RU2822663C1 - Antibody-drug conjugate targeting trop2, and method for preparing and using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising an anti-TROP2 antibody-drug conjugate, a method of producing an anti-TROP2 antibody-drug conjugate, a pharmaceutical composition for the treatment and/or prevention of TROP2-positive gastric cancer, use of the conjugate or pharmaceutical composition in the manufacture of a TROP2 protein inhibitor, use of the conjugate or pharmaceutical composition for treating and/or preventing TROP2-positive gastric cancer.

EFFECT: invention extends the range of TROP2 binding agents.

12 cl, 1 dwg, 7 tbl, 5 ex

Description

TECHNICAL FIELD

[1] The present invention relates to the field of biomedicine and, in particular, relates to a TROP2-targeting antibody-drug conjugate, a method for its preparation and its use.

BACKGROUND OF THE ART

[2] Tumor-associated calcium signal transducer 2 (Trop2, TROP2) is a type I transmembrane glycoprotein encoded by the TACSTD2 gene, which is a calcium signal transducer associated with the regulation of intracellular calcium signals. Expression of Trop2 can activate ERK. Under normal conditions, Trop2 is mainly expressed in trophoblast cells and plays an important role in the development of embryonic organs. Trop2 is overexpressed in a variety of human epithelial tumor tissues, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, ovarian cancer, etc., and the level of expression is associated with worsening and tumor prognosis. Genomic analysis of breast cancer patients and results from current clinical trials indicate that Trop2 is a clinically validated therapeutic target. Trop2 has received widespread attention due to its high expression on the surface of various tumor cells. The level of Trop2 on the surface of a single cell ranges from tens of thousands to hundreds of thousands. Epidemiological studies have proven that Trop2 is associated with the development and prognosis of various tumors, and Trop2 has become a target for the development of antibody drugs. Once the anti-Trop2 antibody binds to the Trop2 protein, its endocytic efficiency becomes very high, which is suitable for the development of antibody-drug conjugates.

[3] Antibody-drug conjugate (ADC) is a new generation of targeted therapeutic drugs mainly used to treat cancer tumors. ADC-based drugs are composed of three parts: a small molecule cytotoxic drug (drug), an antibody (antibody), and a linker (linker) that connects the antibody to the cytotoxic drug. The small molecule cytotoxic drug is linked to the antibody protein by chemical coupling. ADC-based therapeutics utilize antibodies to specifically recognize and target small molecule drugs to target cancer cells expressing cancer-specific antigens and enter cancer cells through endocytosis. The linker part is destroyed under the influence of an intracellular environment with a low pH value or lysosomal protease, releasing low molecular weight cytotoxic drugs, thereby providing the effect of specifically killing cancer cells without damaging normal tissue cells. Therefore, ADC-based drugs simultaneously have the characteristics of antibody targeting specificity and high toxicity of small molecule toxins against cancer cells, greatly expanding the therapeutic window of drug efficacy. Clinical studies have proven that ADC-based drugs are highly effective and relatively stable in the blood, and can effectively reduce the toxicity of small molecule cytotoxic drugs (chemotherapeutic drugs) in the circulatory system and healthy tissues, and they are currently a hot spot in the development of anticancer drugs around the world.

CONTENTS OF THE PRESENT INVENTION

[4] The technical problem solved by the present invention is to overcome the current shortage of limited types of antibody-drug conjugates, and the present invention provides a TROP2-targeting antibody-drug conjugate, a method for its preparation and its use.

[5] The present invention has developed an antibody-drug conjugate with good targeting ability, strong inhibitory effect on tumor cells that are positive for TROP2 expression, and good druggability and high safety. The antibody-drug conjugate has an inhibitory effect on TROP2 and also has a good inhibitory effect on at least one of NCI-N87, MDA-MB-468 and BXPC3 cells.

[6] The present invention solves the above technical problem by the following technical solution.

[7] The present invention provides an antibody-drug conjugate of Formula I , a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof;

;

I

[8] wherein Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody;

[9] the light chain amino acid sequence of the anti-TROP2 antibody is shown as SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 2;

[10] the anti-TROP2 antibody variant is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the anti-TROP2 antibody;

[11] m ranges from 2 to 8;

[12] D is a cytotoxic drug that is a topoisomerase inhibitor;

[13] R ¹ is C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with one or more 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 heteroatom in the 5-14 membered heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4; wherein each of R ^1-1 , R ^1-2 and R ^1-3 independently represents C ₁ -C ₆ alkyl;

[14] L ₁ independently represents one or more of a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a glutamic acid 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 and a valine residue; p is from 2 to 4;

[15] L ₂ represents , , , , or where n is independently from 1 to 12, the c-terminus is attached to L ₁ via a carbonyl group, and the f-terminus is attached to the d-terminus of L ₃ ;

[16] L ₃ represents , where the b-end is attached to Ab, and the d-end is attached to the f-end of L ₂ .

[17] In a preferred embodiment of the present invention, certain groups of the antibody-drug conjugate are defined as follows, and the definition of any unspecified groups is as described in any of the above embodiments (this paragraph hereinafter referred to as "in the preferred embodiment embodiment of the present invention"):

[18] The b-terminus of L ₃ is preferably attached to the sulfhydryl group of the antibody in the form of a thioester bond. Taking as an example, contact form with a cysteine residue in the antibody is .

[19] In a preferred embodiment of the present invention, if D is a cytotoxic topoisomerase inhibitor drug, the cytotoxic topoisomerase inhibitor drug is preferably or ; wherein each of R ² and R ⁵ independently represents H, C ₁ -C ₆ alkyl or halogen; each of R ³ and R ⁶ independently represents H, C ₁ -C ₆ alkyl or halogen; R ⁴ and R ⁷ are each independently C ₁ -C ₆ alkyl.

[20] In a preferred embodiment of the present invention, if R ² and R ⁵ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl.

[21] In a preferred embodiment of the present invention, when each of R ² and R ⁵ independently represents a halogen, the halogen is preferably fluorine, chlorine, bromine or iodine, further preferably fluorine.

[22] In a preferred embodiment of the present invention, if R ³ and R ⁶ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl.

[23] In a preferred embodiment of the present invention, if R ³ and R ⁶ are each independently halogen, the halogen is preferably fluorine, chlorine, bromine or iodine, further preferably fluorine.

[24] In a preferred embodiment of the present invention, if R ⁴ and R ⁷ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably ethyl.

[25] In a preferred embodiment of the present invention, R ² and R ⁵ are each independently C ₁ -C ₆ alkyl.

[26] In a preferred embodiment of the present invention, R ³ and R ⁶ are each independently halogen.

[27] In a preferred embodiment of the present invention, R ⁴ and R ⁷ are ethyl.

[28] In a preferred embodiment of the present invention, D is or .

[29] In a preferred embodiment of the present invention, if D is or , the antibody-drug conjugate may be or .

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

[31] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with more than one -NR ^1-1 R ^1-2 , "more than one" is two or three.

[32] In a preferred embodiment of the present invention, if R ^1-1 and R ^1-2 are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl or tert-butyl, most preferably methyl.

[33] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , -NR ^1-1 R ^1-2 is preferably -N(CH ₃ ) ₂ .

[34] In a preferred embodiment of the present invention, if R ¹ is a C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 , a C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^{1 -2} , represents .

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

[36] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with more than one R ^1-3 S(O) ₂ -, "more than one" is two or three.

[37] In a preferred embodiment of the present invention, if R ^1-3 is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert -butyl, most preferably methyl.

[38] In a preferred embodiment of the present invention, if R ¹ is C ₁ -C ₆ alkyl substituted with one R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl substituted with one R ^1-3 S( O) ₂ -, represents .

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

[40] In a preferred embodiment of the present invention, m is an integer (such as 2, 3, 4, 5, 6, 7 or 8) or a non-integer, preferably 3 to 5, further preferably 3.5 to 4, 5, even more preferably 3.8 to 4.2, such as 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4.05, 4. 10, 4.12 or 4.13.

[41] In a preferred embodiment of the present invention, L ₁ is preferably one or more of a phenylalanine residue, an alanine residue, a glycine residue, an isoleucine residue, a leucine residue, a proline residue and a valine residue; more preferably one or more than one of a phenylalanine residue, an alanine residue, a glycine residue or a valine residue; further preferably a valine residue and/or an alanine residue; "more than one" preferably equals two or three; p is preferably 2.

[42] In a preferred embodiment of the present invention, (L ₁ ) _p is further preferably , where the g-terminus is attached to the c-terminus of L ₂ via a carbonyl group.

[43] In a preferred embodiment of the present invention, n is preferably from 8 to 12, such as 8, 9, 10, 11 and 12, optionally such as 8 or 12.

[44] In a preferred embodiment of the present invention, if R ^1-1 , R ^1-2 and R ^1-3 are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is preferably C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl.

[45] In a preferred embodiment of the present invention, R ¹ is preferably C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl or C ₃ -C ₁₀ cycloalkyl; more preferably C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ² -, or C ₁ -C ₆ alkyl; further preferably C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , or C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ² -; most preferably C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ² -.

[46] In the present invention, if the Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody, the anti-TROP2 antibody or a variant of an anti-TROP2 antibody is a residue of an anti-TROP2 antibody (a group formed by replacing a hydrogen in one of the sulfhydryl groups in the anti-TROP2 antibody ) or a residue of a variant of an anti-TROP2 antibody (a group formed by replacing a hydrogen in one of the sulfhydryl groups in a variant of an anti-TROP2 antibody).

[47] In a preferred embodiment of the present invention, the compound of formula I is represented by any of the following schemes:

[48] scheme I:

[49] Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody;

[50] D represents or R ² and R ⁵ are each independently H, C ₁ -C ₆ alkyl or halogen; each of R ³ and R ⁶ independently represents H, C ₁ -C ₆ alkyl or halogen; R ⁴ and R ⁷ are each independently C ₁ -C ₆ alkyl; D is preferably or ;

[51] L ₁ independently represents one or more of a phenylalanine residue, an alanine residue, a glycine residue, an isoleucine residue, a leucine residue, a proline residue, and a valine residue;

[52] scheme II:

[53] Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody;

[54] D represents or R ² and R ⁵ are each independently C ₁ -C ₆ alkyl; each of R ³ and R ⁶ independently represents a halogen, D preferably represents or ;

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

[56] L ₁ independently represents one or more of a phenylalanine residue, an alanine residue, a glycine residue and a valine residue;

[57] scheme III:

[58] Ab is an antibody to TROP2;

[59] D represents or R ² and R ⁵ are each independently C ₁ -C ₆ alkyl; each of R ³ and R ⁶ independently represents a halogen, D preferably represents or ;

[60] m is from 3.5 to 4.5,

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

[62] L ₁ is independently a valine residue and/or an alanine residue;

[63] scheme IV:

[64] Ab is an antibody to TROP2;

[65] D represents or ,

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

[67] L ₁ is independently a valine residue and/or an alanine residue;

[68] circuit V:

[69] Ab is an antibody to TROP2;

[70] D represents or ,

[71] m is from 3.5 to 4.5,

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

[73] L ₁ is independently a valine residue and/or an alanine residue.

[74] In a preferred embodiment of the present invention, the antibody-drug conjugate is preferably , or

.

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

[76] In a preferred embodiment of the present invention, the Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody, wherein the light chain amino acid sequence of the anti-TROP2 antibody is preferably represented by SEQ ID NO: 1; the amino acid sequence of the heavy chain in the antibody to TROP2 is presented under SEQ ID NO: 2; D represents or ; L ₁ is a valine residue and/or an alanine residue, p is 2, (L ₁ )p is preferably ; R ¹ is C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ₂ - , or C ₁ -C ₆ alkyl, preferably C ₁ -C ₆ alkyl substituted with one or more -NR ^1-1 R ^1-2 , or C ₁ -C ₆ alkyl substituted with one or more R ^{1- 3} S(O) ₂ -, further preferably C ₁ -C ₆ alkyl substituted with one or more 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 one or more -NR ^1-1 , R ^1-2 is preferably ; C ₁ -C ₆ alkyl substituted with one or more R ^1-3 S(O) ₂ - is preferably ; L ₂ represents ; L ₃ represents .

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

, , , , , , , , , or ;

[78] where Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody, and m is from 3.8 to 4.2, preferably 3.88, 3.90, 3.96, 3.97, 3.98, 4 .00, 4.03, 4.05, 4.10, 4.12 or 4.13; the light chain amino acid sequence of the anti-TROP2 antibody variant is preferably presented as SEQ ID NO: 1, the heavy chain amino acid sequence is preferably presented as SEQ ID NO: 2.

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

, , , , , , , , , or ;

[80] wherein Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody, preferably an anti-TROP2 antibody; the light chain amino acid sequence of the anti-TROP2 antibody variant is preferably presented as SEQ ID NO: 1, and the heavy chain amino acid sequence is preferably presented as SEQ ID NO: 2.

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

, or ;

[82] wherein Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody; m is preferably 3.90, 4.00 or 4.10; the light chain amino acid sequence of the anti-TROP2 antibody variant is preferably presented as SEQ ID NO: 1, and the heavy chain amino acid sequence is preferably presented as SEQ ID NO: 2.

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

, or ;

[84] where Ab is an anti-TROP2 antibody or a variant of an anti-TROP2 antibody.

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

wherein Ab is an anti-TROP2 antibody and m is preferably 3.90;

wherein Ab is an anti-TROP2 antibody and m is preferably 4.10;

wherein Ab is an anti-TROP2 antibody, and m is preferably 4.00;

wherein Ab is an anti-TROP2 antibody and m is preferably 4.12;

wherein Ab is an anti-TROP2 antibody, and m is preferably 4.03;

wherein Ab is an anti-TROP2 antibody and m is preferably 3.98;

wherein Ab is an anti-TROP2 antibody and m is preferably 3.96;

wherein Ab is an anti-TROP2 antibody and m is preferably 4.13;

wherein Ab is an anti-TROP2 antibody and m is preferably 3.88;

wherein Ab is an anti-TROP2 antibody, and m is preferably 4.05;

wherein Ab is an anti-TROP2 antibody and m is preferably 4.00.

[86] The present invention also provides a method for producing an antibody-drug conjugate, comprising the following step: carrying out a coupling reaction between a compound of formula II and Ab-hydrogen, as shown below;

[87] where L ₁ , L ₂ , L ₃ , R ¹ , p and Ab are defined above.

[88] In the present invention, the coupling reaction conditions and procedures for carrying out the coupling reaction may be conditions and procedures for carrying out the coupling reaction generally accepted in the art.

[89] The present invention also provides a pharmaceutical composition comprising substance X and a pharmaceutically acceptable excipient, wherein substance X is an antibody-drug conjugate, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof.

[90] In a pharmaceutical composition, substance X may be used in a therapeutically effective amount.

[91] The present invention also provides the use of substance X or a pharmaceutical composition in the manufacture of a TROP2 protein inhibitor. The present invention also provides for the use of substance X or a pharmaceutical composition in the manufacture of a medicament for treating and/or preventing a tumor, and the tumor is preferably a TROP2-positive tumor.

[92] When used, the TROP2-positive tumor is preferably one or more of TROP2-positive gastric cancer, triple-negative breast cancer and human pancreatic cancer.

[93] In some embodiments of the present invention, in the case of gastric cancer, the gastric cancer cells are NCI-N87 cells;

[94] in some embodiments of the present invention, in the case of triple negative breast cancer, the triple negative breast cancer cells are MDA-MB-468 cells;

[95] in some embodiments of the present invention, in the case of pancreatic cancer, the pancreatic cancer cells are BXPC3 cells.

[96] Unless otherwise specified, the following terms have the following meanings when found in the specification and claims of the present invention.

[97] The pharmaceutical excipient may be an excipient widely used in the field of pharmaceutical production. The excipient is primarily used to provide a safe, stable and functional pharmaceutical composition, and may also provide a method for dissolving the active ingredient at a desired rate after administration or facilitating effective absorption of the active ingredient in a subject after the subject has undergone administration of the composition. The pharmaceutical excipient may be an excipient or may provide a specific function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient of the composition. Pharmaceutical excipients may include one or more of the following excipients: buffers, chelating agents, preservatives, solubilizers, stabilizers, carrier media, surfactants, coloring agents, flavoring agents and sweeteners.

[98] The native or native TROP2 sequence may be isolated from nature or obtained through recombinant DNA techniques, chemical synthesis, or a combination of the above and similar techniques.

[99] An antibody is herein defined in the broadest sense as one that can specifically bind to a target through at least one antigen recognition region located in the variable region of an immunoglobulin molecule, such as a carbohydrate, polynucleotide, fat, polypeptide, etc. B in particular, it includes complete monoclonal antibodies, polyclonal antibodies, bispecific antibodies and antibody fragments, if they have the required biological activity. The antibody variants of the present invention relate to mutant amino acid sequences and covalent derivatives of natural polypeptides, provided that biological activity equivalent to that of natural polypeptides is retained. The difference between mutant amino acid sequences and natural amino acid sequences is typically that one or more amino acids in the natural amino acid sequence are replaced or one or more amino acids are removed from and/or inserted into the polypeptide sequence. Deletion mutants include natural polypeptide fragments and N-terminal and/or C-terminal truncation mutants. Typically, the mutant amino acid sequence is at least 70% (eg, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the native sequence.

[100] Antibodies of the present invention can be produced by techniques well known in the art, such as hybridoma technologies, recombinant DNA techniques, phage display techniques, synthesis techniques, or combinations thereof, or other techniques known in the art.

[101] Description of the term drug-antibody ratio (DAR). L-D represents a reactive group with a conjugation site on the antibody, L represents a linker, D represents a cytotoxic agent further associated with the antibody attached to L. In the present invention, D represents Dxd. The DAR numeric value for each antibody ultimately bound to a D is represented by m, or m can also represent the number of Ds bound to a single antibody. In some embodiments, m is actually the average of 2 to 8, 3 to 7, or 3 to 5, or m is an integer of 2, 3, 4, 5, 6, 7, or 8; in some embodiments, m is the average of 3.5 to 4.5 (e.g., 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4, 05, 4.10, 4.12 or 4.13); in other embodiments, m is the average of 2, 3, 4, 5, 6, 7, or 8.

[102] Linker refers to a direct or indirect connection between an antibody and a drug. The linker can be attached to the mAb in a variety of ways, such as through surface lysine, reductive binding to oxidized carbohydrates, and through cysteine residues released through reduction of interchain disulfide bonds. A variety of ADC coupling systems are known in the art, including hydrazone, disulfide, and peptide coupling variants.

[103] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively non-toxic and pharmaceutically acceptable acid or base. In case the compound of the present invention contains relatively acidic functional groups, a base addition salt can be prepared by contacting a sufficient amount of a pharmaceutically acceptable base with the neutral form of the compound in a pure solution or suitable inert solvent. Pharmaceutically acceptable base addition salt includes, but is not limited to, lithium salt, sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, zinc salt, bismuth salt, ammonium salt and diethanolamine salt. If a compound of the present invention contains relatively basic functional groups, an acid addition salt can be prepared by contacting a sufficient amount of a pharmaceutically acceptable acid with a neutral form of the compound in a pure solution or suitable inert solvent. The pharmaceutically acceptable acid includes inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, orthophosphoric acid, phosphoric acid, sulfuric acid, etc. The pharmaceutically acceptable acid includes organic acid, and organic acid includes, but is not limited to, acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, trans-butenedioic acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p- toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. e. 4,4′-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acid (for example, glutamic acid and arginine), etc. If the compound of the present invention contains relatively acidic functional groups and relatively basic functional groups group, it can be converted to a base addition salt or an acid addition salt. For details, see Berge et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science 66: 1–19 (1977) or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed. , Wiley-VCH, 2002).

[104] The term “solvate” refers to the substance formed when a compound of the present invention is combined with a stoichiometric or non-stoichiometric amount of a solvent. Solvent molecules in a solvate can exist in ordered or disordered arrangements. The solvent includes, but is not limited to, water, methanol, ethanol, and the like.

[105] The term “treatment” or an equivalent expression when used in relation to, for example, cancer, refers to a procedure or process to reduce or eliminate a number of cancer cells in a patient's body or to alleviate the symptoms of cancer. “Treating” a cancer or other proliferative disorder does not necessarily mean that the cancer cells or other disorders will actually be eliminated, but that the number of cells or disorders will actually be reduced, or the symptoms of the cancers or other disorders will actually be alleviated. In general, a method of treating cancer will be carried out even if there is only a low probability of success, but it is still believed that an overall beneficial course of action is induced, taking into account the patient's medical history and the estimated predicted survival rate.

[106] The term “prevention” refers to reducing the risk of acquiring or developing a disease or disorder.

[107] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group containing from three to twenty carbon atoms (eg, C ₃ -C ₆ cycloalkyl), including monocyclic and polycyclic cycloalkyl. Cycloalkyl contains from 3 to 20 carbon atoms, preferably from 3 to 10 carbon atoms and more preferably from 3 to 6 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl.

[108] The term "alkyl" refers to a straight or branched chain alkyl group with a specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl and similar alkyl groups.

[109] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[110] The term "heteroaryl" refers to an aryl group (or aromatic ring) containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, which may be a monocyclic, bicyclic or tricyclic aromatic system such as furyl . , benzisoxazolyl, quinolyl, isoquinolyl etc.

[111] The term "aryl" refers to any stable monocyclic or bicyclic carbocycle where all rings are aromatic rings. Examples of the aryl moiety include phenyl or naphthyl.

[112] The above preferred conditions can be optionally combined to obtain preferred embodiments of the present invention without violating the generally known knowledge in the prior art.

[113] Unless otherwise specified, room temperature in the present invention refers to a temperature of 20 to 30°C.

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

[115] The positive progressive effects provided by the present invention are as follows.

[116] 1. The antibody-drug conjugate containing an anti-TROP2 antibody provided by the present invention has good targeting ability and good suppressive effect on various tumor cells expressing TROP2, etc.

[117] 2. In vivo studies have shown that the antibody-drug conjugate of the present invention has better cytotoxicity in vitro and antitumor activity in vivo.

[118] 3. The antibody-drug conjugate of the present invention is characterized by good solubility and good druggability. Abnormal phenomena such as precipitation were not observed during the coupling procedure, which is very favorable for the production of antibody-drug conjugate.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[119] Figure 1 shows the construction of expression vectors for the light and heavy chains of the FDA018 antibody; where Ab-L represents the light chain of an antibody, and Ab-H represents the heavy chain of an antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[120] The present invention will be further described below with reference to examples, but the present invention is not limited by the scope of the examples. Experimental methods without specific conditions in the following examples are selected in accordance with generally accepted methods and conditions or in accordance with commercial specifications.

[121] Description of abbreviations

[122] PCR - polymerase chain reaction

[123] CHO - Chinese hamster ovary cells

[124] HTRF - homogeneous time-resolved fluorescence

[125] PB - phosphate buffer

[126] EDTA - ethylenediaminetetraacetic acid

[127] TECP - tris(2-carboxyethyl)phosphine

[128] DMSO - dimethyl sulfoxide

[129] DMF - N,N-dimethylformamide

[130] His - histidine

[131] HATU - 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[132] v/v - volume/volume, volume ratio

[133] UV - ultraviolet light

[134] ELISA - enzyme-linked immunosorbent assay

[135] BSA - bovine serum albumin

[136] rpm - revolutions per minute

[137] FBS - fetal bovine serum

[138] Example 1: Preparation of Anti -TROP2 Antibody

[139] In the present invention, a monoclonal antibody FDA018 with high affinity and specific targeting of TROP2, the light chain amino acid sequence of which is shown as SEQ ID NO: 1, and the heavy chain amino acid sequence of which is shown as SEQ ID NO: 2, was selected. The nucleotide sequences of the light and FDA018 heavy chains were obtained by total gene synthesis (Suzhou Genewiz). They were individually constructed to be incorporated into the pV81 vector (as shown in Figure 1) by double digestion with EcoR I and Hind III (purchased from TAKARA), and then transformed into competent Trans 1-T1 cells (purchased from Beijing TransGen Biotech, product number: CD501-03) by ligation, which were collected for cloning, identified by PCR, and submitted for sequencing confirmation. Positive clones were cultured and propagated for plasmid extraction, thereby obtaining the eukaryotic antibody light chain expression plasmid FDA018-L/pV81 and the eukaryotic antibody heavy chain expression plasmid FDA018-H/pV81. These two plasmids were linearized by digestion with XbaⅠ (purchased from Takara, product number: 1093S). Eukaryotic light and heavy chain expression plasmids were transformed into CHO cells adapted for suspension growth (purchased from ATCC) at a ratio of 1.5/1 by electroporation. After electroporation, cells were seeded at 2000-5000 cells/well in a 96-well plate. After 3 weeks of culture, the expression level was measured by the HTRF (Homogeneous Time Resolved Fluorescence) method. The ten best cell pools with respect to expression level were selected for expansion and cryopreservation. Cells were recovered in a 125 ml shake flask (culture volume 30 ml) and cultured at 37°C, 5.0% CO ₂ and 130 rpm with vibration for 3 days, then expanded into a 1000 ml shake flask (with a culture volume of 300 ml) and cultured at 37°C, 5.0% CO ₂ and 130 rpm using vibration. Starting from day 4, fresh culture medium was added every day in an amount of 5 to 8% of the original culture volume. The culture was completed between days 10 and 12 and the collected liquid was centrifuged at 9500 rpm for 15 minutes to remove the cell sediment. The supernatant was collected and filtered through a 0.22 μm membrane filter. The treated sample was purified using a MabSelect affinity chromatography column (purchased from GE) to obtain antibody FDA018.

[140] The amino acid sequence of the light chain of FDA018 is shown below:

SEQ ID NO: 1:

DIQLTQSPSS LSASVGDRVS ITCKASQDVS IAVAWYQQKP GKAPKLLIYS 50

ASYRYTGVPD RFSGSGSGTD FTLTISSLQP EDFAVYYCQQ HYITPLTFGA 100

GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150

DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 200

LSSPVTKSFN RGEC 214

[141] The amino acid sequence of FDA018 heavy chain is shown below:

SEQ ID NO: 2:

QVQLQQSGSE LKKPGASVKV SCKASGYTFT NYGMNWVKQA PGQGLKWMGW 50

INTYTGEPTY TDDFKGRFAF SLDTSVSTAY LQISSLKADD TAVYFCARGG 100

FGSSYWYFDV WGQGSLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV 150

KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ 200

TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK 250

PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300

NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP 350

QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 400

VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 450

K 451

[142] Example 2: Synthesis of linker-drug conjugates

[143] Example 2-1. Synthesis of LE12

[144] Synthesis of intermediate 2

[145] Dissolve (S)-2-azidopropionic acid (10 g, 86.9 mmol) and 4-aminobenzyl alcohol (21.40 g, 173.8 mmol) in a mixed solvent of 300 ml dichloromethane and methanol (v/v 2:1) and then 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (21.49 g, 86.9 mmol) was added thereto. The reaction was carried out at room temperature for 5 hours. The solvent was then evaporated under reduced pressure and the resulting crude product was purified by silica gel column chromatography [dichloromethane:ethyl acetate = 1:1 (v/v)] to give intermediate 2 (16.3 g, 85% yield), ESI -MS mass/charge: 221 (M+H).

[146] Synthesis of intermediate 3

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

[148] Synthesis of intermediate 5

[149] Intermediate 3 (10 g, 36.1 mmol) was mixed with polyformaldehyde (1.63 g, 54.2 mmol) and dissolved in 150 ml of anhydrous dichloromethane. Trimethylchlorosilane (6.28 g, 57.76 mmol) was added slowly and the reaction was carried out at room temperature for 2 hours to give a crude solution of intermediate 4. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with using methanol. After completion of the reaction, the reaction mixture was filtered and then tert-butyl hydroxyacetate (9.54 g, 72.2 mmol) and triethylamine (10 mL, 72.2 mmol) were added to the filtrate and the reaction was allowed to continue at room temperature for 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure, and then 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).

[150] Synthesis of intermediate 6

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

[152] Synthesis of intermediate 8

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

[154] The crude product obtained in the previous step was dissolved in 50 ml of anhydrous N,N-dimethylformamide, and then Fmoc-L-valine hydroxysuccinimide ester (8.3 g, 19.1 mmol) and triethylamine (5 ml) were added thereto ) and the reaction was carried out at room temperature for 2 hours. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure, and then 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).

[155] Synthesis of intermediate 9

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

[157] Synthesis of compound LE12

[158] Intermediate 9 (1 g, 0.929 mmol) was dissolved in 20 ml of anhydrous DMF, then 0.5 ml of 1,8-diazabicyclo[5.4.0]undec-7-ene was added and the reaction was carried out at room temperature in within 1 hour. After completion of the reaction of the starting materials, N-succinimidyl-6-maleimidohexanoate (428.5 mg, 1.39 mmol) was directly added and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed by distillation under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography [chloroform:methanol = 8:1 (v/v)] to obtain the title compound (0.7 g, yield: 73 %), ESI-MS mass/charge: 1035 (M+H).

[159] Example 2-2. Synthesis LE13 connections

[160] Synthesis of intermediate 14

[161] Commercially available intermediate 12 (267 mg, 0.8 mmol) was mixed with paraformaldehyde (50 mg, 1.6 mmol) and dissolved in 20 ml of anhydrous dichloromethane. Trimethylchlorosilane (0.3 mL, 3.4 mmol) was then added slowly. After addition was completed, the reaction was carried out at room temperature for 2 hours. The reaction was then monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After completion of the reaction, the reaction mixture was filtered and then tert-butyl-2-hydroxyacetate (211 mg, 1.6 mmol) and pempidine (0.5 ml) were added to the filtrate and the reaction was allowed to continue at room temperature for approximately 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 [dichloromethane:methanol = 20:1 (v/v)] to give intermediate 14 (260 mg, 68% yield ), ESI-MS mass/charge: 479 (M+H).

[162] Synthesis of intermediate 15

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

[164] Synthesis of intermediate 16

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

[166] Synthesis of intermediate 17

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

[168] Synthesis of compound LE13

[169] Intermediate 17 (120 mg, 0.15 mmol), obtained according to the previous synthesis method, was mixed with commercially available starting material MC-V (102 mg, 0.33 mmol) in 40 ml of dichloromethane and added a condensing agent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) allowing the reaction to occur overnight at room temperature. After completion of the reaction, 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 obtain compound LE13 (116 mg, 70% yield), ESI- MS mass/charge: 1106.5 (M+H).

[170] Example 2-3. Synthesis of compound LE14

[171] Synthesis of intermediate 19

[172] Commercially available intermediate 18 (300 mg, 0.8 mmol) was mixed with polyformaldehyde (50 mg, 1.6 mmol) and dissolved in 20 ml of anhydrous dichloromethane. Trimethylchlorosilane (0.3 mL, 3.4 mmol) was then slowly added to the resulting mixture, and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After completion of the reaction, the reaction mixture was filtered and then tert-butyl-2-hydroxyacetate (211 mg, 1.6 mmol) and triethylamine (0.22 m, 1.6 mmol) were added to the filtrate. Allow the reaction to continue at room temperature for approximately 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 [dichloromethane:methanol = 20:1 (v/v)] to give intermediate 19 (349 mg, 85 yield %), ESI-MS mass/charge: 514 (M+H), ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.13 (s, 1H), 7.56 (d, J = 7.5 Hz, 2H), 7.35 (s, 2H), 5.14 (s, 2H), 4.91 (s, 2H), 4.25 (q, J = 7.1 Hz, 1H), 3.99 ( d, J = 42.5 Hz, 2H), 3.85 (t, J = 6.2 Hz, 2H), 3.40 (dd, J = 18.5, 7.6 Hz, 2H), 2, 89 (d, J = 48.6 Hz, 3H), 1.65 (d, J = 6.8 Hz, 3H), 1.46 (s, 9H).

[173] Synthesis of intermediate 20

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

[175] The resulting crude product was mixed with exatecan methanesulfonate (170 mg, 0.30 mmol) in 5 ml of anhydrous N,N-dimethylformamide and then 2-(7-azabenzotriazol-1-yl)-N,N,N was added thereto ',N'-tetramethyluronium hexafluorophosphate (341 mg, 0.90 mmol) and 0.60 ml triethylamine and the reaction was carried out at room temperature for 2 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography [dichloromethane:methanol = 20:1 (v/v)] to obtain intermediate 20 (212 mg, yield 81 %), ESI-MS mass/charge: 875 (M+H). ^1H NMR (400 MHz, CDCl ₃ ) δ 8.27 (d, J = 34.7 Hz, 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 Hz, 3H ), 2.43-2.15 (m, 5H), 2.04 (s, 1H), 1.94-1.75 (m, 2H), 1.62 (d, J = 6.6 Hz, 3H), 1.11-0.89 (m, 3H).

[176] Synthesis of intermediate 21

[177] Intermediate 20 (77 mg, 0.09 mmol) was dissolved in 12 ml of anhydrous tetrahydrofuran and 3 ml of water was added to the resulting, then 0.3 ml of an aqueous solution of 1 mol/L triethylphosphine was added to the resulting, and the reaction was carried out at room temperature for 4 hours. After completion of the reaction, the reaction mixture 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 for extraction. The resulting organic phase was dried and evaporated under reduced pressure to remove the solvent. The resulting crude product was purified by silica gel column chromatography [dichloromethane:methanol = 10:1 (v/v)] to give intermediate 21 (53 mg, 69% yield), ESI-MS wt/charge: 849 (M +H). ^1H NMR (400 MHz, DMSO) δ 8.52 (s, 1H), 7.79 (d, J = 10.8 Hz, 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 Hz, 3H), 2.39 (s , 3H), 2.28-2.04 (m,2H), 2.00-1.73 (m, 2H), 1.22 (d, J = 6.6 Hz, 3H), 0.96- 0.70 (m, 3H).

[178] Synthesis of compound LE14

[179] Intermediate 21 (134 mg, 0.16 mmol) was mixed with commercially available starting material MC-V (102 mg, 0.33 mmol) in 40 ml dichloromethane and added the condensing agent 2-ethoxy-1-ethoxycarbonyl-1 ,2-dihydroquinoline (82 mg, 0.33 mmol) allowing the reaction to occur overnight at room temperature. After completion of the reaction, 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 obtain compound LE14 (137 mg, 75% yield), ESI- MS mass/charge: 1141.4 (M+H). ^1H NMR (400 MHz, 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 Hz, 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 Hz, 3H) , 1.23-1.11 (m, 2H), 0.93-0.77 (m, 9H).

[180] Example 2-4. Synthesis of compounds LE15-LE20

[181] Intermediate VI can be prepared by replacing the methylamine hydrochloride in step b with the corresponding commercially available amino compound using Fmoc-L-valyl-L-alanine as the starting material and referring to steps a and b in the method for the synthesis of intermediate 3 in example 2-1. Subsequent steps were carried out starting with intermediate VI, and using the same methods as steps c, d, f and h of Example 2-1, to obtain intermediate IX, similar to intermediate 9. Then, in accordance with those The same steps i and j as in Example 6 removed the amino protecting group and condensed using various commercially available maleimide compounds to obtain the final product. The structures of the amino compounds and maleimides used are shown in Table 1. Compound LE15: grey-white solid, ESI-MS wt/charge: 1121.2 (M+H); compound LE16: light yellow solid, ESI-MS wt/charge: 1167.1 (M+H); compound LE17: yellow solid, ESI-MS wt/charge: 1132.3 (M+H); compound LE18: light yellow solid, ESI-MS wt/charge: 1305.4 (M+H); compound LE19: light yellow solid, ESI-MS wt/charge: 1307.4 (M+H); compound LE20: light yellow solid, ESI-MS wt/charge: 1337.6 (M+H).

[182] Table 1. Intermediates used for the synthesis of LE15-LE20

, , , , ,

[183] Example 2-5. Synthesis of compounds LE21 and LE22

[184] Synthesis of compound DXD-1

[185] Commercially available exatecan methanesulfonate (0.568 g, 1 mmol) was mixed with commercially available 2-(tert-butyldimethylsilyloxy)acetic acid (CAS: 105459-05-0, 0.38 g, 2 mmol) in 20 ml of anhydrous dichloromethane. HATU condensing agent (0.76 g, 2 mmol) and 1 ml of pyridine were added to the resulting mixture and 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 obtain the title compound DXD-1 (0.55 g , yield 90%), ESI-MS mass/charge: 608.1 (M+H). ^1H NMR (400 MHz, CDCl ₃ ) δ 7.73 (d, J = 10.5 Hz, 1H), 7.64 (s, 1H), 7.05 (d, J = 9.2 Hz, 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 Hz, 3H), 0.80 (s, 9H), 0.11 (s, 3H), 0.03 (s, 3H).

[186] Preparation of intermediate V

[187] Intermediate V can be prepared by replacing the methylamine hydrochloride in step b with the corresponding commercially available amino compound in accordance with the method for preparing compound 4 in Example 2-1.

[188] Synthesis of LE21-LE22

[189] Intermediate V was reacted with DXD-1 and then treated with 10% trifluoroacetic acid/dichloromethane to give intermediate X. Intermediate X was then reacted according to subsequent steps e, g, i and j for compound 5 in Example 2-1: Intermediate X was reduced to give the amino compound and the amino compound was then condensed with Fmoc-L-valine hydroxysuccinimide ester. The amino group Fmoc was then removed from the resulting product, and the resulting amino product was then reacted with N-succinimidyl-6-maleimidohexanoate to obtain the final product. Compound LE21: yellow solid, ESI-MS wt/charge: 1141.2 (M+H); compound LE22: yellow solid, ESI-MS wt/charge: 1106.6 (M+H).

,

[190] Example 2-6. Synthesis of compound LS13

[191] Referring to the synthesis method of LE15 in Examples 2-4, SN-38 (7-ethyl-10-hydroxycamptothecin) was reacted with intermediate VII (R ¹ is methylsulfonylethyl) to obtain compound LS13 after deprotection, condensation and other stages: ¹ H NMR (400 MHz, DMSO) δ 9.92 (d, J = 22.4 Hz, 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).

[192] Example 2-7. Synthesis of compound GGFG-Dxd

[193] Compound GGFG-Dxd was prepared according to a known synthesis method described in WO2015146132A1. ESI-MS mass/charge: 1034.5 (M+H), ¹ H-NMR (400 MHz, DMSO _- d6 ) 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).

[194] Example 3: Preparation of Antibody-Drug Conjugates

[195] Anti-TROP2 antibody FDA018 was prepared according to the method of Example 1, but was substituted with 50 mM PB/1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column. 5 equivalents of TECP were added to the resulting mixture and the mixture was stirred at 25°C for 1 hour to fully open the disulfide interchain bonds of the antibody. Citric acid was then used to adjust the pH of the reconstituted antibody solution to 5.0 and the sample was exchanged with 20 mM citrate buffer and 1 mM EDTA buffer (pH 5.0) using a G25 desalting column. The water bath temperature was maintained at 25°C during the coupling reaction. The linker-drug conjugates LE12-LE22, LS13 and GGFG-Dxd prepared according to Example 2 above were dissolved in DMSO, respectively, and 4.5 equivalents of the linker-drug conjugates were added dropwise to the reconstituted antibody solution. Additional DMSO was added to a final concentration of 5% (v/v) and the reaction mixture was stirred at 25°C for 0.5 hour. After completion of the reaction, the sample was filtered through a 0.22 μm pore membrane. A tangential flow filtration system was used to purify and remove unconjugated small molecules. The buffer was 25 mM His, 6% sucrose solution (pH 6.0). After cleaning, the sample was stored in a refrigerator at -20°C. The absorbance values were measured at 280 nm and 370 nm using UV method, respectively, and the DAR value was calculated. The results are presented in Table 2 below.

[196] The coupling reaction was carried out in the same manner as in this example, and all samples were prepared according to the largest DAR (ie, excess coupling). Precipitate formation was observed during each coupling reaction, and the polymer ratio and release rate after each coupling reaction were calculated. The results are also shown in Table 2.

[197] Table 2. Combination conditions for preparing different antibody-drug conjugates (ADCs)

[198] "/" indicates that the release rate was not calculated

[199] In the procedure for preparing the antibody-drug conjugate of the present invention, no precipitate was formed and the proportion of aggregates was within the normal range, indicating that the linker-drug conjugates of the present invention have good physicochemical properties .

[200] Example 1 applied to the effect. In vitro assessment of killing activity of antibody-drug conjugates

[201] NCI-N87 cells (ATCC) were selected as the cell line for in vitro activity determination. 2000 cells per well were seeded in a 96-well cell culture plate and cultured for a period of 20 to 24 hours. Antibody-drug conjugates prepared according to the method of Example 3 were formulated in test solutions with 11 gradient concentrations of 1000, 166.7, 55.6, 18.6, 6.17, 2.06, 0.69, 0 .23, 0.08, 0.008 and 0 nM using L15 cell culture medium containing 10% FBS. Diluted test solutions were added to a culture plate containing seeded cells at 100 μl/well and incubated for 144 hours at 37°C in a 5% CO ₂ incubator. CellTiter-Glo® Luminescent Cell Viability Assay Reagent (50 µl/well) was added and the plate was shaken at 500 rpm at room temperature for 10 minutes to ensure thorough mixing. Data were read using a SpectraMaxL microplate reader (OD 570 nm, reading at 2 s intervals) and IC50 results were calculated as shown in Table 3.

[202] Using the same method as described above, the cytotoxic activity of each antibody-drug conjugate was tested against MDA-MB-468 and BXPC3 tumor cells purchased from ATCC. The results are shown in Table 3. From the results in Table 3, it can be seen that the antibody-drug conjugates of the present invention exhibit excellent in vitro killing activity against cells such as NCI-N87, MDA-MB-468 and BXPC3. However, the antibody-drug conjugate does not have killing activity against Calu-6 negative cells, indicating that the resulting ADC has specific targeted killing activity.

[203] Table 3. In vitro killing activity of antibody-drug conjugates

[204] Example 2 applied to the effect. In vitro plasma stability assay

[205] In this example, the stability of an antibody-drug conjugate prepared according to the method of Example 3 in human plasma is assessed. Specifically in this example, the antibody-drug conjugate from Example 3 was added to human plasma and placed in a water bath at 37°C for periods of 1, 3, 7, 14, 21 and 28 days. An internal standard (exatecan as an internal standard substance) was added and extracted and then subjected to HPLC determination to detect the release of free drugs. The results are shown in Table 4.

[206] Table 4. Evaluation of the stability of various ADCs in human plasma

Plasma stability results demonstrate that the stability of the ADC produced using the new technical solution is at least as good as FDA018-GGFG-DXD and sometimes even superior. At the same time, the above activity test results also prove that some of the newly derived ADCs have better activity than FDA018-GGFG-DXD.

[208] Example 3 applied to the effect. In vitro enzymatic cleavage experiment of linker-drug conjugates

[209] The linker-drug conjugate (LE14 and GGFG-Dxd) was co-incubated with cathepsin B in three buffers with different pH (5.0, 6.0, 7.0). Samples were collected at different time points and injected into a high-performance liquid chromatography-mass spectrometry instrument. An external standard method (with DXD as external standard) was used to determine the percentage of drug release. The experimental results (shown in Table 5) showed that GGFG-Dxd has a slow enzymatic degradation rate within the applicable pH range, while LE14 of the present invention can be rapidly enzymatically degraded at a pH within the range of 5.0 to 7.0.

[210] Table 5. Enzymatic degradation of LE14 and GGFG-Dxd at different pH values in vitro

[211] Example 4 applied to the effect. In Vitro Enzymatic Digestion Experiment FDA018-LS13

[212] The NCI-N87 cell line was chosen as the experimental cell lines. After incubating the sample in a cathepsin B-based system (100 mM sodium acetate-acetic acid buffer, 4 mM dithiothreitol, pH 5.0) at 37°C for 4 hours, the resulting sample was diluted with culture medium to various concentrations. Eight concentrations (from 1.5 to 10-fold dilution) were established with a concentration of 70 nM to 0.003 nM SN-38. The killing (suppressive) ability of the cell line was observed for 144 hours. The IC50 value was calculated by reading fluorescence data after chemical luminescent staining using the CellTiter-Glo® Luminescent Cell Viability Assay.

[213] The above enzymatic digestion samples obtained by incubating in a cathepsin B based system at 37°C for 4 hours were precipitated with an appropriate amount of ethanol to remove the protein and subjected to detection by high performance liquid chromatography for the release of low molecular weight compounds. The release rate over 4 hours was measured using an equivalent amount of SN-38 as a reference, and the results showed that the release rate reached 99%.

[214] The experimental results (shown in Table 6) demonstrate that after enzymatic digestion treatment, the cytotoxic activity of FDA018-LS13 is almost the same as that of SN-38 at an equivalent dose, which also indicates that FDA018-LS13 provided the almost complete release of SN-38 by cathepsin B was what made the difference. However, FDA018-LS13 may have undergone unpredictable changes when taken up by endocytosis into lysosomes, resulting in SN-38 being unable to function effectively.

[215] Table 6. Changes in the activity of FDA018-LS13 in killing the NCI-N87 cell line before and after enzymatic digestion by the cathepsin B-based system

[216] Example 5 applied to the effect. Testing the antitumor activity of FDA018-LE14 in the NCI-N87 human gastric cancer model

[217] Female Balb/c nude mice, 6 to 8 weeks old, were injected subcutaneously into the right side of the neck and back with 5 × 10 ⁶ human gastric cancer cells (NCI-N87) dissolved in 100 μl of PBS solution. When the tumor had grown to an average volume of 150 to 200 mM ³ , mice were randomly divided into 5 groups according to tumor size and weight of mice with 6 animals in each group. The groups were a blank control group and two dose groups of the antibody-drug conjugates FDA018-GGFG-DXD and FDA018-LE14, respectively. Specifically, group 01 was the blank control group; group 02 was group FDA018-GGFG-DXD at 4.0 mg/kg; group 03 was group FDA018-GGFG-DXD at 2.0 mg/kg; group 04 was group FDA018-LE14 at 4.0 mg/kg; group 05 was group FDA018-LE14 at 2.0 mg/kg; In this case, the administration was carried out intraperitoneally once a week. The body weight and tumor volume of the animals were measured twice a week, and the vital status of the experimental animals was monitored during the experiment. As shown in Table 7, the average tumor volume of mice in the blank control group was 1388.47 mm ³ at the end of treatment. The mean tumor volume in the FDA018-GGFG-DXD treatment group at 2.0 mg/kg was 1235.21 mm ³ on day 14 after completion of treatment, and the mean tumor volume in the FDA018-GGFG-DXD treatment group at 4.0 mg /kg was 721.09 mm ³ on the 14th day after completion of treatment. The mean tumor volume in the FDA018-LE14 treatment group at 2.0 mg/kg was 1342.31 mm ³ on day 14 after completion of treatment, and the mean tumor volume in the FDA018-LE14 treatment group at 4.0 mg/kg was 435 .36 mm ³ on the 14th day after completion of treatment. The experimental results demonstrated that FDA018-LE14 had good antitumor activity in vivo, and all experimental mice had no death or weight loss, indicating that FDA018-LE14 had good safety.

[218] Table 7. Antitumor activity of FDA018-LE14 in the NCI-N87 human gastric cancer model

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LIST OF SEQUENCES

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

<120> ANTIBODY-DRUG CONJUGATE,

TARGETTING TROP2 AND METHOD OF OBTAINING IT

AND ITS APPLICATION

<130> P23412149RU

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 214

<212> PROTEIN

<213> Homo sapiens

<400> 1

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 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> 2

<211> 451

<212> PROTEIN

<213> Homo sapiens

<400> 2

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

<---

Claims (102)

1. Antibody-drug conjugate of formula I to TROP2, a pharmaceutically acceptable salt thereof; ; I where Ab is an antibody to TROP2; the amino acid sequence of the light chain in the anti-TROP2 antibody is shown under SEQ ID NO: 1 and the amino acid sequence of the heavy chain is shown under SEQ ID NO: 2; m is from 3.5 to 4.5; D represents or ; each of R ² and R ⁵ independently represents H, C ₁ -C ₆ alkyl or halogen; each of R ³ and R ⁶ independently represents H, C ₁ -C ₆ alkyl or halogen; R ⁴ and R ⁷ are each independently C ₁ -C ₆ alkyl; R ¹ is C ₁ -C ₆ alkyl substituted with one -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; wherein each of R ^1-1 , R ^1-2 and R ^1-3 independently represents C ₁ -C ₆ alkyl; L ₁ independently represents one of an alanine residue and a valine residue; p is 2; L ₂ represents , , , , or where n is independently from 8 to 12, the c-terminus is attached to L ₁ via a carbonyl group and the f-terminus is attached to the d-terminus of L ₃ ; L ₃ represents , where the b-end is attached to Ab and the d-end is attached to the f-end of L ₂ . 2. Antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to claim 1, where the b-end of L ₃ is attached to the sulfhydryl group of the antibody in the form of a thioester bond; and/or, if R ¹ is C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably ethyl; and/or, if R ^1-1 and R ^1-2 are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; and/or, if R ¹ is C ₁ -C ₆ alkyl substituted with one 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, most preferably ethyl; and/or, if R ^1-3 is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; and/or, if R ¹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl; and/or m is an integer or a non-integer; and/or n is 8; and/or, if R ^1-1 , R ^1-2 and R ^1-3 are independently C ₁ -C ₆ alkyl, 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 methyl. 3. Antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to claim 1, where, if R ² and R ⁵ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl; and/or, if R ² and R ⁵ are each independently halogen, the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine; and/or, if R ³ and R ⁶ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n -butyl, isobutyl or tert-butyl, most preferably methyl; and/or, if R ³ and R ⁶ are each independently halogen, the halogen is fluorine, chlorine, bromine or iodine, further preferably fluorine; and/or, if R ⁴ and R ⁷ are each independently C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl is C ₁ -C ₄ alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n -butyl, isobutyl or tert-butyl, most preferably ethyl; and/or, if R ¹ is C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 , -NR ^1-1 R ^1-2 is -N(CH ₃ ) ₂ ; and/or, if R ¹ is C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 , C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 is ; and/or, if R ¹ is C ₁ -C ₆ alkyl substituted with one R ^1-3 S(O) ₂ -, C ₁ -C ₆ alkyl substituted with one R ^1-3 S(O) ₂ - is yourself ; and/or m is from 3.8 to 4.2, such as 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4.05, 4, 10, 4.12 or 4.13; and/or (L ₁ ) _p represents , where the g-terminus is attached to the c-terminus of L ₂ via a carbonyl group. 4. Antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to claim 1, where R ² and R ⁵ are each independently C ₁ -C ₆ alkyl; and/or R ³ and R ⁶ are each independently halogen; and/or R ⁴ and R ⁷ are ethyl; and/or L ₁ is one of an alanine residue and a valine residue; and/or R ¹ is C ₁ -C ₆ alkyl substituted with one -NR ^1-1 R ^1-2 , or C ₁ -C ₆ alkyl substituted with one R ^1-3 S(O) ₂ -, most preferably C ₁ -C ₆ alkyl substituted with one R ^1-3 S(O) ₂ -; preferably D is or . 5. Antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to any one of claims. 1-4, where the antibody-drug conjugate is represented by any of the following schemes: scheme I: D represents or ; L ₁ independently represents one of an alanine residue and a valine residue; scheme II: R ² and R ⁵ are each independently C ₁ -C ₆ alkyl; R ³ and R ⁶ are each independently halogen; D is preferably or ; L ₁ independently represents one of an alanine residue and a valine residue; scheme III: Ab is an anti-TROP2 antibody; R ² and R ⁵ are each independently C ₁ -C ₆ alkyl; R ³ and R ⁶ are each independently halogen; D is preferably or ; m is from 3.5 to 4.5; L ₁ independently represents a valine residue or an alanine residue; scheme IV: Ab is an anti-TROP2 antibody; D represents or ; L ₁ independently represents a valine residue or an alanine residue; diagram V: Ab is an anti-TROP2 antibody; D represents or ; m is from 3.5 to 4.5; L ₁ independently represents a valine residue or an alanine residue. 6. An anti-TROP2 antibody-drug conjugate, a pharmaceutically acceptable salt thereof according to claim 1, wherein the anti-TROP2 antibody-drug conjugate is any of the following: , , , , , , , , , or ; where Ab is an antibody to TROP2 and m is from 3.8 to 4.2, preferably 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4, 05, 4.10, 4.12 or 4.13; the light chain amino acid sequence of the anti-TROP2 antibody is preferably provided by SEQ ID NO: 1 and the heavy chain amino acid sequence is preferably presented by SEQ ID NO: 2. 7. Antibody-drug conjugate to TROP2, its pharmaceutically acceptable salt according to claim 1, where the antibody-drug conjugate is represented by any of the following schemes: diagram A: , , , , , , , , , or ; wherein Ab is an anti-TROP2 antibody, preferably an anti-TROP2 antibody; the light chain amino acid sequence of the anti-TROP2 antibody is preferably represented by SEQ ID NO: 1 and the heavy chain amino acid sequence is preferably presented by SEQ ID NO: 2; diagram B: , or , where Ab is an antibody to TROP2; m is preferably 3.90, 4.00 or 4.10; the light chain amino acid sequence of the anti-TROP2 antibody is preferably represented by SEQ ID NO: 1 and the heavy chain amino acid sequence is preferably presented by SEQ ID NO: 2; diagram C: , or ; where Ab is an antibody to TROP2; diagram D: , wherein Ab is an antibody to TROP2 and m is preferably 3.90; , wherein Ab is an antibody to TROP2 and m is preferably 4.10; , wherein Ab is an antibody to TROP2 and m is preferably equal to 4.00; , wherein Ab is an antibody to TROP2 and m is preferably 4.12; , wherein Ab is an antibody to TROP2 and m is preferably equal to 4.03; , wherein Ab is an antibody to TROP2 and m is preferably 3.98; , wherein Ab is an antibody to TROP2 and m is preferably 3.96; , wherein Ab is an antibody to TROP2 and m is preferably equal to 4.13; , wherein Ab is an antibody to TROP2 and m is preferably equal to 3.88; , wherein Ab is an antibody to TROP2 and m is preferably equal to 4.05; , wherein Ab is an anti-TROP2 antibody and m is preferably 4.00. 8. A method for producing an antibody-drug conjugate to TROP2 according to any one of claims. 1-7, comprising the following steps: carrying out a coupling reaction between a compound of formula II and Ab-hydrogen, as shown below; 9. A pharmaceutical composition containing substance X and a pharmaceutically acceptable excipient; wherein substance X is an antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to any one of claims. 1-7, wherein the amount of substance X is a therapeutically effective amount, wherein the pharmaceutical composition is used for the treatment and/or prevention of TROP2-positive gastric cancer. 10. Use of substance X or the pharmaceutical composition according to claim 9 in the manufacture of a TROP2 protein inhibitor; wherein substance X is an antibody-drug conjugate to TROP2, a pharmaceutically acceptable salt thereof according to any one of claims. 1-7. 11. Use of substance X or the pharmaceutical composition according to claim 9 in the manufacture of a medicinal product for the treatment and/or prevention of TROP2-positive gastric cancer. 12. Use of substance X or the pharmaceutical composition according to claim 11, wherein the gastric cancer cells are NCI-N87 cells.