Linkers for ligand drug conjugates

Abstract

The present disclosure relates to linkers for ligand drug conjugates, in particular, to a linker for ligand drug conjugates and ligand drug conjugates prepared from the linker, and use of the ligand drug conjugates in the treatment of tumors or other diseases.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to a linker for ligand-drug conjugates, ligand-drug conjugates prepared from the linker, and the use of the ligand-drug conjugates in the treatment of tumors or other diseases. Background Technology

[0002] The statements herein are provided for relevant background information only and do not necessarily constitute prior art.

[0003] Ligand-drug conjugates, including antibody-drug conjugates (ADCs), are a new class of targeted therapeutic drugs, primarily used in the treatment of cancer, autoimmune diseases, and infections. Ligand-drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that conjugates the ligand and drug. The mechanism of action of ligand-drug conjugates is as follows: the antibody or antibody-like ligand specifically recognizes and binds to cell surface antigens; the resulting conjugate enters the cell via endocytosis, simultaneously carrying the small molecule drug into the cell; subsequently, the antibody is enzymatically cleaved or the linker itself breaks down, releasing the small molecule drug in the form of an appropriate active ingredient, which kills the target cell.

[0004] Linkers are the core component of ADC drug design and are crucial for achieving targeted drug release. Highly hydrophobic ADC molecules can cause various problems, affecting their efficacy and safety, specifically in several aspects: (1) Highly hydrophobic drug loading may lead to ADC molecule aggregation in vivo, thus affecting drug stability and efficacy; (2) Highly hydrophobic ADC molecules may exhibit higher clearance rates and shorter half-lives, thus affecting drug stability and efficacy; (3) Highly hydrophobic ADC molecules are more easily taken up by non-targeted tissues, increasing drug toxicity in non-targeted tissues. Therefore, one challenge in ADC design and development is finding suitable hydrophilic linkers for antibody-drug conjugation. On the one hand, by using such hydrophilic linkers to adjust the overall hydrophobicity of the molecule, higher drug loading can be achieved, and localized dense distribution of drugs (especially hydrophobic drugs) can be avoided, preventing hydrophobic aggregation and thus reducing efficacy. On the other hand, the hydrophilic linkers used must not affect the overall function and effect of the ADC molecule, that is, they must not affect the normal cleavage of the linker in vivo or the normal release of the drug at the target site.

[0005] In WO2015 / 057699A2 and WO2016 / 059377A1, the preparation of ADCs loaded with 8-36 drugs using linkers containing polyethylene glycol (PEG) moieties orthogonally linked to linkers was reported. PEG is well known for its advantages such as non-toxicity, good water solubility, non-immunogenicity, and non-antigenicity, which can improve the hydrophilicity and stability of drugs, proteins, bioconjugates, and nanoparticles, and increase drug retention time in vivo, and is often used for drug delivery and modification. However, some studies have shown that PEG may also trigger immune responses, leading to the production of anti-PEG antibodies and affecting drug efficacy. In some cases, PEG may accumulate in vivo, leading to potential toxic reactions.

[0006] Therefore, in view of the shortcomings of the existing technology in preparing antibody-drug conjugates, there is still a need in the field to develop new linker technologies to make the obtained conjugates more stable and more effective. Summary of the Invention

[0007] Firstly, this disclosure provides ligand drug conjugates of formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof. (I) in Ab is a ligand; L represents the connector unit; D represents the drug or payload; n represents the average number of connections, and n is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8; PA has the following structure: -L P -[(AA1) s -(AA2) t ] p -R a Where L P It is a key or connecting unit, and L P It forms a covalent connection with L; preferably, L P Selected from -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene -C(O)-, -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene-N(R) 1 )-、-C(O)-C 1-6 Alkylenes -C(O)-, -C(O)-, -C(O)O-, -C(O)-C 1-10 Alkylene-N(R)1 )-、-C(O)-C 1-10 Alkylene-C(O)-N(R) 1 )-、-C(O)-C 1-10 Alkylene-N(R) 1 -C(O)-、-C 1-10 Alkylene-N(R) 1 )-、-C 1-10 Alkylene-C(O)-N(R) 1 )-、-C 1-10 Alkylene-N(R) 1 )-C(O)-, or =N-(O or N)-C 1-10 Alkylene-N(R) 1 )-; R 1 For H or C 1-6 alkyl; AA1 and AA2 together form a peptide chain, and the first AA1 binds with L... P Covalent bonds are formed between them, such as amide bonds; AA1 and AA2 are different and are each independently selected from proline, hydroxyproline or alanine; s and t are each independently 1, 2, 3 or 4, preferably 1 or 2; p is an integer from 1 to 20, preferably an integer from 5 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; R a The group is absent or is a capped group, such as -CR b -OR b -N(R) b )2、-C(O)-R b -C 1-6 Alkylenes -C(O)OH, -CN, -NR b -C(O)-R b -C 1-6 Alkyl-C(O)-R b -C(O)-N(R) b )2、-SO3 - -SO3H, -S(O)2R b -OS(O)2OR b -S(O)2NR b -S(O)R b -OP(O)(OR) b )2、-P(O)(OR b )2、-PO3 --PO3H2, -C(=S)R b -CO2R b -CO2, -C(=S)OR b -C(O)SR b -C(=S)SR b C(=O)N(R) b 2. C(=S)N(R) b )2 or C(=NR) b )N(R b )2, where R b Independently selected from -H, -C 1-20 Alkyl, -C 6-20 Aryl, -C 3-14 cycloalkyl, or -C 3-14 Heterocyclic groups, the -C 1-20 Alkyl groups can be modified with glycosyl groups; preferably, R a Selected from -NH2, C 1-6 Alkyl-, Glycosyl-C 1-6 Alkyl group -, -C(O)OH, -OH or -CH3.

[0008] In some embodiments, the glycosyl group is a monosaccharide group, exemplary of which are glucosyl, galactosyl, glucuronic acid, and galacturonic acid. In some embodiments, the glycosyl group is a disaccharide group composed of two monosaccharides independently selected from glucose, galactose, glucuronic acid, and galacturonic acid. In some embodiments, the glycosyl group is an oligosaccharide group, which is composed of 1-6, 1-5, 1-4, or 1-3 monosaccharides each independently selected from glucose, galactose, glucuronic acid, galacturonic acid, mannose, fructose, arabinose, and xylose.

[0009] In some implementations, the R a It is a monosaccharide-C 1-6 Alkyl group, such as glucosyl-C 1-6 Alkyl-. In some embodiments, the R a It has the following structure: The wavy line indicates the connection point with the rest of PA. In some embodiments, the R... a Selected from -NH2, glucosyl-CH2-C(O)-, CH3-C(O)- and -OH.

[0010] In some embodiments, this disclosure provides the ligand drug conjugate of formula (I), its isomers, pharmaceutically acceptable salts, or mixtures thereof, wherein PA has the following structure: -L P -[(AA1) s-(AA2) t ] p -R a , Where s is 1 and t is 1; or s is 1 and t is 2; or s is 2 and t is 1; s is 2 and t is 2, Lp, AA1, AA2, p and R a As defined above.

[0011] In some embodiments, this disclosure provides a ligand drug conjugate of formula (I), an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein AA1 is proline or hydroxyproline and AA2 is alanine; or AA1 is alanine and AA2 is proline or hydroxyproline.

[0012] In some embodiments, this disclosure provides ligand drug conjugates of formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein L P Selected from -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene -C(O)-, -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene-N(R) 1 )-、-C(O)-C 1-6 Alkylene-C(O)-. In some embodiments, this disclosure provides ligand drug conjugates of formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein L P Selected from -CH2-N(CH3)-CO-(CH2)2-CO-, -CH2-N(CH3)-CO-(CH2)-N(CH3)-, or -CO-(CH2)2-CO-.

[0013] In some embodiments, this disclosure provides a ligand drug conjugate of formula (I), its isomers, pharmaceutically acceptable salts or mixtures thereof, wherein L comprises a natural or non-natural amino acid or a short peptide, said short peptide being composed of a peptide chain formed of 2-10, preferably 2-5, more preferably 2, 3 or 4 natural or non-natural amino acids; wherein each of said natural or non-natural amino acids is independently an amino acid having 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups on its side chain, and wherein at least one reactive functional group of the side chain of at least one of said natural or non-natural amino acids is covalently linked to PA.

[0014] In some embodiments, the natural or non-natural amino acid is selected from, for example, lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine, more preferably lysine or 2,3-diaminopropionic acid; in some embodiments, the natural or non-natural amino acid is selected from lysine, arginine, and 2,3-diaminopropionic acid.

[0015] In some embodiments, this disclosure provides a ligand drug conjugate of formula (I), an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the L comprises a PAB structure, and the PA is covalently coupled to a phenyl group of the PAB.

[0016] In some embodiments, this disclosure provides ligand drug conjugates of formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein the L in formula (I) has a -L1-L2- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , , , , , , , , , , , , , and , where ** represents the junction with the ligand and * represents the junction with L2; L2 is a splittable linker, in which the two ends of the L2 main chain are connected to L1 and D respectively, and PA is coupled to L2, thus forming the structure shown in equation (IA): (IA) Where n is defined as in equation (I) above.

[0017] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, wherein the L1 is coupled to a thiol or amino group of the ligand. In some embodiments, the L1 is coupled to a sulfur atom of a cysteine ​​residue of the ligand. In some embodiments, the cysteine ​​residue is naturally occurring and derived from an interchain disulfide bond. In some embodiments, the cysteine ​​residue is not naturally occurring and is derived from a cysteine ​​residue introduced into the ligand. In some embodiments, the L1 is coupled to an ε-amino group on the side chain of a lysine residue of the ligand.

[0018] In some embodiments, the L2 comprises natural or non-natural amino acids or short peptides, the short peptides being composed of peptide chains formed from 2 to 10, preferably 2 to 5, more preferably 2, 3 or 4 natural or non-natural amino acids; wherein each of the natural or non-natural amino acids is an amino acid whose side chain has 1 to 5, preferably 1 to 3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA.

[0019] In some embodiments, the natural or non-natural amino acid is selected from, for example, lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine, more preferably lysine or 2,3-diaminopropionic acid; in some embodiments, the natural or non-natural amino acid is selected from lysine, arginine, and 2,3-diaminopropionic acid.

[0020] In some embodiments, L2 comprises a PAB structure, wherein the PA is covalently coupled to the phenyl group of the PAB.

[0021] In some implementations, the L2 includes the following structure: , where the wavy line indicates the connection point with the remaining part of the connector, and # indicates the connection point with D.

[0022] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L2 has a -L3-L4-L5-L6- structure, wherein: L3 represents a bond or spacer unit, such as -(CH2). m -C(=O)-, where m is an integer from 0 to 10, preferably an integer from 0 to 5, such as 0, 1, 2, 3, 4, or 5; or L3 is -C(O)-, -C1-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-S-, C1-C 10Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a bond or a parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2-10, preferably 2-5, more preferably 2, 3, or 4 natural or non-natural amino acids; wherein each of the natural or non-natural amino acids is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; preferably, the natural or non-natural amino acid is selected from, for example, lysine (Lys), arginine (Arg), 2,3-diaminopropionic acid (Dap), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), or ornithine (Orn), more preferably Lys or Dap; L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine (Val), citrulline (Cit), lysine (Lys), glycine (Gly), phenylalanine (Phe), alanine (Ala), proline (Pro), isoleucine (Ile), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), and 2,3-diaminopropionic acid (Dap); L6 is a bond or selected from the following structures: or ; Where * represents the connection point with L5, # represents the connection point with D, and PA is as defined above; The condition is that L4 and L6 are not both keys.

[0023] In some implementations, L3 is -C(O)- or -Cl-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 alkylene-C(O)-, wherein R 1 For H or C 1-6 Alkyl; y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; in some embodiments, L3 is -C1-C6 alkylene-C(O)- or -C(O)-.

[0024] In some embodiments, the side-chain reactive functional group in the ligand drug conjugate, its isomers, pharmaceutically acceptable salts or mixtures thereof described above is selected from: amino, carboxylic acid, activated carboxylic acid (e.g., acyl chloride), hydroxyl, amide, carboxylic acid ester, thiol, Michael receptor (e.g., maleimide), halogen (e.g., bromine), azide, alkyne, isocyanate, etc.

[0025] In some embodiments, the side-chain reactive functional group in the ligand-drug conjugate, its isomers, pharmaceutically acceptable salts or mixtures thereof described above is selected from amino groups.

[0026] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, L4 is a parallel linking unit, which is a natural or non-natural amino acid; wherein each of the natural or non-natural amino acids is independently an amino acid having one or two reactive functional groups on its side chain, and at least one reactive functional group on the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; wherein the natural or non-natural amino acid is selected from lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine. In some embodiments, the natural or non-natural amino acid shown is selected from lysine, arginine, and 2,3-diaminopropionic acid.

[0027] In some embodiments, in the ligand drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, L5 is a natural amino acid or a non-natural amino acid or a short peptide, said short peptide being composed of a peptide chain formed of 2-5 natural or non-natural amino acids; said natural or non-natural amino acid is independently selected from: valine (V), citrulline (Cit), lysine (K), glycine (G), phenylalanine (F), alanine (A), proline (P), isoleucine (I), serine (S), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), and 2,3-diaminopropionic acid (Dap). In some embodiments, L5 is selected from -valine-alanine (VA)-, -valine-citrulline (VC)-, -glycine-glycine-phenylalanine-glycine (GGFG)-, -alanine-alanine-alanine-glycine (AAAG)-, -glycine-glycine-glycine-glycine (GGGG)-, -valine-alanine-glycine (VAG)-, -valine-citrulline-glycine (VCG)-, -alanine-alanine-glycine (AAG)-, -alanine-alanine-alanine (AAA)-, -alanine-alanine-glycine (AAG)-, -alanine-alanine-alanine (AAA)-, -alanine-alanine-p- Short peptides of amino acids (AA), -glutamic acid-alanine-glycine-glycine (EAGG), -glycine-glutamic acid-alanine-glycine (GEAG), -glycine-glutamic acid-glycine-glycine (GEGG), -glutamic acid-glycine-glycine (EGG), -glutamic acid-alanine-glycine (EAG), -valine-lysine-glycine (VKG), -glycine-glutamic acid-glycine (GEG), -glutamic acid-alanine (EA), -glutamic acid-glycine (EG), and -glycine-glutamic acid (GE).

[0028] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , , , , , , , , , , , , and , where ** represents the junction with the ligand and * represents the junction with L3; L3 represents a bond or spacer unit, such as -C(O)- or -Cl-C. 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 --C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a parallel linker unit, which is a natural amino acid, a non-natural amino acid, or a short peptide. The short peptide is composed of a peptide chain formed by 2-5 natural or non-natural amino acids. Each of the natural or non-natural amino acids is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups. At least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA. Preferably, each of the natural or non-natural amino acids is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn. ​​More preferably, the natural or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which is optionally covalently linked to PA. L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; L6 is a bond or selected from the following structures: , Where * represents the connection point with L5, # represents the connection point with D, and PA is defined as in equation (I) above.

[0029] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , , , , , , , , , , , , and , where ** represents the junction with the ligand and * represents the junction with L3; L3 represents a bond or spacer unit, such as -C(O)- or -Cl-C. 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is the key; L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; L6 is selected from the following structures: Preferably, L6 is selected from: or , Where * represents the connection point with L5, # represents the connection point with D, and PA is defined as in equation (I) above.

[0030] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is selected from or , where ** represents the junction with the ligand and * represents the junction with L3; L3 is a spacer unit, such as -C(O)- or -C1-C. 10 Alkylene-C(O)-; L4 is a bond, or a parallel linking unit, wherein the parallel linking unit is a natural amino acid or a non-natural amino acid, wherein the natural amino acid or non-natural amino acid is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and wherein at least one reactive functional group of the side chain of at least one of the natural amino acid or non-natural amino acids is covalently linked to PA; preferably, the natural amino acid or non-natural amino acid is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn; more preferably, the natural amino acid or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which optionally is covalently linked to PA; L5 is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; more preferably, L5 is a valine-alanine dipeptide; L6 is a bond, or can be selected from the following structures: or , Where * represents the connection point with L5, # represents the connection point with D, and PA is defined as in equation (I) above.

[0031] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is selected from or , where ** represents the junction with the ligand and * represents the junction with L3; L3 is a spacer unit, such as -C(O)- or -C1-C. 10 Alkylene-C(O)-; L4 is a parallel linker unit, wherein the parallel linker unit is a natural amino acid or a non-natural amino acid, wherein the natural amino acid or non-natural amino acid is an amino acid having 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups on its side chain, and wherein at least one reactive functional group of the side chain of at least one of the natural amino acid or non-natural amino acids is covalently linked to PA; preferably, the natural amino acid or non-natural amino acid is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn; more preferably, the natural amino acid or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which optionally is covalently linked to PA; L5 is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; more preferably, L5 is a valine-alanine-dipeptide. L6 is a bond, or can be selected from the following structures: , Where * represents the connection point with L5, # represents the connection point with D, and PA is defined as in equation (I) above.

[0032] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is selected from or , where ** represents the junction with the ligand and * represents the junction with L3; L3 is a spacer unit, such as -C(O)- or -C1-C. 10 Alkylene-C(O)-; L4 is the key; L5 is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; more preferably, L5 is a valine-alanine dipeptide; L6 is a bond, or can be selected from the following structures: , Where * represents the connection point with L5, # represents the connection point with D, and PA is defined as in equation (I) above.

[0033] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... ; L3 represents a bond or spacer unit, such as -(CH2). m -C(=O)-, where m is an integer from 0 to 10, preferably an integer from 0 to 5; L4 is a bond or parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, and the short peptide is composed of a peptide chain formed by 2-5 natural amino acids or non-natural amino acids; preferably, each of the natural amino acids or non-natural amino acids is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn; preferably, the natural amino acid or non-natural amino acid is Lys or Dap; L5 is as defined above; L6 is as defined above; The condition is that L4 and L6 are not both keys.

[0034] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, L4 is a parallel linking unit, said parallel linking unit being a natural or non-natural amino acid; preferably, said natural or non-natural amino acid is Lys or Dap, wherein the reactive functional group of said side chain is ε-amino or β-amino, and wherein at least one ε-amino or β-amino group is associated with L in PA. P An amide bond is formed.

[0035] In some implementations, L6 has the following structure in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above: or , PA is defined as above.

[0036] In some implementations, L is a ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof described above. P -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene -C(O)-, -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene-N(R) 1 - or -C(O)-C 1-6 alkylene-C(O)-, where R 1 For H or C 1-6 alkyl.

[0037] In some implementations, L is a ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof described above. P It is -CH2-N(CH3)-CO-(CH2)2-CO-, -CH2-N(CH3)-CO-(CH2)-N(CH3)-, or -CO-(CH2)2-CO-.

[0038] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, wherein PA is selected from the following structures: , , , , , , , , or , Among them, L P and R a As defined above.

