is if the structure of L ₁ is as shown in formula II, L ₂ is if the structure of L ₁ is as shown in formula IV, L ₂ is 2. The antibody-drug conjugate of claim 1, wherein the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing; the light chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 3 in the sequence listing, and the heavy chain amino acid sequence of the anti-Trop2 RS7 antibody is preferably shown under SEQ ID No. 4 in the sequence listing; the anti-HER2 antibody trastuzumab variant has at least 95%, 98%, or 99% homology compared to the anti-HER2 antibody trastuzumab; the anti-B7-H3 P2E5 variant has at least 95%, 98% or 99% homology compared to the anti-B7-H3 P2E5 antibody; the Trop2 RS7 antibody variant has at least 90%, 95%, 98%, or 99% homology compared to the Trop2 RS7 antibody; the anti-claudin antibody 18.2 IMAB362 variant has at least 90%, 95%, 98% or 99% homology compared to the anti-claudin antibody 18.2 IMAB362; and/or if R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably ethyl; each of R ^1-1 and R ^1-2 is independently preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl; and/or if R ¹ is C ₁ -C ₆ alkyl substituted by R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, more preferably ethyl; R ^1-3 is preferably C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl; and/or if R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl or ethyl; and/or m is 4-8, preferably 7-8, eg 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0; and/or n is preferably 8-12. 3. The antibody-drug conjugate according to claim 1 or 2, where L is a valine residue or an alanine residue, and p is preferably 2; (L)p is more preferably where the amino end of (L)p is connected to the carbonyl end in the formula III; and/or R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - or C 1 -C ₆ alkyl, preferably C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , or C _{1 -C 6} alkyl substituted with R ^1-3 S(O) ₂ -, more preferably C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -; if R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably methyl or ethyl; wherein C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - is preferably C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 is preferably preferably is or and/or L ₃ is and/or if the structure L₁ is as shown in formula I, L₂ represents L₃ preferably is and/or if the structure of L ₁ is as shown in formula II, L ₃ is preferably and/or if the structure of L ₁ is as shown in formula III, L ₂ is preferably more preferably even more preferable L ₃ is preferably and/or if the structure of L ₁ is as shown in formula IV, L ₂ is L ₃ is preferably 4. The antibody-drug conjugate of claim 1 wherein D is a cytotoxic drug m is 2-8; the structure of L ₁ is as shown in formula I, II, III or IV, L ₂ is or n is independently 8-12; if the structure of L ₁ is as shown in formula I, L ₂ is if the structure of L ₁ is as shown in formula II, L ₂ is if the structure of L ₁ is as shown in formula IV, L ₂ is L ₃ is L is independently a valine residue or an alanine residue; p is from 2 to 4; R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - or C ₁ -C ₆ alkyl; each of R ^1-1 , R ^1-2 and R ^1-3 independently represents C ₁ C ₆ alkyl; wherein the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably represented under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 7 in the sequence listing and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing. 5. Conjugate antibody-drug according to any one of paragraphs. 1-4, where D represents m is 7-8; the structure of L ₁ is as shown in formula I or III, if the structure of L ₁ is as shown in formula I, L ₂ is or n is independently 8-12; if the structure of L ₁ is as shown in formula III, L ₂ is or n is independently 8-12; L ₃ is L is independently a valine residue or an alanine residue; p is from 2 to 4; R ¹ is C ₁ -C ₄ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₄ alkyl substituted with R ^1-3 S(O) ₂ -, or C ₁ -C ₄ alkyl; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₄ alkyl; the light chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of the anti-HER2 antibody trastuzumab is preferably shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is preferably shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is preferably shown under SEQ ID No. 2 in the sequence listing. 