Enzyme-degradable linker and ligand-eribulin conjugate containing the same

Abstract

The present disclosure relates to the field of biopharmaceutical technology, particularly to an enzymatically degradable linker and a ligand-eribulin conjugate comprising the same, more particularly to the application of VAGGF as an enzymatically degradable linker.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a patent application filed on October 20, 2022, bearing application number "CN202211287874.0" and entitled "Enzyme-degradable linker and ligand-eribulin conjugate containing the same," and to a patent application filed on July 12, 2023, bearing application number "CN202310853999.3," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biopharmaceutical technology, and in particular to an enzyme-degradable linker and a ligand-eribulin conjugate comprising the same. [Background technology]

[0003] Antibody-drug conjugates (ADCs) are created by attaching a biologically active small molecule drug to a monoclonal antibody (single antibody) via a linker. Currently, most ADCs are composed of a tumor antigen-targeting antibody linked to a highly cytotoxic small molecule drug via a linker. Taking advantage of the specific binding properties of the antibody and the target antigen, the small molecule drug is delivered to tumor cells, killing the tumor. Proteins and polypeptides with specific binding properties similar to those of antibodies, such as specific binding ligands, antigens, and polypeptides, can also be attached to the linker toxin.

[0004] The linker is one of the key factors that determine the stability and therapeutic window of a drug. First, the linker must have a certain level of stability, which ensures the integrity of the ADC during blood circulation before reaching tumor cells and avoids off-target toxicity due to premature release of the toxin, which can affect the therapeutic window of the ADC drug. After entering the target cell, the linker must ensure the effective release of the toxin and exert its killing effect. Polypeptide linkers are the more common degradable linkers. As of September 2022, more than 10 ADC drugs have been approved for marketing. Most of them use a polypeptide linker to attach the toxin to a cysteine ​​residue on a monoclonal antibody, which is cleaved by cathepsins in tumor cells to release the toxin, thereby producing antitumor effects.

[0005] Currently, the most commonly used linker in clinical studies is valine-citrulline (VC), and the commercially available drug Adcetris uses this linker to connect MMAE to antibodies. Other ADCs use valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG), including SGN-CD33A, loncastuximab tesirine, and DS-8201a.

[0006] Eribulin, an antibody-toxin conjugate, has a large molecular weight, complex structure, and good hydrophilicity. The large molecular weight requires screening for an appropriate linker length. Because of the complex structure, different linkers may have different enzymatic cleavage release efficiencies due to steric hindrance. Therefore, eribulin is suitable for preparing high-conjugation conjugates in which one antibody molecule binds multiple toxins. PEG, a commonly used linker component, has good hydrophilicity and stability. The commonly used PEG length is 2-8, but its light absorption is unclear and its synthesis is more complex. Summary of the Invention

[0007] This disclosure identifies for the first time the application of VAGGFG as an enzymatically degradable linker. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate has the general formula TL-(LD)n; wherein T L is a targeting ligand, L is a linker unit, D is eribulin, and n is a positive integer of 1 to 20; L contains the short peptide VAGGFG as an enzymatically cleavable linker. The present disclosure further relates to methods for preparing, pharmaceutical compositions and pharmaceutical uses of such ligand-drug conjugates.

[0008] The newly discovered linker of the present disclosure is expected to be applied in ligand-drug conjugates due to its significantly improved drug release efficiency and stronger killing ability against tumor cells. [Brief explanation of the drawings]

[0009] In order to more clearly describe the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly describe the drawings that need to be used in the description of the specific embodiments or the prior art. The drawings in the following description are some embodiments of the present disclosure, and it will be obvious to those skilled in the art that other drawings can also be obtained based on these drawings without any creative efforts. [Figure 1] 1 shows the results of a cellular activity assay of antibodies 7B7-H04 and 7B7 linked to eribulin via different linker units, as provided in one example of the present disclosure. [Figure 2]1 shows the results of the conjugation ratio and purity of antibody 7B7-H04 and 7B7 conjugated to eribulin via different linker units, as provided in one example of the present disclosure, where A shows the DAR assay result for 7B7 naked antibody, B shows the DAR assay result for 7B7-H04 MC-VA-PAB-Eribulin, C shows the result of SEC-HPLC purity assay for 7B7-H04 MC-VA-PAB-Eribulin, D shows the result of DAR assay for 7B7-H04 MC-GGFG-PAB-Eribulin, E shows the result of SEC-HPLC purity assay for 7B7-H04 MC-GGFG-PAB-Eribulin, F shows the result of DAR assay for 7B7-H04 MC-VAGGFG-PAB-Eribulin, and G shows the result of SEC-HPLC purity assay for 7B7-H04 1 shows the results of a SEC-HPLC purity assay of MC-VAGGFG-PAB-Eribulin, H shows the results of a DAR assay of 7B7 MC-VAGGFG-PAB-Eribulin, and I shows the results of a SEC-HPLC purity assay of 7B7 MC-VAGGFG-PAB-Eribulin. [Figure 3] 1 shows the results of an in vivo antitumor experiment of antibody 7B7 linked to eribulin via different linker units, provided in one example of the present disclosure. [Figure 4] FIG. 1 shows the binding ratio and SEC-HPLC purity results of the antibody Trastuzumab bound to MC-VAGGFG-PAB-Eribulin provided in one example of the present disclosure, where A shows the DAR assay result for the naked Trastuzumab antibody, B shows the DAR assay result for Trastuzumab MC-VAGGFG-PAB-Eribulin, and C shows the SEC-HPLC purity assay result for Trastuzumab MC-VAGGFG-PAB-Eribulin. [Figure 5]FIG. 1 shows the binding ratio and SEC-HPLC purity results of the antibody Bemarituzumab bound to MC-VAGGFG-PAB-Eribulin provided in one example of the present disclosure, where A shows the DAR assay result for Bemarituzumab naked antibody, B shows the DAR assay result for Bemarituzumab MC-VAGGFG-PAB-Eribulin, and C shows the SEC-HPLC purity assay result for Bemarituzumab MC-VAGGFG-PAB-Eribulin. Specific Embodiments

[0010] Reference will now be made in detail to one or more examples of the present invention, with reference to the embodiments thereof. Each example is provided by way of explanation, not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield still a further embodiment. Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present invention are the same as those commonly understood by those skilled in the art to which the present invention belongs. With further guidance, the following definitions are used to better understand the teachings of the present invention. In this specification, the terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0011] As used herein, the term "and / or," "or / and," and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and said any and all combinations include any two related listed items, any more related listed items, or all combinations of the related listed items. It should be understood that when at least three items are connected by a conjunction combination selected from at least two of "and / or," "or / and," and "and / or," the technical solution in this application definitely includes technical solutions all connected by "logical AND," and also definitely includes technical solutions all connected by "logical OR." For example, "A and / or B" includes three parallel schemes: A, B, and A+B. For example, a technical solution of "A, and / or, B, and / or, C, and / or D" includes any of A, B, C, and D (i.e., all technical solutions connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, i.e., includes any two or three combinations of A, B, C, and D, and further includes four combinations of A, B, C, and D (i.e., all technical solutions connected by "logical AND").

[0012] As used in this disclosure, the terms "containing," "including," and "comprises" are synonymous, inclusive, or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0013] Numerical ranges expressed in this disclosure by endpoints include all numbers, fractions, and recited endpoints subsumed within that range.

[0014] This disclosure relates to concentration values, and the meaning includes variations within a certain range. For example, it can vary within a corresponding precision range. For example, 2% can allow for variations within ±0.1%. For larger values ​​or values ​​that do not require as much control, the meaning can also include larger variations. For example, 100 mM can allow for variations within ±1%, ±2%, ±5%, etc. In the case of molecular weight, the meaning can also include variations of ±10%.

[0015] In this disclosure, references to the terms "plurality," "various," and the like, unless otherwise limited, refer to a number greater than or equal to two.

[0016] In this disclosure, technical features that are openly described include closed technical solutions consisting of the listed features, and also open technical solutions that include the listed features.

[0017] It should be understood that in this disclosure, "preferred," "better," "superior," and "desirable" are used only to describe more effective embodiments or examples, and do not limit the scope of protection of the present disclosure. In this disclosure, "optionally," "optional," and "optional" mean that there may be or may not be, that is, any one selected from two parallel schemes of "yes" or "no." When there are multiple "options" in one technical solution, each "option" is independent unless there is a special description, contradiction, or mutual constraint.

[0018] As used herein, the term "eribulin" refers to a synthetic analog of halichondrin B (a macrocyclic compound originally isolated from Halichondria okadais), whose CAS number is 253128-41-5. Eribulin is a microtubule dynamics inhibitor that is believed to cause cell cycle arrest at the G2 / M phase by binding to microtubule proteins and inhibiting mitotic spindle assembly. The term "eribulin methanesulfonate" refers to a compound with the CAS number 441045-17-6 and sold under the trade name Halaven. TM This refers to the methanesulfonate salt of eribulin sold in the US.

[0019] All documents mentioned in this disclosure are incorporated herein by reference as if each document were incorporated by reference in its entirety. The entire contents of the cited documents to which this disclosure relates are incorporated for all purposes, unless they contradict the inventive objectives and / or technical solutions of this application. When this disclosure relates to a cited document, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated herein. When this disclosure relates to a cited document, examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application by reference, but only to the extent that this disclosure can be implemented. It should be understood that when the cited content contradicts the description of this application, it should be governed by or adaptively modified in accordance with the description of this application.

[0020] The present disclosure relates to the application of VAGGFG as an enzymatically degradable linker.

[0021] The short peptides that can be used are X 1 X 2 GGFG, where X 1 X 2 are all amino acid residues, and X 1 and X 2are each independently selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, aspartic acid, glutamine, asparagine, lysine, arginine, glutamic acid, and histidine. 1 X 2 is one of VC, VA, AC, AV, AA, and VV. 1 X 2 Any one of the amino acids of GGFG may be substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl.

[0022] The present disclosure further relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate has the general formula TL-(LD)n: wherein T L is a targeting ligand, L is a linker unit, D is eribulin, and n is a positive integer of 1 to 20; L contains the short peptide VAGGFG as an enzymatically cleavable linker.

[0023] Here, the value of n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and is preferably 1 to 8, and more preferably 4 to 8.

[0024] The term "targeting ligand" refers to a polymeric compound that can specifically recognize and bind to an antigen or receptor associated with a target cell. The role of the ligand is to present a drug to a group of target cells that bind to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, polypeptides with binding ability, or other molecules capable of binding to cells. In embodiments of the present disclosure, the targeting ligand is designated as TL, and the targeting ligand can form a linker bond with the linker unit via a heteroatom on the ligand.

[0025] The term "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present disclosure and means a salt acceptable for administration to a patient (e.g., a mammal) (for a given dosing scheme, it is a salt containing a counterion with acceptable mammalian safety). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. The ligand-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfonate, hydrogen sulfite, citrate, acetate, succinate, ascorbate, oxalate, nitrate, piriate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate. In particular, when the conjugated drug is eribulin, the preferred pharmaceutically acceptable salt is eribulin methanesulfonate.

[0026] The term "solvate," as used in this disclosure, refers to a combination of a compound of the present disclosure with a solvent molecule formed by solvation. In some cases, the solvate refers to a hydrate, i.e., the solvent molecule is a water molecule, and the combination of a compound of the present disclosure with water forms a hydrate.

[0027] In some embodiments, the linker unit is -L 1 -L 2 -L 3 -L 4 - and L 1 is concatenated to TL, and L 4 is connected to D, where: L 1 teeth, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a group required for click chemistry (preferably), L 2 is -NC(R 1 R 2 )C(O), -NR 3 (CH2) o C(O)-, -NR 3 (CH2CH2O) o CH2C(O)-, -S(CH2) p C(O)- or a chemical bond, where o is selected from an integer from 0 to 20 and p is selected from an integer from 0 to 20; R 1 , R 2 are each independently selected from hydrogen, deuterium, alkyl, substituted alkyl, deuteroalkyl, heteroalkyl, carboxy, amino, and substituted amino; R 3 is selected from hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuteroalkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl; L 1 and L 2 shares an N atom, L 3 is VAGGFG, L 4 is -NR 4 (CR 5 R 6 ) q -, -C(O)NR 4 -, -C(O)NR 4 (CH2) q - or a chemical bond, and q is selected from an integer of 0 to 6; R 4 , R 5 and R 6 are each independently selected from hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuteroalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl, or heteroaryl. In some embodiments, L 4 is -NR 4 -aryl-(CR 5 R 6 ) q -OC(O)-, -NR 4 (CR 5 R 6 ) q -OC(O)-, where q is selected from an integer of 0 to 6; R 4 , R 5 and R 6 are each independently selected from hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuteroalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl, or heteroaryl.

[0028] In some embodiments, o may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0029] In some embodiments, p may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0030] In some embodiments, q may be 0, 1, 2, 3, 4, 5, or 6.

[0031] "L 1 and L 2 "share N atom" means that the defined L 1 L defined as the right terminal group of the group 2 When both left terminal groups of the groups contain an N atom, it means that the two groups share the N atom.

[0032] Obviously, L 1 By simply replacing one with another, it is possible to conjugate to other natural amino acid sites, unnatural amino acid sites, terminal sites, glycosylation sites of TL, or to link via chelation or click chemistry. Examples of conjugation via click chemistry include cycloaddition reactions, nucleophilic ring-opening reactions, carbonyl chemistry of non-alcohol aldehydes, or carbon-carbon multiple bond addition reactions.

[0033] In some instances, TL may be a functional group F 1 L 1 has or is a complementary functional group thereof. Preferably, the reactive group L 1 and functional group F 1 can react in bioorthogonal reactions because they do not interfere with the biomolecules present in the reaction. Bioorthogonal reactions and suitable functional groups therein are known to those skilled in the art, for example, Gong and Pan, Tetrahedron Lett. 2015, 56, 2123-2132, including Staudinger ligation and copper-free click chemistry. Thus, L 1 is preferably selected from 1,3-dipoles, alkynes, (hetero)cyclooctynes, cyclooctenes, tetrazines, ketones, aldehydes, alkoxyamines, hydrazines and triphenylphosphine. For example, F 1 is an azide group, the linking of the azide-modified antibody to the linker-adduct is preferably carried out by a cycloaddition reaction. 1is preferably selected from alkynyl, with terminal alkynyl and (hetero)cycloalkynyl being preferred. For example, F 1 is a ketone group, the linkage of the ketone-modified antibody to the linker-adduct is preferably carried out by selective coupling with a hydroxylamine derivative or hydrazine to form an oxime or hydrazone, respectively. 1 is preferably a primary amino group (e.g., -NH2 group), an aminooxy group (e.g., -O-NH2), or a hydrazine group (e.g., -N(H)NH2). For example, F 1 When the functional group L is alkynyl, the linkage between the alkyne-modified antibody and the linker-adduct is preferably carried out by a cycloaddition reaction, more preferably by a 1,3-dipolar cycloaddition reaction. 1 is preferably a 1,3-dipole such as an azide, a nitroketone or a nitrile oxide. 1 and L 1 The positions of the can also be swapped.

[0034] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl containing 1 to 12 carbon atoms, more preferably an alkyl containing 1 to 10 carbon atoms, and most preferably an alkyl containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-amyl, 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, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 ,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof.More preferably, it is a lower alkyl containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-amyl, 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, and the like. The alkyl may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available point of attachment, and said substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo groups.

[0035] The term "substituted alkyl" refers to an alkyl having a hydrogen atom replaced with a substituent, and unless otherwise specified, alkyl substituents include -halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -COR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)R', -NH- R', R" and R"' may each independently represent a hydrogen atom, an unsubstituted C(NH2)=NH, an unsubstituted C(NH2)=NR' ... 1-8Alkyl, unsubstituted aryl, aryl substituted with 1 to 3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy or unsubstituted aryl-C 1-4 When R' and R" are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6-, or 7-membered ring together with the nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0036] The term "heteroalkyl" refers to an alkyl containing one or more heteroatoms selected from N, O, or S, where alkyl is defined as above.

[0037] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl are defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0038] The term "alkoxyalkyl" means that alkyl is substituted with one or more alkoxy groups, preferably one alkoxy group, where alkyl is defined above and alkoxy is defined above.

[0039] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, where the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyls include spirocyclic, fused-ring, and bridged-ring cycloalkyls.

[0040] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m (where m is an integer of 0 to 2), but does not include the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1, 2, 3, or 4 are heteroatoms, and more preferably, the heterocyclic ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyls include spirocyclic, fused-ring, and bridged-ring heterocyclyls.

[0041] The term "cycloalkylalkyl" means that an alkyl is substituted with one or more cycloalkyls, preferably one cycloalkyl, where alkyl is defined above and cycloalkyl is defined above.

[0042] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group containing 6 to 14 carbon ring atoms, i.e., a polycyclic (i.e., rings having adjacent pairs of carbon atoms) group having a conjugated π-electron system. An aryl may be monocyclic or polycyclic (i.e., it may contain one or more rings). In the case of a polycyclic aromatic ring, only one ring in the polycyclic ring system need be aromatic; the remaining rings may be saturated, partially saturated, or unsaturated. An aryl is preferably a 6- to 10-membered group such as phenyl or naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or heterocyclic group ring, where the ring connected to the parent structure is an aryl ring.

[0043] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms include oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10-membered. More preferably, it is 5- or 6-membered, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, oxazolyl, isoxazolyl, etc., and the heteroaryl ring may be fused to an aryl, heterocyclyl, or cyclic group ring, where the ring connected to the parent structure is a heteroaryl ring. Heteroaryl may be optionally substituted or unsubstituted.

[0044] The term "deuteroalkyl" means that a hydrogen on an alkyl has been replaced with one or more deuterium atoms, where alkyl is defined above.

[0045] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0046] "Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1, 2 or 3 hydrogen atoms, are independently replaced with a substituent. Substituents are present only at their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions without much effort (by experiment or theory). For example, an amino or hydroxyl having a free hydrogen atom may become unstable when bonded to a carbon atom having an unsaturated (e.g., alkene) bond.

[0047] In some embodiments, -L 1 -L 2 -teeth, [ka] and s1 is 2, 3, 4, 5, 6, 7 or 8.

[0048] In some embodiments, L 4 is p-aminobenzyloxycarbonyl (PAB).

[0049] In some embodiments, the targeting ligand is an antibody or an antigen-binding fragment thereof.

[0050] As used herein, the term "antibody" is used in its broadest sense to refer to a protein or polypeptide, including immunoglobulins or other types of molecules that contain one or more specific antigen-binding structural domains and exhibit binding specificity to a specific antigen. Specific examples of antibodies include intact antibodies (e.g., classical four-chain antibody molecules), single-chain antibodies, single-domain antibodies, bispecific antibodies, and multispecific antibodies. Classical antibody molecules are typically tetramers consisting of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on conserved differences in amino acid sequence, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is responsible for antigen recognition and binding, while the constant region (e.g., Fc fragment) is responsible for initiating downstream effects such as antibody-dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions with high variability in amino acid composition and sequence order, which are also called complementarity-determining regions (CDRs) because they are key locations for antibody binding to antigens. The amino acid sequences of CDRs can be readily determined using art-recognized numbering schemes such as Kabat, Chothia, IMGT, AbM, or Contact. The antibody may be an IgG, IgM, IgD, IgE, or IgA antibody.

[0051] An "antigen-binding fragment" of an antibody refers to any of the amino acid fragments in the antibody molecule that are involved in antigen-specific binding, such as F(ab')2, Fab, and scFv.

[0052] The term "F(ab')2" refers to a fragment obtained by digesting a full-length antibody with pepsin to remove most of the Fc region while retaining a portion of the hinge region. Since the F(ab')2 fragment contains two antigen-binding Fab portions linked by disulfide bonds, it is a bivalent antibody. For example, the molecular weight of an F(ab')2 fragment prepared from an IgG antibody is approximately 110 kDa.

[0053] The term "Fab" refers to an antibody structure that is still capable of binding to antigen; it is monovalent and does not contain an Fc portion. After digestion of a full-length antibody with papain protease, two Fab fragments and one Fc fragment are generated, each of which is approximately 50 kDa.

[0054] The term "scFv" refers to a single peptide chain formed by linking the heavy and light chain variable regions of an antibody with a short peptide. When correctly folded, the variable regions from the heavy and light chains form an Fv segment through non-covalent interactions, allowing scFv to better retain its affinity activity for its antigen.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof is selected from a rabbit-derived antibody, a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0056] A "rabbit-derived antibody / mouse-derived antibody" refers to an antibody derived from a rabbit immunoglobulin sequence / mouse immunoglobulin sequence, if a variable region and a constant region are present. A rabbit-derived antibody / mouse-derived antibody can be easily obtained by immunizing a rabbit / mouse (including a mouse or rat) with the corresponding antigen and isolating the desired antibody therefrom. Alternatively, it can be obtained by immunizing a rabbit / mouse with the corresponding antigen, followed by isolating and culturing cells (e.g., B cells) expressing the desired antibody. Alternatively, a rabbit / mouse can be immunized with the corresponding antigen, followed by isolating and culturing cells expressing the desired antibody, and then fusing these with immortalized cells such as myeloma cells to obtain hybridoma cells. By culturing the hybridoma cells, the desired antibody (e.g., monoclonal antibody) can be obtained over a long period of time and in large quantities.

[0057] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody derived from a first animal with the constant region of an antibody derived from a second animal. To establish a chimeric antibody, first establish a hybridoma secreting a specific single antibody derived from the first animal. Then, clone the variable region genes from the hybridoma cells, and optionally clone the constant region genes of an antibody derived from a second animal. The variable region genes from the first animal and the constant region genes from the second animal are ligated to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the first animal origin is rabbit or mouse, and the second animal origin is preferably human, thereby reducing the immune response induced by the first animal-derived antibody. The antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa chain, lambda chain, or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof. Mutations due to the subtype of the antibody constant region, different human isomers, and changes in the functional effects of the constant region do not affect the preparation of antibody conjugates. Linker-toxin binding sites, such as cysteine, lysine, glutamine, the peptide chain carboxy terminus, and glycosylation sites, include natural sites and engineered sites, and these sites are commonly used for binding.

[0058] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences derived from a first animal onto a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. This can overcome the xenogeneic reaction induced by chimeric antibodies carrying a large amount of protein components derived from the first animal. Such framework sequences can be obtained from open DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (www.mrccpe.com.ac.uk / vbase) and Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition. To avoid a decrease in activity due to reduced immunogenicity, minimal back mutations or reverse mutations can be performed on the human antibody variable region framework sequences to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies after affinity maturation of CDRs using bacteriophage. In a preferred embodiment of the present disclosure, the first animal source is a rabbit or mouse. The human antibody variable region framework is selected by design. To avoid reduced activity due to reduced immunogenicity, minimal back mutations can be performed on the human antibody variable region to maintain activity.

[0059] A fully human antibody is one that has been produced by introducing human antibody-encoding genes into a genetically engineered animal lacking the antibody gene using genetic recombination or transchromosomal techniques, thereby enabling the animal to express human antibodies and achieving full humanization of the antibody.

[0060] In some embodiments, the antibody is selected from an anti-CD3 antibody, an anti-FOLR1 antibody, an anti-ROR1 antibody, an anti-TNFα antibody, an anti-tissue factor (TF) antibody, an anti-EpCAM antibody, an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-Claudin 6 antibody, an anti-Claudin 9 antibody, an anti-Claudin 18.2 antibody, an anti-Mesothelin antibody, an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 (ErbB3) antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-Nectin 4 antibody, an anti-TROP-2 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FRα antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin Av antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD47 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody, and an anti-CD123 antibody.

[0061] In some specific embodiments, the antibody is the anti-GD2 antibody 3F8, Abagovomab, Abciximab, ACZ885 (canakinumab), Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Aselizumab, Atezolizumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab, Bivatuzumab mertansine, blinatumomab, brentuximab vedotin, briakinumab, canakinumab, cantuzumab mertansine, capromab pendetide, catumaxomab, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clenoliximab, clivatuzumabTetraxetan, CNTO 148 (golimumab), CNTO 1275 (ustekinumab), Conatumumab, Dacetuzumab, Daclizumab, Denosumab, Detumomab, Dorlimomab aritox, dorlixizumab, durvalumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elsilimomab, enlimomab pegol pegol, epitumomabcituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, exbivirumab, fanolesomab, faralimomab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gavilimomab, Gemtuzumab ozogamicin ozogamicin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan, Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Ribivirumab, Lintuzumab, Lucatumumab ab), Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab-CD3 (Muromonab_CD3), MY0-029 (stamulumab, nacolomab tafenatox, naptumomab estafenatox, natalizumab, nebacumab, necitumumab, nerelimomab, nimotuzumab, nivolumab, nofetumomab merpentane) merpentan, ocrelizumab, odulimomab, ofatumumab, omalizumab, oportuzumabmonatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pembrolizumab, Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab, PRO 140, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab, Ruplizumab, Satumomab Satumomab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, tefibazumab, telimomabAritox, Tenatumomab, Teneliximab, Teplizumab, TGN1412, Ticilimumab, Tremelimumab, Tigatuzumab, TNX-355 (Ibalizumab), TNX-650, TNX-901 (Talizumab), Tocilizumab, Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and Zolimomab aritox.

[0062] In some embodiments, the antibody i) Trastuzumab antibody, ii) Bemarituzumab antibody, iii) an anti-B7-H3 antibody (whose heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 1 to 3, respectively, and whose light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4 to 6, respectively).

[0063] In some embodiments, the anti-B7-H3 antibody has a heavy chain variable region HCVR as set forth in SEQ ID NO:7 and a light chain variable region LCVR as set forth in SEQ ID NO:8.

[0064] In some embodiments, the anti-B7-H3 antibody has a heavy chain constant region as set forth in SEQ ID NO:9 or 10 and a light chain constant region as set forth in SEQ ID NO:11.

[0065] Variants of the above amino acid sequences are also within the scope of the present invention, each of which may contain up to three amino acid mutations occurring in at least one CDR region compared to any of the polypeptides of SEQ ID NO:1 to SEQ ID NO:6, or may contain up to three or more mutations across the entire sequence of SEQ ID NO:7 to SEQ ID NO:15, for example, a sequence having at least 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity to any of the polypeptides of SEQ ID NO:7 to SEQ ID NO:15. The mutations may be amino acid substitutions, deletions, or additions, or any combination thereof; preferably, the mutations are conservative substitutions.

[0066] "Conservative substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation and rigidity, etc.), often allowing for changes without altering the biological activity of the protein.

[0067] Substitutions generally considered to be conservative include intersubstitutions between the aliphatic amino acids Ala, Val, Leu, and Ile, interchanging the hydroxyl residues Ser and Thr, interchanging the acidic residues Asp and Glu, interchanging the amide residues Asn and Gln, interchanging the basic residues Lys and Arg, and interchanging the aromatic residues Phe and Tyr. As known to those skilled in the art, single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to destroy biological activity.

[0068] In some embodiments, the structure of the ligand-drug complex is: [ka] is.

[0069] The present disclosure further relates to a method for preparing such a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the method comprising: The target ligand is reduced and then subjected to a coupling reaction with pre-synthesized -LD to obtain a compound having the general formula TL-LD.

[0070] The reducing agent is preferably TCEP, and is particularly preferred for reducing disulfide bonds on the target ligand.

[0071] The present disclosure further relates to a pharmaceutical composition comprising a ligand-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent, or vector.

[0072] As used in this disclosure, "pharmaceutically acceptable vector, diluent or excipient" includes any material that, when combined with an active ingredient, enables said ingredient to retain biological activity and is non-reactive with the subject's immune system.

[0073] The present disclosure further relates to the use of such a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating a tumor.

[0074] The term "cancer" refers to a physiological condition or disease characterized by uncontrolled cell proliferation in an uncontrolled manner. "Tumor" includes cancer cells. In some embodiments, the tumor is a solid tumor or a blood tumor, such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, and leukemia.

[0075] The present disclosure further relates to a method of treating a medical condition in a subject, comprising administering a safe and effective amount of a ligand-drug conjugate as described above.

[0076] Preferably, the condition is cancer.

[0077] As used herein, the phrase "safe and effective amount" means, within a reasonable pharmaceutical regimen, an amount of a compound or composition large enough to appreciably and effectively alleviate the symptoms or condition being treated, yet small enough (at a reasonable benefit / harm ratio) to avoid serious side effects. The safe and effective amount of the active ingredient in the pharmaceutical compositions used in the methods of the present disclosure will vary depending on these factors, including the specific condition being treated, the age, and physical condition of the patient being treated, the severity of the disease, the duration of treatment, concurrent treatment context, the specific active ingredient used, the specific pharmacologically acceptable excipients used, and the knowledge and skill of the treating physician.

[0078] It is understood that contemplated therapeutic methods further include administering other therapeutic entities, particularly and preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic E. coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), chemotherapeutic agents, antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neo-antigens), stem cell transplants (e.g., allografts or autografts), and tumor-targeted cellular factors (e.g., NHS-IL12, where IL-12 binds to a tumor-targeting antibody or fragment thereof). In some embodiments, contemplated therapeutic methods further include administering radiation therapy to the patient. In some embodiments, contemplated therapeutic methods further include administering surgery, such as tumor resection, to the patient.

[0079] The ligand-drug conjugates may also be administered in combination with and / or co-formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-γ, and / or NSAIDs. Pharmaceutical compositions may include the above therapeutic entities.

[0080] The terms "subject" and "patient" are used interchangeably herein and refer to any animal, including, but not limited to, any mammal, such as a human, non-human primate, rodent, dog, cat, chimpanzee, orangutan, gibbon, macaque, marmoset, pig, horse, panda, and elephant.

[0081] As used herein, "treating" refers to any improvement in the outcome of any disease, such as extending survival, reducing incidence, and / or reducing side effects that are a by-product of alternative treatments. For example, as will be readily understood in the art, complete eradication of the disease is preferred, but is not required for therapeutic action. As used herein, "treating" refers to administering the Ligand-Drug Conjugate to a subject (e.g., a patient). Treating may be curing, curing, palliating, alleviating, altering, treating, ameliorating, attenuating, reversing, or affecting the disease, symptoms of the disease, or predisposition to a disease, such as cancer.

[0082] The pharmaceutical compositions of the present disclosure may be administered by any route as understood by those of skill in the art to which they pertain, hi some examples, the pharmaceutical compositions of the present disclosure are administered intravenously (IV).

[0083] Hereinafter, the embodiments of the present disclosure will be described in detail in conjunction with examples.It should be understood that these examples are only used to explain the present disclosure and are not used to limit the scope of the present disclosure.In the following examples, for the experimental methods for which specific conditions are not specified, the guidelines given in this disclosure shall be used as a guide, and the experimental methods may be based on the experimental handbooks or conventional conditions in the art, or may refer to other experimental methods known in the art, or may be based on the conditions recommended by the manufacturer.

[0084] In the specific examples below, assay parameters for raw material components may vary slightly within the limits of metering accuracy unless otherwise specified. Temperature and time parameters allow for acceptable deviations due to the testing or operating accuracy of the instrument. [Example]

[0085] Example 1 Preparation of Anti-B7-H3 Antibody The expression and purification of the monoclonal antibody was further carried out based on the anti-B7-H3 antibody sequence described in Chinese patent application CN113402610A, with a disclosure date of September 17, 2021.

[0086] Unless otherwise specified, all antibodies in this disclosure can be expressed using the commercially available vector pTT5, in which the antibody sequence can be inserted between the EcoR I and Hind III restriction enzyme cleavage sites following the promoter. The nucleic acid sequence including the signal peptide preceding the antibody light or heavy chain sequence is GAATTC GCCGCCACC ATG GGATGGTCCTGTATTATCCTGTTCCTGGTCGCTACCGCTACCGGTGTCCACTCA can be selected.

[0087] In the above sequence, EcoR I and the start amino acid are underlined.

[0088] After the antibody light or heavy chain sequence: TGAAAGCTT (which is a stop codon and Hind III).

[0089] The antibody amino acid sequence was optimized into a nucleotide sequence and then inserted between the signal peptide and terminator.

[0090] Heavy chain H00 sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAVSWVRQAPGKGLEWVASISGGGIYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGGAGYFDYWGQGTLVTVSS(SEQ ID NO: 7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9)

[0091] Light chain L00 sequence: DSQMTQSPSSLSASVGDRVTITCRGSESVHSYLAWYQQKPGKAPKLLVYNAKTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYGSPPWTFGGGTKVEIK (SEQ ID NO: 8) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11)

[0092] Heavy chain H04 sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAVSWVRQAPGKGLEWVASISGGGIYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGGAGYFDYWGQGTLVTVSS (SEQ ID NO: 7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 10)

[0093] The anti-B7-H3 antibody is cloned 7B7, and the antibody with the light chain L00 and heavy chain H00 is cloned 7B7. The antibody with the light chain L00 and heavy chain H04 is cloned 7B7-H04. The 7B7-H04 antibody is an engineered antibody, which is suitable for generating conjugated products with uniform conjugation ratios and is highly suitable for evaluating different linker toxins.

[0094] The heavy and light chains of the above antibodies were constructed in vectors, and the plasmids were extracted. Suspension-adapted CHO-K1 cells were resuscitated in OPM-CD TransCHO medium (manufacturer: OPM, part number: P83059) and cultured at a density of 2 million cells / ml with a viability of over 95% in a volume of 1000 ml. 0.5 mg of light chain plasmid and 0.5 mg of heavy chain plasmid were dissolved in 10 ml of medium, and 3 mg of PEI (manufacturer: Polyscience, part number: 24765-1) dissolved in medium was diluted in 10 ml of medium. The plasmid and PEI solutions were mixed and left at room temperature for 10 minutes, then added dropwise to 1000 ml of cell culture medium. The mixture was cultured at 37°C for 5 days, then centrifuged at 12,000 g for 15 minutes and the supernatant was collected. The supernatant was purified using HiTrap Mabselect SuRe and eluted with 50 mM acetic acid. The collected peak after neutralization was eluted with PBS pH 7.4 in a 30KD ultrafiltration tube (Manufacturer: Merck, Part Number: UFC9030). The antibody was assayed for absorbance at 280 nm and divided by the theoretical extinction coefficient to obtain the concentration.

[0095] Example 2 Synthesis of Control Linker Toxins 1. Synthesis of linker toxin MC-VA-PAB-Eribulin The structure of the designed linker toxin MC-VA-PAB-Eribulin (LK-322018) is: [ka] and The synthetic route of compound LK-322018 is as follows: [ka] is.

[0096] Synthesis of compound LK-322018: Commercially available compound MC-VA-PABC-PNP (5.9 mg, 0.091 mmol) was dissolved in 1 mL of N,N-dimethylformamide, followed by the addition of N,N-diisopropylethylamine (3.1 mg, 0.024 mmol) and commercially available eribulin (5 mg, 0.007 mmol). The reaction mixture was incubated at room temperature for 16 hours. The reaction mixture was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, 5 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by liquid phase separation to give LK-322018. ESI-MS m / z: 1240 (M−H).

[0097] 2. Synthesis of linker toxin MC-GGFG-PAB-Eribulin The structure of the designed linker toxin MC-GGFG-PAB-Eribulin (LK-322016) is: [ka] and The synthetic route of compound LK-322016 is as follows: [ka] is.

[0098] Synthesis of compound 3: Commercially available compound 1 (50 mg) was dissolved in 2 mL of N,N-dimethylformamide, and Bis-PNP (36 mg, 0.1182 mmol) and N,N-diisopropylethylamine (20.3 mg, 0.006053 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 16 hours. After monitoring the completion of the reaction by liquid chromatography-mass spectrometry, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by liquid-phase separation to give the title compound 3. ESI-MS m / z: 743 (M+H).

[0099] Synthesis of compound LK-322016: Compound 3 (7.26 mg, 0.009079 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.1 mg, 0.02412 mmol) and commercially available eribulin (5 mg, 0.007 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 16 hours. After monitoring the completion of the reaction by liquid chromatography-mass spectrometry, 5 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by liquid-phase separation to give LK-322016. ESI-MS m / z: 1389 (M−H).

[0100] 3. Synthesis of linker toxin Mal-PEG2-VC-PAB-Eribulin For specific synthesis steps, see page 145 of Chinese patent application CN108883198A, published on November 23, 2018, where the synthesis method and steps are the same as those in the patent. (DOI: 10.1158 / 1535-7163.MCT-17-1215, MORAb-202, an Antibody-Drug Conjugate Utilizing Humanized Anti-human FRα Farletuzumab and the Microtubule-Targeting Agent Eribulin, Has Potent Antitumor Activity) describes that Mal-PEG2-VC-PAB-Eribulin and MC-VC-PAB-Eribulin have the same activity against several cell lines, and the two are considered to be essentially the same. Therefore, in the present disclosure, Mal-PEG2-VC-PAB-Eribulin can be used interchangeably with MC-VC-PAB-Eribulin in some cases.

[0101] Example 3: Synthesis of Linker Toxins in Experimental Groups Synthesis of linker toxin MC-VAGGFG-PAB-Eribulin The structure of the designed linker toxin MC-VAGGFG-PAB-Eribulin (LK-322022) is: [ka] and The synthetic route of compound LK-322022 is as follows: [ka] is.

[0102] Synthesis of compound 2: Commercially available compound 1 (100 mg, 0.244 mmol) was dissolved in 3 mL of tetrahydrofuran and cooled to 0 °C. HOSU (33.7 mg, 0.293 mmol) and DCC (60.3 mg, 0.293 mmol) were added and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was monitored for completion by TLC spot plate and liquid chromatography mass spectrometry. The reaction mixture was filtered, the filtrate was concentrated, and the crude product was used directly in the next reaction. ESI-MS m / z: 508 (M+H).

[0103] Synthesis of compound 4: Compound 2 (crude, 0.244 mmol) and commercially available compound 3 (82 mg, 0.244 mmol) were mixed in 3 mL of N,N-dimethylformamide, N,N-diisopropylethylamine (126 mg, 0.976 mmol) was added, and the mixture was allowed to react at room temperature for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry for completion, after which the solvent was removed by vacuum distillation. The crude product was pulped with 10 mL of water and filtered to give the title compound (200 mg, 100% yield). ESI-MS m / z: 729 (M+H).

[0104] Synthesis of compound 6: Compound 4 (200 mg, 0.274 mmol) was dissolved in 5 mL of N,N-dimethylformamide and cooled to 0 °C. Compound 5 (41 mg, 0.329 mmol), N,N-diisopropylethylamine (107 mg, 0.823 mmol), and DEPBT (124 mg, 0.412 mmol) were added in that order and reacted at room temperature for 4 hours. The reaction mixture was monitored for completion by liquid chromatography-mass spectrometry. The reaction mixture was quenched with 10 mL of aqueous ammonium chloride and extracted three times with ethyl acetate (10 mL each time). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography [methylene chloride / methanol = 88:12 (v / v)] to give the title compound 6 (100 mg, 43.8% yield). ESI-MS m / z: 834 (M+H).

[0105] Synthesis of compound 7: Compound 6 (100 mg, 0.120 mmol) was dissolved in 3 mL of N,N-dimethylformamide, piperidine (31 mg, 0.360 mmol) was added, and the mixture was allowed to react at room temperature for 0.5 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry, and the solvent was removed by vacuum distillation. The resulting crude product was purified by silica gel column chromatography [methylene chloride / methanol = 82:18 (v / v)] to give the title compound 7 (76 mg, 100% yield). ESI-MS m / z: 612 (M+H).

[0106] Synthesis of compound 9: Compound 7 (76 mg, 0.124 mmol) and commercially available compound 8 (46 mg, 0.149 mmol) were mixed in 3 mL of N,N-dimethylformamide, N,N-diisopropylethylamine (32 mg, 0.248 mmol) was added, and the mixture was allowed to react at room temperature for 2 hours. The reaction was monitored for completion by liquid chromatography-mass spectrometry, and the solvent was removed by vacuum distillation. The resulting crude product was purified by silica gel column chromatography [methylene chloride / methanol = 82:18 (v / v)] to give the title compound 9 (70 mg, yield: 70%). ESI-MS m / z: 805 (M+H).

[0107] Synthesis of compound 10: Compound 9 (70 mg, 0.087 mmol) was dissolved in 3 mL of N,N-dimethylformamide, and Bis-PNP (40 mg, 0.130 mmol) and N,N-diisopropylethylamine (22 mg, 0.174 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 16 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. The solvent was then removed by vacuum distillation, and the resulting crude product was purified by silica gel column chromatography [methylene chloride / methanol = 90:10 (v / v)] to give the title compound 10 (40 mg, 47.6% yield). ESI-MS m / z: 970 (M+H).

[0108] Synthesis of compound LK-322022: Compound 10 (14 mg, 0.014 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.5 mg, 0.027 mmol) and commercially available eribulin (5 mg, 0.007 mmol) were added, successively, and the mixture was allowed to react at room temperature for 16 hours. The reaction was monitored by liquid chromatography-mass spectrometry for completion. After the addition of 5 mL of water, the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by preparative liquid phase separation to give LK-322022 (5.1 mg, 48.1% yield). ESI-MS m / z: 1560 (M−H).

[0109] Example 4 Antibody Binding 3 mg of antibody 7B7-H04 and antibody 7B7 conjugated in the above examples were each diluted to 3 mg / ml with 10 mM sodium phosphate buffer, pH 7.4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) mother solution was added. The TCEP mother solution was a 10 mM mother solution prepared in 10 mM sodium phosphate buffer, pH 7.4 (TCEP, manufacturer: Sigma, product number: C4706-2G, CAS. 51805-45-9). The molar ratio of the final TCEP concentration to the final antibody concentration was confirmed to be 4:1. The antibody was reduced for 45 minutes at 37°C. The linker toxin mother solution from Examples 2 and 3 was added. The linker toxin mother solution was prepared by dissolving the linker toxin in dimethyl sulfoxide (DMSO) to a final concentration of 5 mM. The molar ratio of the final linker toxin concentration to the final antibody concentration was confirmed to be 6:1. The antibody was conjugated at 2-8°C for 2 hours. The antibody was then transferred to a 30 KD ultrafiltration tube (manufacturer: Merck, product number: UFC9030) and centrifuged at 3,000 g at 2-8°C for liquid exchange. The residual toxin was reduced to 1 / 1,000 of the reaction concentration, after which the tube was sterile filtered, the concentration was assayed, and the tube was dispensed and frozen for storage.

[0110] To obtain products with different conjugation ratios, the molar ratio of antibody:reducing agent:linker toxin was adjusted between 1:1 and 6:2 and 15, while other conditions were unchanged. The DAR values ​​of the final conjugated products were determined based on the assay results.

[0111] Example 5 In vitro comparison of the effects of different ADCs 1. Assay Method 1. Cellular activity assay Human Calu-6 (Procell, product code: CL-327, human lung cancer cells) were resuscitated and seeded at 2500 cells per well in a volume of 150 μl into a 96-well culture plate supplemented with DMEM / F12 (Hyclone, product code: SH30023.01) containing 10% FBS (Gibco, product code: 10099141). The cells were cultured at 37°C in a 5% CO2 incubator. After approximately 20 hours, 50 μl of medium containing different antibody-drug conjugates was added. The final drug concentrations were adjusted to 12 gradient dilutions ranging from 0 to 100 nM (the first well concentration was 100 nM, followed by 10 5-fold dilutions, with the final gradient drug concentration at 0 nM. Each sample was repeated in duplicate). After 4 to 7 days of co-culture, the culture plate was removed and 50 μl of the conjugates were added to each well. The CTG detection reagent (CellTiterGlo, manufacturer: Promega, product number: G7575) was added and reacted for 2 minutes according to the recommended procedure in the reagent kit, and the fluorescence value was assayed using a Tecan Spark. A smooth graph was created using the average of the replicate wells as the Y-axis and the log10 value of the dilution gradient as the X-axis, and the EC / IC was calculated according to four parameters. 50 values ​​were calculated.

[0112] Similar cell activity assay methods can be employed and re-examined by substituting Jurkat, MCF-7, and MDA-MB-468 cell lines.

[0113] 2. Assay and analysis of binding ratio The average number of drugs bound to the antibody (DAR) was assayed using a hydrophobic chromatography (HIC-HPLC) method. The analytical column was a TSKgel Butyl-NPR, 4.6 mm x 10 cm, 2.5 μm column (manufacturer: TOSOH, part number: 042168). Approximately 0.3 mg of the sample to be assayed was taken in a volume of 150 μl, and 150 μl of mobile phase A was added. The mixture was centrifuged at 10,000 g for 5 minutes to collect the supernatant. Mobile phase A was 20 mM sodium phosphate + 1.5 M ammonium sulfate, pH 7.0, and mobile phase B was 20 mM sodium phosphate + 20% (v / v) acetonitrile, pH 7.0. The analytical column was connected to an Agilent 1260 high-performance liquid chromatograph at a flow rate of 0.6 ml / min. It was washed with mobile phase A for over 30 minutes until a stable 280 nm UV baseline was achieved. A 50 μg sample was then injected. After a 2-minute wash with mobile phase A, gradient elution (0% B - 100% B) was performed for 18 minutes, followed by a 5-minute wash with 100% mobile phase B. The percentage of each DAR value was calculated from the peak area. The percentage of each peak was multiplied by the DAR value, the resulting products were added, and the average DAR value was calculated by dividing by the total percentage.

[0114] 3. Purity assay and analysis Purity was assayed by SEC-HPLC using a molecular sieve method. The analytical column was a TSKgel G3000SWXL, 7.8 mm x 30 cm, 5 μm column (manufacturer: TOSOH, part number: 08541). Approximately 0.3 mg of the sample to be assayed was taken and centrifuged at 10,000 g for 5 minutes to recover the supernatant. The mobile phase was 50 mM sodium phosphate + 0.1 M sodium chloride, pH 6.8. The analytical column was connected to an Agilent 1260 high-performance liquid chromatograph at a flow rate of 0.8 ml / min. The column was washed with the mobile phase for at least 30 minutes until a stable 280 nm UV baseline was achieved. A 100 μg sample was then injected and washed with the mobile phase for 20 minutes. The percentages of the individual peaks representing polymers, monomers, and small molecules were calculated from the peak areas.

[0115] 2. Assay results 1. Results of Cellular Activity Assay When antibody 7B7-H04 was bound to MC-VA-PAB-Eribulin, Mal-PEG2-VC-PAB-Eribulin, MC-GGFG-PAB-Eribulin, and MC-VAGGFG-PAB-Eribulin, the EC / IC of the complexes on Calu6 cell activity. 50 As a result of assaying, EC / IC 50 were 115.9 pM, 29.97 pM, 62.32 pM, and 11.40 pM, respectively (FIG. 1), that is, MC-VAGGFG-PAB-Eribulin was significantly more effective than the other groups. When 7B7 was bound to Mal-PEG2-VC-PAB-Eribulin and MC-VAGGFG-PAB-Eribulin, the EC / IC 50 were 50.23 pM and 9.241 pM, respectively (FIG. 1), and MC-VAGGFG-PAB-Eribulin still achieved a superior technical effect.

[0116] EC / IC obtained by assaying other cell lines except Calu-6 cell line 50 The trends were similar to those above, and MC-VAGGFG-PAB-Eribulin was the most effective in both cases.

[0117] 2. Binding ratio and purity results The average DAR value and SEC-HPLC purity results for each group of conjugates are shown in Figure 2 and the table below.

[0118] [Table 1]

[0119] The average DAR values ​​of the complexes were 3.33, 3.27, and 3.31 (FIG. 2), indicating that the VAGGFG polypeptide linker had higher activity than the GGFG and VA polypeptide linkers when the binding ratios were similar.

[0120] Using the same method as above, we also tested several other common tumor cell lines (e.g., MCF-7 and MDA-MB-468) and found that the VAGGFG linker-containing conjugates showed the best EC / IC values ​​compared to other conjugates. 50 The results showed a consistent trend of expression, stable mean DAR values, and SEC-HPLC purity, meeting the requirements for antibody conjugates.

[0121] Example 6 In vivo pharmacodynamic analysis Bioduro (www.bioduro-sundia.com) conducted an in vivo pharmacodynamic evaluation in which a well-cultured human breast cancer tumor cell line (MDA-MB-468) was inoculated into immunodeficient mice (B-NDG) at 10 million cells per mouse, with tumors growing to 100 mm. 3 After the tumors grew to 100 μg / mL, they were randomly assigned to groups, and the next day, each group was administered 3 mg / kg twice a week. The tumor size was measured twice a week until the end of the experiment. The entire process was approved by the Animal Care and Use Committee.

[0122] The experimental results are shown in FIG. 3, and the results demonstrate that the 7B7 MC-VAGGFG-PAB-Eribulin group is effective in suppressing tumor volume.

[0123] The same animal model was used, but the cell lines were replaced with Calu6 and MCF-7. Each group was independently selected from 1 to 10 mg / kg (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 mg / kg). The results were consistent with the experimental trend of MDA-MB-468, which showed the best tumor volume inhibitory effect.

[0124] Example 7 Stability Test The stability of the 7B7 MC-VAGGFG-PAB-Eribulin conjugate was tested in PBS buffer, cynomolgus monkey plasma, and mouse plasma at a concentration of 100 nM at 37°C for 0, 4, 7, 10, and 14 days. The samples were then assayed for killing activity against Jurkat cells. The release rate of eribulin (Y-axis) was found to increase with time (X-axis), with a maximum release rate of 0.1-5%. This demonstrated excellent stability of the linker plasma.

[0125] Example 8 Binding test of other antibodies The experimental results of the above examples were all verified based on the antibodies 7B7-H04 and 7B7.

[0126] Trastuzumab heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 12)

[0127] Trastuzumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 13)

[0128] Bemarituzumab heavy chain sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYIFTTYNVHWVRQAPGQGLEWIGSIYPDNGDTSYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 14)

[0129] Bemarituzumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQGVSNDVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 15)

[0130] Furthermore, ADCs of the linker toxin MC-VAGGFG-PAB-Eribulin, which conjugates trastuzumab and bemarituzumab, were prepared and validated according to the above examples. The average DAR values ​​of the conjugation ratios were 2-3, and the purity was >95% (Figures 4 and 5). Activity assays were performed using SK-BR-3 cell lines (human breast cancer cells) and KATO III cell lines (human gastric cancer cells), and the EC / IC values ​​of cellular activity were measured using the same method as Calu-6. 50 The values ​​were 32 pM and 97 pM.

[0131] Example 9 Binding test of other drugs To test the differences between different toxins, different polypeptide linker toxins were synthesized, and conjugates were prepared according to the relevant experimental methods in the above examples, and the activity was assayed. The results showed that the different polypeptide linkers tested for monomethyl auristatin EMMAE and exatecan did not show significant differences at the cellular level, which differs from the results in the above examples using eribulin as the toxin.

[0132] The compound MC-VC-PAB-MMAE is commercially available, and it has been reported that MC-VC-PAB-MMAE is not significantly different from MC-VC-PAB-MMAE.

[0133] The synthetic route of the compound MC-GGFG-PAB-MMAE is as follows: [ka] and Synthesis steps: Commercially available compound 1 (50 mg, 0.0865 mmol) was dissolved in 2 mL of N,N-dimethylformamide, and Bis-PNP (36 mg, 0.1182 mmol) and N,N-diisopropylethylamine (20.3 mg, 0.006053 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 16 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. After that, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by liquid phase separation to obtain compound 3 (50 mg, 77.8% yield). Compound 3 (7.26 mg, 0.009 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.1 mg, 0.02412 mmol) and commercially available MMAE (7.9 mg, 0.011 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 15 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. After the addition of 5 mL of water, the mixture was extracted three times with ethyl acetate (10 mL each time). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative liquid phase separation to give the desired compound (8.5 mg, 62% yield). ESI-MS m / z: 1378 (M+H).

[0134] The synthetic route of the compound MC-VAGGFG-PAB-MMAE is as follows: [ka] and Step 1: Compound 1 (synthetic route the same as in the experimental group) (9.7 mg, 0.01 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.0 mg, 0.02 mmol) and commercially available MMAE (7.9 mg, 0.011 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 15 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. After the addition of 5 mL of water, the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative liquid phase separation to give the target compound (9.3 mg, 60% yield). ESI-MS m / z: 1548 (M+H).

[0135] The compound MC-VA-PAB-Exatecan is commercially available.

[0136] The synthetic route of the compound MC-GGFG-PAB-Exatecan is as follows: [ka] and Step: Commercially available compound 1 (50 mg, 0.0865 mmol) was dissolved in 2 mL of N,N-dimethylformamide, and Bis-PNP (36 mg, 0.1182 mmol) and N,N-diisopropylethylamine (20.3 mg, 0.006053 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 16 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. After that, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by liquid phase separation to obtain compound 3 (50 mg, yield 77.8%). Compound 3 (7.26 mg, 0.009079 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.1 mg, 0.02412 mmol) and commercially available Exatecan (5 mg, 0.011 mmol) were added in that order. The reaction mixture was incubated at room temperature for 15 hours. The reaction mixture was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, 5 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative liquid phase separation to give the desired compound (6.0 mg, 61% yield). ESI-MS m / z: 1096 (M+H).

[0137] The synthetic route of the compound MC-VAGGFG-PAB-Exatecan is as follows: [ka] and Step 1: Compound 1 (synthesized using the same synthetic route as in the experimental group) (9.7 mg, 0.01 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.0 mg, 0.02 mmol) and commercially available Extecan (5 mg, 0.011 mmol) were added in that order. The reaction was allowed to proceed at room temperature for 15 hours. The completion of the reaction was monitored by liquid chromatography-mass spectrometry. After this, 5 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL each time). After separation, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative liquid phase separation to give the target compound (7.3 mg, 58% yield). ESI-MS m / z: 1266 (M+H).

[0138] The above compounds were conjugated to antibody 7B7 according to Example 4 to prepare conjugates with conjugation ratios of 3 to 4, and the activity against the cell line MCF-7 was assayed according to Example 5. The results were: 1) The IC of activity of the conjugates conjugated to MMAE toxin for the VC, GGFG, and VAGGFG polypeptide linkers was 50 were 0.49, 0.41, and 0.27 nM, and 2) the IC of activity of the complexes bound to Extecan toxin for the VA, GGFG, and VAGGFG polypeptide linkers. 50 were 242, 270, and 307 nM.

[0139] The above examples merely represent some embodiments of the present disclosure, and although the descriptions are more specific and detailed, they should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present disclosure, and all of these are included in the scope of protection of the present disclosure. Therefore, the scope of protection of the patent of the present disclosure shall be subject to the scope of the attached claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. Application of VAGGFG as an enzymatically degradable linker.

2. A ligand-drug conjugate having the general formula TL-(LD)n, or a pharmaceutically acceptable salt or solvate thereof, TL is a targeting ligand, L is a linker unit, D is eribulin, and n is a positive integer from 1 to 20; L is a ligand-drug conjugate containing the short peptide VAGGFG as an enzymatically degradable linker, or a pharmaceutically acceptable salt or solvate thereof.

3. The linker unit is -L 1 -L 2 -L 3 -L 4 - and L 1 is concatenated to TL, and L 4 is connected to D, where: L 1 teeth, 【Chemical 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 or a group required for click chemistry (preferably), L 2 teeth, -NC(R 1 R 2 )C(O), -NR 3 (CH 2 ) o C(O)-, -NR 3 (CH 2 CH 2 O) o CH 2 C(O)-, -S(CH 2 ) p C(O)- or a chemical bond, where o is selected from an integer from 0 to 20 and p is selected from an integer from 0 to 20; R 1 , R 2 are each independently selected from hydrogen, deuterium, alkyl, substituted alkyl, deuteroalkyl, heteroalkyl, carboxy, amino, and substituted amino; R 3 is selected from hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuteroalkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl; L 1 and L 2 shares an N atom, L 3 is VAGGFG, L 4 is -NR 4 (CR 5 R 6 ) q -, -C(O)NR 4 -, -C(O)NR 4 (CH 2 ) q - or a chemical bond, and q is selected from an integer from 0 to 6; R 4 , R 5 and R 6 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a substituted alkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclyl, an aryl, a substituted aryl, or a heteroaryl, or a pharmaceutically acceptable salt or solvate thereof.

4. -L 1 -L 2 -teeth, 【Chemistry 10】 and s1 is 2, 3, 4, 5, 6, 7, or 8, or a pharmaceutically acceptable salt or solvate thereof.

5. The linker unit is -L 1 -L 2 -L 3 -L 4 - and L 1 is concatenated to TL, and L 4 is connected to D, where: L 1 teeth, 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 or a group required for click chemistry (preferably), L 2 teeth, -NC(R 1 R 2 )C(O), -NR 3 (CH 2 ) o C(O)-, -NR 3 (CH 2 CH 2 O) o CH 2 C(O)-, -S(CH 2 ) p C(O)- or a chemical bond, where o is selected from an integer from 0 to 20 and p is selected from an integer from 0 to 20; R 1 , R 2 are each independently selected from hydrogen, deuterium, alkyl, substituted alkyl, deuteroalkyl, heteroalkyl, carboxy, amino, and substituted amino; R 3 is selected from hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuteroalkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl; L 1 and L 2 shares an N atom, L 3 is VAGGFG, L 4 is -NR 4 -aryl-(CR 5 R 6 ) q -OC(O)-, -NR 4 (CR 5 R 6 ) q -OC(O)-, q is selected from an integer from 0 to 6; R 4 , R 5 and R 6 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a substituted alkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclyl, an aryl, a substituted aryl, or a heteroaryl, or a pharmaceutically acceptable salt or solvate thereof.

6. -L 1 -L 2 -teeth, 【Chemistry 20】 and s1 is 2, 3, 4, 5, 6, 7 or 8, or a pharmaceutically acceptable salt or solvate thereof.

7. L 4 The ligand-drug complex or a pharmaceutically acceptable salt or solvate thereof according to claim 5, wherein is p-aminobenzyloxycarbonyl (PAB).

8. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 2 to 7, wherein the targeting ligand is an antibody or an antigen-binding fragment thereof.

9. The ligand-drug complex or a pharmaceutically acceptable salt or solvate thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof is selected from a rabbit-derived antibody, a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

10. The antibody of the ligand-drug conjugate according to claim 9, or a pharmaceutically acceptable salt or solvate thereof, is selected from an anti-CD3 antibody, an anti-FOLR1 antibody, an anti-ROR1 antibody, an anti-TNFα antibody, an anti-TF antibody, an anti-EpCAM antibody, an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-Claudin 6 antibody, an anti-Claudin 9 antibody, an anti-Claudin 18.2 antibody, an anti-Mesothelin antibody, an anti-HER2 antibody, an anti-EGFR antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-Nectin 4 antibody, an anti-TROP-2 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FRα antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin Av antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD47 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody, and an anti-CD123 antibody.

11. The antibody is i) Trastuzumab antibody, ii) Bemarituzumab antibody, iii) The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 8, which is selected from anti-B7-H3 antibodies (whose heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 1 to 3, respectively, and whose light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4 to 6, respectively).

12. The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 11, wherein the anti-B7-H3 antibody has a heavy chain variable region HCVR as shown in SEQ ID NO:7 and a light chain variable region LCVR as shown in SEQ ID NO:

8.

13. The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 12, wherein the anti-B7-H3 antibody has a heavy chain constant region as shown in SEQ ID NO:9 or 10 and a light chain constant region as shown in SEQ ID NO:

11.

14.

21. The ligand-drug complex or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 2, 5 to 7, and 9 to 13, wherein the structure of the ligand-drug complex is:

15. The method for preparing the ligand-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 2 to 14, wherein the targeting ligand is reduced and then subjected to a binding reaction with a pre-synthesized -LD to obtain a compound having the general formula TL-LD.

16. A pharmaceutical composition comprising the ligand-drug conjugate of any one of claims 2 to 14 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent, or vector.

17. 15. Use of the ligand-drug conjugate of any one of claims 2 to 14 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating a tumor.

18. 18. The use according to claim 17, wherein the tumor is a solid tumor or a blood tumor such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

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