[0039] In some embodiments, PA is selected from the following structures among the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above: , , , , , , , , , , , , , , , , or , in: p is as defined above; R a End-capping groups, such as -R b -CR b -OR b -N(R) b )2、-C 1-6 -C(O)OH, -CN, -NR b -C(O)-R b -C(O)-Rb -C 1-6 -C(O)-R b -C(O)-N(R) b )2、-SO3 - -SO3H, -S(O)2R b -OS(O)2OR b -S(O)2NR b -S(O)R b -OP(O)(OR) b )2、-P(O)(OR b )2、-PO3 - -PO3H2, -C(=S)R b -CO2R b -CO2, -C(=S)OR b -C(O)SR b -C(=S)SR b C(=O)N(R) b 2. C(=S)N(R) b )2 or C(=NR) b )N(R b )2, where R b Independently selected from -H, -C 1-20 Alkyl, -C 6-20 aryl, or -C 3-14 Heterocyclic group, wherein the alkyl group can be modified with a glycosyl group; preferably, R a Selected from -NH2, C 1-6 Alkyl group -, -OH, glucosyl group -C 1-6 Alkyl group -, -C(O)OH, -H or -CH3; The wavy line indicates the connection point with L.

[0040] In some embodiments, PA is selected from the following structures among the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above: , , , , , , , , , , , , , , or , in: p is as defined above; R a End-capping groups, such as -R b -CR b -OR b -N(R) b )2、-C 1-6 -C(O)OH, -CN, -NR b -C(O)-R b -C(O)-R b -C 1-6 -C(O)-R b -C(O)-N(R) b )2、-SO3 - -SO3H, -S(O)2R b -OS(O)2OR b -S(O)2NR b -S(O)R b -OP(O)(OR) b )2、-P(O)(OR b )2、-PO3 - -PO3H2, -C(=S)R b -CO2R b -CO2, -C(=S)OR b -C(O)SR b -C(=S)SR b C(=O)N(R) b 2. C(=S)N(R) b )2 or C(=NR) b )N(R b )2, where R b Independently selected from -H, -C 1-20 Alkyl, -C 6-20 aryl, or -C 3-14 Heterocyclic group, wherein the alkyl group can be modified with a glycosyl group; preferably, R a Selected from -NH2, C 1-6 Alkyl group -, -OH, glucosyl group -C 1-6 Alkyl group -, -C(O)OH, -H or -CH3; The wavy line indicates the connection point with L.

[0041] In some embodiments, this disclosure provides ligand drug conjugates of formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein the L in formula (I) has a -L1-L2- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , , , , , , , , , , , , , and , where ** represents the junction with the ligand and * represents the junction with L2; L2 is a splittable linker, in which the two ends of the L2 main chain are connected to L1 and D respectively, and PA is coupled to L2, thus forming the structure shown in equation (IA): (IA) Where n is defined as in equation (I) above.

[0042] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, wherein the L1 is coupled to a thiol or amino group of the ligand. In some embodiments, the L1 is coupled to a sulfur atom of a cysteine ​​residue of the ligand. In some embodiments, the cysteine ​​residue is naturally occurring and derived from an interchain disulfide bond. In some embodiments, the cysteine ​​residue is not naturally occurring and is derived from a cysteine ​​residue introduced into the ligand. In some embodiments, the L1 is coupled to an ε-amino group on the side chain of a lysine residue of the ligand.

[0043] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L2 has a -L3-L4-L5-L6- structure, wherein: L3 represents a bond or spacer unit, such as -(CH2). m -C(=O)-, where m is an integer from 0 to 10, preferably an integer from 0 to 5, such as 0, 1, 2, 3, 4, or 5; or L3 is -C(O)-, -C1-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a bond or a parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2-10, preferably 2-5, more preferably 2, 3, or 4 natural or non-natural amino acids; wherein each of the natural or non-natural amino acids is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; preferably, the natural or non-natural amino acid is selected from, for example, lysine (Lys), arginine (Arg), 2,3-diaminopropionic acid (Dap), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), or ornithine (Orn), more preferably Lys or Dap; L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine (Val), citrulline (Cit), lysine (Lys), glycine (Gly), phenylalanine (Phe), alanine (Ala), proline (Pro), isoleucine (Ile), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), and 2,3-diaminopropionic acid (Dap); L6 is the key, or L6 is selected from -N(R) 1 )-C1- 10 Alkylene-, -C(O)-C1- 10 Alkylene-C(O)-, -N(R) 1 )-C1- 10 Alkylene-N(R) 1 -C(O)- or -N(R) 1 )-C1- 10 Alkylene-N(R) 1 )-C1- 10 Alkylene; preferably, L6 is -N(R 1 -C1-6 alkylene-, for example -N(R 1 )-CH2-; where R 1 For H or C 1-6 Alkyl; or L6 is selected from , , , , , , , , , , and , Where a * Indicates the connection point with L5, b * This represents the connection point with D, and PA is as defined above; The conditions are: L4 and L6 are not simultaneously bonds, and at least one of L4 and L6 is covalently connected to PA.

[0044] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the L3 is -C(O)-, -Cl-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 alkylene-C(O)-, wherein R 1 For H or C 1-6 Alkyl; y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; preferably, L3 is a -C1-C6 alkylene-C(O)- or -C(O)-.

[0045] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, L4 is a parallel linking unit, which is a natural or non-natural amino acid; wherein each of the natural or non-natural amino acids is an amino acid having one or two reactive functional groups on its side chain, and at least one reactive functional group on the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; preferably, the natural or non-natural amino acid is selected from lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine; more preferably, the natural or non-natural amino acid is selected from lysine, arginine, and 2,3-diaminopropionic acid.

[0046] In some embodiments, in the ligand drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the L5 is a natural amino acid or a non-natural amino acid or a short peptide, said short peptide being composed of a peptide chain formed from 2, 3, 4, 5, 6, 7, or 8 natural amino acids or non-natural amino acids, preferably 2 or 3 natural amino acids or non-natural amino acids; each natural amino acid or non-natural amino acid is independently selected from: valine (V), citrulline (Cit), lysine (K), glycine (G), phenylalanine (F), alanine (A), proline (P), leucine (L), isoleucine (I), serine (S), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), and 2,3-diaminopropionic acid (Dap); preferably, the L5 is selected from -valine-alanine (VA)-, -valine-citrulline (VC)-, -glycine-glycine-phenylalanine-glycine (GGFG)-, -Alanine-Alanine-Alanine-Glycine (AAAG)-, -Glycine-Glycine-Glycine-Glycine (GGGG)-, -Valine-Alanine-Glycine (VAG)-, -Valine-Citriline-Glycine (VCG)-, -Alanine-Alanine-Glycine (AAG)-, -Alanine-Alanine-Alanine (AAA)-, -Alanine-Alanine (AA)-, -Glutamic acid-Alanine-Glycine-Glycine (EAGG)-, -Glycine Short peptides of glutamic acid-alanine-glycine (GEAG), glycine-glutamic acid-glycine-glycine (GEGG), glycine-glycine-glycine (EGG), glycine-alanine-glycine (EAG), valine-lysine-glycine (VKG), glycine-glutamic acid-glycine (GEG), glycine-alanine (EA), glycine-glycine (EG), and glycine-glutamic acid (GE).

[0047] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L6 is -N(R 1 -C1-6 alkylene-, for example -N(R 1 )-CH2-; where R 1 For H or C 1-6 alkyl.

[0048] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit conjugated to the antibody; preferably, L1 is selected from... , , , , , , , , , , , , , and , where ** represents the binding point with the antibody, and * represents the binding point with L3; L3 represents a bond or spacer unit, such as -C(O)- or -Cl-C. 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)- or -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a parallel linker unit, which is a natural amino acid, a non-natural amino acid, or a short peptide. The short peptide is composed of 2-5 natural or non-natural amino acids, wherein each natural or non-natural amino acid is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA. Preferably, each natural or non-natural amino acid is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn. ​​More preferably, the natural or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which is covalently linked to PA. L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine (Val), citrulline (Cit), lysine (Lys), glycine (Gly), phenylalanine (Phe), alanine (Ala), proline (Pro), leucine (Leu), isoleucine (Ile), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), and 2,3-diaminopropionic acid (Dap); L6 is the key, or L6 is selected from -N(R) 1 )-C1- 10 Alkylene-, -C(O)-C1- 10 Alkylene-C(O)-, -N(R) 1 )-C1- 10 Alkylene-N(R) 1 -C(O)- or -N(R) 1 )-C1- 10 Alkylene-N(R) 1 )-C1- 10 Alkylene; preferably, L6 is -N(R 1 -C1-6 alkylene-, for example -N(R 1 )-CH2-; where R 1 For H or C 1-6 Alkyl; or L6 is selected from: , , , , , , , , , , and , Where a * Indicates the connection point with L5, b * This indicates the connection point with D.

[0049] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, wherein the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is selected from or , where ** represents the binding point with the antibody, and * represents the binding point with L3; L3 is a spacer unit, such as -C(O)- or -C1-C. 10 Alkylene-C(O)-; L4 is a parallel linker unit, which is a natural or non-natural amino acid. The natural or non-natural amino acid is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA. Preferably, the natural or non-natural amino acid is independently selected from Lys, Arg, Dap, Glu, Gln, Asp, Asn, Ser, Thr, Cys, or Orn. ​​More preferably, the natural or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which is covalently linked to PA. L5 is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; more preferably, L5 is a valine-alanine-dipeptide. L6 is the key; or L6 is -N(R) 1 )-C 1-10 Alkylene-, -C(O)-C 1-10 Alkylene-C(O)-, -N(R) 1 )-C 1-10 Alkylene-N(R) 1 -C(O)- or -N(R) 1 )-C 1-10 Alkylene-N(R) 1 )-C 1-10 Alkylene; preferably, L6 is -N(R 1 )-C 1-6 Alkylene, for example -N(R) 1 )-CH2-; where R 1 For H or C 1-6 Alkyl; or L6 is selected from: , , , , , , , , , , and , Where a * Indicates the connection point with L5, b * This indicates the connection point with D.

[0050] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the linker group in formula (I) Select from the following structure, where ** represents the connection point with Ab, and * represents the connection point with D: , , , , , , , , , , or .

[0051] In some embodiments, in the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the linker group in formula (I) Select from the following structure, where ** represents the connection point with Ab, and * represents the connection point with D: , , or .

[0052] In some embodiments, among the ligand drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the antibody drug conjugate of formula (I) has the structure of formula (IB): (IB) Wherein, Ab, L1 and L2 are each independently defined as above, D1, D2 and D3 are different drugs, and n1, n2 and n3 are each independently an integer or decimal from 0 to 10, preferably an integer or decimal from 1 to 8; The condition is that at least two of n1, n2, and n3 are not zero. In some embodiments, the drug is selected from anticancer drugs, such as cytotoxic drugs, immunostimulants, or radioisotopes, among the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above.

[0053] In some embodiments, the drug is selected from glucocorticoids, among the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above.

[0054] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.

[0055] In some embodiments, the microtubule inhibitor is selected from orlistatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, maytansine, or dolastatin and its derivatives).

[0056] In some embodiments, the DNA topoisomerase inhibitor is selected from camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, such as ixotecan, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotecone, and homosilatecan.

[0057] In some embodiments, the drug is Exatecan, having the following structure: .

[0058] In some embodiments, the drug is MMAE and has the following structure: .

[0059] In some implementations, the immune enhancer is a STING agonist.

[0060] In some embodiments, the glucocorticoid drug is BD0260, whose structure is shown below: .

[0061] In some embodiments, the ligand in the ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof described above is an antibody.

[0062] In some embodiments, in the ligand-drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof described above, wherein the Ab is selected from antibodies that specifically bind to one or more of the following antigens: TROP2, HER2, AXL, BAFFR, BCMA, BCR-list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, differentiation antigen cluster 40 (C D40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2. CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, C XCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAp, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, type II HLA (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR1), IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, integrin (Integri) ns), LAG3, LIFR, MAG / Siglec-4 (sialic acid-binding immunoglobulin-like lectin-4), MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1 (sialic acid-binding immunoglobulin-like lectin-1), Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list components / assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, tumor necrosis factor α (TNFα), TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-γ, and VTCN1.

[0063] In some embodiments, the antibody described above, in the ligand-drug conjugate, its isomers, pharmaceutically acceptable salts or mixtures thereof, is an antibody that specifically binds to TROP2.

[0064] In some embodiments, the antibody that specifically binds to TROP2 comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; and The light chain variable region includes LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

[0065] In some embodiments, the antibody that specifically binds to TROP2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85% sequence identity with it; and The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85% sequence identity with it.

[0066] In some embodiments, the antibody that specifically binds to TROP2 comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 85% sequence identity with it; and The light chain comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 85% sequence identity with it.

[0067] In some embodiments, the antibody described above, in the ligand-drug conjugate, its isomers, pharmaceutically acceptable salts or mixtures thereof, is an antibody that specifically binds to HER2.

[0068] In some embodiments, the antibody that specifically binds to HER2 comprises: CDR regions of the heavy chain variable region as shown in SEQ ID NO: 16 and CDR regions of the light chain variable region as shown in SEQ ID NO: 17; or CDR regions of the heavy chain variable region as shown in SEQ ID NO: 18 and CDR regions of the light chain variable region as shown in SEQ ID NO: 19.

[0069] In some embodiments, the antibody that specifically binds to HER2 comprises: As shown in SEQ ID NO: 16, the heavy chain variable region is shown in SEQ ID NO: 17, and the light chain variable region is shown in SEQ ID NO: 17; or The heavy chain variable region is shown in SEQ ID NO: 18 and the light chain variable region is shown in SEQ ID NO: 19.

[0070] In some embodiments, the antibody that specifically binds to HER2 comprises: As shown in SEQ ID NO: 12, the heavy chain, and as shown in SEQ ID NO: 13, the light chain; or Heavy chains as shown in SEQ ID NO: 14 and light chains as shown in SEQ ID NO: 15.

[0071] In some embodiments, the antibody is an antibody that specifically binds to IL-4R, in the ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof described above.

[0072] In some embodiments, the antibody that specifically binds to IL-4R is BR206, which is prepared with reference to the H4H098P antibody in patent WO2008054606A2.

[0073] In some embodiments, the antibody that specifically binds to IL-4R comprises: a CDR region of the heavy chain variable region as shown in SEQ ID NO: 20, 21 and 22 and a CDR region of the light chain variable region as shown in SEQ ID NO: 23, 24 and 25.

[0074] In some embodiments, the antibody that specifically binds to IL-4R includes a heavy chain variable region as shown in SEQ ID NO: 26 and a light chain variable region as shown in SEQ ID NO: 27.

[0075] In some embodiments, the antibody that specifically binds to IL-4R comprises a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29.

[0076] In some embodiments, the ligand-drug conjugate described above, its isomers, pharmaceutically acceptable salts, or mixtures thereof, is selected from: , , Wherein Ab is a ligand, D, D1, and D2 are all drugs, and D1 and D2 are different drugs, n is 3.3-4.3; the sum of n1 and n2 is 3.3-4.3; Preferably, the ligand-drug conjugate is selected from: , and ; The antibody mentioned above is: An anti-Trop2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 11 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 10; An anti-HER2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 12; or An anti-HER2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 15 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 14; n is 3.3-4.3; the sum of n1 and n2 is 3.3-4.3.

[0077] In some embodiments, the ligand-drug conjugate described above, its isomers, pharmaceutically acceptable salts, or mixtures thereof, is selected from: or , Where Ab refers to ligands, D refers to drugs, and n is 3.3-4.3.

[0078] In some embodiments, among the ligand-drug conjugates, their isomers, pharmaceutically acceptable salts, or mixtures thereof described above, the ligand-drug conjugate is: ; The antibody mentioned is an anti-IL-4R antibody, with n being 3.7-4.3.

[0079] In a preferred embodiment, the antibody that specifically binds to IL-4R is BR206.

[0080] Secondly, this disclosure provides drug-linker molecules of formula (II), their isomers, pharmaceutically acceptable salts, or mixtures thereof. (II) L, D, and PA are as defined in the first aspect above; R b1 The leaving group on the terminal L unit, such as F, Cl, Br, I, p-nitrophenoxy, N-hydroxysuccinimide (NHS), substituted N-hydroxysuccinimide, phenoxy, phenylthio, dinitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, trifluoromethanesulfonate, imidazole, dichlorophenoxy, tetrachlorophenoxy, trichlorophenoxy, 1-hydroxybenzotriazole, toluenesulfonate, methanesulfonate, 2,4-dinitrophenoxy, 2-ethyl-5-phenylisoxazonium-3'-sulfonate, sulfone or sulfoxide, or R b1Together with the terminal L unit, they form Michael acceptors, such as maleimide groups; z is an integer from 1 to 5, such as 1, 2, 3, 4 or 5.

[0081] In some embodiments, the drug linker molecule of formula (II) has the structure of formula (II-A): (II-A) in R b1 The leaving group on the terminal L1 unit, such as F, Cl, Br, I, p-nitrophenoxy, N-hydroxysuccinimide (NHS), substituted N-hydroxysuccinimide, phenoxy, phenylthio, dinitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, trifluoromethanesulfonate, imidazole, dichlorophenoxy, tetrachlorophenoxy, trichlorophenoxy, 1-hydroxybenzotriazole, toluenesulfonate, methanesulfonate, 2,4-dinitrophenoxy, 2-ethyl-5-phenylisoxazonium-3'-sulfonate, sulfone or sulfoxide, or R b1 Together with the terminal L1 unit, they form Michael acceptors, such as maleimide groups; z is an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L1, L2, D, and PA are as defined in the first aspect above.

[0082] In some embodiments, the drug linker molecule of formula (II) is selected from the following compounds: .

[0084] In some embodiments, the drug linker molecule of formula (II) is selected from the following compounds: .

[0086] Thirdly, this disclosure also provides pharmaceutical compositions comprising the ligand drug conjugate, its isomers, pharmaceutically acceptable salts or mixtures thereof as described in the first aspect, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0087] Fourthly, this disclosure also provides the use of the ligand drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, as described in the first aspect, or the pharmaceutical compositions described in the third aspect, in the preparation of medicaments for treating and / or preventing diseases. In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the cancer is breast cancer or pancreatic cancer.

[0088] Fifthly, this disclosure also provides methods for treating and / or preventing diseases, comprising administering to an individual in need a ligand-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, as described in the first aspect, or a pharmaceutical composition as described in the third aspect. In some embodiments, the disease is cancer or an autoimmune disease.

[0089] In a sixth aspect, this disclosure also provides ligand drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, as described in the first aspect, or pharmaceutical compositions as described in the third aspect, for use as pharmaceuticals. In some embodiments, they are used as pharmaceuticals for treating or preventing diseases. In some embodiments, they are used as pharmaceuticals for treating cancer or autoimmune diseases. Attached Figure Description

[0090] Figure 1A The chromatogram of ADC-2 is shown after 1 hour of reaction; Figure 1B The chromatogram of ADC-3 is shown after 1 hour of reaction; Figure 1C The chromatogram of ADC-1 is shown after 1 hour of reaction, where peak 1 is the unreacted product, peak 2 is the fully reacted product, and peak 3 is the product with a high DAR value. Figure 1D The chromatogram of ADC-1 is shown after 4 hours of reaction, where peak 1 is the unreacted product, peak 2 is the fully reacted product, and peak 3 is the product with a high DAR value. Figure 1E The chromatogram of ADC-1 after the reaction was allowed to proceed overnight is shown, with peak 2 being the product of the complete reaction and peak 3 being the product with a high DAR value.

[0091] Figure 2A The graph shows the PK results of the total antibody detection. Figure 2B The image shows the PK detection results of the ADC.

[0092] Figure 3 shows the FKBP5 expression in PBMCs induced by IL-4R-ADC, where, Figure 3A The expression of FKBP5 in ADC-40-induced monocytes was shown. Figure 3B This shows FKBP5 expression in B cells induced by ADC-40.

[0093] definition The terminology used herein is for descriptive purposes only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0094] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0095] It should be understood that the ordinal numbers “first”, “second”, “Formula 1”, “Formula 2”, “1)”, “2)”, etc. in this application are only used to distinguish different technical features, elements, components and steps, and are not intended to impose restrictions on level, order or quantity.

[0096] Ligands are any large molecule (peptide, protein, peptide, typically antibody) commonly used in ligand-drug conjugate (e.g., antibody-drug conjugate) technology, or a small molecule (such as folic acid or aptamers) that can be covalently conjugated to the linker or drug-linker disclosed herein using bioconjugation techniques (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally selected compounds based on their targeting ability. A non-exhaustive list of ligands includes: proteins, peptides, peptides, antibodies, full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or any other cell-binding molecule or substance. The primary class of ligands used to prepare ligand-drug conjugates is antibodies.

[0097] End-capping group: An end-capping group is any chemical group that terminates one end of a peptide bond. This group is typically covalently linked to an amino or carboxyl (or carbonyl) group at the end of the peptide chain. Exemplary end-capping groups include: alkyl, aryl, alkyl, amino, alkoxy, alkoxyalkyl, alkyl ester, alkyl orthoester, aryloxy, aryl ester, arylalkoxycarbonyl, tertiary amine, hydroxy, aldehyde, or glycosyl-O-alkylyl-.

[0098] "Antigen" refers to a molecule or part thereof that can be selectively recognized or bound by antigen-binding molecules (such as the antibody-drug conjugate and anti-TROP2 antibody of this application). An antigen may have one or more epitopes.

[0099] An epitope is a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformational epitopes). Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Antibodies or their antigen-binding fragments that bind to specific epitopes can be screened using methods routine in the art, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, and chemical modification of the antigen.

[0100] The term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL). The light chains are either κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε groups, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In both the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, while the heavy chain also includes "D" regions of approximately 10 or more amino acids. (For general information, see *Basic Immunology*, Paul, W., ed.) , 2nd ed. Raven Press, NY, 1989, Ch. 7, all cited herein for various purposes.

[0101] "Specific binding" refers to the phenomenon where an antibody or its antigen-binding fragment binds to a targeted antigen or epitope with a higher affinity than other antigens or epitopes. Typically, this is approximately 1 × 10⁻⁶. -7 A binding constant of M or smaller (KD) is considered "specific". KD can be measured using known methods such as FACS or surface plasmon resonance. "Specific binding" does not preclude cross-reactivity with homologous antigens from other species (e.g., cynomolgus monkeys). Macaca fascicularis ),chimpanzee( Pan troglodytes ) or marmoset ( Callithrix jacchus ).

[0102] "Affinity" describes the strength of the non-covalent interaction between an antibody or its antigen-binding fragment and its antigen or epitope. Affinity is usually expressed by the equilibrium dissociation constant (KD). "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, while "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. "KD" is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration.

[0103] "Fully human antibodies" include antibodies that contain both variable and constant regions of human immunoglobulin sequences.

[0104] "Humanized antibody" refers to an antibody produced by transplanting a non-human CDR sequence into the variable region framework of a human antibody.

[0105] "Chimeric antibodies" are antibodies formed by fusing the variable region of an antibody from one species (such as a mouse or rabbit) with the constant region of an antibody from another species (such as a human).

[0106] An "antibody fragment" or "antigen-binding fragment" is different from a complete antibody molecule, but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0107] “Fab” consists of a light chain, a heavy chain CH1, and a variable region.

[0108] The “Fab” fragment is a Fab fragment with one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0109] The “F(ab′)2 fragment” is a divalent fragment formed by connecting two Fab' fragments through a disulfide bridge in the hinge region.

[0110] "Fab'-SH" refers to a Fab' that has a free thiol group on the cysteine ​​residue in the constant region.

[0111] A "single-chain Fab fragment" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; wherein the linker is a peptide linker. In some embodiments, the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.

[0112] "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) that are linked together in the same polypeptide chain.

[0113] A “linear antibody” contains a pair of tandem Fd fragments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions.

[0114] The “Fv fragment” contains the VL and VH domains of one arm of the antibody.

[0115] A "single-domain antibody" is an antibody fragment that contains all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain.

[0116] Single-chain antibodies (scFv) are antibodies composed of variable regions of heavy and light chains linked together by linkers.

[0117] "Fc" contains two heavy chain segments, each containing a CH2 and a CH3 domain. The two heavy chain segments are held together by disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0118] "Variable region" refers to the domain in an antibody or antigen-binding fragment that binds to the antigen.

[0119] The "complementarity-determining region," "hypervariable region," or "CDR" refers to the region within the variable region that primarily facilitates antigen binding. Both VH and VL contain four frame regions (FRs) and three complementarity-determining regions (CDRs). VH contains three CDRs: HCDR1, HCDR2, and HCDR3; VL contains three CDRs: LCDR1, LCDR2, and LCDR3. From N-terminus to C-terminus, each VH and VL region is named as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0120] "Frame" or "FR" refers to the variable domain residues outside the hypervariable region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0121] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the Kabat numbering system (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278).

[0122] The correspondences between various numbering systems are well known to those skilled in the art. In other words, when a CDR sequence and its location in an antibody are provided under one numbering system, those skilled in the art are able to determine the corresponding CDR sequence and its location in an antibody under another numbering system. Technical solutions corresponding to different numbering systems are considered equivalent technical solutions.

[0123] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise stated, the Fc region is numbered using the EU index. The C-terminus of the Fc region can be a full C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may comprise an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of intact antibodies may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of intact antibodies has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0124] Antibody-drug conjugates (ADCs) are molecules formed by linking an antibody (or its antigen-binding fragment) to a drug (such as a cytotoxic agent) via a linker.

[0125] The terms "drug fraction," "D," or "drug" refer to any compound having the desired biological activity and reactive functional group, which can be used to link the drug to the linker disclosed herein. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing diseases in humans or other animals. In one embodiment, the drug fraction (D) may be a cytotoxic drug or an immune enhancer (e.g., a STING agonist). Such cytotoxic drugs include microtubule inhibitors (e.g., alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics. Examples of such cytotoxic drugs include orisstatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, maytansine, or dolastatin and their derivatives), or camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, such as eczetidine, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotecone, and homosilatecan.

[0126] "Cytotoxic drugs" are chemical molecules that disrupt cell growth within cells. In principle, cytotoxic drugs can kill cells at sufficient concentrations; however, due to their lack of specificity, while killing tumor cells, they can also cause apoptosis or death of normal cells, leading to side effects. Cytotoxic drugs include, but are not limited to, microtubule inhibitors (such as alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.

[0127] The term "STING agonist" refers to a compound capable of binding to and activating the STING (Stimulator of interferon genes) receptor and STING signaling. For example, upon contact with the STING receptor, a STING agonist induces one or more of the following: (1) stimulation or activation of the STING receptor, (2) upregulation of the IRF3 and NFkB signaling pathways, and / or (3) induction of IFN-β and other cytokines. STING agonist activity can be measured in vitro by various assays known in the art, such as, but not limited to, measurements of cell signaling, cell proliferation, markers of immune cell activation, and cytokine production. STING agonist activity can also be measured in vivo by various assays measuring surrogate endpoints, such as, but not limited to, measurements of T cell proliferation or innate immune-related cytokine production, particularly type I interferon.

[0128] The term "glucocorticoid drugs" generally refers to naturally occurring or synthetic steroid hormones that interact with glucocorticoid receptors, as well as their derivatives or analogues. Exemplary examples include prednisolone hemisuccinate, methylprednisolone hemisuccinate, dexamethasone hemisuccinate, allogestrelone hemisuccinate, beclomethasone 21-hemisuccinate, betamethasone 21-hemisuccinate, pevidone hemisuccinate, prednisolone sodium hemisuccinate, prednisolone 21-hemisuccinate, nandrolone hemisuccinate, 19-nortestosterone hemisuccinate, deoxycorticosterone 21-hemisuccinate, dexamethasone hemisuccinate, dexamethasone hemisuccinate:semine, corticosterone hemisuccinate, and 11-deoxycorticosterone hemisuccinate.

[0129] The function of the "ligand-coupled extension unit" is to connect the ligand unit to the linker unit. In this regard, the extension unit has functional groups capable of forming bonds with the functional groups of the ligand. Useful functional groups on the ligand can be naturally present or provided by chemical manipulation, including but not limited to, thiol (-SH), amino, hydroxyl, carboxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl groups. For example, the functional groups of the ligand are thiol and amino. The extension unit may contain, for example, maleimide groups, aldehydes, ketones, carbonyl groups, or haloacetamides for linking to the ligand. In some embodiments, the ligand is an antibody, and said antibody is linked to the extension unit (L1) via the sulfur atom of its cysteine ​​residue.

[0130] The term "linker unit," "linker," or "connector" refers to a chemical structural fragment or bond that is linked at one end to a ligand (e.g., an antibody or its antigen-binding fragment) and at the other end to a drug. It may also be linked to other linkers before being attached to the drug. Linkers can be cleavable or non-cleavable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable cleavable linkers include, for example, enzymatically cleavable linkers, including peptide-containing linkers that can be cleaved by intracellular proteases (e.g., lysosomal proteases or endosomal proteases, such as cathepsins), or glycolinkers, such as glucuronidase-containing linkers. Peptide-containing linkers can include, for example, dipeptides such as valine-citrullyl, phenylalanyl-lysyl, or valine-alanyl; or tripeptides such as glycyl-phenylalanyl-glycyl; or tetrapeptides such as glycyl-glycyl-phenylalanyl-glycyl. Other suitable cleavable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that cleave under reducing conditions (e.g., disulfide linkers). Non-cleavable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0131] Before being linked to a ligand (e.g., an antibody), the linker has a reactive group capable of reacting with certain amino acid residues, and the linking is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counterion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide linked to the antibody via thiosuccinimide.

[0132] In this context, the term "peptide residue" refers to a short peptide formed by the linear linkage of amino acid residues through amide bonds.

[0133] The term "amino acid" refers to an organic compound whose molecular structure contains both an amino group and a carboxyl group, with both groups directly attached to a -CH- structure. When amino acids combine with each other, a water molecule is lost due to the participation of some groups in the formation of an amide bond; therefore, the amino acid unit in a peptide is called an "amino acid residue." In this application, the amino acid can be an "L-configuration" amino acid or a "D-configuration" amino acid. For clarity, when any chemical structure listed in this application shows an L-configuration amino acid, D-configuration amino acids are also included. This also applies to peptides formed from multiple amino acids, where each amino acid residue can be independently selected from L-amino acids and D-amino acids.

[0134] The term "side chain" refers to all atoms of an amino acid residue that are not included in the "main chain" portion of the amino acid residue. The "main chain" refers to a structure formed by a series of peptide bonds connecting amino acids, wherein the α-nitrogen atom of an α-amino acid, the β-nitrogen atom of a β-amino acid, the γ-nitrogen atom of a γ-amino acid residue, the δ-nitrogen atom of a δ-amino acid, the ε-nitrogen atom of an ε-amino acid, or the ω-nitrogen atom of an ω-amino acid is attached to the C1 carbonyl atom of the preceding amino acid.

[0135] The term "reactive functional group" refers to any chemical moiety that is reactive to covalently bondable groups, and is reactive under defined reaction conditions compared to an inert group. Exemplary reactive functional groups include carboxylic acids; primary amines; secondary amines; tertiary amines; hydroxyl groups; halogens; activated esters such as N-hydroxysuccinimide esters, perfluorinated esters, nitrobenzene esters, azido-benzotriazole and benzotriazole activated esters, acylureas; alkynyl groups; alkenyl groups; azides; isocyanates; isothiocyanates; aldehydes; thiols; reactive moieties such as maleimides, halogenated maleimides, halogenated acetyl groups, pyridyl disulfides; thiols; acrylates; methanesulfonates; toluenesulfonates; trifluoromethanesulfonates; hydroxylamines; chlorosulfonyl groups; and so on.

[0136] The term "PAB" refers to the para-aminobenzyloxycarbonyl group, which undergoes spontaneous 1,6-elimination upon cleavage by a protease, such as cathepsin B, thereby releasing the drug or payload. Its structure is as follows: , where # and * represent connection points with the rest of the molecule.

[0137] The term "covalent bond" refers to the connection between two atoms of two separate parts via a covalent bond. Different types of bonds are formed depending on the type of reactive functional groups involved. For example, the connection between an amine-containing part and a carboxylic acid-containing part results in an amide bond (-C(O)-N-, or -NC(O)-), the connection between an isothiocyanate-containing part and an amine-containing part results in a thiourea bond (-NC(S)-N-), and the connection between a part containing an unsaturated carbon atom and a part containing a thiol group (-C-SH) results in a thioether bond (-CSC). Table 1 below lists common covalent bonds in the ADC field.

[0138] Table 1

[0139] In some embodiments disclosed herein, the reactive functional groups of the main chain or side chain used as connectors and the covalent connections they form are shown in Table 2 below.

[0140] Table 2

[0141] The term "activated carboxylic acid" refers to a carboxylic acid group having the general formula -CO-X, where X is a leaving group. For example, the activated form of the carboxylic acid group may include, but is not limited to, acyl chlorides, symmetrical or asymmetrical acid anhydrides, and esters.

[0142] Natural amino acids are those encoded by genes and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Natural amino acids are identified by their standard three-letter codes and single-letter abbreviations, as shown in Table 3.

[0143] Table 3. Natural Amino Acids and Their Abbreviations

[0144] Non-natural amino acids are any kind of compounds that contain both amino and carboxyl groups and are not natural amino acids, such as Dap, i.e., 2,3-diaminopropionic acid. Table 4 shows some examples of natural and non-natural amino acids used in this disclosure.

[0145] Table 4. Names and structures of some natural and non-natural amino acids

[0146] The term "isomer" includes structural isomers and stereoisomers. Structural isomers are molecules with the same molecular formula but different sequences of atoms or groups. Stereoisomers are molecules with the same molecular formula and atomic sequence but different spatial arrangements of atoms. Stereoisomers include cis-trans isomers, optical isomers, and trans-blocking isomers. Alternatively, stereoisomers can be classified into enantiomers and diastereomers. Enantiomers are mirror images of each other and cannot be superimposed; their configurations at the chiral centers are different, thus allowing plane-polarized light to be deflected by equal angles in different directions. Diastereomers refer to all stereoisomers that are not enantiomers, i.e., optical isomers that are not mirror images of each other, including meso compounds, cis-trans isomers, conformational isomers, and stereoisomers with one or more chiral centers but not mirror images of each other. The isomers described herein include any single enantiomer or diastereomer, and mixtures thereof in any proportion, including racemic mixtures.

[0147] The term "single bond" refers to a covalent bond in a compound molecule formed by two atoms sharing a pair of electrons. In the chemical structure described in this application, the bond... "Indicates no configuration is specified, that is, if a chiral carbon atom is present in the chemical structure, the bond is " "can be " "or" , or both contain " "and" "Two forms. For all carbon-carbon double bonds, even if only one configuration is shown or named, the Z-type and E-type are included."

[0148] The term "alkyl" refers to a saturated, straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms. Alkyl groups can be substituted or unsubstituted; when substituted, they can be substituted at any valence-permissible point. Substituents are preferably selected from one or more of deuterium, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl groups. Alkyl groups include, for example, C14. 1-20 Alkyl, C 1-10 Alkyl, C1-6 Alkyl, C 1-4 Alkyl groups, etc., where the subscript indicates the number of carbon atoms. The term "C" is used in this context. 1-20 Alkyl group, C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 "alkyl" and the term "C1-C" 20 Alkyl group, C1-C 10 The terms “alkyl”, “C1-C6 alkyl”, and “C1-C4 alkyl” are used interchangeably and have the same meaning.

[0149] Term "C" 1-20 "alkyl" and the term "C" 1-10 "Alkyl" refers to alkyl groups containing 1 to 20 carbon atoms or 1 to 10 carbon atoms, including but not limited to C14 and C24. 1-3 Alkyl, C 1-4 Alkyl, C 1-5 Alkyl, C 1-6 Alkyl, C 1-8 Alkyl and C 1-9 Alkyl group. The term "C" 1-6 Alkyl groups are lower alkyl groups containing 1 to 6 carbon atoms. 1-6 Non-limiting examples of "alkyl" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted.

[0150] The term "halogenated C" 1-6 "alkyl" refers to C 1-6 The hydrogen atom on the alkyl group is replaced by one or more halogens, wherein C 1-6 Alkyl groups are as defined above.

[0151] The term "cycloalkyl" refers to a cyclic hydrocarbon substituent that is saturated or partially unsaturated, either monocyclic or polycyclic. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl. Polycyclic cycloalkyl groups include spirocyclic, fused-ring, or bridged-ring cycloalkyl groups.

[0152] Term "C" 3-14 "Cycloalkyl" and "C" 3-7"Cycloalkyl" refers to cycloalkyl groups as defined above that contain 3 to 14 or 3 to 7 cyclic carbon atoms.

[0153] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy, etc. Alkoxy groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable link. Substituents are preferably selected from one or more of deuterium, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0154] Term "C" 1-6 "Alkoxy" refers to an alkoxy group containing 1 to 6 carbon atoms, as defined above.

[0155] The term "alkylene" refers to a divalent alkyl group, wherein the alkyl group, as defined above, has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C2). 1-20 Alkylene). In some embodiments, the alkylene is an alkylene having 1 to 10 carbon atoms (i.e., C10). 1-10 Alkylenes), in some embodiments being alkylenes having 1 to 6 carbon atoms (i.e., C164-C ... 1-6 Alkylenes). Non-limiting examples include: -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH2CH3)-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. 0-6 Alkylene refers to a group that is absent or has a carbon group. 1-6 Alkylene.

[0156] The term "alkanoyl" refers to an alkyl group -C(O)-, where the alkyl group is as defined above. 1-6 Alkyl group refers to C 1-6 Alkyl-C(O)-.

[0157] The term "aromatic acyl" refers to aryl-C(O)-, where the aryl group is defined below. 6-10 Aromatic acyl group refers to C 6-10 Aryl-C(O)-.

[0158] The term "aryl alkoxy carbonyl" refers to aryl-alkoxy-C(O)-, such as benzyloxy carbonyl.

[0159] The term "glycosyl" refers to monosaccharide groups such as glucosyl, galactosyl, glucuronic acid, galacturonic acid, mannose, fructose, arabinose, and xylose, or combinations thereof, as well as disaccharide groups or oligosaccharide groups. An exemplary glycosyl structure is shown below: , Glucosyl glucuronic acid group, where discontinuous dashed lines indicate connection sites.

[0160] In some embodiments, the glycosyl group is a disaccharide, with an exemplary structure as follows: , In this context, discontinuous dashed lines represent connection points.

[0161] The term “leaving group” is the same as the concept known to those skilled in the art (Advanced Organic Chemistry: reactions, mechanisms and structure, 4th ed., Jerry March, John Wiley and Sons; 1992, pp. 351-357), referring to a chemical functional group attached to any reactant that moves when the reactant is subjected to a substitution reaction (displacement reaction), such as a nucleophilic substitution reaction. A good leaving group is a leaving group that moves readily during a nucleophilic substitution reaction. Exemplary good leaving groups include halogens (F, Cl, Br and I), toluenesulfonates / esters, methanesulfonates / esters, trifluoromethanesulfonates / esters, acetates, trifluoromethyl acetates, camphorsulfonates / esters, 2-thionylbenzo[d]thiazolyl-3(2H)-yl, N-hydrogenated succinimide, N-aryl oxides, and aryl oxides substituted with one or more electron-withdrawing groups (EWG), but this disclosure is not limited thereto. For example, aryl oxides substituted with one or more electron-withdrawing groups include 2-nitrobenzene oxide, 4-nitrobenzene oxide, 2,4-dinitrophenylene oxide, pentafluorophenylene oxide, 2-chloro-4-nitrobenzene oxide, 2,4-dichlorophenylene oxide, and 2,4,6-chlorophenylene oxide, and the electron-withdrawing groups include, for example, halogens (F, Cl, Br, or I), -NO2, -CN, and -C(O)(C 1-6 Alkyl), -C(O)(aryl), -C(O)O(C 1-6 Alkyl groups, -C(O)O (aryl groups), etc.

[0162] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aryl) or a polycyclic aromatic ring system (i.e., polycyclic aryl) having a conjugated π-electron system, having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 6 to 14-membered aryl). The aryl is preferably an aryl having 6 to 10 ring atoms (i.e., 6 to 10-membered aryl). The monocyclic aryl is, for example, phenyl. Non-limiting examples of the polycyclic aryl include naphthyl, anthraceneyl, phenanthrene, etc. The polycyclic aryl further includes fusion of the phenyl with one or more heterocyclic groups or cycloalkyl groups, or fusion of the naphthyl with one or more heterocyclic groups or cycloalkyl groups, wherein the bonding point is on the phenyl or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples including: wait.

[0163] The aryl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0164] The term "heteroaryl" refers to a monocyclic heteroaryl ring (i.e., monocyclic heteroaryl) or a polycyclic heteroaryl ring system (i.e., polycyclic heteroaryl) having a conjugated π-electron system, wherein the ring contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-), having 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 5 to 14-membered heteroaryl). The heteroaryl is preferably a heteroaryl having 5 to 10 ring atoms (i.e., 5 to 10-membered heteroaryl), more preferably a heteroaryl having 5 or 6 ring atoms (i.e., 5 or 6-membered heteroaryl).

[0165] The term “cycloalkyloxy” refers to cycloalkyl-O-, where the cycloalkyl group is as defined above.

[0166] The term “heterocyclic oxy group” refers to the heterocyclic group -O-, where the heterocyclic group is as defined above.

[0167] The term "aryloxy group" refers to aryl-O-, where the aryl group is as defined above.

[0168] The term “heteroaryloxy” refers to heteroaryl-O-, where the heteroaryl group is as defined above.

[0169] The term "alkylthio" refers to alkyl-S-, where the alkyl group is as defined above.

[0170] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0171] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0172] The term "haloalkoxy" refers to an alkoxy group that is substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0173] The term "hydroxyalkyl" refers to an alkyl group that is replaced by one or more hydroxyl groups, wherein the alkyl group is as defined above.

[0174] The term "hydroxyl group" refers to the -OH group.

[0175] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0176] The term "amino" refers to -NH2.

[0177] The term "cyano" refers to -CN.

[0178] The term "carbonyl" refers to C=O.

[0179] The term "carboxyl group" refers to -C(O)OH.

[0180] N-Ethyldiisopropylamine is abbreviated as DIEA.

[0181] N,N-Dimethylformamide is abbreviated as DMF.

[0182] O-(7-azabenzotriazol-1-yl)-N,',N',N'-tetramethylureonium hexafluorophosphate, abbreviated as HATU.

[0183] 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0184] 2-Bromoacetic anhydride is abbreviated as BrAc2O.

[0185] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions without much effort (through experimentation or theory). For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0186] The term "drug loading" refers to the average amount of drug conjugated to each ligand in a ligand-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of ligand (e.g., antibody). Drug loading can range from 1 to 20, preferably 1 to 10 drugs (e.g., cytotoxic agents), per ligand (e.g., antibody). In embodiments disclosed herein, drug loading is expressed as n, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the average of any two values ​​(which can be integers or decimals). n is preferably 1-10, more preferably 3-8, or an integer or decimal of 1-5. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, CE-SDS (monoclonal antibody molecular size variant assay), and HPLC characterization. While the drug-to-ligand ratio has a precise value for a particular conjugate molecule, it should be understood that when used to describe samples containing many molecules, this value will often be an average. In some implementations, the drug loading rate (DAR) is between about 1 and about 6, and typically about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0. In some implementations, 'approximately x' DAR means that the measured DAR value is within 20% of x.

[0187] The loading of ligand-drug conjugates can be controlled using the following non-limiting methods, including: (1) Controlling the molar ratio of drug linker fragments to ligands, (2) Control the reaction time and temperature. (3) Choose different reaction reagents.

[0188] The antibodies or antigen-binding fragments disclosed herein can be generated using recombinant methods.

[0189] In one specific implementation, host cells containing the expression vector are cultured under conditions suitable for antibody expression, and the antibody is recovered (optionally purified) from the host cells (or their culture).

[0190] "Pharmaceutical composition" means a mixture containing one or more antibody-drug conjugates disclosed herein and other chemical components, such as pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0191] "Pharmaceutically acceptable carriers, diluents, buffers, or excipients" refer to components in a pharmaceutical preparation that are different from the active ingredient and are non-toxic to the subjects. Pharmaceutically acceptable carriers, diluents, buffers, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0192] "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a drug-linker, or a ligand-drug conjugate). The compound may contain at least one amino group and is therefore capable of forming an acid addition salt with the amino group. Exemplary salts include, but are not limited to: sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pyrates (i.e., 1,1'-methylenebis(2-hydroxy-3-naphthylcarbamate)). Pharmaceutically acceptable salts may contain another molecule of, for example, acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic component that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. An example where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Therefore, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counterions.

[0193] "Subject" or "individual" refers to an animal, preferably a mammal. Depending on the specific implementation plan, the subject is a mammal, including, for example, a camel, horse, cow, pig, sheep, cat, dog, rabbit, rat, guinea pig, mouse, or primate (e.g., human). In a specific implementation plan, the subject is a human.

[0194] In the specific implementation plan, the subjects are individuals who are susceptible to, suspected of having, or already have the disease.

[0195] "Treatment" means providing a subject with the antibody-drug conjugate or a pharmaceutical composition thereof disclosed herein. The subject has symptoms of one or more diseases. Typically, the antibody-drug conjugate is administered to the treated subject or a group of subjects in an amount that effectively relieves disease symptoms.

[0196] In specific implementation schemes, those skilled in the art can determine the selection of an effective dose based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to those skilled in the art.

[0197] The effective dosage in a specific implementation can be obtained from dose-response curves derived from animal model testing systems, and can be determined based on the physician's judgment and the individual subject's condition. As an example, the amount of drug required for a single administration to a subject can be conveniently obtained by calculating the product of the subject's weight and the dose per unit body weight required for that single administration. For instance, in the drug preparation process, it is generally assumed that an adult's weight is 50-70 kg, and the dosage can initially be determined using the equivalent dose conversion relationship between experimental animals and human body weight doses. For example, it can be determined according to guidelines issued by drug regulatory agencies such as the SFDA and FDA. In some implementations, the dosage for humans and mice can be converted using a conversion factor of 0.0026 based on the body surface area of ​​humans and mice.

[0198] The active ingredient (e.g., antibody-drug conjugate) described herein may be administered in a single dose or divided into many smaller unit doses administered at regular time intervals. It should be understood that the dosage, duration, and intervals of treatment are functions of the disease being treated and can be inferred from animal or clinical trial data. Administration may include a single dose or two or more doses at appropriate time intervals. Intervals between consecutive doses may be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0199] The terms “cancer” and “cancerous” refer to, or describe, a physiological condition or disease in mammals that is typically characterized by disordered cell growth. A “tumor” contains one or more cancer cells.

[0200] In this article, "autoimmune disease" refers to a disease or disorder that originates from and targets an individual's own tissues or proteins.

[0201] It should be understood that when a range of values ​​is publicly mentioned, such as “A to B”, it is a concise way of writing that, although not every point value in the range is given, is regarded as the integers and decimals in the range being clearly disclosed.

[0202] "And / or", for example "A and / or B", should be understood as meaning "A and B" or "A or B".

[0203] "Optional" or "optionally" means that the events or circumstances described below may, but do not necessarily, occur. The singular forms "a" and "an" as used in the specification and claims include their corresponding plural references, unless otherwise clearly stated in the text. Detailed Implementation

[0204] The following examples are used to illustrate representative embodiments of this disclosure. However, it should be understood that the methods, pharmaceuticals, and chemical structures described below are merely exemplary and do not limit the scope of this disclosure. Those skilled in the art can make various changes and modifications to the technical features of the embodiments without departing from the spirit and intent of this disclosure, and the resulting new embodiments will still be within the scope of this disclosure.

[0205] 1. Exemplary drugs and structures The exemplary drugs used in the examples are the cytotoxic drug eczema, MMAE, the STING agonist Sting3, and the glucocorticoid BD0260, the structures of which are as follows: Exatecan: MMAE: Sting3 can be prepared with reference to patent application WO2024137619 A1, and its structure is as follows: .

[0206] BD0260: .

[0207] Example 1. Synthesis of BD0260 200 mg (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 66 mg (0.531 mmol) of compound 7A (p-fluorobenzaldehyde) was added, and the flask was placed in an ice bath. 0.239 g (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0260-A with a mixed configuration. C18 preparative chromatography was used for purification to obtain 0.083 g of compound BD0260.

[0208] MS-ESI of compound BD0260: m / z 483.2 [M + H] + .

[0209] 1 H NMR (400 MHz, DMSO) δ 7.57 – 7.48 (m, 2H), 7.32 (d, J = 10.1 Hz,1H), 7.26 – 7.17 (m, 2H), 6.17 (dd, J = 10.1, 1.9 Hz, 1H), 5.94 (s, 1H), 5.47(s, 1H), 5.09 (t, J = 5.9 Hz, 1H), 4.94 (d, J = 4.9 Hz, 1H), 4.80 (d, J = 3.2 Hz, 1H), 4.53 (dd, J = 19.5, 6.4 Hz, 1H), 4.34 – 4.27 (m, 1H), 4.19 (dd, J = 19.5, 5.5 Hz, 1H), 2.55 (dd, J = 13.7, 8.7 Hz, 1H), 2.32 (dd, J = 13.3, 2.6 Hz, 1H),2.19 – 2.08 (m, 1H), 2.06 – 1.98 (m, 1H), 1.84 – 1.58 (m, 5H), 1.40 (s, 3H),1.14 – 0.97 (m, 2H), 0.87 (s, 3H).

[0210] Example 2. Preparation of drug-linker 2.1 PL-1: DiBr-VA-PAB-Exatecan To a stirred solution of 1,350 mg 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid in anhydrous dichloromethane, N-hydroxysuccinimide (230 mg) was added, followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (400 mg). The mixture was stirred at room temperature for 30 minutes, and the reaction was concentrated to dryness under reduced pressure. The residue was purified directly by RP-HPLC and lyophilized to give compound 2 as a white solid (337 mg).

[0211] MS: 452.0 [M+H + ].

[0212] To an anhydrous N,N-dimethylformamide (3 mL) solution of compound 26 (purchased from Medchemexpress, catalog number HY-36136, 136 mg), eczemab mesylate (106 mg) and diisopropylethylamine (0.07 mL) were added, and the mixture was stirred at room temperature for 16 hours. Then piperidine (300 μL) was added, and the mixture was stirred for another 10 min. The mixture was then directly purified by RP-HPLC to give compound 27, trifluoroacetate (138 mg).

[0213] Compound 2 (25 mg) was added to a solution of compound 27 (43 mg) in N,N-dimethylformamide (2 mL), followed by N-ethyldiisopropylamine (0.04 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 28, PL-1, as a yellow solid (42 mg).

[0214] MS: 1091.3 [M+H + ].

[0215] 2.2 PL-2: DiBr-VA-PAB(PA5)-Exatecan Ammonium formate (0.6 g) and zinc powder (1.0 g) were added to a solution of compound 3 in acetonitrile (10 mL) / water (10 mL) (synthesized according to the method reported in Cancer Discovery, 2023; 13:950-73, 300 mg, 1 mmol). The mixture was stirred for 30 min. Then water (20 mL) was added, and the mixture was extracted with ethyl acetate (100 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give compound 4 as a white solid (250 mg).

[0216] Fmoc-L-Ala-OH (300 mg) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (250 mg) were added to a solution of compound 4 (250 mg) in a mixture of dichloromethane (4 mL) and methanol (2 mL). The mixture was stirred at room temperature for 16 hours. The solvent was evaporated under vacuum, and the resulting residue was dissolved in 3 mL of N,N-dimethylformamide. Piperidine (0.6 mL) was then added, and the mixture was stirred for 10 min. The mixture was then purified by RP-HPLC to give compound 5 as trifluoroacetate (360 mg).

[0217] Fmoc-L-Val-OH (270 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (420 mg), and diisopropylethylamine (0.6 mL) were added to an anhydrous N,N-dimethylformamide (4 mL) solution of compound 5 (360 mg). The mixture was stirred at room temperature for 15 min. Then water (50 mL) was added. The precipitate was collected by filtration to give compound 6 as a white solid (0.47 g).

[0218] To a solution of compound 6 (200 mg) in anhydrous N,N-dimethylformamide (4 mL), bis(4-nitrophenyl) carbonate (95 mg) and diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 24 hours. Then, 2 mL of anhydrous N,N-dimethylformamide solution containing eczemab mesylate (165 mg) and diisopropylethylamine (0.1 mL) was added, and the mixture was stirred for 16 hours. Then, ether (15 mL) / hexane (10 mL) was added. The precipitate was collected by filtration to give compound 7 as a brown solid (310 mg).

[0219] Compound 7 (310 mg) was dissolved in a mixture of dichloromethane (6 mL) and trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 10 min, then the solvent was removed under vacuum, and the residue was purified by RP-HPLC to give compound 8, a trifluoroacetate salt (120 mg).

[0220] Compound 12 (50 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (35 μL) were added to a solution of compound 8 (trifluoroacetate, 57 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 10 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 13, trifluoroacetate (54 mg).

[0221] Compound 12 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0222] Compound 2 (10 mg) was added to a solution of compound 13 (37 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 14, PL-2, as a yellow solid (36 mg).

[0223] MS: 1037.9 [M+2H] + ] / 2.

[0224] 2.3 PL-3: DiBr-VA-PAB(HyPA5)-Exatecan Compound 15 (55 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (35 μL) were added to a solution of compound 8 (trifluoroacetate, 57 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 16, trifluoroacetate (57 mg).

[0225] Compound 15 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Hyp(OtBu)-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0226] Compound 2 (10 mg) was added to a solution of compound 16 (39 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 17, PL-3, as a yellow solid (40 mg).

[0227] MS: 1077.8 [M+2H] + ] / 2.

[0228] 2.4 PL-4: DiBr-VA-PAB(SAR10)-Exatecan Ammonium formate (0.6 g) and zinc powder (1.0 g) were added to a solution of compound 3 in acetonitrile (10 mL) / water (10 mL) (synthesized according to the method reported in Cancer Discovery, 2023; 13:950-73, 300 mg, 1 mmol). The mixture was stirred for 30 min. Then water (20 mL) was added, and the mixture was extracted with ethyl acetate (100 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give compound 4 as a white solid (250 mg).

[0229] Fmoc-L-Ala-OH (300 mg) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (250 mg) were added to a solution of compound 4 (250 mg) in a mixture of dichloromethane (4 mL) and methanol (2 mL). The mixture was stirred at room temperature for 16 hours. The solvent was evaporated under vacuum, and the resulting residue was dissolved in 3 mL of N,N-dimethylformamide. Piperidine (0.6 mL) was then added, and the mixture was stirred for 10 min. The mixture was then purified by RP-HPLC to give compound 5 as trifluoroacetate (360 mg).

[0230] Fmoc-L-Val-OH (270 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (420 mg), and diisopropylethylamine (0.6 mL) were added to an anhydrous N,N-dimethylformamide (4 mL) solution of compound 5 (360 mg). The mixture was stirred at room temperature for 15 min. Then water (50 mL) was added. The precipitate was collected by filtration to give compound 6 as a white solid (0.47 g).

[0231] To a solution of compound 6 (200 mg) in anhydrous N,N-dimethylformamide (4 mL), bis(4-nitrophenyl) carbonate (95 mg) and diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 24 hours. Then, 2 mL of anhydrous N,N-dimethylformamide solution containing eczemab mesylate (165 mg) and diisopropylethylamine (0.1 mL) was added, and the mixture was stirred for 16 hours. Then, ether (15 mL) / hexane (10 mL) was added. The precipitate was collected by filtration to give compound 7 as a brown solid (310 mg).

[0232] Compound 7 (310 mg) was dissolved in a mixture of dichloromethane (6 mL) and trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 10 min, then the solvent was removed under vacuum, and the residue was purified by RP-HPLC to give compound 8, a trifluoroacetate salt (120 mg).

[0233] Compound 9 (80 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (52 mg), and diisopropylethylamine (70 μL) were added to a solution of compound 8 (trifluoroacetate, 120 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (300 μL) was added, and the mixture was stirred for another 10 min. The mixture was then directly purified by RP-HPLC to give compound 10, trifluoroacetate (113 mg).

[0234] Compound 9 was obtained by solid-phase peptide synthesis using Cl-Trt resin, Fmoc-Sar-OH, and Fmoc-Sar-Sar-OH as raw materials, and the final product was purified by RP-HPLC.

[0235] Compound 2 (10 mg) was added to a solution of compound 10 (33 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 11, PL-4, as a yellow solid (32 mg).

[0236] MS: 1886.8 [M+H + ].

[0237] 2.5 PL-5: DiBr-VA-PAB(SAR8-Glc)-Exatecan Compound 30 (42 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (35 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 57 mg). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 31, trifluoroacetate (69 mg).

[0238] Compound 30 was obtained by solid-phase peptide synthesis using Cl-Trt resin, Fmoc-Sar-OH, Fmoc-Sar-Sar-OH, and compound 29 (synthesized according to the method reported in Tetrahedron: Asymmetry (2008), 19(16), 1919-1933) as raw materials. The acetyl group was removed in methanol with sodium methoxide, and the final product was purified by RP-HPLC.

[0239] Compound 2 (10 mg) was added to a solution of compound 31 (34 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 32, PL-5, as a yellow solid (32 mg).

[0240] MS: 1922.8 [M+H + ].

[0241] To investigate whether PA can be used for conjugation of different drugs and antibodies, the following drug-linker molecules were synthesized: 2.6 PL-6: DiBr-VA-PAB(PEG12)-Exatecan To an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 35 mg), mPEG12-COOH (18 mg, ChemScene, catalog number CS-0114277), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (17 mg), and diisopropylethylamine (17 μL) were added. The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then directly purified by RP-HPLC to give compound 34, trifluoroacetate (40 mg).

[0242] Compound 2 (10 mg) was added to a solution of compound 34 (30 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 35, PL-6, as a yellow solid (31 mg).

[0243] MS: 1704.5 [M+H + ].

[0244] 2.7 PL-7: DiBr-VA-PAB-MMAE To an anhydrous N,N-dimethylformamide (3 mL) solution of compound 26 (136 mg), MMAE (142 mg) and diisopropylethylamine (0.07 mL) were added, and the mixture was stirred at room temperature for 24 hours. Then piperidine (300 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 36 as trifluoroacetate (150 mg).

[0245] Compound 2 (25 mg) was added to a solution of compound 36 (57 mg) in N,N-dimethylformamide (2 mL), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 37, PL-7, as a white solid (58 mg).

[0246] MS: 1373.5 [M+H + ].

[0247] 2.8 PL-8: DiBr-VA-PAB(PA5)-MMAE To a solution of compound 6 (200 mg) in anhydrous N,N-dimethylformamide (4 mL), bis(4-nitrophenyl) carbonate (95 mg) and diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 24 hours. Then, a solution of MMAE (215 mg) and diisopropylethylamine (0.05 mL) in anhydrous N,N-dimethylformamide was added, and the mixture was stirred for 40 hours. Then, diethyl ether (15 mL) / hexane (10 mL) was added. The resulting precipitate was collected by filtration and dissolved in a mixture of dichloromethane (6 mL) and trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 10 min, then the solvent was removed under vacuum, and the residue was purified by RP-HPLC to give compound 38 as a trifluoroacetate salt (210 mg).

[0248] Compound 12 (50 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 39, trifluoroacetate (86 mg).

[0249] Compound 2 (6 mg) was added to a solution of compound 39 (21 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 40, PL-8, as a white solid (19 mg).

[0250] MS: 1178.7 [M+2H + ] / 2.

[0251] 2.9 PL-9: DiBr-VA-PAB(HyPA5)-MMAE Compound 15 (57 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 41, trifluoroacetate (88 mg).

[0252] Compound 2 (6 mg) was added to a solution of compound 41 (22 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 42, PL-9, as a white solid (22 mg).

[0253] MS: 1218.7 [M+2H + ] / 2.

[0254] 2.10 PL-10: DiBr-VA-PAB(SAR10)-MMAE Compound 9 (40 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 43, trifluoroacetate (92 mg).

[0255] Compound 2 (6 mg) was added to a solution of compound 43 (20 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 44, PL-10, as a white solid (20 mg).

[0256] MS: 1085.6 [M+2H + ] / 2.

[0257] 2.11 PL-11: DiBr-VA-PAB(PEG12)-MMAE To a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL), mPEG12-COOH (30 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 45, trifluoroacetate (79 mg).

[0258] Compound 2 (6 mg) was added to a solution of compound 45 (18 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 46, PL-11, as a white solid (18 mg).

[0259] MS: 1987.0 [M+H + ].

[0260] 2.12 PL-12: DiBr-VA-PAB-Pz-Sting3 Compound 47 (prepared according to patent application WO2024137619 A1, 116 mg) and diisopropylethylamine (0.07 mL) were added to an anhydrous N,N-dimethylformamide (3 mL) solution of compound 26 (68 mg), and the mixture was stirred at room temperature for 4 hours. Then piperidine (200 μL) was added, and the mixture was stirred for another 10 min. The mixture was then directly purified by RP-HPLC to give compound 48 as trifluoroacetate (125 mg).

[0261] Compound 2 (11 mg) was added to a solution of compound 48 (30 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 49, PL-12, as a white solid (28 mg).

[0262] MS: 1474.5 [M+H + ].

[0263] 2.13 PL-13: DiBr-VA-PAB(PA5)-Pz-Sting3 To a solution of compound 6 (200 mg) in anhydrous N,N-dimethylformamide (4 mL), bis(4-nitrophenyl) carbonate (95 mg) and diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 24 hours. Then, a solution of compound 47 (trifluoroacetate, 340 mg) and diisopropylethylamine (0.21 mL) in anhydrous N,N-dimethylformamide was added, and the mixture was stirred for 6 hours. Then, diethyl ether (50 mL) / hexane (10 mL) was added. The resulting precipitate was collected by filtration and dissolved in a mixture of dichloromethane (6 mL) and trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 30 min, then the solvent was removed under vacuum, and the residue was purified by RP-HPLC to give compound 50, trifluoroacetate (305 mg).

[0264] Compound 12 (50 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (50 μL) were added to a solution of compound 50 (trifluoroacetate, 87 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 51, trifluoroacetate (103 mg).

[0265] Compound 2 (6 mg) was added to a solution of compound 51 (24 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 52, PL-13, as a white solid (20 mg).

[0266] MS: 1229.5 [M+2H + ] / 2.

[0267] 2.14 PL-14: DiBr-VA-PAB(HyPA5)-Pz-Sting3 Compound 15 (56 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (50 μL) were added to a solution of compound 50 (trifluoroacetate, 87 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 53 as trifluoroacetate (105 mg).

[0268] Compound 2 (6 mg) was added to a solution of compound 53 (25 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 54, PL-14, as a white solid (22 mg).

[0269] MS: 1269.6 [M+2H + ] / 2.

[0270] 2.15 PL-15: DiBr-VA-PAB(SAR10)-Pz-Sting3 Compound 9 (40 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (50 μL) were added to a solution of compound 50 (trifluoroacetate, 87 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then directly purified by RP-HPLC to give compound 55 as trifluoroacetate (109 mg).

[0271] Compound 2 (6 mg) was added to a solution of compound 55 (23 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 56, PL-15, as a white solid (23 mg).

[0272] MS: 1134.0 [M+2H + ] / 2.

[0273] 2.16 PL-16: DiBr-VA-PAB(PEG12)-Pz-Sting3 To a solution of compound 50 (trifluoroacetate, 87 mg) in anhydrous N,N-dimethylformamide (2 mL), mPEG12-COOH (30 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (50 μL) were added. The mixture was stirred at room temperature for 20 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 57, trifluoroacetate (98 mg).

[0274] Compound 2 (6 mg) was added to a solution of compound 57 (21 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 58, PL-16, as a white solid (20 mg).

[0275] MS: 1044.5 [M+2H + ] / 2.

[0276] To investigate the effect of PA length and the combination of two amino acids in PA on ADC, the following drug-linker molecule was synthesized: 2.17 PL-17: DiBr-VA-PAB(PA2)-MMAE Compound 59 (23 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 30 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 60, trifluoroacetate (76 mg).

[0277] Compound 59 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0278] Compound 2 (6 mg) was added to a solution of compound 60 (16 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 61, PL-17, as a white solid (16 mg).

[0279] MS: 1852.0 [M+H + ].

[0280] 2.18 PL-18: DiBr-VA-PAB(PA8)-MMAE Compound 62 (75 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 63 as trifluoroacetate (114 mg).

[0281] Compound 62 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0282] Compound 2 (6 mg) was added to a solution of compound 63 (27 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 6 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 64, PL-18, as a white solid (26 mg).

[0283] MS: 1431.2 [M+2H + ] / 2.

[0284] 2.19 PL-19: DiBr-VA-PAB(PA12)-MMAE Compound 65 (108 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 60 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 66 as trifluoroacetate (155 mg).

[0285] Compound 65 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0286] Compound 2 (6 mg) was added to a solution of compound 66 (33 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 67, PL-19, as a white solid (32 mg).

[0287] MS: 1768.0 [M+2H + ] / 2.

[0288] 2.20 PL-20: DiBr-VA-PAB(PPA5)-MMAE Compound 68 (74 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to a solution of compound 38 (trifluoroacetate, 71 mg) in anhydrous N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 60 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 69, trifluoroacetate (113 mg).

[0289] Compound 68 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0290] Compound 2 (6 mg) was added to a solution of compound 69 (27 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 70, PL-20, as a white solid (25 mg).

[0291] MS: 1421.6 [M+2H + ] / 2.

[0292] 2.21 PL-21: DiBr-VA-PAB(PAA5)-MMAE Compound 71 (68 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 38 (trifluoroacetate, 71 mg). The mixture was stirred at room temperature for 60 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 20 min. The mixture was then purified directly by RP-HPLC to give compound 72, trifluoroacetate (113 mg).

[0293] Compound 71 was obtained by solid-phase peptide synthesis using Rink resin, Fmoc-Ala-OH, Fmoc-Pro-OH and cyclosuccinic anhydride as raw materials, and the final product was purified by RP-HPLC.

[0294] Compound 2 (6 mg) was added to a solution of compound 72 (25 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 73, PL-21, as a white solid (24 mg).

[0295] MS: 1356.6 [M+2H] + ] / 2.

[0296] 2.22 PL-22: DiBr-VA-PAB(AP5)-MMAE 2.23 PL-23: DiBr-VA-PAB(AP5-Glc)-MMAE To investigate the effect of PA coupling position on ADC, the following drug-linker molecules were synthesized: 2.24 PL-24: DiBr-Lys(PA5)-VA-PAB-Pz-Sting3 2.25 PL-25: DiBr-Dap(PA5)-VA-PAB-Pz-Sting3 2.26 PL-26: DiBr-Dap(PEG12)-VA-PAB-Pz-Sting3 To investigate the effect of coupling methods on ADCs, drug-linker molecules using different drugs were synthesized, and their structures are as follows: 2.27 PL-27: MC-VA-PAB-MMAE Compound 74 (18 mg) was added to a solution of compound 36 (57 mg) in N,N-dimethylformamide (2 mL), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 3 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 75, PL-27, as a white solid (54 mg).

[0297] MS: 1230.9 [M+H + ].

[0298] 2.28 PL-28: MC-VA-PAB(PA5)-MMAE Compound 74 (18 mg) was added to a solution of compound 39 (107 mg) in N,N-dimethylformamide (2 mL), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 5 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 86, PL-28, as a white solid (103 mg).

[0299] MS: 1107.7 [M+2H + ] / 2.

[0300] To investigate the effect of PA length and coupling with different toxins on ADCs, the following drug-linker molecules were synthesized: 2.29 PL-29: DiBr-VA-PAB(PA2)-Exatecan Compound 59 (24 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 58 mg). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then directly purified by RP-HPLC to give compound 76, trifluoroacetate (62 mg).

[0301] Compound 2 (6 mg) was added to a solution of compound 76 (14 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 77, PL-29, as a yellow solid (14 mg).

[0302] MS: 1569.5 [M+H + ].

[0303] 2.30 PL-30: DiBr-VA-PAB(PA8)-Exatecan Compound 62 (74 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 58 mg). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 78, trifluoroacetate (110 mg).

[0304] Compound 2 (6 mg) was added to a solution of compound 78 (24 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 79, PL-30, as a yellow solid (22 mg).

[0305] MS: 1289.5 [M+2H + ] / 2.

[0306] 2.31 PL-31: DiBr-VA-PAB(PA12)-Exatecan Compound 65 (108 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 58 mg). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 80, trifluoroacetate (135 mg).

[0307] Compound 2 (6 mg) was added to a solution of compound 80 (30 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 81, PL-31, as a yellow solid (29 mg).

[0308] MS: 1626.4 [M+2H + ] / 2.

[0309] 2.32 PL-32: DiBr-VA-PAB(PPA5)-Exatecan Compound 68 (73 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 58 mg). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 82, trifluoroacetate (102 mg).

[0310] Compound 2 (6 mg) was added to a solution of compound 82 (24 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 83, PL-32, as a yellow solid (22 mg).

[0311] MS: 1230.1 [M+2H + ] / 2.

[0312] 2.33 PL-33: DiBr-VA-PAB(PAA5)-Exatecan Compound 71 (66 mg), (7-azabenzotriazol-1-oxy)tripyrrolidinylphosphonium hexafluorophosphate (28 mg), and diisopropylethylamine (30 μL) were added to an anhydrous N,N-dimethylformamide (2 mL) solution of compound 8 (trifluoroacetate, 58 mg). The mixture was stirred at room temperature for 40 min. Then piperidine (100 μL) was added, and the mixture was stirred for another 10 min. The mixture was then purified directly by RP-HPLC to give compound 84, trifluoroacetate (93 mg).

[0313] Compound 2 (6 mg) was added to a solution of compound 84 (22 mg) in N,N-dimethylformamide (1 mL), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature for 4 hours, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 85, PL-33, as a yellow solid (22 mg).

[0314] MS: 1215.1 [M+2H + ] / 2.

[0315] 2.34 Synthesis of LD-1, the drug linker of glucocorticoids LD-1: DiBr-Dap(Glc-AP5-Sar)-VA-pF-Prednisolone Preparation of intermediate compound 6: 8 g of intermediate compound 1, N-[fluorenylmethoxycarbonyl]-L-valine-L-alanine (purchased from Jier Biochemical (Shanghai) Co., Ltd.), was added to a reaction flask, dissolved in 200 mL of dichloromethane and 40 mL of DMF. Then, 5.088 g of glycine tert-butyl ester (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), 16 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and 12.84 mL of 2,4,6-trimethylpyridine were added, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, 400 mL of dichloromethane was added for dilution, and the mixture was washed 5 times with water. After separation, the mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 10.144 g of intermediate compound 2.

[0316] MS (ESI) m / z: 524.3 [M + H] + .

[0317] 10.144 g of intermediate compound 2 was dissolved in 70 mL of dichloromethane, and 70 mL of trifluoroacetic acid was added. After the reaction was completed by HPLC monitoring, ethyl acetate was added and stirred for 2 h. The mixture was filtered and dried under vacuum to obtain 8.819 g of intermediate compound 3.

[0318] MS (ESI) m / z: 468.2 [M + H] + .

[0319] 8.819 g of intermediate compound 3, 18 mg of copper acetate, and 2.56 mL of acetic acid were added to the reaction flask. 9.960 g of lead tetraacetate (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) was dissolved in 180 mL of DMF and added dropwise to the reaction flask. The reaction system was purged with nitrogen, heated to 70 °C, and reacted for 4 hours. After the reaction was complete, ethyl acetate was added, the mixture was filtered, the filtrate was washed with water, separated, and the organic phase was concentrated to obtain 4.177 g of intermediate compound 4, with a yield of 46.1%.

[0320] MS (ESI) m / z: 482.2 [M + H] + .

[0321] Add 4.165 g of BD0260 prepared in Example 1 and 4.177 g of intermediate compound 4 to the reaction flask, dissolve in 300 mL of tetrahydrofuran, dissolve 1.954 g of potassium tert-butoxide in 90 mL of tetrahydrofuran to obtain a tetrahydrofuran solution of potassium tert-butoxide, purge the reaction system with nitrogen, and add the tetrahydrofuran solution of potassium tert-butoxide dropwise to the reaction flask under ice bath. After the addition is complete, continue the reaction under ice bath for 2 hours. After the reaction is complete, quench with water, extract with 80 mL of ethyl acetate, concentrate the organic phase, and obtain 4.012 g of intermediate compound 5, with a yield of 51.4%.

[0322] MS (ESI) m / z: 904.4 [M + H] + .

[0323] 4.012 g of intermediate compound 5 was added to the reaction flask, dissolved in 60 mL of dichloromethane, and then 13 mL of piperidine was added. The reaction was carried out at room temperature for 4 hours. After the reaction was complete, 630 mL of diethyl ether was added, stirred and slurried, filtered, and dried under vacuum to obtain 2.993 g of intermediate compound 6, with a yield of 98.5%.

[0324] MS (ESI) m / z: 681.3 [M + H] + .

[0325] Preparation of intermediate compound 7 To a solution of 2-tert-butylhydrazine-1,2-dicarboxylic acid ester (76.8 mg, 0.33 mmol) and tert-butyl 3-bromo-2-(bromomethyl)propionate (200 mg, 0.66 mmol) in anhydrous tetrahydrofuran (THF), NaH (60%, 80 mg, 2.0 mmol) was added. The mixture was stirred at room temperature for 15 minutes, and then the reaction was quenched with 1 mL of water containing 60 μL AcOH. The mixture was then purified by RP-HPLC. The purified fraction was lyophilized to give intermediate compound 8 as 204 mg of white solid.

[0326] MS m / z: 373.6 [M+H]+.

[0327] To 8.0 mL of acetic acid (AcOH) solution containing intermediate compound 8 (204 mg, 0.55 mmol), 3,4-dibromofuran-2,5-dione (140 mg, 0.55 mmol) was added. The mixture was refluxed and stirred under an argon atmosphere for 11 days, then concentrated to 3 mL and purified by RP-HPLC to give 43 mg of intermediate compound 7 as a white solid, yield: 22%.

[0328] MS m / z: 354.8 [M+H] + .

[0329] The synthetic route for LD-1 is as follows: Intermediate compound 19 (purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd., 2.503 g, 2.43 mmol) and intermediate compound 20 ((S)-2-(((9)) were added to the reaction flask. H tert-butyl fluorene-9-yl)methoxy)carbonyl)amino)-3-aminopropionate hydrochloride (purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., 1.075 g, 2.55 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., 1.100 g, 2.92 mmol), and 2,4,6-trimethylpyridine (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., 0.883 g, 7.29 mmol) were dissolved in 50 mL of N,N-dimethylacetamide (DMAC) and reacted at room temperature for 2 h. After the reaction was completed, 200 mL of ethyl acetate was added for dilution, and the mixture was washed 5 times with water. The organic phases were separated, combined, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to give 2.105 g of intermediate compound 21, with a yield of 61.2%.

[0330] MS-ESI: m / z = 1394.6 [M + H] + .

[0331] Intermediate compound 21 (2.105 g, 1.4 mmol) was dissolved in 30 mL of dichloromethane (DCM) in a reaction flask, and 10 mL of trifluoroacetic acid was added dropwise to the reaction solution. The reaction was carried out at room temperature for 16 h. After the reaction was completed, the crude product was concentrated. The crude product was prepared by C18 chromatography to give 1.370 g of intermediate compound 22, with a yield of 78.8%.

[0332] MS-ESI: m / z = 1238.4 [M + H] + .

[0333] Intermediate compound 12 (0.667 g, 1.17 mmol) and intermediate compound 22 (1.370 g, 1.10 mmol), along with DIPEA (1.200 mL, 7.0 mmol), were dissolved in 30 mL of DMAC in a reaction flask and reacted at room temperature for 2 h. After the reaction was complete, the mixture was diluted with 200 mL of ethyl acetate, washed five times with water, separated, and the organic phases were combined and concentrated to obtain the crude product. The crude product was then subjected to C18 reverse-phase reaction to obtain 1.502 g of intermediate compound 23, with a yield of 78.9%.

[0334] MS-ESI: m / z = 1626.8 [M + H] + .

[0335] Intermediate compound 6 (0.573 g, 0.85 mmol), intermediate compound 23 (1.377 g, 0.85 mmol), HATU (0.355 g, 0.94 mmol), and DIPEA (0.48 mL, 2.56 mmol) were dissolved in 20 mL of DMF in a reaction flask and stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was reversed in a C18 phase to prepare 0.805 g of intermediate compound 24, with a yield of 41.0%.

[0336] MS-ESI: m / z = 2290.3 [M + H] + .

[0337] Intermediate compound 24 (0.800 g, 0.35 mmol) was dissolved in 8 mL of methanol and 2 mL of water in a reaction flask. Lithium hydroxide hydrate (0.018 g, 0.43 mmol) was added under ice bath conditions, and the reaction was carried out for 1 h under ice bath conditions. After the reaction was completed, the reaction solution was reversed using a C18 phase to prepare 0.553 g of intermediate compound 25, with a yield of 82.7%.

[0338] MS-ESI: m / z = 1899.9 [M + H] + .

[0339] Intermediate compound 25 (0.380 g, 0.2 mmol), intermediate compound 7 (0.100 g, 0.3 mmol), 2-bromo-1-ethylpyridine tetrafluoroborate (BEP, purchased from Shanghai Yichang Chemical Co., Ltd., 0.082 g, 0.3 mmol), and DIPEA (0.100 mL, 0.6 mmol) were dissolved in 10 mL of DMF in a reaction flask and reacted at room temperature in the dark for 1 h. After the reaction was completed, the reaction solution was reversed in a C18 phase to prepare 0.205 g of compound LD-1, with a yield of 44.8%.

[0340] MS-ESI: m / z = 2233.8 [M + H] + .

[0341] 2.35 Synthesis of LD-2, the drug linker of glucocorticoids The synthesis route is as follows: Compound 26 (purchased from Hangzhou Gutuo Biotechnology, 2.695 g, 2.43 mmol) and compound 20 ((S)-2-(((9)) were added to the reaction flask. H 1.075 g (2.55 mmol) of tert-butyl fluorene-9-yl)methoxy)carbonyl)amino)-3-aminopropionate hydrochloride (purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd.), 1.100 g (2.92 mmol) of HATU (purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd.), and 0.883 g (7.29 mmol) of 2,4,6-trimethylpyridine (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) were dissolved in 50 mL of N,N-dimethylacetamide (DMAC) and reacted at room temperature for 2 h. After the reaction was completed, the mixture was diluted with 200 mL of ethyl acetate, washed five times with water, separated, and the organic phases were combined and concentrated to obtain the crude product. The crude product was subjected to column chromatography to give 2.235 g of compound 27, with a yield of 62.4%.

[0342] MS-ESI: m / z = 1474.7 [M + H] + .

[0343] Compound 27 (2.235 g, 1.5 mmol) was dissolved in 30 mL of dichloromethane (DCM) in a reaction flask, and 10 mL of trifluoroacetic acid was added dropwise to the reaction solution. The reaction was carried out at room temperature for 16 h. After the reaction was completed, the crude product was concentrated. The crude product was prepared by C18 chromatography to give 1.45 g of compound 28, with a yield of 73.3%.

[0344] MS-ESI: m / z = 1318.6 [M + H] + .

[0345] Compound 12 (0.667 g, 1.17 mmol), compound 28 (1.45 g, 1.10 mmol), and DIPEA (1.200 mL, 7.0 mmol) were dissolved in 30 mL of DMAC in a reaction flask and reacted at room temperature for 2 h. After the reaction was complete, the mixture was diluted with 200 mL of ethyl acetate, washed five times with water, separated, and the organic phases were combined and concentrated to obtain the crude product. The crude product was then subjected to C18 reverse-phase reaction to prepare 1.585 g of compound 29, with a yield of 84.4%.

[0346] MS-ESI: m / z = 1706.4 [M + H] + .

[0347] Compound 6 (0.633 g, 0.93 mmol), compound 29 (1.585 g, 0.93 mmol), HATU (0.389 g, 1.02 mmol), and DIPEA (0.49 mL, 2.79 mmol) were dissolved in 20 mL of DMF in a reaction flask and stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was reversed in a C18 phase to prepare 0.865 g of compound 30, with a yield of 39.3%.

[0348] MS-ESI: m / z = 2370.2 [M + H] + .

[0349] Compound 30 (0.865 g, 0.36 mmol) was dissolved in 8 mL of methanol and 2 mL of water in a reaction flask. Lithium hydroxide hydrate (0.022 g, 0.54 mmol) was added under ice bath conditions, and the reaction was carried out for 1 h under ice bath conditions. After the reaction was completed, the reaction solution was reversed in a C18 phase to prepare 0.603 g of compound 31, with a yield of 84.7%.

[0350] MS -ESI: m / z =1979.9[M +H]+.

[0351] Compound 31 (0.396 g, 0.2 mmol), compound 7 (0.100 g, 0.3 mmol), 2-bromo-1-ethylpyridine tetrafluoroborate (BEP, purchased from Shanghai Yichang Chemical Co., Ltd., 0.082 g, 0.3 mmol), and DIPEA (0.100 mL, 0.6 mmol) were dissolved in 10 mL of DMF in a reaction flask and reacted at room temperature in the dark for 1 h. After the reaction was completed, the reaction solution was reversed in a C18 phase to prepare 0.215 g of compound LD-2, with a yield of 46.4%.

[0352] MS-ESI: m / z = 2313.8 [M + H] + .

[0353] Example 3. Antibodies used in this disclosure For illustrative purposes only, the first antibody used in this application embodiment is the anti-Trop2 antibody RS7, whose amino acid sequence is shown in Table 5 below: Table 5. Sequence of anti-Trop2 antibody RS7

[0354] The second antibody used in this application is the anti-HER2 antibody Trastuzumab, the antibody sequence of which is described below: Heavy chain of Trastuzumab (SEQ ID NO: 12): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Light chain of Trastuzumab (SEQ ID NO: 13): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain variable region of Trastuzumab (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS Light chain variable region of Trastuzumab (SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK The third antibody used in the present application is the anti-HER2 antibody Pertuzumab, and its antibody sequence is described as follows: Heavy chain of Pertuzumab (SEQ ID NO: 14): EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Light chain of Pertuzumab (SEQ ID NO: 15): DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain variable region of Pertuzumab (SEQ ID NO: 18): EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS The light chain variable region of Pertuzumab (SEQ ID NO: 19): DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK The fourth antibody used in this application is the anti-IL-4R antibody BR206, which was prepared with reference to the H4H098P antibody in patent WO2008054606A2.

[0355] The amino acid sequence of BR206 is as follows: Table 5-1. Amino acid sequence of BR206

[0356] Example 4. Synthesis of ADC The disclosed antibody was reacted with a drug-linker molecule (PL) to prepare an ADC drug, and the DAR value of the ADC was determined.

[0357] In simple terms, antibodies are treated with reducing agents (such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT)) to reduce some or all of the cysteine ​​disulfide residues, forming highly nucleophilic cysteine ​​thiol groups (-CH2SH). This partially or completely reduced antibody then reacts with the electrophilic functional groups of the drug conjugate (such as maleimide or bromoacetyl) to ultimately prepare the ADC.

[0358] DAR value determination method: The DAR values ​​of the ADCs disclosed herein were analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADCs were separated in the column using a MabPac HIC-Butyl analytical column (4.6 × 100 mm, 5 μm, catalog number 088558, Thermo Fisher, USA).

[0359] Chromatographic conditions: Mobile phase A was 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and mobile phase B was 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile.

[0360] Gradient elution: 0-2 min 15% buffer B, 2-22 min 15%-85% buffer B, 22-25 min 85% buffer B, 25-28 min 85%-15% buffer B, 28-30 min 15% buffer B.

[0361] The flow rate and temperature were set to 0.5 mL / min and 25 °C, respectively. Drug distribution in the ADC was detected at 214 nm and 280 nm to calculate the DAR value.

[0362] 4.1 Preparation of ADC-1 (Trop2_RS7-DiBr-VA-PAB-Exatecan-DAR-4) Antibody RS7 dissolved in pH 8.0 was mixed with 2 mM EDTA and reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, drug linker PL-1 was added to the reduced antibody (drug linker:antibody molar ratio 6:1). The mixture was purified and desalted by elution with G25 resin overnight at room temperature, and then filtered through a 0.2 μm filter under aseptic conditions to obtain ADC-1, which was then frozen and stored.

[0363] Using the above-described hydrophobic interaction chromatography-high performance liquid chromatography analysis, the average DAR value of the ADC was determined to be between 3.7 and 4.3. Its structure is as follows: Where Ab represents RS7 antibody, and n is 3.7-4.3.

[0364] 4.2 Preparation of ADC-2 (Trop2_RS7-DiBr-VA-PAB-(PA)5-Exatecan-DAR4) Antibody RS7 dissolved in pH 8.0 was mixed with 2 mM EDTA and reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, drug linker PL-2 was added to the reduced antibody (drug linker:antibody molar ratio 6:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour. The purified ADC-2 was then filtered through a 0.2 μm filter under aseptic conditions and stored frozen.

[0365] Using the above-described hydrophobic interaction chromatography-high performance liquid chromatography analysis, the average DAR value of the ADC was determined to be between 3.7 and 4.3. Its structure is as follows: , Where Ab represents RS7 antibody, and n is 3.7-4.3.

[0366] 4.3 Preparation of ADC-3, ADC-4 and ADC-5: ADC-3, ADC-4, and ADC-5 were prepared using the same method as ADC-2. Analysis using the aforementioned hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR values ​​of the ADCs ranged from 3.7 to 4.3. Their structures are as follows: ADC-3: ; ADC-4: ; ADC-5: ; Where Ab represents RS7 antibody, and n is 3.7-4.3.

[0367] 4.6 Preparation of ADC-29 to ADC-33 Add 2 mM EDTA to antibody RS7 dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody is 8:1). After incubation at 37°C for approximately 120 minutes, add drug-linker PL29:DiBr-VA-PAB(PA2)-Exatecan, PL30:DiBr-VA-PAB(PA8)-Exatecan, PL31:DiBr-VA-PAB(PA12)-Exatecan, PL32:DiBr-VA-PAB(PPA5)-Exatecan, or PL33:DiBr-VA-PAB(PAA5)-Exatecan to the reduced antibody (molar ratio of drug-linker to antibody is 6:1), and react at room temperature for 1 hour. After the reaction was complete, the mixture was purified and desalted by elution with G25 resin. The resulting ADCs were filtered through a 0.2 μm filter under aseptic conditions to obtain ADC-29, ADC-30, ADC-31, ADC-32, and ADC-33, which were then frozen and stored. The average DAR value of the ADCs was determined to be between 3.7 and 4.3. Their structures are as follows: ADC-29: ADC-30: ADC-31: ADC-32: ADC-33: , Where Ab represents RS7 antibody, and n is 3.7-4.3.

[0368] 4.7 Preparation of ADCs coupled with Trastuzumab Add 2 mM EDTA to the antibody Trastusumab dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody is 10:1). After incubation at 37°C for approximately 120 minutes, the drug-linker PL7:DiBr-VA-PAB-MMAE, PL8:DiBr-VA-PAB(PA5)-MMAE, PL9:DiBr-VA-PAB(HyPA5)-MMAE, PL10:DiBr-VA-PAB(SAR10)-MMAE, PL11:DiBr-VA-PAB(PEG12)-MMAE, PL17:DiBr-VA-PAB(PA2)-MMAE, PL18:DiBr-VA-PAB(PA8)-MMAE, PL19:DiBr-VA-PAB(PA12)-MMAE, PL20:DiBr-VA-PAB(PPA5)-MMAE, or PL21:DiBr-VA-PAB(PAA5)-MMAE were added to the reduced antibody (the molar ratio of drug linker to antibody was 6:1), and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the mixture was filtered through a 0.2 μm filter under aseptic conditions to obtain ADC-7, ADC-8, ADC-9, ADC-10, ADC-11, ADC-17, ADC-18, ADC-19, ADC-20, and ADC-21, which were then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography determined that the average DAR value of the ADCs was between 3.7 and 4.3. Their structures are as follows: ADC-7: , ADC-8: , ADC-9: , ADC-10: , ADC-11: , ADC-17: , ADC-18: , ADC-19: , ADC-20: , ADC-21: , Where Ab represents Trastusumab, and n is 3.7-4.3.

[0369] 4.8 Preparation of ADCs coupled with Pertuzumab Add 2 mM EDTA to the Pertuzumab antibody dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody is 10:1). After incubation at 37°C for approximately 120 minutes, add the drug-linker PL12:DiBr-VA-PAB-Pz-Sting3, PL13:DiBr-VA-PAB(PA5)-Pz-Sting3, PL14:DiBr-VA-PAB(HyPA5)-Pz-Sting3, PL15:DiBr-VA-PAB(SAR10)-Pz-Sting3, or PL16:DiBr-VA-PAB(PEG12)-Pz-Sting3 to the reduced antibody (molar ratio of drug-linker to antibody is 6:1). Incubate PL12 at room temperature for 4 hours, and the other linkers at room temperature for 1 hour. After the reaction was complete, the mixture was purified and desalted by elution with G25 resin. The resulting ADCs were filtered through a 0.2 μm filter under aseptic conditions to obtain ADC-12, ADC-13, ADC-14, ADC-15, and ADC-16, which were then frozen and stored. The average DAR value of the ADCs was determined to be between 3.7 and 4.3. Their structures are as follows: ADC-12: , ADC-13: , ADC-14: , ADC-15: , ADC-16: Where Ab represents Pertuzumab monoclonal antibody, and n is 3.7 to 4.3.

[0370] 4.9 Preparation of ADCs conjugated with IL-4R antibodies Preparation of ADC-40: The IL-4R antibody BR206 was dissolved in PBS at pH 8.0, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 12:1). After incubation overnight at 37°C, the tris(2-carboxyethyl)phosphine hydrochloride reducing agent was removed by purification with G25 resin. The drug linker LD-1 was added to the reducing antibody (molar ratio of drug linker to antibody was 5:1), and the reaction was carried out at room temperature for 1 hour. After elution and desalting with G25 resin, the solution was filtered through a 0.2 μm filter under aseptic conditions to obtain IL-4R-ADC, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography determined that the average DAR value of IL-4R-ADC was between 3.7 and 4.3. Its structure is as follows: .

[0371] Using the same experimental method, LD-1 was conjugated with the control antibody hIgG4 to obtain IgG4-ADC, which served as an isotype control ADC. Its average DAR value was between 3.7 and 4.3. Its structure was the same as ADC-40, with only the antibody portion being different.

[0372] Similarly, coupling LD-2 with BR206 yields ADC-41, with an average DAR value between 3.7 and 4.3.

[0373] 4.10 Preparation of BiADC (DiBr-VA-PAB(PA5)-Exatecan + DiBr-VA-PAB(PA5)-MMAE) Antibody Trop2_RS7 dissolved in PBS at pH 8.0 was mixed with 2 mM EDTA and reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 37°C for approximately 120 minutes, drug linkers PL-2 and PL-8 were mixed and added to the reduced antibody (molar ratio of total drug linker to antibody was 6:1). After the reaction was complete, the mixture was purified and desalted by elution with G25 resin and filtered through a 0.2 μm filter under aseptic conditions to obtain BiADC, which was then frozen and stored.

[0374] Using the above hydrophobic interaction chromatography-high performance liquid chromatography analysis, the average total DAR value of BiADC was determined to be between 3.3 and 4.3, i.e., n1+n2=3.3~4.3. Its structure is as follows: .

[0375] For example, the ADCs synthesized in this disclosure are shown in Table 6 below.

[0376] Table 6. ADCs in the embodiments of this application

[0377] Table 6-1. ADCs in the embodiments of this application

[0378] Test case Test Example 1. Detection of Coupling Reaction by ADC Add 2 mM EDTA to antibody RS7 dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody 10:1). After incubation at 37°C for approximately 120 minutes, add drug-linker PL1:DiBr-VA-PAB-Exatecan, PL2:DiBr-VA-PAB-(PA)5-Exatecan, or PL3:DiBr-VA-PAB-(HyPA)5-Exatecan to the reduced antibody (molar ratio of drug-linker to antibody 6:1), react at room temperature for 1 hour, 4 hours, or overnight, and take samples for analysis using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to determine the degree of completion of the coupling reaction.

[0379] After the reaction was complete, the mixture was eluted and purified using G25 resin, then filtered through a 0.2 μm filter under aseptic conditions to obtain ADC-1, ADC-2, and ADC-3, which were then frozen and stored. The coupling results for each ADC are shown in the figure below. Figures 1A-1E .

[0380] The results showed that ADC-2 and ADC-3 completed the coupling reaction after 1 hour at room temperature; however, 29% of ADC-1 remained incompletely reacted after 1 hour at room temperature; and 6% of ADC-1 remained incompletely reacted after 4 hours at room temperature. The coupling reaction was only fully completed after overnight reaction at room temperature. This clearly demonstrates that the coupling speed between linkers containing PA and HyPA side chains and antibodies is significantly faster than that between linkers without PA and HyPA side chains.

[0381] Test Example 2. Detection of hydrophilicity and aggregate content of ADC 2.1 Hydrophilicity The hydrophilicity of the ADC disclosed herein was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADC was separated in the column using a MabPac HIC-Butyl analytical column (4.6 × 100 mm, 5 μm, catalog number 088558, ThermoFisher, USA).

[0382] The chromatographic conditions are as follows: Mobile phase A is a 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and mobile phase B is a 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile.

[0383] Gradient elution: 0-2 min 15% buffer B, 2-12 min 15%-85% buffer B, 12-15 min 85% buffer B, 15-18 min 85%-15% buffer B, 28-30 min 15% buffer B.

[0384] The flow rate and temperature were set to 0.5 mL / min and 25 °C.

[0385] The elution time of the ADC was detected at 280nm. The earlier the elution, the higher the hydrophilicity of the ADC and the more stable the ADC is in vivo.

[0386] 2.2 Aggregates The aggregate content of the ADCs disclosed herein was analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). The ADCs were separated in the column using a Biozen dSEC-2 analytical column (4.6 × 150 mm, 3 μm, catalog number 00F-4788-E0, Phenomenex, USA).

[0387] The chromatographic conditions are as follows: The mobile phase was 100 mM sodium phosphate buffer (pH 6.8) containing 10% acetonitrile. The flow rate and temperature were set to 0.35 mL / min and 25 °C, respectively.

[0388] The ADC elution time was detected at 280 nm. The ADC aggregates, with their relatively large molecular weight, elute before the main peak. The aggregate content was calculated. The results are shown in Table 7.

[0389] Table 7. Hydrophilicity and Aggregates of ADCs

[0390] The results showed that ADC-2 and ADC-3 with PA or HyPA side chains had the fewest aggregates, while ADC-5 and ADC-4 with hydrophilic polysarcosine side chains both contained a certain amount of aggregates (4-5%). ADC-1, without hydrophilic side chains, had the highest hydrophobicity and the highest aggregate content, exceeding 10%. This clearly demonstrates that using PA hydrophilic side chains can significantly reduce the formation of ADC aggregates. Moreover, the aggregate reduction effect of PA hydrophilic side chain linkers is better than that of polysarcosine side chain linkers.

[0391] Test Example 3. Pharmacokinetic Detection of ADC ADC-1 and ADC-2 were intravenously injected at a dose of 5 mpk into Sprague Dawley rats (purchased from Charles River, train code 001), with two rats in each group. Whole blood was collected via jugular vein at 5 minutes, 1 hour, 12 hours, day 1, day 2, day 7, day 14, and day 21 after ADC injection. After clotting for 30 minutes, serum was obtained by centrifugation at 10,000 rpm for 30 minutes at 4°C. Total antibodies and ADC levels in the serum were measured using ELISA.

[0392] When detecting total antibodies, the ADC specifically binds to the coated hTrop2 antigen (purchased from Acrobiosystems Cat# TR2-H5223). Then, Peroxidase-conjugated AffiniPure Goat anti-Human IgG (purchased from Jackson Immuno Research Laboratories, INC Cat# 109-035-190) is added to specifically bind the antibody portion of the ADC. Finally, a substrate is added to perform a colorimetric reaction. The color intensity is positively correlated with the ADC concentration within a certain range.

[0393] In the ADC detection, the ADC specifically binds to the coated hTrop2 antigen (purchased from Acrobiosystems, Cat# TR2-H5223). Then, mouse anti-Exatecan / DXD antibody (purchased from Acrobiosystems, Cat# DXD-M684) is added to specifically bind to the sample containing Exatecan. Next, Peroxidase-conjugated AffiniPure Goat anti-Mouse IgG (purchased from Jackson Immuno Research Laboratories, INC, Cat# 115-035-164) is added to bind to the mouse Exatecan antibody. Finally, the substrate is added for a colorimetric reaction. The color intensity is positively correlated with the ADC concentration within a certain range. The results are shown in Table 8. Figure 2A and Figure 2B .

[0394] Table 8. Half-life of ADCs

[0395] The results showed that the total antibody t1 / 2 of ADC-2 was 8.1 days, higher than that of ADC-1 (6.64 days). The ADC half-life showed a similar trend, with the t1 / 2 of ADC-2 being 7.4 days, higher than that of ADC-1 (7.0 days). These results indicate that ADCs with hydrophilic PA side chains have a longer in vivo half-life than ADCs without hydrophilic chains.

[0396] Test Example 4: Antibody-Drug Conjugate (ADC) Killing Experiment on Tumor Cells This experiment used CellTiterGlo2 (Promega) to evaluate cell death induced by drugs conjugated with antibodies using different linkers.

[0397] The tumor cell lines BxPC-3 (human pancreatic cancer cells, CRL-1687, ATCC) and HCC1954 (human breast cancer cells, CRL-2338, ATCC) used in the experiment were both purchased from ATCC. The culture medium for both BxPC-3 and HCC1954 cells, as well as the test medium, was Gibco RPMI-1640 medium containing 10% fetal bovine serum and 100 units of penicillin-0.1 mg / mL streptomycin. The tumor cells were cultured at 37°C in a 5% CO2 incubator.

[0398] The experimental procedure is as follows: Logarithmic growth phase BxPC-3 and HCC1954 tumor cells were dissociated with 0.25% Trypsin, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in fresh culture medium. Cell counts were performed twice, the cells were mixed, and the cell density was adjusted to 4-10 × 10⁻⁶ cells / cm². 4 Cells / mL were seeded at 50 μL / well in 96-well white transparent substrate cell culture plates. After 24 hours of culture, antibody-drug conjugates diluted with different concentrations of culture medium were added at 50 μL / well, for a final volume of 100 μL / well. Each concentration was replicated, and corresponding solvent control wells were included. After culturing at 37°C in a 5% CO2 incubator for 144 hours, 100 μL of CellTiterGlo2 was added to each well, and the mixture was placed on a shaker at room temperature for 15 minutes. The luminescence value was then measured. After exporting the data, the IC50 of the test drug against various tumor cells was calculated using Prism10. 50 Value (nM), IC 50 See Table 9 for a summary.

[0399] Table 9. Inhibition IC50 of Trop2 antibody-drug conjugates on the proliferation of various tumor cells 50 Value (nM)

[0400] The results showed that the cytotoxic activity of the Trop2 antibody-drug conjugates using different linkers disclosed in this study was comparable against BxPC-3 and HCC1954 tumor cells, with no significant difference. This demonstrates that using linkers with hydrophilic PA side chains does not affect the overall function and cellular activity of the ADC molecule.

[0401] Table 9-1. IC50 of Trop2 antibody-drug conjugate on the inhibitory effect of HCC1954 cell proliferation. 50 Value (nM)

[0402] The results showed that the Trop2 antibody-drug conjugates ADC29, ADC2, ADC30, and ADC31, with 2–12 PA linkers, exhibited comparable cytotoxic activity against HCC1954 tumor cells to those of ADC1, which lacked hydrophilic side chains. This demonstrates that using linkers with hydrophilic PA side chains does not affect the overall function and cellular activity of the ADC molecule.

[0403] Meanwhile, the antibody-drug conjugates ADC32 and ADC33, containing PPA and PAA side chains, exhibited comparable killing activity against HCC1954 tumor cells, and were comparable to those of the antibody-drug conjugate ADC1, which lacked hydrophilic side chains. This indicates that the addition of PPA and PAA hydrophilic side chains does not affect the overall function and cellular activity of the ADC molecule.

[0404] Test Example 5. Effects of Linkers of Different Lengths and Structures on the Hydrophilicity and Aggregate Content of ADCs 5.1 Coupling Reaction Add 2 mM EDTA to antibody RS7 dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody 10:1). After incubation at 37°C for approximately 120 minutes, add drug-linker PL29:DiBr-VA-PAB(PA2)-Exatecan, PL30:DiBr-VA-PAB(PA8)-Exatecan, PL31:DiBr-VA-PAB(PA12)-Exatecan, PL32:DiBr-VA-PAB(PPA5)-Exatecan, or PL33:DiBr-VA-PAB(PAA5)-Exatecan to the reduced antibody (molar ratio of drug-linker to antibody 6:1), react at room temperature for 1 hour, and then sample for analysis using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to determine the completeness of the coupling reaction. After the reaction was complete, the mixture was eluted and purified with G25 resin and desalted. The mixture was then filtered through a 0.2 μm filter under sterile conditions to obtain ADC-30, ADC-31, ADC-32, ADC-33 and ADC-34, which were then frozen and stored.

[0405] 5.2 Detection of hydrophilicity and aggregate content of ADC The hydrophilicity of the ADC disclosed herein was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC), and the aggregate content of the ADC disclosed herein was analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). The results are shown in Table 10.

[0406] Table 10. Hydrophilicity and Aggregates of ADCs

[0407] The results showed that the peak times of ADC-30, ADC-31, ADC-32, and ADC-33, which have 2 to 12 PA and 5 PPA or PAA side chains, were similar to those of ADC-2 with 5 PA side chains, and the aggregate content was around 2.0%. This fully demonstrates that using PA hydrophilic side chains of different lengths, or PPA and PAA side chains composed of proline and alanine, can significantly improve the hydrophilicity of ADC molecules and reduce the formation of ADC aggregates.

[0408] Test Example 6. Effect of PA linkers on the hydrophilicity and aggregate content of ADCs conjugated with different antibodies and toxins. 6.1 Coupling Reaction Add 2 mM EDTA to the antibody Trastusumab dissolved in PBS at pH 8.0, and reduce the antibody with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody is 10:1). After incubation at 37°C for approximately 120 minutes, the drug-linker PL7:DiBr-VA-PAB-MMAE, PL8:DiBr-VA-PAB(PA5)-MMAE, PL9:DiBr-VA-PAB(HyPA5)-MMAE, PL17:DiBr-VA-PAB(PA2)-MMAE, PL18:DiBr-VA-PAB(PA8)-MMAE, PL19:DiBr-VA-PAB(PA12)-MMAE, PL20:DiBr-VA-PAB(PPA5)-MMAE, or PL21:DiBr-VA-PAB(PAA5)-MMAE were added to the reduced antibody (the molar ratio of drug linker to antibody was 6:1). After reacting at room temperature for 1 hour, the reaction was analyzed by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to determine the degree of completion of the coupling reaction. After the reaction was complete, the mixture was eluted and purified with G25 resin and desalted. The mixture was then filtered through a 0.2 μm filter under sterile conditions to obtain ADC-7, ADC-8, ADC-9, ADC-17, ADC-18, ADC-19, ADC-20, and ADC-21, which were then frozen and stored.

[0409] 6.2 Detection of hydrophilicity and aggregate content of ADC The hydrophilicity of the ADC was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC), and the aggregate content of the ADC was analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). The results are shown in Table 11.

[0410] Table 11. Hydrophilicity and Aggregates of ADCs

[0411] The results showed that the peak times of ADCs with 2 to 12 PA and 5 PPA or PAA side chains were significantly earlier than those of ADC-7 without side chains, indicating that the use of PA hydrophilic side chains in ADCs can significantly improve the hydrophilicity of ADC molecules.

[0412] Test Example 7. Killing Experiment of Antibody-Drug Conjugate on Tumor Cells HT29 This experiment used CellTiterGlo2 (Promega) to evaluate cell death induced by drugs conjugated with Trastuzumab antibody and linked by different linkers to small molecule MMAE.

[0413] The tumor cell line HT29 (human colon cancer cells, HTB-38, ATCC) used in the experiment was purchased from ATCC. Both the culture medium for HT29 tumor cells and the test medium were Gibco RPMI-1640 medium containing 10% fetal bovine serum and 100 units of penicillin-0.1 mg / mL streptomycin. Tumor cells were cultured at 37°C in a 5% CO2 incubator.

[0414] The experimental procedure is as follows: HT29 tumor cells in logarithmic growth phase were dissociated with 0.25% Trypsin, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in fresh culture medium. Cell counts were performed twice, the cells were mixed, and the cell density was adjusted to 4-10 × 10⁻⁶ cells / cm². 4 CellTiterGlo2 was seeded at 50 μL / well in 96-well white transparent substrate cell culture plates. After 24 hours of culture, different concentrations of medium diluted with the Her2 antibody-drug conjugate were added at 50 μL / well, for a final volume of 100 μL / well. Each concentration was replicated, and corresponding solvent control wells were included. After culturing at 37°C in a 5% CO2 incubator for 96 hours, 100 μL of CellTiterGlo2 was added to each well, and the mixture was placed on a fixed-track shaker at room temperature for 15 minutes. The luminescence value was measured, and the data were exported and used to calculate the IC50 of the test drug against various tumor cells using Prism10. 50 Value (nM), IC 50 See Table 12 for a summary.

[0415] Table 12. Inhibition IC50 of Trop2 antibody-drug conjugate on HT29 tumor cell proliferation 50 Value (nM)

[0416] The results showed that Her2 antibody-drug conjugates ADC17, ADC8, ADC18, and ADC19, with 2–12 PA linkers, exhibited superior cytotoxic activity against HT29 tumor cells compared to ADC7, which lacked hydrophilic side chains. The more PA repeats, the higher the activity. This demonstrates that using linkers with hydrophilic PA side chains can enhance the overall function and cellular activity of ADC molecules.

[0417] Meanwhile, the antibody-drug conjugates ADC20, ADC21, and ADC9, which contain PPA, PAA, and HyPA side chains, exhibited comparable cytotoxic activity against HT29 tumor cells, and all showed superior activity compared to the antibody-drug conjugate ADC7, which lacked hydrophilic side chains. This indicates that the addition of hydrophilic side chains can enhance the overall function and cellular activity of ADC molecules.

[0418] Test Example 8. Effect of PA linkers on the hydrophilicity and aggregate content of ADCs conjugated with different antibodies and non-toxic drugs. 8.1 Coupling Reaction 2 mM EDTA was added to the antibody Pertuzumab dissolved in PBS at pH 8.0, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 37°C for approximately 120 minutes, drug-linkers PL12:DiBr-VA-PAB-Pz-Sting3, PL13:DiBr-VA-PAB(PA5)-Pz-Sting3, and PL14:DiBr-VA-PAB(HyPA5)-Pz-Sting3 were added to the reduced antibody (molar ratio of drug-linker to antibody was 6:1). PL12 was reacted at room temperature for 4 hours, and the other linkers were reacted at room temperature for 1 hour. The reaction was then analyzed by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to determine the completeness of the coupling reaction. After the reaction was complete, the mixture was eluted and purified with G25 resin and desalted. The mixture was then filtered through a 0.2 μm filter under sterile conditions to obtain ADC-12, ADC-13, and ADC-14, which were then frozen and stored.

[0419] 8.2 Detection of hydrophilicity and aggregate content of ADC The hydrophilicity of ADC-12, ADC-13, and ADC-14 was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The aggregate content of ADC-12, ADC-13, and ADC-14 was analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). The results are shown in Table 13.

[0420] Table 13. Hydrophilicity and Aggregates of ADCs

[0421] The results showed that the conjugation of the linker-drug load containing PA and HyPA side chains to the antibody was completed within 1 hour, significantly faster than that of the linker-drug load without PA and HyPA side chains (completed within 4 hours). The aggregates of ADC-13 and ADC-14 with PA and HyPA side chains were reduced, while the aggregates of ADC-12 without hydrophilic side chains were the highest, reaching 7.6%. This indicates that using PA and HyPA hydrophilic side chains in ADCs can accelerate the conjugation reaction and significantly reduce the formation of ADC aggregates.

[0422] Test Example 9. Immune Cell Activation Assay with STING Agonists and Antibody-Drug Conjugates The tumor cell lines used in the experiment were HCC1954 (human breast cancer cells, CRL-2338, ATCC) which highly expressed the Her2 antigen, and HT29 (human colon cancer cells, HTB-38, ATCC) which lowly expressed the Her2 antigen. The Invivogen THP1-Dual-KI-hSTING-R232 reporter cell line and QUANTI-Luc™ 4 Lucia / Gaussia reagent were used to evaluate the activation of the IRF pathway in immune cells by STING agonists and their antibody-drug conjugates. Tumor cells HCC1954 and HT29 were cultured in Gibco RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The reporter cell line was cultured in Gibco RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 100 µg / mL Normocin™, 0.1 mg / mL streptomycin, 100 units of penicillin, 10 µg / mL methimazole, and 100 µg / mL bleomycin in a 5% CO2 incubator at 37°C.

[0423] The experimental procedure is as follows: Tumor cells at 2×10 3 -5×10 3 Seed cells at a density of 50 μL / well in a 96-well plate and cultured for 24 h. Then add 5 × 10⁶ reporter cells. 4 25 μL of antibody-drug conjugate diluted with different concentrations of culture medium per well, for a final volume of 100 μL per well. Each concentration was replicated, and corresponding solvent control wells were included. After incubation at 37°C in a 5% CO2 incubator for 48 h, 10 μL of supernatant was collected per well of the 96-well plate, and 15 μL of QUANTI-Luc™ 4 Lucia / Gaussia reagent was added per well. The luminescence value was measured, and the EC50 of the antibody-drug conjugate for immune cell activation was calculated. 50 Value (nM). The results are shown in Table 14.

[0424] Table 14. Effects of antibody-drug conjugates on EC activation of immune cells 50 Value (nM)

[0425] The results showed that antibody-drug conjugates can effectively activate the IRF pathway in reporter cells after antigen-antibody binding, thus more effectively activating immune cells. ADC-12, along with ADC-13 and ADC-14 (which have hydrophilic side chains), showed comparable activity in activating the STING pathway; this indicates that the hydrophilic side chains do not affect the activity of the antibody-drug conjugates, and all can effectively activate the STING pathway in effector cells through target cells.

[0426] Test Example 10. IL-4R ADC Activity Test 10.1 Affinity detection of antibody-drug conjugates and monoclonal antibodies with human IL-4Rα antigen protein Affinity assays were performed using a Gator affinity analyzer (Gator Bio). First, the anti-His probe (GatorBio, Cat: 160009) was immersed in PBS for 300 seconds to establish a baseline signal. Then, the probe was loaded with 5 µg / mL human IL-4Rα protein (Sino Biological, Cat: 10402-H08H), pre-diluted in PBS, for approximately 180 seconds, followed by a 30-second PBS wash. Next, the bound IL-4Rα was interacted with 400 nM to 6.25 nM IL-4R-ADC (ADC-40) or BR206 antibody for 180 seconds, followed by a 300-second dissociation step. Binding and dissociation curves were analyzed using GatorPrime software (Gator Bio, version 1.0), and binding kinetic parameters (koff, kon, and KD) were calculated. The results are shown in Table 15.

[0427] The results showed that the binding activity of IL-4R-ADC (ADC-40) with hydrophilic side chains to human IL-4Rα antigen protein was comparable to that of naked anti-BR206.

[0428] Table 15. Affinity of IL-4R antibody or ADC-40 to human IL-4Rα protein

[0429] 10.2 ADC-induced FKBP5 expression in PBMCs Peripheral blood mononuclear cells (PBMCs) from healthy donors were seeded into 96-well plates (Costar, Corning, Cat: 3595) at a density of 200,000 cells per well. The cells were incubated with BR206 monoclonal antibody or IL-4R-ADC (ADC-40) (initial concentration 800 nM, serially diluted 1:4) at 37°C for 24 hours. After culture, the PBMCs were washed with PBS and stained for viable cells using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher, Cat: L34957). Surface staining was performed using the following markers: CD3 (BioLegend, Cat: 300408), CD14 (BioLegend, Cat: 325608), CD16 (BioLegend, Cat: 302008), and CD19 (BioLegend, Cat: 302228). For intracellular staining, cells were fixed at 4°C for 45 min using True-Nuclear™ Transcription Factor Fixation Buffer (BioLegend, Cat: 424401). After fixation, cells were washed twice with True-Nuclear™ Permeabilization Buffer (BioLegend, Cat: 424402) and resuspended in the same buffer. Cells were then incubated at room temperature in the dark for 45 min with Alexa Fluor 647-labeled anti-FKBP51 antibody (Abcam, Cat: ab228086) or isotype control antibody (Abcam, Cat: ab199093). After incubation, cells were washed twice again with buffer containing permeabilizing agent and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% BSA). Fluorescence signals were detected using a flow cytometer (Agilent Technologies, NovoCyte), and data analysis was performed using FlowJo software (FlowJo, LLC). Unstained samples and isotype control samples were used to determine background fluorescence and the location of negative cell populations. Results are as follows: Figure 3A and 3B As shown.

[0430] The results showed that IL-4R-ADC (ADC-40) could induce FKBP5 expression in monocytes and B cells, indicating that the drug load of IL-4R-ADC could effectively activate the transduction of downstream hormone receptor signals.

Claims

1. Ligand drug conjugates of formula (I), their isomers, pharmaceutically acceptable salts or mixtures thereof, (I) in: Ab is a ligand; L represents the connector unit; D represents the drug or payload; n represents the average number of connections, and n is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8; PA has the following structure: -L P -[(AA1) s -(AA2) t ] p -R a Where L P It is a key or connecting unit, and L P It forms a covalent connection with L; preferably, L P Selected from -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene -C(O)-, -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene-N(R) 1 )-、-C(O)-C 1-6 Alkylenes -C(O)-, -C(O)-, -C(O)O-, -C(O)-C 1-10 Alkylene-N(R) 1 )-、-C(O)-C 1-10 Alkylene-C(O)-N(R) 1 )-、-C(O)-C 1-10 Alkylene-N(R) 1 -C(O)-、-C 1-10 Alkylene-N(R) 1 )-、-C 1-10 Alkylene-C(O)-N(R) 1 )-、-C 1-10 Alkylene-N(R) 1 )-C(O)-, or =N-(O or N)-C 1-10 Alkylene-N(R) 1 )-; R 1 For H or C 1-6 alkyl; AA1 and AA2 together form a peptide chain, and the first AA1 binds with L... P Covalent bonds are formed between them, such as amide bonds; AA1 and AA2 are different and are each independently selected from proline, hydroxyproline or alanine; s and t are each independently 1, 2, 3 or 4, preferably 1 or 2; p is an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably an integer from 5 to 15; R a The group is absent or is a capped group, such as -CR b -OR b -N(R) b )2、-C(O)-R b -C 1-6 Alkylenes -C(O)OH, -CN, -NR b -C(O)-R b -C 1-6 -C(O)-R b -C(O)-N(R) b )2、-SO3 - -SO3H, -S(O)2R b -OS(O)2OR b -S(O)2NR b -S(O)R b -OP(O)(OR) b )2、-P(O)(OR b )2、-PO3 - -PO3H2, -C(=S)R b -CO2R b -CO2, -C(=S)OR b -C(O)SR b -C(=S)SR b C(=O)N(R) b 2. C(=S)N(R) b )2 or C(=NR) b )N(R b )2, where R b Independently selected from -H, -C 1-20 Alkyl, -C 6-20 Aryl, -C 3-14 cycloalkyl, or -C 3-14 Heterocyclic groups, the -C 1-20 Alkyl groups can be modified with glycosyl groups; preferably, R a Selected from -NH2, C 1-6 Alkyl-, Glycosyl-C 1-6 Alkyl group -, -C(O)OH, -OH or -CH3.

2. The ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 1, wherein AA1 is proline or hydroxyproline, and AA2 is alanine; or AA1 is alanine, and AA2 is proline or hydroxyproline.

3. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to claim 1 or 2, wherein the L in formula (I) has a -L1-L2- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , , , , , , , , , , , , and , where ** represents the junction with the ligand and * represents the junction with L2; L2 is a splittable linker, in which the two ends of the L2 main chain are connected to L1 and D respectively, and PA is coupled to L2, thus forming the structure shown in equation (IA): (IA) Wherein Ab, D and n are as defined in claim 1.

4. The ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 3, wherein the L2 has a -L3-L4-L5-L6- structure, wherein: L3 represents a bond or spacer unit, such as -(CH2). m -C(=O)-, where m is an integer from 0 to 10, preferably an integer from 0 to 5, such as 0, 1, 2, 3, 4, or 5; or L3 is -C(O)-, -C1-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)- or -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a bond, or a parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2-10, preferably 2-5, more preferably 2, 3, or 4 natural or non-natural amino acids; wherein each of the natural or non-natural amino acids is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one of the reactive functional groups of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; preferably, the natural or non-natural amino acid is selected from, for example, lysine (Lys), arginine (Arg), 2,3-diaminopropionic acid (Dap), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), or ornithine (Orn), more preferably lysine or 2,3-diaminopropionic acid; L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine, citrulline, lysine, glycine, phenylalanine, alanine, proline, isoleucine, serine, glutamic acid, aspartic acid, glutamine, asparagine, and 2,3-diaminopropionic acid; L6 is the key, or L6 is selected from the following structures: or , Where * represents the connection point with L5, # represents the connection point with D, and PA is as defined in claim 1; The condition is that L4 and L6 are not both keys.

5. The ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 3, wherein the L2 has a -L3-L4-L5-L6- structure, wherein: L3 represents a bond or spacer unit, such as -(CH2). m -C(=O)-, where m is an integer from 0 to 10, preferably an integer from 0 to 5, such as 0, 1, 2, 3, 4, or 5; or L3 is -C(O)-, -C1-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(=O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)-, -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a bond or a parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2-10, preferably 2-5, more preferably 2, 3, or 4 natural or non-natural amino acids; wherein each of the natural or non-natural amino acids is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA; preferably, the natural or non-natural amino acid is selected from, for example, lysine (Lys), arginine (Arg), 2,3-diaminopropionic acid (Dap), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), or ornithine (Orn), more preferably Lys or Dap; L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of a peptide chain formed by 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine (Val), citrulline (Cit), lysine (Lys), glycine (Gly), phenylalanine (Phe), alanine (Ala), proline (Pro), isoleucine (Ile), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), and 2,3-diaminopropionic acid (Dap); L6 is the key; or L6 is selected from -N(R) 1 )-C1- 10 Alkylene-, -C(O)-C1- 10 Alkylene-C(O)-, -N(R) 1 )-C1- 10 Alkylene-N(R) 1 -C(O)- or -N(R) 1 )-C1- 10 Alkylene-N(R) 1 )-C1- 10 Alkylene; preferably, L6 is -N(R 1 -C1-6 alkylene-, for example -N(R 1 )-CH2-; where R 1 For H or C 1-6 Alkyl; or L6 is selected from , , , , , , , , , , and , Where a * Indicates the connection point with L5, b * The connection point with D is indicated, and PA is as defined in claim 1; The conditions are: L4 and L6 are not simultaneously bonds, and at least one of L4 and L6 is covalently connected to PA.

6. The ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 4, wherein the reactive functional group is selected from: amino, carboxylic acid, activated carboxylic acid (e.g., acyl chloride), hydroxyl, amide, carboxylic acid ester, thiol, Michael receptor (e.g., maleimide), halogen (e.g., bromine), azide, alkyne, isocyanate, etc.

7. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-4 and 6, wherein the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit coupled to a ligand; preferably, L1 is selected from... , where ** represents the junction with the ligand and * represents the junction with L3; L3 represents a bond or spacer unit, such as -C(O)- or -Cl-C. 10 Alkylene-C(O)-; L4 is a bond or parallel linking unit, wherein the parallel linking unit is a natural amino acid, a non-natural amino acid, or a short peptide, and the short peptide is composed of a peptide chain formed by 2-5 natural amino acids or non-natural amino acids; wherein each of the natural amino acids or non-natural amino acids is independently selected from lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine; preferably, the natural amino acid or non-natural amino acid is lysine or 2,3-diaminopropionic acid; L5 is as defined in claim 4; L6 is as defined in claim 4; The condition is that L4 and L6 are not both keys.

8. The ligand-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 7, wherein L4 is a parallel linking unit, said parallel linking unit being a natural or non-natural amino acid or a short peptide, said short peptide being composed of a peptide chain formed of 2-5 natural or non-natural amino acids; wherein said natural or non-natural amino acid is lysine or 2,3-diaminopropionic acid, wherein said reactive functional group of the side chain is ε-amino or β-amino, and wherein at least one ε-amino or β-amino group is associated with L in PA. P An amide bond is formed.

9. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 4 and 6-8, wherein L6 has the following structure: or , Where * represents the connection point with L5, # represents the connection point with D, and PA is as defined in claim 1.

10. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-9, wherein L P -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene -C(O)-, -C 0-6 Alkylene-N(R) 1 )-C(O)-C 1-6 Alkylene-N(R) 1 )-、-C(O)-C 1-6 Alkylene-N(R) 1 - or -C(O)-C 1-6 alkylene-C(O)-, where R 1 For H or C 1-6 alkyl.

11. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-10, wherein L P is -CH2-N(CH3)-CO-(CH2)2-CO-, -CH2-N(CH3)-CO-(CH2)-N(CH3)-, -C(O)-CH2-N(CH3)-, or -CO-(CH2)2-CO-.

12. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-11, wherein PA is selected from the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or , in: p is as defined in claim 1. R a As defined in claim 1, preferably, R is the end-capping group. a Selected from -NH2, C 1-6 Alkyl-, Glucose-C 1-6 Alkyl group -, -C(O)OH, -OH or -CH3, The wavy line indicates the connection point with group L.

13. The antibody-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 5, wherein the L3 is -C(O)-, -Cl-C 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 alkylene-C(O)-, wherein, R 1 For H or C 1-6 Alkyl; y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; preferably, L3 is a -C1-C6 alkylene-C(O)- or -C(O)-.

14. The antibody-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 5, wherein L4 is a parallel linking unit, the parallel linking unit being a natural amino acid or a non-natural amino acid; wherein each of the natural amino acids or non-natural amino acids is an amino acid having one or two reactive functional groups on its side chain, and at least one reactive functional group on the side chain of at least one of the natural amino acids or non-natural amino acids is covalently linked to PA; preferably, wherein the natural amino acid or non-natural amino acid is selected from lysine, arginine, 2,3-diaminopropionic acid, glutamic acid, glutamine, aspartic acid, asparagine, serine, threonine, cysteine, tyrosine, or ornithine; more preferably, the natural amino acid or non-natural amino acid is selected from lysine, arginine, and 2,3-diaminopropionic acid.

15. The antibody-drug conjugate, its isomers, pharmaceutically acceptable salts, or mixtures thereof according to claim 13 or 14, wherein the L5 is a natural amino acid or a non-natural amino acid or a short peptide, wherein each natural amino acid or non-natural amino acid is independently selected from: valine (V), citrulline (Cit), lysine (K), glycine (G), phenylalanine (F), alanine (A), proline (P), leucine (L), isoleucine (I), serine (S), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), and 2,3-diaminopropionic acid (Dap); preferably, the L5 is selected from -valine-alanine (VA)-, -valine-citrulline (VC)-, -glycine-glycine-phenylalanine-glycine (GGFG)-, -alanine-alanine-alanine-glycine (AAAG)-, and -glycine Short peptides of -glycine-glycine-glycine (GGGG)-, -valine-alanine-glycine (VAG)-, -valine-citrulline-glycine (VCG)-, -alanine-alanine-glycine (AAG)-, -alanine-alanine-alanine (AAA)-, -alanine-alanine (AA)-, -glutamic acid-alanine-glycine-glycine (EAGG)-, -glycine-glutamic acid-alanine-glycine (GEAG)-, -glycine-glutamic acid-glycine-glycine (GEGG)-, -glutamic acid-glycine-glycine (EGG)-, -glutamic acid-alanine-glycine (EAG)-, -valine-lysine-glycine (VKG)-, -glycine-glutamic acid-glycine (GEG)-, -glutamic acid-alanine (EA)-, -glutamic acid-glycine (EG)- and -glycine-glutamic acid (GE)-.

16. The antibody-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 13-15, wherein the L in formula (I) has a -L1-L3-L4-L5-L6- structure, wherein: L1 is an extension unit conjugated to the antibody; preferably, L1 is selected from... , , , , , , , , , , , , , and , where ** represents the binding point with the antibody, and * represents the binding point with L3; L3 represents a bond or spacer unit, such as -C(O)- or -Cl-C. 10 Alkylene -C(O)-, -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -NR 1 -(CH2CH2O) y -C 1-10 Alkylene-C(O)-, -C(O)-NR 1 -C 1-10 Alkylene-C(O)-, -NR 1 -C 1-10 Alkylene-C(O)-,-(CH2NR) 1 )-C 1-10 Alkylene -C(O)-, -C 1-10 alkylene-, -C 1-6 Heteroalkylene-, -C 3-8 Carbocyclic group -, -O-(C 1-8 alkyl, -arylene, -C1- 10 alkylene-arylene-, -arylene-C 1-10 alkylene-, -C 1-10 Alkylene-(C 3-8 (carbocyclic)-, -(C 3-8 (Carbocyclic group)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-C(O)-, -C3-C8 carbocyclic-C(O)-, -O-(C1-C8 alkyl)-C(O)-, -arylene-C(O)-, -C1-C 10 alkylene-arylene-C(O)-, -arylene-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(O)-, -C3-C8 heterocyclic-C(O)-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-C(O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(O)-, -C1-C 10 Alkylene -NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclic-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-OC(O)-, -C3-C8 carbocyclic-OC(O)-, -O-(C1-C8 alkyl)-OC(O)-, -arylene-OC(O)-, -C1-C 10 Alkylene-arylene-OC(O)-, -arylene-C1-C 10 Alkylene -OC(O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-OC(O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-OC(O)-, -C3-C8 heterocyclic-OC(O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-OC(O)- or -(C3-C8 heterocyclic)-C1-C 10 alkylene-OC(O)-, where R 1 For H or C 1-6 Alkyl groups, where y is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L4 is a parallel linker unit, which is a natural amino acid, a non-natural amino acid, or a short peptide. The short peptide is composed of 2-5 natural or non-natural amino acids, wherein each natural or non-natural amino acid is an amino acid whose side chain has 1-5, preferably 1-3, more preferably 1 or 2 reactive functional groups, and at least one reactive functional group of the side chain of at least one of the natural or non-natural amino acids is covalently linked to PA. Preferably, each natural or non-natural amino acid is independently selected from lysine (Lys), arginine (Arg), 2,3-diaminopropionic acid (Dap), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), serine (Ser), threonine (Thr), cysteine ​​(Cys), or ornithine (Orn). More preferably, the natural or non-natural amino acid is Lys or Dap, wherein the reactive functional group of the side chain is ε-amino or β-amino, which is covalently linked to PA. L5 is a bond, or a natural amino acid, a non-natural amino acid, or a short peptide, wherein the short peptide is composed of 2, 3, 4, 5, 6, 7, or 8 natural or non-natural amino acids, preferably 2 or 3 natural or non-natural amino acids; preferably, each of the natural or non-natural amino acids is independently selected from: valine (Val), citrulline (Cit), lysine (Lys), glycine (Gly), phenylalanine (Phe), alanine (Ala), proline (Pro), leucine (Leu), isoleucine (Ile), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), and 2,3-diaminopropionic acid (Dap); L6 is the key, or L6 is selected from -N(R) 1 )-C1- 10 Alkylene-, -C(O)-C1- 10 Alkylene-C(O)-, -N(R) 1 )-C1- 10 Alkylene-N(R) 1 -C(O)- or -N(R) 1 )-C1- 10 Alkylene-N(R) 1 )-C1- 10 Alkylene; preferably, L6 is -N(R 1 -C1-6 alkylene-, for example -N(R 1 )-CH2-; where R 1 For H or C 1-6 Alkyl; or L6 is selected from: , , , , , , , , , , and , Where a * Indicates the connection point with L5, b * This indicates the connection point with D.

17. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-16, wherein the linker group in formula (I) Select from the following structure, where ** represents the connection point with Ab, and * represents the connection point with D: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or 。 18. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to claim 1 or 2, wherein the ligand-drug conjugate of formula (I) has the structure of formula (IB). (IB) in, Ab, L1, and L2 are each independently defined as claimed in any one of claims 3-16, D1, D2, and D3 are different drugs, and n1, n2, and n3 are each independently an integer or decimal from 0 to 10, preferably an integer or decimal from 1 to 8; The condition is that at least two of n1, n2, and n3 are not 0.

19. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-18, wherein the drug is a cytotoxic drug, an immunostimulant, or a radioisotope; preferably, the drug is selected from cytotoxic drugs and STING agonists; more preferably, the drug is selected from orlistatine derivatives, camptothecin analogs, DNA topoisomerase I inhibitors and their derivatives, and maytansine alkaloid derivatives.

20. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-18, wherein the drug is a glucocorticoid; preferably, the glucocorticoid is BD0260.

21. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-20, wherein the Ab is an antibody; preferably, the antibody is selected from antibodies that specifically bind to one or more of the following antigens: TROP2, HER2, AXL, BAFFR, BCMA, BCR-list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD137, CD138, CD14, CD163, CD168, CD137, CD138, CD14, CD163, CD168, CD137, CD138, CD14, CD163, CD168, CD137, CD138, CD14, CD15 ... 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, differentiation antigen cluster 40 (C D40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.

2. CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, C XCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, type II HLA (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR1), IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, integrin (Integri) ns), LAG3, LIFR, MAG / Siglec-4 (sialic acid-binding immunoglobulin-like lectin-4), MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1 (sialic acid-binding immunoglobulin-like lectin-1), Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list components / assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, tumor necrosis factor α (TNFα), TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-γ, and VTCN1.

22. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt, or mixture thereof according to any one of claims 1-21, wherein the ligand-drug conjugate is selected from: 、 、 、 or Wherein Ab is a ligand, D, D1, and D2 are all drugs, and D1 and D2 are different drugs, n is 3.3-4.3; the sum of n1 and n2 is 3.3-4.3; Preferably, the ligand-drug conjugate is selected from: 、 、 ， The antibody mentioned above is: An anti-Trop2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 11 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 10; An anti-HER2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 12; or An anti-HER2 antibody comprising a light chain with an amino acid sequence as shown in SEQ ID NO: 15, and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 14; n is 3.3-4.3; the sum of n1 and n2 is 3.3-4.

3.

23. Drug-linker molecules of formula (II), their isomers, pharmaceutically acceptable salts or mixtures thereof, (II) Wherein L, D, and PA are defined by formula (I) of any one of claims 1-22; R b1 The leaving group at the end of the L unit, such as F, Cl, Br, I, p-nitrophenol, N-hydroxysuccinimide (NHS), substituted N-hydroxysuccinimide, phenoxy, phenylthio, dinitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, trifluoromethanesulfonate, imidazole, dichlorophenoxy, tetrachlorophenoxy, trichlorophenoxy, 1-hydroxybenzotriazole, toluenesulfonate, methanesulfonate, 2,4-dinitrophenoxy, 2-ethyl-5-phenylisoxazonium-3'-sulfonate, sulfone or sulfoxide, or R b1 The L-unit does not exist and forms a Michael acceptor, such as a maleimide group, at its end; z is an integer from 1 to 5, such as 1, 2, 3, 4 or 5.

24. The drug-linker molecule, its isomer, pharmaceutically acceptable salt, or mixture thereof according to claim 23, wherein the drug-linker molecule of formula (II) has the structure of formula (II-A): (II-A) in R b1 The leaving group on the L1 unit, such as F, Cl, Br, I, p-nitrophenol, N-hydroxysuccinimide (NHS), substituted N-hydroxysuccinimide, phenol, phenylthio, dinitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, trifluoromethanesulfonate, imidazole, dichlorophenoxy, tetrachlorophenoxy, trichlorophenoxy, 1-hydroxybenzotriazole, toluenesulfonate, methanesulfonate, 2,4-dinitrophenoxy, 2-ethyl-5-phenylisoxazonium-3'-sulfonate, sulfone or sulfoxide, or R b1 The L1 unit does not exist and forms a Michael acceptor, such as a maleimide group, at its end; z is an integer from 1 to 5, such as 1, 2, 3, 4 or 5; L1, L2, D, and PA are as defined in any one of claims 3-22.

25. The drug-linker molecule, its isomer, pharmaceutically acceptable salt, or mixture thereof according to claim 23 or 24, wherein the drug-linker molecule is selected from the group consisting of: 。 26. A pharmaceutical composition comprising a ligand-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, according to any one of claims 1-22, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

27. Use of any ligand-drug conjugate, isomer thereof, pharmaceutically acceptable salt thereof, or mixture thereof, or pharmaceutical composition of claim 26, in the preparation of a medicament for treating and / or preventing disease.

28. A method of treating and / or preventing a disease, the method comprising administering to an individual in need a ligand-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition of claim 26.

29. The ligand-drug conjugate, its isomer, pharmaceutically acceptable salt or mixture thereof, or the pharmaceutical composition of claim 26, as a medicine for treating or preventing disease, according to any one of claims 1-22.

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