6. The antibody-drug conjugate according to claim 1, where Ab is an antibody; the antibody is an anti-HER2 antibody trastuzumab or a variant thereof, an anti-B7-H3 P2E5 antibody or a variant thereof, or an anti-claudin 18.2 IMAB362 antibody or a variant thereof; the HER2 antibody variant trastuzumab has at least 90% homology compared to the HER2 antibody trastuzumab; the variant of the antibody to B7-H3 P2E5 has at least 90% homology compared to the antibody to B7-H3 P2E5; the anti-claudin antibody 18.2 IMAB362 variant has at least 90% homology compared to the anti-claudin antibody 18.2 IMAB362; D represents L ₁ is where L is a valine residue or an alanine residue, p is 2, (L)p is preferably R ¹ is C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with R 1-3 S(O) ₂ -, or C ₁ C ₆ alkyl, preferably C ₁ -C ₆ alkyl substituted with ^{-NR 1-1 R 1-2} , or C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -, more preferably C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ -; R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₄ alkyl, preferably methyl; wherein C ₁ -C ₆ alkyl substituted with -NR ^1-1 R ^1-2 is preferably C ₁ -C ₆ alkyl substituted with R ^1-3 S(O) ₂ - is preferably L ₂ is or where n is preferably 8, L ₂ is preferably L ₃ is 7. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is any of the following: where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0; Ab is anti-HER2 trastuzumab, anti-B7-H3 P2E5, or anti-claudin 18.2 IMAB362; the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing; the light chain amino acid sequence of the anti-B7-H3 antibody P2E5 is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of the anti-B7-H3 antibody P2E5 is shown under SEQ ID No. 8 in the sequence listing; the light chain amino acid sequence of the anti-claudin antibody 18.2 IMAB362 is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of the anti-claudin antibody 18.2 is shown under SEQ ID No. 2 in the sequence listing. 8. Conjugate antibody-drug according to any one of paragraphs. 1-7, wherein the antibody-drug conjugate is any of the compounds below: where Ab, m and R ¹ are as defined in any of paragraphs. 1-7. 9. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is any of the following: or where Ab is an antibody to HER2 trastuzumab; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing; where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0; or the antibody-drug conjugate is any of the compounds below: where Ab is an antibody to HER2 trastuzumab; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 5 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 6 in the sequence listing; or the antibody-drug conjugate is any of the compounds below: where Ab is an antibody to B7-H3 P2E5; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 8 in the sequence listing; where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0; or the antibody-drug conjugate is any of the compounds below: where Ab is an antibody to B7-H3 P2E5; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 7 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 8 in the sequence listing; or the antibody-drug conjugate is any of the compounds below: where Ab is an antibody to claudin 18.2 IMAB362; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 2 in the sequence listing; where m is 2-8, preferably 7-8, such as 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 8.0; or the antibody-drug conjugate is any of the compounds below: where Ab is an antibody to claudin 18.2 1MAB362; or the light chain amino acid sequence of Ab is shown under SEQ ID No. 1 in the sequence listing, and the heavy chain amino acid sequence of Ab is shown under SEQ ID No. 2 in the sequence listing. 10. The linker-drug conjugate, wherein the general structural formula of the linker-drug conjugate is L ₄ -L ₂ -L ₁ -D; where L ₄ represents L ₂ , L ₁ and D are as defined in any of paragraphs. 1-9, the f-terminus of L ₂ is connected to the d-terminus of L ₄ ; if L ₄ is if L ₁ is L ₂ is not 11. The linker-drug conjugate of claim 10, wherein the linker-drug conjugate is any of the following: where R ¹ is as defined in any of paragraphs. 1-9. 12. The linker-drug conjugate of claim 10, wherein the linker-drug conjugate is any of the following: 13. A method of obtaining an antibody-drug conjugate according to any one of paragraphs. 1-9, wherein the method includes the following step of carrying out the coupling of the linker-drug conjugate according to any one of paragraphs. 10-12 with an antibody according to any one of paragraphs. 1-9. 14. A pharmaceutical composition containing an effective amount of an antibody-drug conjugate according to any one of paragraphs. 1-9 and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is used to treat gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer, or pancreatic cancer. 15. The use of an antibody-drug conjugate according to any one of paragraphs. 1-9 in obtaining a drug for the prevention and/or treatment of cancer; the cancer is gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer, or pancreatic cancer. 16. Connection shown below: where R ¹ is as defined in any of paragraphs. 1-9; R ² is -N ₃ , -NH ₂ , 17. A compound according to claim 16, wherein the compound is any of the compounds below: