WO2024082934A1 - 酶裂解连接子及包含其的配体-艾瑞布林偶联物 - Google Patents

酶裂解连接子及包含其的配体-艾瑞布林偶联物 Download PDF

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WO2024082934A1
WO2024082934A1 PCT/CN2023/121374 CN2023121374W WO2024082934A1 WO 2024082934 A1 WO2024082934 A1 WO 2024082934A1 CN 2023121374 W CN2023121374 W CN 2023121374W WO 2024082934 A1 WO2024082934 A1 WO 2024082934A1
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antibody
group
ligand
cancer
pharmaceutically acceptable
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PCT/CN2023/121374
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French (fr)
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尚玉栓
邱均专
王振生
夏明德
陈如雷
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英诺湖医药(杭州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present disclosure relates to the field of biomedicine technology, and in particular, to an enzyme-cleavable linker and a ligand-eribulin conjugate comprising the same.
  • Antibody-Drug Conjugate is produced by coupling a small molecule drug with biological activity to a monoclonal antibody (mAb) through a linker.
  • mAb monoclonal antibody
  • most ADCs are made by coupling an antibody targeting a tumor antigen with a small molecule chemical drug with high cytotoxicity through a linker.
  • the small molecule drug is delivered to tumor cells by using the specific binding characteristics of the antibody and the target antigen to kill the tumor.
  • proteins and peptides that have specific binding properties similar to antibodies, such as specifically bound ligands, antigens and peptides, coupled to linker toxins.
  • Linkers are one of the key factors related to drug stability and therapeutic window. First of all, linkers need to have a certain degree of stability, so as to ensure the integrity of ADC during blood circulation before reaching tumor cells, avoid premature release of toxins leading to off-target toxicity, and affect the therapeutic window of ADC drugs. After entering the target cells, the linker must ensure the effective release of toxins to play a killing role. Peptide linkers are a more common cleavable linker. As of September 2022, more than ten ADC drugs have been approved for marketing. Most of them couple toxins to monoclonal antibody cysteine residues through peptide linker connectors. The peptides can be cut off by the tissue proteases of tumor cells to release toxins and produce anti-tumor effects.
  • valine-citrulline is the most commonly used in clinical research, and the marketed drug Adcetris That is, this linker is used to connect MMAE to the antibody.
  • Some ADCs drugs also use valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG), such as SGN-CD33A, Loncastuximab tesirine and DS-8201a.
  • Eribulin As a toxin conjugated to an antibody, Eribulin has a large molecular weight, a complex structure, and good hydrophilicity. Large molecular weight requires the selection of a linker of appropriate length, and complex structures may have different release efficiencies due to steric hindrance. Good hydrophilicity is more suitable for preparing a high coupling ratio conjugate of one antibody molecule coupled to multiple toxins.
  • PEG a commonly used component of a linker, has good hydrophilicity and stability. The commonly used PEG length is 2 to 8, but its light absorption is not obvious and its synthesis is relatively complicated.
  • the present disclosure is the first to discover the use of VAGGFG as an enzyme-cleavable linker.
  • TL is a targeting ligand
  • L is a linker unit
  • D is eribulin
  • n is a positive integer ranging from 1 to 20;
  • L contains the short peptide VAGGFG as an enzymatically cleavable linker.
  • the present disclosure also relates to a preparation method, a pharmaceutical composition and a medical use of the ligand-drug conjugate as described above.
  • the newly discovered linker disclosed in the present invention has significantly improved drug release efficiency, stronger ability to kill tumor cells, and has good application prospects in ligand-drug conjugates.
  • FIG1 is a cell activity test result of antibodies 7B7-H04 and 7B7 provided in one embodiment of the present disclosure coupled to Eribulin via different linker units.
  • FIG2 shows the coupling ratio and purity results of antibodies 7B7-H04 and 7B7 coupled to Eribulin through different linker units provided in one embodiment of the present disclosure
  • FIG3 shows the in vivo anti-tumor experimental results of antibody 7B7 provided in one embodiment of the present disclosure coupled to Eribulin via different linker units.
  • FIG4 shows the coupling ratio and SEC-HPLC purity results of the antibody trastuzumab coupled to MC-VAGGFG-PAB-Eribulin provided in one embodiment of the present disclosure
  • A DAR test results of Trastuzumab naked antibody
  • FIG5 is a coupling ratio and SEC-HPLC purity result of the antibody Bemarituzumab coupled to MC-VAGGFG-PAB-Eribulin provided in one embodiment of the present disclosure
  • A DAR test results of Bemarituzumab naked antibody
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").
  • the present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range.
  • fluctuations can be allowed within a corresponding precision range.
  • 2% a fluctuation within the range of ⁇ 0.1% can be allowed.
  • the meaning is also allowed to include greater fluctuations.
  • 100mM fluctuations within the range of ⁇ 1%, ⁇ 2%, ⁇ 5%, etc. can be allowed.
  • the meaning is allowed to include fluctuations of ⁇ 10%.
  • the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
  • eribulin refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the sea sponge Halichondria okadais, with a CAS of 253128-41-5. Eribulin is a microtubule dynamics inhibitor that is believed to bind to tubulin and cause cell cycle arrest in the G2/M phase by inhibiting mitotic spindle assembly.
  • eribulin mesylate CAS 441045-17-6, refers to the mesylate salt of eribulin, which is sold under the trade name Halaven TM .
  • the present disclosure relates to the use of VAGGFG as an enzymatically cleavable linker.
  • the short peptides that can be used also include X1X2GGFG , wherein X1X2 are both amino acid residues, and X1 and X2 are 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; in some embodiments , X1X2 is any one of VC , VA, AC, AV, AA, VV.
  • any one of the amino acids in X1X2GGFG can be substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl.
  • the present disclosure also relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the general formula of the ligand-drug conjugate is TL-(L-D)n;
  • TL is a targeting ligand
  • L is a linker unit
  • D is eribulin
  • n is a positive integer ranging from 1 to 20;
  • L contains the short peptide VAGGFG as an enzymatically cleavable linker.
  • n can also be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19; preferably 1-8, more preferably 4-8.
  • targeting ligand refers to a macromolecular compound that can target and recognize and bind to antigens or receptors associated with target cells.
  • the function of the ligand is to present the drug to the target cell population bound to the ligand.
  • These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies, polypeptides with binding ability, or other molecules that can bind to cells.
  • the targeting ligand The target ligand is represented as TL, and the targeting ligand can form a connection bond with the linker unit through a heteroatom on the ligand.
  • pharmaceutically acceptable salt refers to a salt of the ligand-drug conjugate of the present disclosure, meaning an acceptable salt for administration to a patient (e.g., a mammal) (for a given dosage regimen, it is a salt containing a counterion with acceptable mammalian safety).
  • Such salts may be derived from a pharmaceutically acceptable inorganic base or organic base, as well as from a pharmaceutically acceptable inorganic acid or organic acid.
  • the ligand-drug conjugate of the present disclosure contains at least one amino group, and therefore may form a salt with an acid
  • pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • the coupled drug is eribulin
  • its preferred pharmaceutically acceptable salt is eribulin mesylate.
  • solvate used in the present disclosure refers to a combination of a compound of the present disclosure and a solvent molecule formed by solvation.
  • the solvate refers to a hydrate, that is, the solvent molecule is a water molecule, and the combination of the compound of the present disclosure and water forms a hydrate.
  • the linker unit is -L 1 -L 2 -L 3 -L 4 -, L 1 is connected to TL, and L 4 is connected to D;
  • L 1 is selected from:
  • L2 is selected from:
  • R 1 and R 2 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a heteroalkyl group, a carboxyl group, an amino group, and a substituted amino group;
  • R3 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, an aryl group, a substituted aryl group or a heteroaryl group;
  • L 1 and L 2 share a N atom
  • L 3 is VAGGFG
  • L 4 is selected from: -NR 4 (CR 5 R 6 ) q -, -C(O)NR 4 -, -C(O)NR 4 (CH 2 ) q - or a chemical bond, q is selected from an integer of 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 group, a substituted alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group or a heteroaryl group.
  • L 4 can also be selected from: -NR 4 -aryl-(CR 5 R 6 ) q -OC(O)-, -NR 4 (CR 5 R 6 ) q -OC(O)-, q is an integer selected 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 group, a substituted alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group or a heteroaryl group.
  • o can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
  • p can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
  • q can be 0, 1, 2, 3, 4, 5, 6.
  • L1 and L2 share a nitrogen atom means that when the right terminal group of the defined L1 group and the left terminal group of the defined L2 group both contain nitrogen atoms, the two share the nitrogen atom.
  • L1 can be simply replaced with other, and then coupled to other natural amino acid sites, non-natural amino acid sites, terminal sites, glycosylation sites of TL, or connected by chelation or click chemistry, etc.
  • click chemistry coupling are cycloaddition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, or addition reactions of carbon-carbon multiple bonds.
  • TL has a functional group F1
  • L1 has or is a complementary functional group thereof.
  • the reactive group L1 and the functional group F1 are capable of reacting in a bioorthogonal reaction, because those reactions do not interfere with the biomolecules present during the reaction.
  • Bioorthogonal reactions and suitable functional groups therein are known to those skilled in the art, for example, as known in Gong and Pan, Tetrahedron Lett. 2015, 56, 2123-2132, including Staudinger connection and copper-free click chemistry.
  • L1 is selected from 1,3-dipole, alkyne, (hetero)cyclooctyne, cyclooctene, tetrazine, ketone, aldehyde, alkoxyamine, hydrazine and triphenylphosphine.
  • F1 is an azido group
  • the connection of the azide-modified antibody and the linker-conjugate is preferably carried out by a cycloaddition reaction.
  • the functional group L1 is preferably selected from an alkynyl group, preferably a terminal alkynyl group and a (hetero)cycloalkynyl group.
  • the connection of the keto-modified antibody to the linker-conjugate is preferably performed by selective conjugation with a hydroxylamine derivative or a hydrazine to produce an oxime or a hydrazone, respectively.
  • the functional group L 1 is preferably a primary amino group (e.g., a -NH 2 group), an aminooxy group (e.g., -O-NH 2 ) or a hydrazine group (e.g., -N(H)NH 2 ).
  • connection of the alkyne-modified antibody to the linker-conjugate is preferably performed by a cycloaddition reaction, preferably a 1,3-dipolar cycloaddition reaction.
  • the functional group L 1 is preferably a 1,3-dipole, such as an azide, a nitrone or a nitrile oxide .
  • the positions can also be interchanged.
  • 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 group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.
  • Non-limiting examples 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, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,
  • lower alkyl groups having 1 to 6 carbon atoms are 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, and the like.
  • the alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • substituted alkyl refers to an alkyl group in which a hydrogen atom is replaced by a substituent.
  • R', R" and R"' independently refer to hydrogen, unsubstituted C 1-8 alkyl, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 alkyl, C 1-8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6- or 7-membered ring with the nitrogen atom.
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • groups which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio,
  • alkoxyalkyl refers to an alkyl group substituted with one or more alkoxy groups, preferably with one alkoxy group, wherein alkyl is as defined above, and wherein alkoxy is as defined above.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein 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, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), but excluding -O-O-, -O-S- or -S-S- ring moieties, and the remaining ring atoms are carbon.
  • 3 to 12 ring atoms are contained, of which 1, 2, 3 or 4 are heteroatoms; more preferably, the heterocyclyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.
  • cycloalkylalkyl refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above and wherein cycloalkyl is as defined above.
  • aryl refers to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group containing 6 to 14 carbon ring atoms, a polycyclic (i.e., rings with adjacent pairs of carbon atoms) group with a conjugated ⁇ electron system.
  • Aryl can be monocyclic or polycyclic (i.e., it can contain more than one ring). In the case of a polycyclic aromatic ring, only one ring in the polycyclic system is required to be an aromatic ring, and the remaining rings can be saturated, partially saturated or unsaturated rings.
  • Aryl is preferably 6 to 10 yuan, such as phenyl and naphthyl.
  • the aryl ring can be fused to a heteroaryl, heterocyclyl or cyclyl ring, wherein the ring connected to the parent structure is an aryl ring.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms include oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members. More preferably, it is 5 or 6 members, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl,
  • the heteroaryl ring may be fused to an aryl, heterocyclyl or cyclyl ring, wherein the ring connected to the parent structure is the heteroaryl ring.
  • the heteroaryl group may be optionally substituted or unsubstituted.
  • deuterated alkyl refers to an alkyl group in which hydrogen atoms are replaced by one or more deuterium atoms, wherein alkyl is as defined above.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • Substituted means that one or more hydrogen atoms, preferably up to 5, more preferably 1, 2 or 3 hydrogen atoms in the group are replaced independently of each other by substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (such as olefinic) bonds.
  • -L 1 -L 2 - is And s1 is 2, 3, 4, 5, 6, 7 or 8.
  • L 4 is p-aminobenzyloxycarbonyl (PAB).
  • the targeting ligand is an antibody or an antigen-binding fragment thereof.
  • antibody as used herein is used in its broadest sense, including immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and are proteins or polypeptides that exhibit binding specificity to a specific antigen.
  • Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, bispecific antibodies, multispecific antibodies, and the like.
  • Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds.
  • variable region located at the amino terminus and a constant region (C) located at the carboxyl terminus.
  • the variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • variable regions of the heavy and light chains there are three local regions, respectively, with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody-antigen binding, and are therefore also referred to as complementary determining regions (CDRs).
  • CDRs complementary determining regions
  • the amino acid sequence of a CDR 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.
  • the "antigen-binding fragment" of an antibody refers to an amino acid fragment of an antibody molecule that participates in antigen-specific binding, for example, one of F(ab') 2 , Fab and scFv.
  • F(ab') 2 is obtained by digesting the entire full-length antibody with pepsin to remove most of the Fc region while leaving some of the hinge region intact.
  • the F(ab') 2 fragment has two antigen-binding Fab parts linked together by a disulfide bond, so the F(ab') 2 fragment is a bivalent antibody.
  • the F(ab') 2 prepared from an IgG antibody has a molecular weight of about 110 kDa.
  • Fab refers to the antibody structure that can still bind to the antigen, is monovalent and does not contain the Fc portion. Papain digestion of a full-length antibody yields two Fab fragments and one Fc fragment, each of which is approximately 50 kDa.
  • scFv is composed of the variable regions of the heavy chain and light chain of an antibody connected into a peptide chain by a short peptide. Through correct folding, the variable regions from the heavy chain and light chain interact through non-covalent bonds to form an Fv segment, so that scFv can better retain its affinity activity for the antigen.
  • the antibody or antigen-binding fragment thereof is selected from a rabbit antibody, a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • “Rabbit antibody/mouse antibody” refers to an antibody whose variable region and constant region (if present) are derived from rabbit/mouse immunoglobulin sequences.
  • Rabbit antibody/mouse antibody can be conveniently obtained by immunizing rabbit/mouse (including mouse or rat) with the corresponding antigen and isolating the target antibody therefrom.
  • cells such as B cells
  • cells expressing the target antibody are isolated and cultured to obtain the target antibody.
  • cells expressing the target antibody are isolated and cultured, and the cells are fused with immortalized cells such as myeloma cells to obtain hybridoma cells. Cultivating hybridoma cells can obtain the target antibody (such as monoclonal antibody) for a long time and in large quantities.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a first animal-derived antibody with the constant region of a second animal-derived antibody.
  • a hybridoma that secretes a first animal-derived specific monoclonal antibody must first be established, and then the variable region gene must be cloned from the hybridoma cell, and then the constant region gene of the second animal-derived antibody must be cloned as needed, and the first animal-derived variable region gene and the second animal-derived constant region gene must be connected into a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the first animal-derived source is rabbit or mouse-derived
  • the second animal-derived source is preferably human-derived, which can reduce 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 ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the subtype of the antibody constant region, the isoforms of different people, and the mutations caused by the changes in the effector function of the constant region do not affect the preparation of the antibody conjugate.
  • Linker toxin coupling sites such as cysteine, lysine, glutamine, peptide chain carboxyl terminus and glycosylation site, including natural sites and engineered sites, are generally used for coupling.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of the first animal origin into the human antibody variable region framework, that is, different types of human germline antibody framework sequences.
  • the heterologous reaction induced by chimeric antibodies due to carrying a large amount of protein components from the first animal origin can be overcome.
  • Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be obtained in the "VBase" human germline sequence database (www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition.
  • the human antibody variable region framework sequence may be subjected to a minimum reverse mutation or back mutation to maintain activity.
  • the humanized antibodies disclosed herein also include humanized antibodies after CDR affinity maturation is further performed by phage display.
  • the first animal source is rabbit or mouse.
  • the human antibody variable region framework is selected by design.
  • the human antibody variable region may be subjected to a minimum reverse mutation to maintain activity.
  • a fully humanized antibody refers to a human antibody gene that is transferred into a genetically engineered antibody gene-deficient animal through transgenic or chromosome transfer technology, so that the animal expresses human antibodies and achieves the purpose of fully humanized antibodies.
  • the antibody is selected from: anti-CD3 antibody, anti-FOLR1 antibody, anti-ROR1 antibody, anti-TNF ⁇ antibody, anti-tissue factor (TF) antibody, anti-EpCAM antibody, anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-Cl audin 6 antibody, anti-Claudin 9 antibody, anti-Claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2(ErbB2) antibody, anti-EGFR antibody, anti-c ⁇ MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GP
  • the antibody is selected from the group consisting of the following antibodies:
  • 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 vedo
  • the antibody is selected from:
  • Anti-B7-H3 antibody Its heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1 to 3, and its light chain complementary determining regions LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4 to 6.
  • the heavy chain variable region HCVR of the anti-B7-H3 antibody is as shown in SEQ ID NO:7
  • the light chain variable region LCVR is as shown in SEQ ID NO:8.
  • the heavy chain constant region of the anti-B7-H3 antibody is as shown in SEQ ID NO:9 or 10
  • the light chain constant region is as shown in SEQ ID NO:11.
  • variants of the above amino acid sequences are also within the scope of the present invention, and the corresponding variants respectively contain up to 3 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, the variants may contain 3 or more mutations relative to the overall sequence of SEQ ID NO:7 to SEQ ID NO:15, for example, sequences having at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identity compared to any of the polypeptides of SEQ ID NO:7 to SEQ ID NO:15.
  • the mutations may be substitutions, deletions or additions of amino acids or any combination thereof; preferably, the mutations are conservative substitutions.
  • Constant substitution refers to the replacement 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.), such that changes can frequently be made without altering the biological activity of the protein.
  • substitutions that are generally considered conservative substitutions are substitutions between aliphatic amino acids Ala, Val, Leu and Ile, interchanges of hydroxyl residues Ser and Thr, exchanges of acidic residues Asp and Glu, substitutions between amide residues Asn and Gln, exchanges of basic residues Lys and Arg, and substitutions between aromatic residues Phe and Tyr.
  • substitutions of amino acids with similar structures or functions are unlikely to destroy biological activity.
  • the structure of the ligand-drug conjugate is as follows:
  • the present disclosure also relates to a method for preparing the ligand-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, comprising:
  • a coupling reaction is carried out with the pre-synthesized -L-D to obtain a compound represented by the general formula TL-L-D.
  • the reducing agent is preferably TCEP, and in particular, preferably reduces disulfide bonds on the targeting ligand.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the ligand-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, diluent or carrier.
  • pharmaceutically acceptable carrier includes any material that, when combined with the active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • the present disclosure also relates to use of the ligand-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof in preparing a drug for treating tumors.
  • cancer refers to a physiological condition or disease characterized by unregulated cell growth.
  • Tumor includes cancer cells.
  • the tumor is a solid tumor or hematological 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 (such as non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, multiforme glioma, sarcoma, lymphoma and leukemia.
  • the present disclosure also 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.
  • the medical condition is cancer
  • safe and effective amount means an amount of a compound or composition that is large enough to significantly and effectively alleviate the symptoms or conditions being treated, but small enough to avoid serious side effects (at a reasonable benefit/risk ratio) within reasonable medical regulation.
  • the safe and effective amount of the active ingredient in the pharmaceutical composition used in the method of the present disclosure varies depending on the specific symptoms being treated, the age and physical condition of the patient being treated, the severity of the disease, the duration of treatment, concurrent treatment, use
  • the dosage form may vary, depending on the particular active ingredient employed, the particular pharmaceutically acceptable excipient employed, and such factors including the knowledge and skill of the treating physician.
  • the envisioned treatment methods will also include the administration of other therapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic E.
  • viral cancer vaccines e.g., adenoviral vectors encoding cancer-specific antigens
  • bacterial cancer vaccines e.g., non-pyrogenic E.
  • the envisioned treatment methods also include radiotherapy of the patient.
  • the envisioned treatment methods also include surgery of the patient, such as tumor resection surgery.
  • the ligand-drug conjugates may also be administered and/or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN- ⁇ and/or NSAIDs.
  • antiviral agents antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN- ⁇ and/or NSAIDs.
  • antiviral agents antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN- ⁇ and/or NSAIDs.
  • antiviral agents antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN- ⁇ and/or NSAIDs.
  • the above therapeutic entities may be included in the pharmaceutical composition.
  • subject and “patient” are used interchangeably herein and refer to any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, dogs, cats, chimpanzees, orangutans, gibbons, macaques, marmosets, pigs, horses, pandas, and elephants, among others.
  • treatment refers to any improvement in any consequence of a disease, such as prolonged survival, less morbidity, and/or reduced side effects as a byproduct of alternative treatment modalities. As is readily understood in the art, complete eradication of the disease is preferred, but not a prerequisite for therapeutic action.
  • treatment refers to administering the ligand-drug conjugate to a subject (e.g., a patient). Treatment can be to cure, heal, alleviate, mitigate, alter, cure, improve, attenuate, improve, or affect a condition, a symptom of a condition, or a predisposition to a disease, such as cancer.
  • compositions of the present disclosure may be administered by any route, as will be appreciated by those skilled in the art.
  • pharmaceutical compositions of the present disclosure are administered intravenously (IV).
  • the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified.
  • acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
  • the commercial vector pTT5 can be used for the expression of all antibodies in the present disclosure, and the antibody sequence can be inserted between the EcoR I and Hind III restriction enzyme sites after the promoter.
  • the nucleic acid sequence including the signal peptide before the antibody light chain or heavy chain sequence can be selected from the following sequences:
  • the antibody light chain or heavy chain sequence is followed by:
  • TGAAAGCTT (which is the stop codon and Hind III).
  • the antibody amino acid sequence is optimized into a nucleotide sequence and then inserted between the above-mentioned signal peptide and terminator.
  • Anti-B7-H3 antibody is clone 7B7, the antibody with light chain L00 and heavy chain H00 is 7B7.
  • the antibody with light chain L00 and heavy chain H04 is 7B7-H04.
  • 7B7-H04 antibody is an engineered antibody, which is suitable for producing conjugated products with uniform conjugation ratios, and is very suitable for evaluating different linker toxins.
  • the above antibody light and heavy chains were constructed in the vectors respectively, and the plasmids were extracted.
  • the suspended domesticated CHO-K1 cells were revived in OPM-CD TransCHO medium (manufacturer: OPM, catalog number P83059), cultured to a density of 2 million cells/ml, a viability of more than 95%, and a volume of 1000ml.
  • 0.5mg of the light chain plasmid and 0.5mg of the heavy chain plasmid were combined and dissolved in 10ml of culture medium, 3mg of PEI (manufacturer: Polyscience, catalog number: 24765-1) dissolved in the culture medium was diluted in 10ml of culture medium, the plasmid and PEI solutions were mixed and placed at room temperature for 10 minutes, and then added dropwise to 1000ml of cell culture medium, cultured at 37°C for 5 days, and centrifuged at 12000g for 15 minutes to harvest the supernatant. The supernatant was purified with HiTrap Mabselect SuRe and eluted with 50 mM acetic acid.
  • the collected peak after neutralization was replaced with PBS pH 7.4 using a 30KD ultrafiltration tube (Merck, catalog number UFC9030).
  • the antibody was measured for absorbance at 280 nm, and the absorbance divided by the theoretical extinction coefficient was the concentration value.
  • linker toxin MC-VA-PAB-Eribulin (LK-322018) structure is as follows:
  • linker toxin MC-GGFG-PAB-Eribulin (LK-322016) structure is as follows:
  • the designed linker toxin MC-VAGGFG-PAB-Eribulin (LK-322022) has the following structure:
  • reaction solution was quenched with 10 ml of ammonium chloride aqueous solution, extracted three times with ethyl acetate (10 ml each time), and the organic phases were combined after separation, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated.
  • TCEP mother solution is 10 mM mother solution (TCEP manufacturer Sigma, item number C4706-2G, CAS.51805-45-9) prepared with 10 mM sodium phosphate buffer pH 7.4, ensure that the final molar concentration of TCEP is 4:1 with the final molar concentration of antibody, and place the antibody at 37°C for reduction for 45 minutes.
  • the mother solution is the conjugate toxin dissolved in dimethyl sulfoxide DMSO to 5 mM, ensure that the final molar concentration of the conjugate toxin is 6:1 with the final molar concentration of the antibody, and place the antibody at 2-8°C for coupling for 2 hours.
  • the antibody was then transferred to a 30KD ultrafiltration tube (Merck, catalog number UFC9030), placed at 3000g centrifugal force at 2-8°C for liquid exchange to reduce the residual toxin to one thousandth of the reaction concentration, followed by sterile filtration, determination of concentration, and aliquoting for cryopreservation.
  • the molar ratio of antibody: reducing agent: linker toxin was adjusted between 1:1 and 6:2 and 15, and the other conditions remained unchanged.
  • the DAR value of the final coupling product was based on the test results.
  • Human Calu-6 (Procell, Catalog No. CL-327, human lung cancer cells) was revived and inoculated into a 96-well culture plate.
  • the culture medium was DMEM/F12 (Hyclone, Catalog No. SH30023.01) containing 10% FBS (Gibco, Catalog No. 10099141), 2500 cells per well, 150 ⁇ l, and cultured in a 37°C, 5% carbon dioxide incubator. After about 20 hours, 50 ⁇ l of culture medium containing different antibody-drug conjugates was added to make the final drug concentration between 0 and 100 nM in 12 dilution concentrations (the first well concentration was 100 nM, followed by 10 gradients of 5-fold dilutions, and the last gradient drug was 0, and each sample was repeated).
  • CTG detection reagent CellTiterGlo, Promega, Catalog No. G7575
  • Tecan Spark The average value of the duplicate wells was taken as the Y-axis, and the log10 value of the dilution gradient was used as the X-axis to make a smooth curve graph, and the EC/ IC50 value was calculated according to the four parameters.
  • the cell lines can be replaced with Jurket, MCF-7 and MDA-MB-468 for re-validation.
  • the average value (DAR) of the number of drugs attached to the antibody was detected by the hydrophobic chromatography HIC-HPLC method.
  • the analytical column was TSKgel Butyl-NPR, 4.6mm ⁇ 10cm, 2.5 microns, manufacturer TOSOH, item number 042168.
  • the volume is 150 ⁇ l, add 150 ⁇ l of mobile phase A, centrifuge at 10000g for 5 minutes and take the supernatant.
  • Mobile phase A is 20mM sodium phosphate + 1.5M ammonium sulfate pH7.0
  • mobile phase B is 20mM sodium phosphate + 20% (v/v) acetonitrile pH7.0.
  • the analytical column is connected to an Agilent 1260 high-performance liquid chromatograph with a flow rate of 0.6ml/min.
  • the mobile phase A is rinsed for more than 30 minutes until the UV baseline at 280nm is stable. 50 micrograms of sample are injected. After rinsing with mobile phase A for 2 minutes, a gradient elution (0%B–100%B) is performed for 18 minutes, and the 100% mobile phase B rinse is continued for 5 minutes before the end.
  • the percentage of each DAR value is calculated based on the peak area of each DAR value. The percentage of each peak is multiplied by the DAR value, then added up and divided by the total percentage to obtain the average DAR value.
  • Antibody 7B7-H04 was conjugated to MC-VA-PAB-Eribulin, Mal-PEG2-VC-PAB-Eribulin, MC-GGFG-PAB-Eribulin and MC-VAGGFG-PAB-Eribulin.
  • the EC/IC 50 of the conjugates against Calu6 cells showed that the EC/IC 50 were 115.9 pM, 29.97 pM, 62.32 pM and 11.40 pM, respectively ( Figure 1), indicating that the effect of MC-VAGGFG-PAB-Eribulin was significantly better than that of the other groups.
  • the average DAR values of the conjugates were 3.33, 3.27 and 3.31 ( FIG. 2 ), indicating that the peptide linker of VAGGFG was more active than the peptide linkers of GGFG and VA when the conjugation ratios were close.
  • Bioduro www.bioduro-sundia.com was commissioned to conduct an in vivo efficacy evaluation.
  • Well-cultured human breast cancer cell lines (MDA-MB-468) were inoculated into immunodeficient mice (B-NDG), with 10 million cells per mouse.
  • B-NDG immunodeficient mice
  • the mice were randomly divided into groups after the tumors grew to 100 mm 3. The next day, each group was given 3 mg/kg for a total of 2 times, once a week, and the tumor size was measured twice a week until the end of the experiment. The entire process was approved by the Experimental Animal Committee.
  • Each group was independently selected to be administered at 1 to 10 mg/kg (such as 2, 3, 4, 5, 6, 7, 8 or 9 mg/kg), which was consistent with the experimental trend of MDA-MB-468 and had the best effect on inhibiting tumor volume.
  • 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, and placed at 37°C for 0, 4, 7, 10, and 14 days, and then the killing activity of the samples against Jurket cells was determined. It was found that the release rate of Eribulin (Y axis) increased with time (X axis), with a maximum of 0.1-5% Eribulin released, indicating that the plasma stability of the linker was very good.
  • ADCs of trastuzumab and bemarituzumab coupled to the linker toxin MC-VAGGFG-PAB-Eribulin were prepared for verification.
  • the results showed that the average DAR value of the coupling ratio was 2 to 3, and the purity was >95% ( Figures 4 and 5).
  • SK-BR-3 cell line human breast cancer cell
  • KATO III cell line human gastric cancer cell
  • the compound MC-VC-PAB-MMAE is commercially available, and MC-VA-PAB-MMAE has been reported to have no significant difference from MC-VC-PAB-MMAE.
  • the synthesis steps are as follows: dissolve the commercially available compound 1 (50 mg, 0.0865 mmol) in 2 ml N, N-dimethylformamide, add Bis-PNP (36 mg, 0.1182 mmol) and N, N-diisopropylethylamine (20.3 mg, 0.006053 mmol) in sequence, react at room temperature for 16 hours, monitor the reaction of the raw materials by liquid chromatography-mass spectrometry, add 15 ml of water, extract three times with ethyl acetate (10 ml each time), separate the organic phases, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate and concentrate, and purify the crude product by preparative liquid phase to obtain compound 3 (50 mg, yield: 77.8%).
  • the compound MC-VA-PAB-Exatecan is commercially available.
  • the above compound was conjugated to antibody 7B7 according to Example 4 to prepare a conjugate with a conjugation ratio of 3 to 4, and the activity against the cell line MCF-7 was determined according to Example 5.
  • the results are as follows: 1) The conjugates conjugated to MMAE toxin, with VC, GGFG and VAGGFG polypeptide linkers, had an activity IC 50 of 0.49, 0.41 and 0.27 nM; 2) The conjugates conjugated to Extecan toxin, with VA, GGFG and VAGGFG polypeptide linkers, had an activity IC 50 of 242, 270 and 307 nM.

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Abstract

本公开涉及生物医药技术领域，具体而言，涉及一种酶裂解连接子及包含其的配体-艾瑞布林偶联物。更具体的，涉及VAGGFG作为酶裂解连接子的应用。

Description

酶裂解连接子及包含其的配体-艾瑞布林偶联物
相关申请的交叉引用
本申请要求于2022年10月20日提交的申请号为“CN202211287874.0”、发明名称为“酶裂解连接子及包含其的配体-艾瑞布林偶联物”，以及于2023年07月12日提交的申请号为“CN202310853999.3”的专利申请的优先权，其内容通过引用整体并入本文。
技术领域
本公开涉及生物医药技术领域，具体而言，涉及一种酶裂解连接子及包含其的配体-艾瑞布林偶联物。
背景技术
抗体偶联药物(Antibody-Drug Conjugate，ADC)是通过连接子(linker)将具有生物活性的小分子药物偶联至单克隆抗体(单抗)上而产生的。目前绝大部分ADC是由靶向肿瘤抗原的抗体通过连接子与高效细胞毒性的小分子化学药物偶联而成，利用抗体与靶抗原特异性结合的特点，将小分子药物靶向递送至肿瘤细胞进而发挥杀伤肿瘤的作用。也有将特异性结合的配体、抗原和多肽等具有与抗体相似的特异性结合性质的蛋白及多肽偶联连接子毒素的。
连接子是关系到药物稳定性和治疗窗口的关键之一。首先，连接子需要具备一定的稳定性，从而在未达到肿瘤细胞之前，能确保ADC在血液循环过程中的完整性，避免提前释放毒素导致脱靶毒性，影响ADC药物的治疗窗口。而在进入靶细胞后，连接子要确保毒素的有效释放，发挥杀伤作用。多肽连接子是一种较为常见的可裂解连接子。截止2022年9月，已经有十个以上ADC药物被批准上市，多数通过将毒素经多肽连接子接头偶联于单克隆抗体半胱氨酸残基上，多肽可被肿瘤细胞的组织蛋白酶切断从而释放毒素，产生抗肿瘤作用。
现在临床研究中最常用的是缬氨酸-瓜氨酸(VC)，已上市药物Adcetris 即采用这种连接子将MMAE与抗体相连。还有一些ADCs药物采用缬氨酸-丙氨酸(VA)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(GGFG)，如SGN-CD33A，Loncastuximab tesirine和DS-8201a。
艾瑞布林(艾日布林，Eribulin)作为抗体偶联物的毒素，其分子量大，结构复杂，亲水性较好。分子量大需要筛选合适长度连接子，结构复杂可能不同的连接子由于空间位阻酶切释放效率不同，亲水性好比较适合制备一个抗体分子偶联多个毒素的高偶联比值偶联物。PEG作为一种连接子常用的组件，其亲水性好、稳定性好，常用PEG长度为2～8个，但是其光吸收不明显，合成较为复杂。
发明内容
本公开首次发现了VAGGFG作为酶裂解连接子的应用。
配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物的通式为TL-(L-D)n；
其中，TL为靶向配体，L为接头单元，D为艾瑞布林，n为1～20的正整数；
L中包含短肽VAGGFG作为酶裂解连接子。
本公开还涉及如上所述的配体-药物偶联物的制备方法、药物组合物及医药用途。
本公开所新发现的连接子，其具有显著提升的药物释放效率，对肿瘤细胞的杀伤能力更强，在配体-药物偶联物中具有很好的应用前景。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开一个实施例所提供的抗体7B7-H04和7B7通过不同接头单元偶联艾瑞布林的细胞活性检测结果。
图2为本公开一个实施例所提供的抗体7B7-H04和7B7通过不同接头单元偶联艾瑞布林的偶联比值和纯度结果；
A：7B7裸抗体(naked antibody)的DAR检测结果；
B：7B7-H04 MC-VA-PAB-Eribulin的DAR检测结果；
C：7B7-H04 MC-VA-PAB-Eribulin的SEC-HPLC纯度检测结果；
D：7B7-H04 MC-GGFG-PAB-Eribulin的DAR检测结果；
E：7B7-H04 MC-GGFG-PAB-Eribulin的SEC-HPLC纯度检测结果；
F：7B7-H04 MC-VAGGFG-PAB-Eribulin的DAR检测结果；
G：7B7-H04 MC-VAGGFG-PAB-Eribulin的SEC-HPLC纯度检测结果；
H：7B7 MC-VAGGFG-PAB-Eribulin的DAR检测结果；
I：7B7 MC-VAGGFG-PAB-Eribulin的SEC-HPLC纯度检测结果。
图3为本公开一个实施例所提供的抗体7B7通过不同接头单元偶联艾瑞布林的体内抗肿瘤实验结果。
图4为本公开一个实施例所提供的抗体曲妥珠单抗(Trastuzumab)偶联MC-VAGGFG-PAB-Eribulin的偶联比值、SEC-HPLC纯度结果；
A：曲妥珠单抗裸抗体(Trastuzumab naked antibody)的DAR检测结果；
B：Trastuzumab MC-VAGGFG-PAB-Eribulin的DAR检测结果；
C：Trastuzumab MC-VAGGFG-PAB-Eribulin的SEC-HPLC纯度检测结果。
图5为本公开一个实施例所提供的抗体贝马珠单抗(Bemarituzumab)偶联MC-VAGGFG-PAB-Eribulin的偶联比值、SEC-HPLC纯度结果；
A：贝马珠单抗裸抗体(Bemarituzumab naked antibody)的DAR检测结果；
B：Bemarituzumab MC-VAGGFG-PAB-Eribulin的DAR检测结果；
C：Bemarituzumab MC-VAGGFG-PAB-Eribulin的SEC-HPLC纯度检测结果。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以 用于另一实施方式中，来产生更进一步的实施方式。
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本公开中所使用的术语“含有”、“包含”和“包括”是同义词，其是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。
本公开中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。
本公开中涉及浓度数值，其含义包括在一定范围内的波动。比如，可以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。
本公开中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。
本公开中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本公开中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本公开保护范围的限制。本公开中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可 选“”各自独立。
如本文所用，术语“艾瑞布林”是指软海绵素B(最初从海绵冈田软海绵(Halichondria okadais)分离的大环化合物)的合成类似物，其CAS为253128-41-5。艾瑞布林为微管动力学抑制剂，认为其结合微管蛋白并通过抑制有丝分裂纺锤体组件，在G2/M期引起细胞周期停滞。术语“艾瑞布林甲磺酸盐”，CAS为441045-17-6，是指艾瑞布林的甲磺酸盐，其以商品名HalavenTM出售。
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的发明目的和/或技术方案相冲突，否则，本公开涉及的引用文献以全部内容、全部目的被引用。本公开中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本公开中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本公开为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。
本公开涉及VAGGFG作为酶裂解连接子的应用。
可采用的短肽还包括X1X2GGFG，其中，X1X2均为氨基酸残基，X1和X2各自独立的选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、酪氨酸、色氨酸、丝氨酸、苏氨酸、半胱氨酸、蛋氨酸、天冬氨酸、谷氨酰胺、天冬酰胺、赖氨酸、精氨酸、谷氨酸和组氨酸；在一些实施方式中，X1X2为VC、VA、AC、AV、AA、VV中的任一种。进一步的，X1X2GGFG中的任一个氨基酸可以被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代。
本公开还涉及一种配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物的通式为TL-(L-D)n；
其中，TL为靶向配体，L为接头单元，D为艾瑞布林，n为1～20的正整数；
L中包含短肽VAGGFG作为酶裂解连接子。
其中，n的值还可以取2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19；优选1～8，更优选4～8。
术语“靶向配体”是能靶向性识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体、具有结合能力的多肽或其他能与细胞结合的分子。在本公开的实施方式中，靶向配 体表示为TL，靶向配体可通过配体上的杂原子与连接单元形成连接键。
术语“药学上可接受的盐”是指本公开配体-药物偶联物的盐，意指对于给药至患者(例如哺乳动物)是可接受的盐(对于给定的剂量方案，其为包含具有可接受的哺乳动物安全性的抗衡离子的盐)。这种盐可衍生自药学上可接受的无机碱或有机碱，以及衍生自药学上可接受的无机酸或有机酸。本公开的配体-药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。尤其当所偶联的药物为艾瑞布林时，其优选的药学上可接受的盐是艾瑞布林甲磺酸盐。
本公开所用术语“溶剂化物”是指通过溶剂化作用形成的本公开化合物与溶剂分子的组合。在某些情况下，溶剂化物指水合物，即溶剂分子为水分子，本公开化合物与水的组合形成水合物。
在一些实施方式中，所述接头单元为-L1-L2-L3-L4-，L1与TL相连，L4与D相连；
其中，
L1选自：
或点击化学所需基团(优选)；
L2选自：
-NC(R1R2)C(O)、-NR3(CH2)oC(O)-、-NR3(CH2CH2O)oCH2C(O)-、-S(CH2)pC(O)-或化学键，其中o选自0～20的整数；p选自0～20的整数；
R1、R2各自独立地选自氢原子、氘原子、烷基、取代烷基、氘代烷基、杂烷基、羧基、氨基、取代氨基；
R3选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
L1与L2共用N原子；
L3为VAGGFG；
L4选自：-NR4(CR5R6)q-、-C(O)NR4-、-C(O)NR4(CH2)q-或者化学键，q选自0～6的整数；
R4、R5和R6各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
在一些实施方式中，L4还可以选自：-NR4-芳基-(CR5R6)q-OC(O)-、-NR4(CR5R6)q-OC(O)-，q选自0～6的整数；
R4、R5和R6各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
在一些实施方式中，o可为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20。
在一些实施方式中，p可为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20。
在一些实施方式中，q可为0、1、2、3、4、5、6。
“L1与L2共用N原子”是指，当所定义的L1基团右侧端基、所定义的L2基团左侧端基均含N原子时，二者共用N原子。
很明显，可以通过将L1简单替换为其它，进而偶联到TL的其它天然氨基酸位点、非天然氨基酸位点、末端位点、糖基化位点，或通过螯合或点击化学等方式进行连接。点击化学偶联的实例如环加成反应、亲核开环反应、非醇醛的羰基化学或碳碳多键的加成反应。
在一些实例中，TL具有官能团F1，L1具有或为其互补官能团。优选地，反应性基团L1和官能团F1能够在生物正交反应中反应，因为那些反应不会干扰在该反应过程中存在的生物分子。生物正交反应和其中适合的官能团是本领域技术人员已知的，例如已知于Gong和Pan，Tetrahedron Lett.2015，56，2123‐2132，包括Staudinger连接和无铜点击化学。因此，优选L1选自1，3‐偶极子、炔烃、(杂)环辛炔、环辛烯、四嗪、酮、醛、烷氧基胺、肼和三苯基膦。例如，当F1是叠氮基时，叠氮化物修饰的抗体和接头‐缀合物的连接优选通过环加成反应进行。然后，官能团L1优选选自炔基，优选末端炔基和(杂)环炔基。例如，当F1是酮基时，酮修饰的抗体与接头‐缀合物的连接优选通过与羟胺衍生物或肼的选择性缀合进行，分别产生肟或腙。然后，官能团L1优选为伯氨基(例如，‐NH2基团)、氨氧基(例如，‐O‐NH2)或肼基(例如，‐N(H)NH2)。例如，当F1是炔基时，炔烃修饰的抗体与接头‐缀合物的连接优选通过环加成反应，优选1，3‐偶极环加成反应进行。然后，官能团L1优选为1，3‐偶极子，例如叠氮化物、硝酮或氧化腈。F1和L1的 位置也可以互换。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1‐二甲基丙基、1,2‐二甲基丙基、2,2‐二甲基丙基、1‐乙基丙基、2‐甲基丁基、3‐甲基丁基、正己基、1‐乙基‐2‐甲基丙基、1,1,2‐三甲基丙基、1,1‐二甲基丁基、1,2‐二甲基丁基、2,2‐二甲基丁基、1,3‐二甲基丁基、2‐乙基丁基、2‐甲基戊基、3‐甲基戊基、4‐甲基戊基、2,3‐二甲基丁基、正庚基、2‐甲基己基、3‐甲基己基、4‐甲基己基、5‐甲基己基、2,3‐二甲基戊基、2,4‐二甲基戊基、2,2‐二甲基戊基、3,3‐二甲基戊基、2‐乙基戊基、3‐乙基戊基、正辛基、2,3‐二甲基己基、2,4‐二甲基己基、2,5‐二甲基己基、2,2‐二甲基己基、3,3‐二甲基己基、4,4‐二甲基己基、2‐乙基己基、3‐乙基己基、4‐乙基己基、2‐甲基‐2‐乙基戊基、2‐甲基‐3‐乙基戊基、正壬基、2‐甲基‐2‐乙基己基、2‐甲基‐3‐乙基己基、2,2‐二乙基戊基、正癸基、3,3‐二乙基己基、2,2‐二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1‐二甲基丙基、1,2‐二甲基丙基、2,2‐二甲基丙基、1‐乙基丙基、2‐甲基丁基、3‐甲基丁基、正己基、1‐乙基‐2‐甲基丙基、1,1,2‐三甲基丙基、1,1‐二甲基丁基、1,2‐二甲基丁基、2,2‐二甲基丁基、1,3‐二甲基丁基、2‐乙基丁基、2‐甲基戊基、3‐甲基戊基、4‐甲基戊基、2,3‐二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“取代烷基”指烷基中的氢被取代基团取代，除非文中另有说明，烷基的取代基可以是选自下组的多种基团：‐卤素、‐OR’、‐NR’R”、‐SR’、‐SiR’R”R”’、‐OC(O)R’、‐C(O)R’、‐CO2R’、‐CONR’R”、‐OC(O)NR’R”、‐NR”C(O)R’、‐NR’‐C(O)NR”R”’、‐NR”C(O)2R’、‐NH‐C(NH2)＝NH、‐NR’C(NH2)＝NH、‐NH‐C(NH2)＝NR’、‐S(O)R’、‐S(O)2R’、‐S(O)2NR’R”、‐NR’S(O)2R”、‐CN和‐NO2，取代基数量为0至(2m’+1)，其中m’为该基团中碳原子的总数。R’、R”和R”’各自独立的指代氢、未取代的C1‐8烷基、未取代的芳基、由1‐3个卤素取代的芳基、未取代的C1‐8烷基、C1‐8烷氧基或C1‐8硫代烷氧基、或未取代的芳基‐C1‐4烷基。R’和R”连接于同一个氮原子时，它们可与该氮原子一起形成3‐，4‐，5‐，6‐或7‐元环。例如，‐NR’R”包括1‐吡咯烷基和4‐吗啉基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性示例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“烷氧基烷基”指烷基被一个或多个烷氧基取代，优选被一个烷氧基取代，其中烷基如上所定义，其中烷氧基如上所定义。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括‐O‐O‐、‐O‐S‐或‐S‐S‐的环部分，其余环原子为碳。优选包含3至12个环原子，其中1、2、3或4个是杂原子；更优选杂环基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“芳基”是指含有6至14个碳环原子的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，具有共轭的π电子体系的多环(即其带有相邻对碳原子的环)基团。芳基可以是单环或多环的(即，可以含有一个以上的环)。在多环芳环的情况下，只要求多环系统中的一个环是芳香环，而其余的环可以是饱和、部分饱和或不饱和环。芳基优选为6至10元的，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环基或环基环上，其中与母体结构连接在一起的环为芳基环。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子包括氧、硫和氮。杂芳基优选为5至10元。更优选是5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N‐烷基吡咯基、嘧啶基、 吡嗪基、咪唑基、四唑基、噁唑基、异噁唑基等，所述杂芳基环可以稠合于芳基、杂环基或环基环上，其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是任选取代的或未取代的。
术语“氘代烷基”指烷基上的氢被一个或多个氘原子取代，其中烷基如上所定义。
术语“卤素”指氟、氯、溴或碘。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1个、2个或3个氢原子彼此独立地被取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯)键的碳原子结合时可能是不稳定的。
在一些实施方式中，-L1-L2-为且s1为2、3、4、5、6、7或8。
在一些实施方式中，L4为对氨基苄氧羰基(PAB)。
在一些实施方式中，所述靶向配体为抗体或其抗原结合片段。
本文使用的术语“抗体”以它的最广泛意义使用，包括包含一个或更多个特异性结合抗原的抗原结合结构域的免疫球蛋白或其他类型分子，为对特定抗原表现出结合特异性的蛋白质或多肽。抗体的具体实例可包括完整抗体(例如经典四链抗体分子)、单链抗体、单域抗体、双特异性抗体、多特异性抗体等。经典抗体分子通常为由2个相同重链和2个相同轻链通过二硫键相互连接组成的四聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。可变区用于识别和结合抗原，恒定区(如Fc片段)用于启动下游效应，比如抗体依赖性细胞介导的细胞毒作用(ADCC)。在重链和轻链的可变区内，分别有三个局部区域的氨基酸组成和排列顺序具有更高的变异程度，为抗体与抗原结合的关键位置，因而也称为互补决定区(CDR)。CDR的氨基酸序列可以使用本领域公认的编号方案，例如Kabat、Chothia、IMGT、AbM或Contact容易地确定。抗体可以为IgG、IgM、IgD、IgE或IgA抗体。
抗体的“抗原结合片段”指抗体分子中参与抗原特异性结合的氨基酸片段，例如，F(ab')2、Fab以及scFv中的一种。
术语“F(ab')2”是由胃蛋白酶消化整个全长抗体去除大部分Fc区同时完整保留一些铰链区后得到的。F(ab')2片段具有通过二硫键连接在一起的两个抗原结合Fab部分，因此F(ab')2片段为双价抗体，以IgG抗体制备得到的F(ab')2为例，分子量为约110kDa。
术语“Fab”是仍可与抗原结合的抗体结构，其为单价并且不含Fc部分。木瓜蛋白酶消化全长抗体后得到两个Fab片段以及一个Fc片段，每个Fab片段均为约50kDa。
术语“scFv”是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。
在一些实施方式中，所述抗体或其抗原结合片段选自兔源抗体、鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
“兔源抗体/鼠源抗体”是指可变区和恒定区(如果存在的话)衍生自兔/鼠免疫球蛋白序列的抗体。兔源抗体/鼠源抗体可方便地以相应抗原免疫兔/鼠(包括小鼠或大鼠)并从其分离目的抗体而获得。或者，在以相应抗原免疫兔/鼠后，分离并培养表达目的抗体的细胞(如B细胞)而获得。又或者，在以相应抗原免疫兔/鼠后，分离并培养表达目的抗体的细胞，将其与永生化细胞如骨髓瘤细胞融合而获得杂交瘤细胞，培养杂交瘤细胞则可长期和大量获得目的抗体(如单克隆抗体)。
术语“嵌合抗体(chimeric antibody)”，是将第一动物源性抗体的可变区与第二动物源性的抗体的恒定区融合而成的抗体。建立嵌合抗体，要先建立分泌第一动物源性特异性单抗的杂交瘤，然后从杂交瘤细胞中克隆可变区基因，再根据需要克隆第二动物源性抗体的恒定区基因，将第一动物源性可变区基因与第二动物源性恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，第一动物源性为兔源或鼠源，第二动物源性优选为人源，可以减轻第一动物源性抗体诱发的免疫应答反应。所述嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。抗体恒定区的亚型、不同人的异构体和基于恒定区效应功能的变化产生的突变不影响抗体偶联物的制备。连接子毒素偶联位点比如半胱氨酸、赖氨酸、谷氨酰胺、肽链羧基末端和糖基化位点等，包括天然位点和工程化改造位点，这些位点一般用于偶联。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将第一动物源性的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量第一动物源性蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(www.mrccpe.com.ac.uk/vbase)获得，以及在Kabat，E.A. 等人，1991，Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本公开一个优选的实施方案中，第一动物源性为兔源或鼠源。人的抗体可变区框架经过设计选择。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
全人源化抗体(human antibody)是指将人类抗体基因通过转基因或转染色体技术，将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达人类抗体，达到抗体全人源化的目的。
在一些实施方式中，所述抗体选自：抗CD3抗体、抗FOLR1抗体、抗ROR1抗体、抗TNFα抗体、抗组织因子(tissue factor，TF)抗体、抗EpCAM抗体、抗EGFRvIII抗体、抗DLL‐3抗体、抗PSMA抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM‐1抗体、抗LRRC15抗体、抗LIV‐1抗体、抗CanAg/AFP抗体、抗Claudin 6抗体、抗Claudin 9抗体、抗Claudin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c‐MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7‐H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP‐2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD70抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P‐cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD20抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体和抗CD123抗体。
在一些具体的实施方式中，所述抗体选自下列抗体所组成的组：
抗GD2抗体3F8、阿巴伏单抗(Abagovomab)、阿昔单抗(Abciximab)、ACZ885(卡那单抗(canakinumab))、阿达木单抗(Adalimumab)、阿德木单抗(Adecatumumab)、阿非莫单抗(Afelimomab)、阿托珠单抗(Afutuzumab)、培化阿珠单抗(Alacizumab pegol)、阿仑单抗(Alemtuzumab)、喷替酸阿妥莫单抗(Altumomab pentetate)、麻安莫单抗(Anatumomab mafenatox)、安芦珠单抗(Anrukinzumab)(IMA-638)、 阿泊珠单抗(Apolizumab)、阿西莫单抗(Arcitumomab)、阿塞珠单抗(Aselizumab)、阿替珠单抗(Atezolizumab)、阿托木单抗(Atorolimumab)、阿维单抗(Avelumab)、巴匹珠单抗(Bapineuzumab)、巴利昔单抗(Basiliximab)、巴土昔单抗(Bavituximab)、贝妥莫单抗(Bectumomab)、贝利木单抗(Belimumab)、桕替木单抗(Bertilimumab)、贝索单抗(Besilesomab)、贝伐单抗(Bevacizumab)、比西单抗(Biciromab)、比伐单抗-DMl(Bivatuzumab mertansine)、兰妥莫单抗(Blinatumomab)、Brentuximab vedotin、Briakinumab、卡那单抗(Canakinumab)、美坎珠单抗(Cantuzumab mertansine)、卡罗单抗喷地肽偶联物(Capromab pendetide)、卡妥索单抗(Catumaxomab)、西利珠单抗(Cedelizumab)、培舍珠单抗(Certolizumabpegol)、西妥昔单抗(Cetuximab)、泊西他珠单抗(Citatuzumab bogatox)、西妥木单抗(Cixutumumab)、克立昔单抗(Clenoliximab)、Clivatuzumab tetraxetan、CNTO148(戈利木单抗(golimumab))、CNTO1275(优特克单抗(ustekinumab))、可那木单抗(Conatumumab)、达西珠单抗(Dacetuzumab)、达克珠单抗(Daclizumab)、地诺单抗(Denosumab)、地莫单抗(Detumomab)、阿托度单抗(Dorlimomab aritox)、Dorlixizumab、度伐单抗(durvalumab)、依美昔单抗(Ecromeximab)、依库珠单抗(Eculizumab)、埃巴单抗(Edobacomab)、依决洛单抗(Edrecolomab)、依法珠单抗(Efalizumab)、依夫单抗(Efungumab)、艾西莫单抗(Elsilimomab)、培戈赖莫单抗(Enlimomab pegol)、西依匹莫单抗(Epitumomabcituxetan)、依帕珠单抗(Epratuzumab)、厄利珠单抗(Erlizumab)、厄马索单抗(Ertumaxomab)、埃达珠单抗(Etaracizumab)、艾韦单抗(Exbivirumab)、法索单抗(Fanolesomab)、法拉莫单抗(Faralimomab)、非维珠单抗(Felvizumab)、非扎奴单抗(Fezakinumab)、芬妥木单抗(Figitumumab)、芳妥珠单抗(Fontolizumab)、福拉韦单抗(Foravirumab)、夫苏木单抗(Fresolimumab)、加利昔单抗(Galiximab)、加维莫单抗(Gavilimomab)、吉妥珠单抗奥佐米星(Gemtuzumab ozogamicin)、戈利木单抗(Golimumab)、戈利昔单抗(Gomiliximab)、Ibalizumab、替伊莫单抗(Ibritumomab tiuxetan)、伊戈伏单抗(Igovomab)、英西单抗(Imciromab)、英利昔单抗(Infliximab)、英妥木单抗(Intetumumab)、伊诺莫单抗(Inolimomab)、伊珠单抗奥佐米星(Inotuzumab ozogamicin)、伊匹木单抗(Ipilimumab)、伊妥木单抗(Iratumumab)、凯利昔单抗(Keliximab)、拉贝珠单抗(Labetuzumab)、来金珠单抗(Lebrikizumab)、来马索单抗(Lemalesomab)、乐地单抗(Lerdelimumab)、来沙木单抗(Lexatumumab)、利韦单抗(Libivirumab)、林妥珠单抗(Lintuzumab)、鲁卡木单抗(Lucatumumab)、鲁昔单抗(Lumiliximab)、马帕木单抗(Mapatumumab)、马司莫单抗(Maslimomab)、马妥珠单抗(Matuzumab)、美泊利单抗(Mepolizumab)、美替木单抗(Metelimumab)、米拉珠单抗(Milatuzumab)、明瑞莫单抗(Minretumomab)、 米妥莫单抗(Mitumomab)、莫罗木单抗(Morolimumab)、莫他珠单抗(Motavizumab)、莫罗单抗-CD3(Muromonab_CD3)、MY0-029(司他莫单抗(stamulumab)、他那可单抗(Nacolomab tafenatox)、他那莫单抗(Naptumomab estafenatox)、那他珠单抗(Natalizumab)、奈巴库单抗(Nebacumab)、奈昔木单抗(Necitumumab)、奈瑞莫单抗(Nerelimomab)、尼妥珠单抗(Nimotuzumab)、纳武单抗(Nivolumab)、巯诺莫单抗(Nofetumomab merpentan)、奥瑞珠单抗(Ocrelizumab)、奥度莫单抗(Odulimomab)、奥法木单抗(Ofatumumab)、奥马珠单抗(Omalizumab)、莫奥珠单抗(Oportuzumab monatox)、奥戈伏单抗(Oregovomab)、奥昔珠单抗(Otelixizumab)、帕昔单抗(Pagibaximab)JQ利珠单抗(Palivizumab)、帕木单抗(Panitumumab)、帕诺库单抗(Panobacumab)、帕考珠单抗(Pascolizumab)、派姆单抗(Pembrolizumab)、帕尼单抗(Pemtumomab)、培妥珠单抗(Pertuzumab)、培克珠单抗(Pexelizumab)、平妥莫单抗(Pintumomab)、普立昔单抗(Priliximab)、普立木单抗(Pritumumab)、PRO 140、雷韦单抗(Rafivirumab)、雷莫芦单抗(Ramucirumab)、雷珠单抗(Ranibizumab)、雷昔库单抗(Raxibacumab)、瑞加韦单抗(Regavirumab)、瑞利珠单抗(Reslizumab)、利妥木单抗(Rilotumumab)、利妥昔单抗(Rituximab)、罗妥木单抗(Robatumumab)、罗利珠单抗(Rontalizumab)、罗维珠单抗(Rovelizumab)、鲁利珠单抗(Ruplizumab)、沙妥莫单抗(Satumomab)、司韦单抗(Sevirumab)、西罗珠单抗(Sibrotuzumab)、西法木单抗(Sifalimumab)、司妥昔单抗(Siltuximab)、西利珠单抗(Siplizumab)、苏兰珠单抗(Solanezumab)、索耐珠单抗(Sonepcizumab)、松妥珠单抗(Sontuzumab)、司他芦单抗(Stamulumab)、硫索单抗(Sulesomab)、他珠单抗(Tacatuzumab tetraxetan)、他度珠单抗(Tadocizumab)、他利珠单抗(Talizumab)、他尼珠单抗(Tanezumab)、帕他莫单抗(Taplitumomab paptox)、替非珠单抗(Tefibazumab)、阿替莫单抗(Telimomab aritox)、替妥莫单抗(Tenatumomab)、替奈昔单抗(Teneliximab)、替利珠单抗(Teplizumab)、TGN1412、替西木单抗(Ticilimumab)、曲美木单抗(tremeIimumab)、替加珠单抗(Tigatuzumab)、TNX-355(伊巴珠单抗(ibalizumab))、TNX-650、TNX-901(他利珠单抗(talizumab))、托珠单抗(Tocilizumab)、托利珠单抗(Toralizumab)、托西莫单抗(Tositumomab)、曲妥珠单抗(Trastuzumab)、曲美木单抗(Tremelimumab)、西莫白介素单抗(Tucotuzumab celmoleukin)、妥韦单抗(Tuvirumab)、乌珠单抗(Urtoxazumab)、优特克单抗(Ustekinumab)、伐利昔单抗(Vapaliximab)、维多珠单抗(Vedolizumab)、维妥珠单抗(Veltuzumab)、维帕莫单抗(Vepalimomab)、维西珠单抗(Visilizumab)、伏洛昔单抗(Volociximab)、伏妥昔单抗(Votumumab)、扎芦木单抗(Zalutumumab)、扎木单抗(Zanolimumab)、齐拉木单抗(Ziralimumab) 以及阿佐莫单抗(Zolimomab aritox)。
在一些实施方式中，所述抗体选自：
i)曲妥珠单抗(Trastuzumab)抗体；
ii)贝马珠单抗(Bemarituzumab)抗体；
iii)抗B7‐H3抗体：其重链互补决定区HCDR1、HCDR2和HCDR3依次如SEQ ID NO:1～3所示，轻链互补决定区LCDR1、LCDR2和LCDR3依次如SEQ ID NO:4～6所示。
在一些实施方式中，所述抗B7‐H3抗体的重链可变区HCVR如SEQ ID NO:7所示，轻链可变区LCVR如SEQ ID NO:8所示。
在一些实施方式中，所述抗B7‐H3抗体的重链恒定区如SEQ ID NO:9或10所示，轻链恒定区如SEQ ID NO:11所示。
上述氨基酸序列的变体也在本发明范围内，相应的变体与SEQ ID NO:1～SEQ ID NO:6任一多肽相比，分别包含发生在至少一个CDR区域的至多3个氨基酸的突变；或者，变体相对于SEQ ID NO:7～SEQ ID NO:15整体序列而言，可包含3个以内或更多的突变，例如与SEQ ID NO:7～SEQ ID NO:15任一多肽相比具有至少80％、85％、90％、93％、95％、97％或99％同一性的序列。突变可以为氨基酸的置换、缺失或添加或其任意组合；优选地，所述突变为保守置换。
“保守置换”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。
通常视为保守置换的置换是在脂肪族氨基酸Ala、Val、Leu和Ile中的彼此置换、羟基残基Ser和Thr的互换、酸性残基Asp和Glu的交换、酰胺残基Asn和Gln之间的置换、碱性残基Lys和Arg的交换以及芳香残基Phe、Tyr间的置换。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
在一些实施方式中，所述配体-药物偶联物的结构如下所示：
本公开还涉及制备如上所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，包括：
将所述靶向配体还原后，与预先合成的-L-D进行偶联反应，得到通式为TL-L-D所示的化合物。
还原剂优选TCEP，特别地，优选还原靶向配体上的二硫键。
本公开还涉及药物组合物，其包含如上所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，以及药学上可接受的赋形剂、稀释剂或载体。
如本公开所用，“药学上可接受载体、稀释剂或赋形剂”包括当与活性组分组合时允许所述组分保持生物学活性并且与受试者的免疫系统不发生反应的任何材料。
本公开还涉及如上所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物在制备用于治疗肿瘤的药物中的用途。
术语“癌症”指的是以失调的细胞生长为特征的生理病症或疾病。“肿瘤”包括癌细胞。在一些实施方式中，所述的肿瘤为乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌(如非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液瘤。
本公开还涉及一种治疗受试者的医学状况的方法，包括施用安全和有效量的如上所述的配体-药物偶联物。
优选其中所述医学状况为癌症。
短语“安全和有效量的”。如本文所用，意指在合理的医药调节范围内化合物或组合物的量大到足以明显有效地缓解所治疗的症状或病症，但小到足以避免严重的副作用(以合理的有益/危险比率)。本公开的方法所用的药物组合物中的活性成份的安全和有效量随所治疗的特定症状、年龄和所治疗患者的身体状况，疾病的严重性、治疗时间、同期治疗情况、使 用的特定活性成份、使用的特定的药物学可接受的赋形剂及包括参与治疗医师的知识和技能在内的这类因素的不同而不同。
应该理解的是，设想的治疗方法还将包括施用其他治疗实体，特别优选为免疫治疗实体，包括病毒癌症疫苗(例如，编码癌症特异性抗原的腺病毒载体)、细菌癌症疫苗(例如，表达一种或多种癌症特异性抗原的非热原性大肠杆菌)、酵母癌症疫苗、N-803(也被称为ALT-803，ALTOR生物科学公司)、化疗药物、抗体(例如，与肿瘤相关抗原或患者特异性肿瘤新抗原结合)、干细胞移植物(例如，异体或自体)和肿瘤靶向细胞因子(例如，NHS-IL12，IL-12与肿瘤靶向抗体或其片段偶联)。在一些实施方式中，设想的治疗方法还包括对所述患者进行放射性治疗。在一些实施方式中，设想的治疗方法还包括对所述患者进行手术，例如肿瘤切除手术。
配体-药物偶联物也可以与抗病毒剂、抗生素、镇痛剂、皮质类固醇、类固醇、氧、抗氧化剂、COX抑制剂、心脏保护剂、金属螯合剂、IFN‐γ和/或NSAID组合施用和/或共配制。药物组合物中可以包含上述治疗实体。
术语“受试者”和“患者”在本文中可互换使用，是指任何动物，例如任何哺乳动物，包括(但不限于)人类、非人类灵长类动物、啮齿类动物、狗、猫、黑猩猩、猩猩、长臂猿、猕猴、狨猴、猪、马、熊猫和大象等等。
如本文所用，“治疗”是指任何疾病后果的任何改善，例如存活期延长、发病率较小和/或作为替代治疗方式的副产物的副作用减轻。如本领域中容易了解，疾病完全根除为优选的，但并不是治疗行为的必要条件。如本文所用，“治疗”是指向受试者(例如患者)施用所述配体-药物偶联物。治疗可以为治愈、医治、缓和、减轻、改变、医治、改善、减弱、好转或影响病症、病症的症状或例如癌症的患病倾向性。
本公开的药物组合物可通过任何途径投与，如所属领域的技术人员将了解。在一些实施例中，本公开的医药组合物通过静脉内(IV)投与。
下面将结合实施例对本公开的实施方案进行详细描述。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，优先参考本公开中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
实施例1 Anti-B7-H3抗体制备
以公开日为2021年09月17日的中国专利申请CN113402610A中anti-B7-H3抗体序列为基础，进一步进行单克隆抗体的表达和纯化。
如无特殊说明，本公开中所有抗体的表达可以选择使用商业化载体pTT5，在其启动子后的EcoR I和Hind III限制性酶切位点之间插入抗体序列，抗体轻链或重链序列前包括信号肽在内的核酸序列可以选用如下序列为：
上述序列下划线为EcoR I和起始氨基酸。
抗体轻链或重链序列后为：
TGAAAGCTT(其为终止密码子和Hind III)。
抗体氨基酸序列优化为核苷酸序列后插入上述信号肽和终止子之间。
重链H00序列：
轻链L00序列：
重链H04序列：

Anti-B7-H3抗体为克隆7B7，轻链L00和重链H00的抗体为7B7。轻链L00和重链H04的抗体为7B7-H04。7B7-H04抗体为工程化改造抗体，工程化改造适合产生均一偶联比值的偶联产物，非常适合评估不同连接子毒素。
将上述抗体轻重链分别构建于载体中，提取质粒。将悬浮驯化CHO-K1细胞复苏于OPM-CD TransCHO培养基(厂家：奥浦迈，OPM，货号P83059)，培养至密度2百万个细胞/ml，活率95％以上，体积1000ml。将轻链质粒0.5mg和重链质粒0.5mg合并且溶于10ml培养基，将3mg溶于培养基的PEI(厂家：Polyscience，货号：24765-1)稀释于10ml培养基中，混合质粒和PEI的溶液并置于室温10分钟，然后滴加至1000ml细胞培养液中，置于37℃培养5天后以12000g离心15分钟收获上清。将上清用HiTrap Mabselect SuRe进行纯化，并用50mM乙酸洗脱，中和后的收集峰用30KD超滤管(厂家Merck，货号UFC9030)置换于PBS pH7.4中。将抗体测定280nm吸光度值，吸光度值除以理论消光系数为浓度值。
实施例2对照连接子毒素的合成
1.连接子毒素MC-VA-PAB-Eribulin的合成
设计的连接子毒素MC-VA-PAB-Eribulin(LK-322018)结构如下：
化合物LK-322018的合成路线为：
化合物LK-322018的合成：将市售化合物MC-VA-PABC-PNP(5.9mg，0.091mmol)溶于1mlN,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.1mg，0.024mmol)和市售艾瑞布林(5mg，0.007mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到LK-322018。ESI-MS m/z:1240(M-H)。
2.连接子毒素MC-GGFG-PAB-Eribulin的合成
设计的连接子毒素MC-GGFG-PAB-Eribulin(LK-322016)结构如下：
化合物LK-322016的合成路线为：
化合物3的合成：将市售化合物1(50mg)溶于2mlN,N-二甲基甲酰胺中，依次加入Bis-PNP(36mg，0.1182mmol)和N,N-二异丙基乙基胺(20.3mg，0.006053mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入15ml水，用乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到标题化合物3。ESI-MS m/z:743(M+H)。
化合物LK-322016的合成：将化合物3(7.26mg，0.009079mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.1mg，0.02412mmol)和市售艾瑞布林(5mg，0.007mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到LK-322016。ESI-MS m/z:1389(M-H)。
3.连接子毒素Mal-PEG2-VC-PAB-Eribulin的合成
具体合成步骤参考公开日为2018年11月23日的中国专利申请CN108883198A第145页，合成方法与步骤均与专利相同。在(DOI: 10.1158/1535-7163.MCT-17-1215，MORAb-202，an Antibody-Drug Conjugate Utilizing Humanized Anti-human FRαFarletuzumab and the Microtubule-targeting Agent Eribulin,has Potent Antitumor Activity)一文中显示Mal-PEG2-VC-PAB-Eribulin与MC-VC-PAB-Eribulin对若干细胞系活性方面相同，认为二者基本一致。因而，在本公开中于某些情况下可将Mal-PEG2-VC-PAB-Eribulin与MC-VC-PAB-Eribulin替换使用。
实施例3实验组连接子毒素的合成
连接子毒素MC-VAGGFG-PAB-Eribulin的合成
设计的连接子毒素MC-VAGGFG-PAB-Eribulin(LK-322022)结构如下：
化合物LK-322022的合成路线为：
化合物2的合成：将市售的化合物1(100mg，0.244mmol)溶于3ml 四氢呋喃中，降温至0℃，加入HOSU(33.7mg，0.293mmol)和DCC(60.3mg，0.293mmol)，室温反应2小时，通过TLC点板和液质联用色谱监测原料反应完毕后，将反应液过滤，滤液浓缩所得粗品直接投入下一步反应。ESI-MS m/z:508(M+H)。
化合物4的合成：将化合物2(crude，0.244mmol)与市售的化合物3(82mg，0.244mmol)混合于3ml N,N-二甲基甲酰胺中，加入N,N-二异丙基乙基胺(126mg，0.976mmol)，室温反应2小时，通过液质联用色谱监测原料反应完毕后，减压蒸馏去除溶剂，粗品用10ml水打浆，过滤得到标题化合物粗品(200mg，收率:100％)。ESI-MS m/z:729(M+H)。
化合物6的合成：将化合物4(200mg，0.274mmol)溶于5mlN,N-二甲基甲酰胺中，降温至0℃，依次加入化合物5(41mg，0.329mmol)、N,N-二异丙基乙基胺(107mg，0.823mmol)和DEPBT(124mg，0.412mmol)，室温反应4小时，通过液质联用色谱监测原料反应完毕后，反应液用10ml氯化铵水溶液淬灭，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品经过硅胶柱层析[二氯甲烷/甲醇＝88:12(v/v)]纯化得到标题化合物6(100mg，收率:43.8％)。ESI-MS m/z:834(M+H)。
化合物7的合成：将化合物6(100mg，0.120mmol)溶于3ml N,N-二甲基甲酰胺中，加入哌啶(31mg，0.360mmol)，室温反应0.5小时，通过液质联用色谱监测原料反应完毕后，减压蒸馏去除溶剂，所得粗品经过硅胶柱层析[二氯甲烷/甲醇＝82:18(v/v)]纯化得到标题化合物7(76mg,收率:100％)。ESI-MS m/z:612(M+H)。
化合物9的合成：将化合物7(76mg，0.124mmol)与市售的化合物8(46mg，0.149mmol)混合于3ml N,N-二甲基甲酰胺中，加入N,N-二异丙基乙基胺(32mg，0.248mmol)，室温反应2小时，通过液质联用色谱监测原料反应完毕后，减压蒸馏去除溶剂，所得粗品经过硅胶柱层析[二氯甲烷/甲醇＝82:18(v/v)]纯化得到标题化合物9(70mg，收率：70％)。ESI-MS m/z:805(M+H)。
化合物10的合成：将化合物9(70mg,0.087mmol)溶于3mlN,N-二甲基甲酰胺中，依次加入Bis-PNP(40mg，0.130mmol)和N,N-二异丙基乙基胺(22mg，0.174mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，减压蒸馏去除溶剂，所得粗品经过硅胶柱层析[二氯甲烷/甲醇＝90:10(v/v)]纯化得到标题化合物10(40mg，收率:47.6％)。ESI-MS m/z:970(M+H)。
化合物LK-322022的合成：将化合物10(14mg，0.014mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.5mg，0.027 mmol)和市售艾瑞布林(5mg，0.007mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到LK-322022(5.1mg，收率:48.1％)。ESI-MS m/z:1560(M-H)。
实施例4抗体偶联
取上述实施例中待偶联的抗体7B7-H04和7B7，各取3mg抗体，用10mM磷酸钠缓冲液pH7.4稀释至3mg/ml，加入三(2-羟乙基)膦盐酸盐TCEP母液，TCEP母液为以10mM磷酸钠缓冲液pH7.4配置10mM母液(TCEP厂家Sigma，货号C4706-2G，CAS.51805-45-9)，确保TCEP摩尔终浓度与抗体摩尔终浓度为4：1，将抗体置于37℃还原45分钟。加入实施例2和3中的连接子毒素母液，母液为以二甲基亚砜DMSO溶解连接子毒素至5mM，确保连接子毒素摩尔终浓度与抗体摩尔终浓度为6：1，将抗体置于2～8℃偶联2小时。后将抗体转入30KD超滤管中(厂家Merck，货号UFC9030)，置于3000g离心力2～8℃换液，将残留毒素降至反应浓度的千分之一，随后无菌过滤，测定浓度，分装冻存。
为获得不同偶联比值DAR的产物，将抗体：还原剂：连接子毒素的摩尔比在1:1～6:2～15间调整，其余条件保持不变，最终偶联产物的DAR值以检测结果为准。
实施例5不同ADC的效果的体外比较
一、检测方法
1.细胞活性检测
将人Calu-6(厂家Procell，货号CL-327，人肺癌细胞)复苏，接种96孔培养板，培养基为DMEM/F12(厂家Hyclone，货号SH30023.01)且含10％FBS(厂家Gibco，货号10099141)，每孔2500个细胞，体积150微升，置于37℃，5％二氧化碳培养箱培养，约20小时后加入含有不同抗体偶联药物的培养基50微升，使药物最终浓度在0～100nM之间的12个稀释浓度(首孔浓度100nM，后续依次5倍稀释10个梯度，最后一个梯度药物为0，每个样品复孔重复)，继续共培养4～7天后取出培养板，每孔加入CTG检测试剂50微升(CellTiterGlo，厂家Promega，货号G7575)，反应2分钟，按试剂盒推荐操作进行，然后用Tecan Spark测定荧光度值。将复孔取平均值并作为Y轴，以稀释梯度log10值为X轴，做平滑曲线图，按照四参数计算EC/IC50值。
采用类似的细胞活性检测方法，可将细胞系替换为Jurket、MCF-7和MDA-MB-468进行再次验证。
2.偶联比值检测分析
抗体所连接药物数量的平均值(DAR)值通过疏水层析HIC-HPLC方法检测，分析柱为TSKgel Butyl-NPR，4.6mm×10cm，2.5微米，厂家TOSOH，货号042168。取待测样品约0.3mg，体积为150微升，加入150微升的流动相A，10000g离心5分钟取上清。流动相A为20mM磷酸钠+1.5M硫酸铵pH7.0，流动相B为20mM磷酸钠+20％(v/v)乙腈pH7.0。分析柱连接在安捷伦1260高效液相色谱仪，流速0.6ml/min，流动相A冲洗30分钟以上至280nm紫外基线平稳，进样50微克样品，流动相A冲洗2分钟后进行18分钟梯度洗脱(0％B–100％B)，继续保持100％流动相B冲洗5分钟后结束。根据每个DAR值的峰面积计算每个DAR值占百分比。将每个峰的百分比与DAR值乘积后累加，然后除以总百分比即为平均DAR值。
3.纯度检测分析
SEC-HPLC纯度通过分子筛方法检测，分析柱为TSKgel G3000SWXL，7.8mm x 30cm，5微米，厂家TOSOH，货号08541。取待测样品约0.3mg，体积为300微升，10000g离心5分钟取上清。流动相为50mM磷酸钠+0.1M氯化钠pH6.8。分析柱连接在安捷伦1260高效液相色谱仪，流速0.8ml/min，流动相冲洗30分钟以上至280nm紫外基线平稳，进样100微克样品，流动相冲洗20分钟。根据峰面积计算高分子、单体、低分子各个峰占百分比。
二、检测结果
1.细胞活性检测结果
抗体7B7-H04偶联MC-VA-PAB-Eribulin，Mal-PEG2-VC-PAB-Eribulin，MC-GGFG-PAB-Eribulin和MC-VAGGFG-PAB-Eribulin，偶联物针对Calu6细胞活性EC/IC50显示，EC/IC50分别是115.9pM、29.97pM，62.32pM和11.40pM(图1)，即MC-VAGGFG-PAB-Eribulin的效果显著优于其他各组。
对于7B7偶联Mal-PEG2-VC-PAB-Eribulin和MC-VAGGFG-PAB-Eribulin，EC/IC50分别是50.23pM和9.241pM(图1)，依然是MC-VAGGFG-PAB-Eribulin取得了更佳的技术效果。
在除Calu-6细胞系外的其他细胞系中检测得到的EC/IC50趋势与上述相似，均是MC-VAGGFG-PAB-Eribulin效果最优。
2.偶联比值和纯度结果
各组的偶联物平均DAR值和SEC-HPLC纯度结果如图2和下表所示：

偶联物平均DAR值为3.33，3.27和3.31(图2)，说明VAGGFG的多肽连接子在偶联比值接近时活性好于GGFG和VA多肽连接子。
采用与上述类似的方法，在其他几种常见的肿瘤细胞系(例如：MCF-7和MDA-MB-468)中进行验证，均有一致的趋势，即相比其他几种偶联物，采用包含VAGGFG连接子的偶联物具有最佳的EC/IC50表现，且平均DAR值和SEC-HPLC纯度表现稳定，满足抗体偶联物要求。
实施例6体内药效学分析
委托Bioduro公司(www.bioduro-sundia.com)进行体内药效评估，取培养良好的人乳腺癌肿瘤细胞系(MDA-MB-468)接种于免疫缺陷型小鼠(B-NDG)，每只接种1000万个细胞，至肿瘤生长至100mm3后随机分组，次日，每组按3mg/kg累计给药2次，每周一次，每周两次测定肿瘤大小，至实验结束。整个过程经过实验动物委员会审批通过。
实验结果如图3所示，从结果可知，7B7 MC-VAGGFG-PAB-Eribulin组在对肿瘤体积的抑制方面有效果。
采用同样的动物模型，将细胞系替换为Calu6和MCF-7进行再次验证，每组独立地选自按1～10mg/kg给药(如2、3、4、5、6、7、8或9mg/kg)，与MDA-MB-468的实验趋势一致，对肿瘤体积的抑制方面效果最佳。
实施例7稳定性测试
将7B7-MC-VAGGFG-PAB-Eribulin偶联物在PBS缓冲液、食蟹猴血浆和小鼠血浆中测试稳定性，浓度100nM，置于37℃放置0、4、7、10、14天，然后测定样品对Jurket细胞的杀伤活性。发现艾瑞布林释放率(Y轴)随着时间(X轴)延长，最高有0.1～5％比例艾瑞布林释放，显示连接子血浆稳定性非常好。
实施例8其它抗体的偶联测试
上述实施例的实验结果均基于抗体7B7-H04和7B7进行验证。
曲妥珠单抗(Trastuzumab)重链序列：
曲妥珠单抗(Trastuzumab)轻链序列：
贝马珠单抗(Bemarituzumab)重链序列：
贝马珠单抗(Bemarituzumab)轻链序列：
进一步的，按照上述实施例，制备曲妥珠单抗(Trastuzumab)和贝马珠单抗(Bemarituzumab)偶联连接子毒素MC-VAGGFG-PAB-Eribulin的ADC进行验证。结果显示偶联比值平均DAR值2～3，纯度>95％(图4、图5)。以SK-BR-3细胞系(人乳腺癌细胞)和KATO III细胞系(人胃癌 细胞)进行活性测定，与Calu-6相同方法测定细胞活性EC/IC50值为32pM和97pM。
实施例9其它药物的偶联测试
为了测试不同毒素的差异，合成了不同多肽连接子毒素，按照上述实施例的相关实验方法进行偶联物制备和活性测定，结果表明：在甲基澳瑞他汀EMMAE和依喜替康Exatecan中，测试的不同多肽连接子在细胞水平未表现出显著差异，与上述实施例中艾瑞布林作为毒素的结果有差异。
化合物MC-VC-PAB-MMAE为市售，MC-VA-PAB-MMAE已有报道与MC-VC-PAB-MMAE无显著差异。
化合物MC-GGFG-PAB-MMAE的合成路线为：
合成步骤为：将市售化合物1(50mg，0.0865mmol)溶于2ml N,N-二甲基甲酰胺中，依次加入Bis-PNP(36mg，0.1182mmol)和N,N-二异丙基乙基胺(20.3mg，0.006053mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入15ml水，用乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到化合物3(50mg，收率:77.8％)。将化合物3(7.26mg，0.009mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基 乙基胺(3.1mg，0.02412mmol)和市售MMAE(7.9mg，0.011mmol)，室温反应15小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到目标化合物(8.5mg，收率:62％)。ESI-MS m/z:1378(M+H)。
化合物MC-VAGGFG-PAB-MMAE的合成路线为：
步骤：将化合物1(合成路线同实验组)(9.7mg，0.01mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.0mg，0.02mmol)和市售MMAE(7.9mg，0.011mmol)，室温反应15小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到目标化合物(9.3mg，收率:60％)。ESI-MS m/z:1548(M+H)。
化合物MC-VA-PAB-Exatecan为市售。
化合物MC-GGFG-PAB-Exatecan的合成路线为：
步骤：将市售化合物1(50mg，0.0865mmol)溶于2ml N,N-二甲基甲酰胺中，依次加入Bis-PNP(36mg，0.1182mmol)和N,N-二异丙基乙基胺(20.3mg，0.006053mmol)，室温反应16小时，通过液质联用色谱监测原料反应完毕后，加入15ml水，用乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到化合物3(50mg，收率:77.8％)。将化合物3(7.26mg，0.009079mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.1mg，0.02412mmol)和市售Exatecan(5mg，0.011mmol)，室温反应15小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到目标化合物(6.0mg,收率:61％)。ESI-MS m/z:1096(M+H)。
化合物MC-VAGGFG-PAB-Exatecan合成路线为：
步骤：将化合物1(合成路线同实验组)(9.7mg，0.01mmol)溶于1ml N,N-二甲基甲酰胺中，依次加入N,N-二异丙基乙基胺(3.0mg，0.02mmol)和市售Extecan(5mg，0.011mmol)，室温反应15小时，通过液质联用色谱监测原料反应完毕后，加入5ml水，乙酸乙酯萃取三次(每次10ml)，分液后合并有机相，饱和食盐水洗涤，无水硫酸钠干燥后并浓缩，所得粗品通过制备液相纯化得到目标化合物(7.3mg，收率:58％)。ESI-MS m/z:1266(M+H)。
将上述化合物按照实施例4偶联抗体7B7，制备偶联比值为3～4的偶联物，并按照实施例5测定针对细胞系MCF-7的活性。结果如下：1)偶联MMAE毒素，为VC、GGFG和VAGGFG多肽连接子的偶联物活性IC50为0.49、0.41、0.27nM；2)偶联Extecan毒素，为VA、GGFG和VAGGFG多肽连接子的偶联物活性IC50为242、270、307nM。
以上所述实施例仅表达了本公开的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本公开构思的前提下，还可以做出若干变形和改进，这些都属于本公开的保护范围。因此，本公开专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。

Claims (18)

  1. VAGGFG作为酶裂解连接子的应用。
  2. 配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物的通式为TL-(L-D)n；
    其中，TL为靶向配体，L为接头单元，D为艾瑞布林，n为1～20的正整数；
    L中包含短肽VAGGFG作为酶裂解连接子。
  3. 根据权利要求2所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述接头单元为-L1-L2-L3-L4-，L1与TL相连，L4与D相连；
    其中，
    L1选自：

    或点击化学所需基团(优选)；
    L2选自：
    -NC(R1R2)C(O)、-NR3(CH2)oC(O)-、-NR3(CH2CH2O)oCH2C(O)-、-S(CH2)pC(O)-或化学键，其中o选自0～20的整数；p选自0～20的整数；
    R1、R2各自独立地选自氢原子、氘原子、烷基、取代烷基、氘代烷基、杂烷基、羧基、氨基、取代氨基；
    R3选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    L1与L2共用N原子；
    L3为VAGGFG；
    L4选自：-NR4(CR5R6)q-、-C(O)NR4-、-C(O)NR4(CH2)q-或者化学键，q选自0～6的整数；
    R4、R5和R6各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
  4. 根据权利要求3所述的配体-药物偶联物或其药学上可接受的盐或溶 剂化物，-L1-L2-为且s1为2、3、4、5、6、7或8。
  5. 根据权利要求2所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述接头单元为-L1-L2-L3-L4-，L1与TL相连，L4与D相连；
    其中，
    L1选自：

    或点击化学所需基团(优选)；
    L2选自：
    -NC(R1R2)C(O)、-NR3(CH2)oC(O)-、-NR3(CH2CH2O)oCH2C(O)-、-S(CH2)pC(O)-或化学键，其中o选自0～20的整数；p选自0～20的整数；
    R1、R2各自独立地选自氢原子、氘原子、烷基、取代烷基、氘代烷基、杂烷基、羧基、氨基、取代氨基；
    R3选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    L1与L2共用N原子；
    L3为VAGGFG；
    L4选自：-NR4-芳基-(CR5R6)q-OC(O)-、-NR4(CR5R6)q-OC(O)-，q选自0～6的整数；
    R4、R5和R6各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
  6. 根据权利要求5所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，-L1-L2-为且s1为2、3、4、5、6、7或8。
  7. 根据权利要求5所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，L4为对氨基苄氧羰基(PAB)。
  8. 根据权利要求2～7任一项所述的配体-药物偶联物或其药学上可接受 的盐或溶剂化物，所述靶向配体为抗体或其抗原结合片段。
  9. 根据权利要求8所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述抗体或其抗原结合片段选自兔源抗体、鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
  10. 根据权利要求9所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述抗体选自：抗CD3抗体、抗FOLR1抗体、抗ROR1抗体、抗TNFα抗体、抗TF抗体、抗EpCAM抗体、抗EGFRvIII抗体、抗DLL‐3抗体、抗PSMA抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM‐1抗体、抗LRRC15抗体、抗LIV‐1抗体、抗CanAg/AFP抗体、抗Claudin 6抗体、抗Claudin 9抗体、抗Claudin 18.2抗体、抗Mesothelin抗体、抗HER2抗体、抗EGFR抗体、抗c‐MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7‐H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP‐2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD70抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P‐cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD20抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体和抗CD123抗体。
  11. 根据权利要求8所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述抗体选自：
    i)Trastuzumab抗体；
    ii)Bemarituzumab抗体；
    iii)抗B7‐H3抗体：其重链互补决定区HCDR1、HCDR2和HCDR3依次如SEQ ID NO:1～3所示，轻链互补决定区LCDR1、LCDR2和LCDR3依次如SEQ ID NO:4～6所示。
  12. 根据权利要求11所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述抗B7‐H3抗体的重链可变区HCVR如SEQ ID NO:7所示，轻链可变区LCVR如SEQ ID NO:8所示。
  13. 根据权利要求12所述的配体-药物偶联物或其药学上可接受的盐或 溶剂化物，所述抗B7‐H3抗体的重链恒定区如SEQ ID NO:9或10所示，轻链恒定区如SEQ ID NO:11所示。
  14. 根据权利要求2、5～7、9～13任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物的结构如下所示：
  15. 制备权利要求2～14任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，包括：
    将所述靶向配体还原后，与预先合成的-L-D进行偶联反应，得到通式为TL-L-D所示的化合物。
  16. 药物组合物，其包含权利要求2～14任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，以及药学上可接受的赋形剂、稀释剂或载体。
  17. 权利要求2～14任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物在制备用于治疗肿瘤的药物中的用途。
  18. 根据权利要求17所述的用途，所述的肿瘤为乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液瘤。
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