Antibody-immune agonist conjugates and their use

Abstract

The present disclosure relates to linking unit molecules of targeting molecule-drug conjugates and the corresponding conjugates, their preparation and uses, and in particular to antibody-immunoagonist conjugates (AIACs), a novel type of cancer therapy.

Description

[Technical Field]

[0001] This disclosure relates to the field of biopharmaceuticals, and more particularly to targeting molecule-immune agonist conjugate linking units, and corresponding conjugates, their preparation processes, and use. [Background technology]

[0002] In development and clinical settings, targeted delivery of therapeutic drugs remains a major challenge for cancer treatment. Current targeted molecule-drug conjugates approved by the FDA are primarily antibody-drug conjugates (ADCs), where the drug (warhead) is typically a small molecule cytotoxic.

[0003] Immunotherapy is a promising new modality in cancer treatment. Immune checkpoint inhibitors, such as CLTA-4 and PD-1 / L1 monoclonal antibodies, are essentially T-cell-based therapies and have been approved for various cancer indications. However, numerous efforts are also underway to explore other immune system mechanisms that fight cancer. Targeting bone marrow cells, primarily macrophages and DCs, is a promising direction. Activating macrophages and DCs with agonists or macrophage checkpoint inhibitors enhances not only their phagocytic ability to eliminate tumor cells but also their antigen-presenting function, leading to a more potent induction of adaptive anti-tumor immunity.

[0004] TLR7 / 8 are two important pattern recognition receptors located in the endosomal membranes of macrophages, DCs, and monocytes. They naturally detect viral ssRNAs and mediate the activation of immune cells and the release of pro-inflammatory cytokines. In many studies, TLR7 / 8 agonists have been demonstrated to have antitumor activity. Imiquimod, a TLR7 agonist, is approved for the treatment of genital warts, superficial basal cell carcinoma, and actinic keratosis by topical administration. Resiquimod, a TLR7 / 8 dual agonist, is approved for the treatment of cutaneous T-cell lymphoma. However, the use of TLR7 / 8 agonists in a wider range of cancers is limited due to the side effects of systemic administration.

[0005] The present invention provides an antibody-immune agonist conjugate (AIAC), a novel type of tumor-targeted therapy.

Summary of the Invention

[0006] In one aspect, a compound of formula (I),

Chemical formula

Chemical formula

[0007] In another embodiment, an antibody-drug conjugate of formula (II), [ka] In the formula, n, Y, Lk, W, Lm2, B2, x, PL, a, and b are as defined in formula (I), z is an integer between 1 and 20. The present invention provides an antibody-drug conjugate in which A is a targeted molecule that is an antibody or an antigen-binding fragment.

[0008] The antibody-immune agonist conjugates (AIACs) of the present invention provide a novel type of tumor-targeted therapy. In vitro experiments have shown that the AIACs can induce higher TNFα production compared to naked, unmodified antibodies. In vivo experiments of the AIACs demonstrate antitumor effects.

[0009] In Figures 23-30, the arrows below the X-axis indicate the time of administration. [Brief explanation of the drawing]

[0010] [Figure 1] Examples of compounds of formula (I') are shown. R7 is as defined herein. [Figure 2a] This is an example of a compound of formula (II'). [Figure 2b] This is an example of a compound of formula (II'). [Figure 3] This is an example of a compound of formula (III'). [Figure 4a] This is an example of a compound of formula (IV'). [Figure 4b] This is an example of a compound of formula (IV'). [Figure 5] These are the connecting unit fragments LU102 to LU110. [Figure 6] TNFα-inducing activity of the conjugate AC102-6-1-1 and the corresponding naked, unmodified antibody Ab0001 (Trastuzumab), as well as the agonist rexiquimod, in a human PBMC-NCI N87 co-culture assay. [Figure 7] TNFα-inducing activity of conjugates AC102-6-1-1, AC102-8-1-1, and their corresponding naked, unmodified antibodies Ab0001 in a human PBMC-NCI N87 co-culture assay. [Figure 8] TNFα-inducing activity of AC102-6-1-1 and antibodies in co-culture of PBMCs with either NCI N87 or MDA-MB-468 cells. [Figure 9] TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and HCC1954 cells. [Figure 10] TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and SK-BR-3 cells. [Figure 11] TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and BT474 cells. [Figure 12] TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and JIMT1 cells. [Figure 13]TNFα induction activities of AC102-8-1-1 and antibodies in co-culture of PBMC and Colo205 cells. [Figure 14] TNFα induction activities of AC102-8-1-1 and antibodies in co-culture of PBMC and MDA-MB-468 cells. [Figure 15] TNFα induction activities of AC102-1-1-1, AC102-1-1-2, AC102-2-1-1, AC102-3-1-1, AC102-4-1-1, and antibodies in co-culture of PBMC and NCI N87 cells. [Figure 16] TNFα induction activities of AC201-1-1-1 and antibodies in co-culture of PBMC and NCI N87 cells. [Figure 17] TNFα induction activities of AC102-1-1-4, AC102-1-1-5, AC102-1-1-6 and antibodies in co-culture of PBMC and NCI N87 cells. [Figure 18] TNFα induction activities of AC102-8-​​​​​​​​​​​​​​Changes in tumor volume over time in NCI N87 CDX model SCID beige mice administered 5 mg / kg of vehicle (PBS pH 6.5), antibody, and conjugates AC102-6-1-1 and AC102-8-1-1. [Figure 24] Changes in tumor volume over time in NCI N87 CDX model SCID beige mice administered AC102-8-1-1 at 0.5, 1, and 3 mg / kg. [Figure 25] Changes in tumor volume over time in JIMT1 CDX model SCID beige mice administered AC102-8-1-1 at 5 mg / kg. [Figure 26] Changes in tumor volume over time in an hHER2-overexpressing MC38 model treated with AC102-6-1-1 at 3 mg / kg and 10 mg / kg. [Figure 27] Changes in tumor volume over time in an hHER2-overexpressing MC38 model treated with AC102-8-1-1 at 3 mg / kg and 10 mg / kg. [Figure 28] Changes in tumor volume over time in an NCI-N87 xenograft model administered AC102-6-2-1 and AC102-8-2-1 at 5 mg / kg. [Figure 29] Changes in tumor volume over time in an MDA-MB-468 xenograft model administered AC102-8-2-1 and AC201-1-2-1 at 3 mg / kg. [Figure 30] Changes in tumor volume over time in NUGC4 models administered AC102-8-3-1 and antibodies at 5 mg / kg. [Modes for carrying out the invention]

[0011] The technical content of this disclosure will be described below with reference to specific embodiments. Those skilled in the art will readily understand other advantages and effects of this disclosure from the content disclosed herein. This disclosure may be implemented or applied in other different specific embodiments. Those skilled in the art can make various modifications and variations without departing from the spirit of this disclosure.

[0012] definition Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "technology" as used herein refers to the technology commonly understood in the art, including obvious modifications and equivalent substitutions to those skilled in the art. Although the following terms are expected to be well understood by those skilled in the art, their definitions are provided below for the purpose of clearly illustrating this disclosure. Trademarks used herein refer to the corresponding products or their active ingredients. All patents, published patent applications, and publications referenced herein are incorporated herein by reference.

[0013] When a particular quantity, concentration, or other value or parameter is described in the form of a range, preferred range, preferred upper limit, or preferred lower limit, it should be understood that any range, whether explicitly stated or not, that is specifically disclosed, is equivalent to any range combining any upper limit or preferred value and any lower limit or preferred value. Unless otherwise specified, numerical ranges enumerated herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range. For example, the expression "i is an integer between 2 and 20" means that i is any integer between 2 and 20, for example, i could be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions should be understood in the same way.

[0014] Unless otherwise specified, singular forms such as "one" and "the" include plural forms. Expressions like "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, 8, and 9 or more.

[0015] When the terms "approximately" and "about" are used in relation to numerical variables, they generally mean that the value of the variable and all values ​​of the variable are within the experimental error range (e.g., within the 95% confidence interval of the mean), within ±10% of a specified value, or within a wider range.

[0016] The term "stoichiometric ratio" refers to the combination of various substances in specific weight amounts. For example, in this disclosure, the active ingredient is mixed with fillers, binders, and lubricants in specified weight ratios.

[0017] The terms “optional” or “at will” mean that the event described thereafter may occur, but is not necessarily to occur, and the expression includes whether or not such event or situation occurs.

[0018] The expression "contains" or similar expressions such as "equips," "contains," and "has" are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consists of..." excludes any elements, steps, or components not explicitly stated. The expression "substantially consists of..." means that the scope is limited to the specified elements, steps, or components, and any elements, steps, or components that exist optionally and do not substantially affect the essential and novel features of the subject matter for which protection is sought. The expression "contains" should be understood to encompass the expressions "substantially consists of..." and "consists of...".

[0019] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., receptors, cell surface proteins, cytokines, etc.). Targeting molecules can deliver payloads to specific sites in the body through targeted delivery. Targeting molecules can recognize one or more targets. The specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a payload to a site containing a large amount of the receptor. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, antigen-binding proteins, antibody mimetic compounds, scaffold proteins with affinity for a given target, ligands, etc.

[0020] As used herein, the term “antibody” is used broadly and includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they possess the desired biological activity. The antibody may be any subtype (IgG, IgE, IgM, IgD, and IgA, etc.) or subclass and may originate from any suitable species. In some embodiments, the antibody is derived from a human or mouse. The antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by recombinant methods.

[0021] In this specification, the term "monoclonal antibody" is used to refer to an antibody obtained from a substantially homogeneous antibody population, meaning that the individual antibodies constituting the population are identical except for a few possible spontaneous variations. Monoclonal antibodies are highly specific to a single antigen site. The term "monoclonal" refers to the fact that the properties of the antibody originate from a substantially homogeneous antibody population and should not be interpreted as requiring any specific method to produce the antibody.

[0022] Intact antibodies or full-length antibodies essentially consist of an antigen-binding variable region and a light chain constant region (C). L ) and heavy chain constant region (C H ) includes the heavy chain steady region (C H ) is C depending on the antibody subtype. H 1, C H 2, C H 3 and C H It may include 4. The antigen-binding variable region (also known as the fragment variable region or Fv fragment) is typically the light chain variable region (V L ) and heavy chain variable region (V H The constant region may be a natural sequence (such as a constant region containing a human natural sequence) or a constant region containing an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region may be recognized by the immune system and interacts with it.

[0023] Antibody fragments may include a portion of an intact antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and V. H and C H The Fab fragment contains a single-domain Fd fragment, an Fv fragment, a single-domain antibody (dAb) fragment, and an isolated complementarity-determining region (CDR). The Fab fragment is an antibody fragment obtained by digesting full-length immunoglobulin with papain, or a fragment having the same structure as one produced by, for example, recombinant expression. The Fab fragment has a light chain (V L and C L The chain includes a heavy chain (V H ) Variable domain and heavy chain (C H 1) contains the constant region domain. The F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of immunoglobulin at pH 4.0-4.5, or a fragment having the same structure as one produced by recombinant expression, for example. The F(ab')2 fragment essentially contains two Fab fragments, and each heavy chain portion contains several additional amino acids, including cysteine, which forms a disulfide bond connecting the two fragments. The Fab' fragment is a fragment containing half of the F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may contain multiple chains linked, for example, via disulfide bonds and / or peptide linking units. Examples of antibody fragments include single-chain Fv(scFv), Fv, dsFv, diabody, Fd and Fd' fragments, and other fragments including modified fragments. Antibody fragments typically contain at least or about 50 amino acids and at least or about 200 amino acids. The antigen-binding fragment may include any antibody fragment that, when inserted into the antibody framework (for example, by substitution of a corresponding region), can produce an antibody that binds immunospecifically to the antigen.

[0024] The antibodies relating to this disclosure can be prepared using techniques known in the art, or combinations thereof, such as recombinant techniques, phage display techniques, or synthesis techniques, or other techniques known in the art. For example, genetically modified recombinant antibodies (or antibody mimes) can be expressed in a suitable culture system (e.g., Escherichia coli or mammalian cells). Such modification may refer to, for example, introducing a ligase-specific recognition sequence to the end.

[0025] HER2 refers to human epidermal growth factor receptor-2 and belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0026] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. TROP2 is an intracellular calcium signaling transducer and is overexpressed in various tumors.

[0027] CLDN18.2 (Claudin 18 isoform 2) is a member of the human claudin family. CLDN18.2 is a pan-oncological target expressed in primary and metastatic lesions of several types of human cancer.

[0028] As used herein, the term “targeting molecule-drug conjugate” is referred to as “conjugate.” Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0029] Small molecule compounds refer to molecules that are comparable in size to organic molecules commonly used in pharmaceuticals. The term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.), but it does include low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of such small molecule compounds may be, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0030] Immune agonists are agonists that can induce or enhance the immune response against tumors through the activation of immune cells, including but not limited to DCs, B cells, macrophages, NK cells, and T cells. Non-exclusive examples of immune agonists known in this field include but not limited to TLR agonists, including TLR7 and / or TLR8 and / or TLR9 agonists (e.g., imiquimod, reximod, 852A, and VTX-2337), and STING agonists (e.g., ADU-S100 and MK-1454).

[0031] A linking unit refers to a functional group that covalently bonds two or more parts of a compound or material. For example, such a linking unit can function to covalently bond the adjuvant portion of a targeted molecule and / or payload.

[0032] A spacer is a structure located between different structural modules that can spatially separate them. The definition of a spacer is not limited by whether it has a specific function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures. Among these, non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples include, but are not limited to, single amino acids such as Leu and Gln, sequences containing multiple amino acids, sequences containing two amino acids such as GA, or sequences such as GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. Other examples of spacers include, for example, self-destructing spacers such as PABC (p-benzyloxycarbonyl).

[0033] The term "alkyl group" refers to a linear or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, where the saturated aliphatic hydrocarbon group is linked to the rest of the molecule by a single bond. An alkyl group may contain 1 to 20 carbon atoms, i.e., C1-C20 These can be alkyl groups, such as C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, and C3-C6 alkyl groups. Non-limiting examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, pentyl groups, hexyl groups, isopropyl groups, isobutyl groups, sec-butyl groups, tert-butyl groups, isopentyl groups, 2-methylbutyl groups, 1-methylbutyl groups, 1-ethylpropyl groups, 1,2-dimethylpropyl groups, neopentyl groups, 1,1-dimethylpropyl groups, 4-methylpentyl groups, 3-methylpentyl groups, 2-methylpentyl groups, 1-methylpentyl groups, 2-ethylbutyl groups, 1-ethylbutyl groups, 3,3-dimethylbutyl groups, 2,2-dimethylbutyl groups, 1,1-dimethylbutyl groups, 2,3-dimethylbutyl groups, 1,3-dimethylbutyl groups, or 1,2-dimethylbutyl groups, or their isomers. A divalent radical is a group obtained by removing one hydrogen atom from a carbon atom that has free valence electrons in the corresponding monovalent radical. A divalent radical has two linkage sites that are linked to the rest of the molecule. For example, an "alkylene group" or "alkylidene group" refers to a straight-chain or branched-chain saturated divalent hydrocarbon group. Examples of alkylene groups include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H 10 -), Hexylene (-C6H 12 This includes, but is not limited to, 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene group, ethylpropylene group, etc.

[0034] As used herein, when one group is combined with another, the linkage of the groups may be linear or branched, as long as a chemically stable structure is formed. The structure formed by such a combination can be linked to other parts of the molecule by any suitable atom in the structure, preferably by specified chemical bonds. For example, C 1-4When describing the combination of an alkylene group with one of the groups containing -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-, C 1-4 The alkylene group can form a linear linkage with the above group, for example, C 1-4 Alkylene-CH2-,C 1-4 Alkylene-NH-,C 1-4 Alkylene-C(O)-, C 1-4 Alkylene-NHC(O)-, C 1-4 Alkylene-C(O)NH-,-CH2-C 1-4 Alkylene, -NH-C 1-4 Alkylene, -C(O)-C 1-4 Alkylene, -NHC(O)-C 1-4 Alkylene, -C(O)NH-C 1-4 Alkylenes can be formed. The resulting divalent structure can be further linked to other parts of the molecule.

[0035] The term “heterocycle” (and variations thereof such as “heterocyclic formula” or “heterocyclyl group”), used alone or in combination with other terms, broadly refers to a single aliphatic ring having typically 3 to 12 ring atoms, containing at least two carbon atoms in addition to one or more, preferably 1 to 3, heteroatoms independently selected from oxygen, sulfur, nitrogen, and phosphorus, and combinations containing at least one of the said heteroatoms. Alternatively, the heterocycle as defined above may be a polycyclic ring system (e.g., bicyclic) in which two or more rings can be fused, bridged, or spirobonded, where at least one of such rings contains one or more heteroatoms independently selected from oxygen, sulfur, nitrogen, and phosphorus.

[0036] The heterocyclyl group may be, for example, a four-membered ring such as an azetidinyl group or an oxetanyl group, or a five-membered ring such as a tetrahydrofuranyl group, dioxolinyl group, pyrrolidinyl group, imidazolidinyl group, pyrazolidinyl group, pyrrolidinyl group, oxopyrrolidinyl group, or 2-oxoimidazolidine-1-yl, or a six-membered ring such as a tetrahydropyranyl group, piperidinyl group, morpholinyl group, dithianyl group, thiomorpholinyl group, piperazinyl group, 1,1-dioxo-1,2-thiadinan-2-yl, or trithianyl group, or a seven-membered ring such as a diazepine ring. Optionally, the heterocyclyl group can be benzo-condensed.

[0037] The heterocyclyl group may be bicyclic without restriction, for example, a five-membered condensed five-membered ring such as hexahydrocyclopenta[c]pyrrole-2(1H)-yl, or a five-membered condensed six-membered bicyclic ring such as hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl.

[0038] As described above, the heterocycle may be unsaturated, that is, it may contain one or more double bonds without limitation. For example, an unsaturated heterocycle containing a nitrogen atom may be 1,6-dihydropyrimidine, 1,2-dihydropyrimidine, 1,4-dihydropyrimidine, 1,6-dihydropyridine, 1,2-dihydropyridine, 1,4-dihydropyridine, 2,3-dihydro-1H-pyrrole, 3,4-dihydro-1H-pyrrole, 2,5-dihydro-1H-pyrrol, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or a 4H-[1,4]thiadinyl ring; an unsaturated heterocycle containing an oxygen atom may be 2H-pyran, 4H-pyran, or 2,3-dihydrofuran; and an unsaturated heterocycle containing a sulfur atom may be 2H-thiopyran or 4H-thiopyran. Heterocycles such as dihydroisoquinolinyl rings can be benzo-condensed without restriction.

[0068] The term “heteroaryl group” should be understood to mean a monovalent monocyclic, bicyclic, or tricyclic aromatic ring system (“5-10 membered heteroaryl group”) having preferably 5, 6, 7, 8, 9, or 10, and particularly 5, 6, 9, or 10, ring atoms, where at least one (preferably 1-4, more preferably 1, 2, or 3) of the ring atoms may be the same or different heteroatoms, such as oxygen, nitrogen, or sulfur ring systems. Furthermore, the heteroaryl group can be benzo-condensed in each case. Specifically, heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiodiazolyl, and their benzo derivatives, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazole, and indolyl groups. The group is selected from the group consisting of isoindolyl groups, pyridyl groups, pyridadinyl groups, pyrimidinyl groups, pyrazinyl groups, triazinyl groups, etc. and their benzo-condensed derivatives, such as quinolinyl groups, quinazolinyl groups, isoquinolinyl groups, etc., or azosinyl groups, indolidinyl groups, purinyl groups, etc. and their benzo-condensed derivatives, or sinnolinyl groups, phthalazinyl groups, quinazolinyl groups, quinoxalinyl groups, naphthilidinyl groups, carbazolyl groups, acridinyl groups, etc.

[0039] Compound of formula (I') In one embodiment, a compound of formula (I') (formula (I'-1) or formula (I'-2)), D1-Y a ―Lk―W b ―A2 p ―Lm2 p ―B2 (I'-1) B1-Lm1 p ―A1 p ―W b ―Lk―Y a ―D2 (I'-2) During the ceremony, D1 and D2 independently contain regions that recognize ligase receptors or donor substrates. B1 and B2 are each independently, 1) C 2-30 an alkylene group, wherein one or more -CH2- structures in the alkylene group are -CR 1 R 2 -, -O-, -C(O)-, -NR 3 -, a cleavable sequence 1, or a spacer Sp1, and 2) a terminal group, where the terminal group is hydrogen or R 7 and, R 7 is a group that can leave when reacting with a group in the payload, R 1 R 2 and R 3 are each independently hydrogen, -OH, -NH2, -C 1-6 alkyl, -O-C 1-6 alkyl, -NH-C 1-6 alkyl, -C 1-6 alkyl-NH2, -N(C 1-6 alkyl)-C 1-6 alkyl, -C 1-6 alkyl-O-C 1-6 alkyl, -C 1-6 alkyl-NH-C 1-6 alkyl, -C(O)-C 1-6 alkyl, -NHC(O)-C 1-6 alkyl, -C(O)-NH2, -C(O)NH-C 1-6 alkyl, -C(O)N(C 1-6 alkyl)-C 1-6 alkyl, -S(=O)2-C 1-6 alkyl, -NHS(=O)2-C 1-6 alkyl, -S(=O)2O-C 1-6 alkyl, -S(=O)2NH-C 1-6 alkyl and -S(=O)2N(C 1-6 alkyl)-C 1-6 alkyl, Lm1 and Lm2 are each independently a ring-opening succinimide moiety, A1 and A2 are each independently conjugated with Lm1 or Lm2 via a disulfide bond, thioether bond, thioester bond, or urethane bond. Lk is a combination of L1-L2-L3, L1 and L3 operate independently of each other. -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, and C 1-4 A combination of an alkylene group and one of the following groups is selected: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-. L2 does not exist, or C 7-34 It is an alkylene group, and one or more (-CH2-) structures in the alkylene group can be optionally replaced by -O-. L1, L2, and L3 are each arbitrary and independent, -OR 4 and -NR 5 R 6 Substituted by one, two, or three substituents selected from, R 4 , R 5 and R 6 These are, independently, hydrogen and -C. 1-6 Alkyl, -C(O)-C 1-6 Alkyl and -S(=O)2-C 1-6 Selected from alkyl groups, Y and W either do not exist independently, or are selected from the cleavable array 2, spacer Sp2, and combinations thereof. Cleavable sequence 1 contains an amino acid sequence that can be cleaved by an enzyme, and cleavable sequence 1 contains 1 to 10 amino acids. The cleavable sequence 2 contains an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 2 contains 1 to 10 amino acids. Sp1 and Sp2 are each independently selected from spacer sequences containing 1 to 20 amino acids, PABC, and combinations thereof. The present invention provides the compound wherein a, b, and p are each independently 0 or 1.

[0040] In one embodiment, both a and b are 0. In one embodiment, a is 1 and b is 0. In one embodiment, p is 0.

[0041] In one embodiment, R 1 and R 2 Both are hydrogen, or both are -C 1-6 It is alkyl. In one embodiment, R 1 and R 2 These are, independently, hydrogen and -C(O)-C 1-6 Alkyl, -C(O)-NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)-C 1-6 Alkyl, -S(=O)2-C 1-6 Alkyl, -S(=O)2O-C 1-6 Alkyl, -S(=O)2NH-C 1-6 Alkyl and -S(=O)2N(C) 1-6 Alkyl)-C 1-6 Selected from alkyl groups, or each independently, -C 1-6 Alkyl, -C(O)-NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)-C 1-6 Alkyl and -S(=O)2-C 1-6 Alkyl, -S(=O)2O-C 1-6 Alkyl, -S(=O)2NH-C 1-6 Alkyl and -S(=O)2N(C) 1-6 Alkyl)-C 1-6 Selected from alkyl. In one embodiment, R 1 and R 2 Both are hydrogen, or both are -C 1-6 It is alkyl, or each is independently hydrogen, -C(O)-C 1-6 Alkyl and -S(=O)2-C 1-6 Selected from alkyl groups, or each independently of -C 1-6 Alkyl, -C(O)-C 1-6 Alkyl and -S(=O)2-C 1-6 Selected from alkyl groups.

[0042] In one embodiment, L 1 , L 2 and L 3 These are independent, -OR 4 and -NR 5 R 6 Substitutions are made by one, two, or three substituents selected from the following. Substitutions include, for example, -CH3, -CH2- or [ka] Structurally, it occurs particularly on -CH2-.

[0043] In one embodiment, L 1 is -NH- or C 1-4 It is a combination of an alkylene group and -NH-. In another embodiment, L 1 is -C(O)- or C 1-4 It is a combination of an alkylene group and -C(O)-.

[0044] In one embodiment, L 3 is -NH- or C 1-4 It is a combination of an alkylene group and -NH-. In another embodiment, L 3 is -C(O)- or C 1-4 It is a combination of an alkylene group and -C(O)-.

[0045] In one embodiment, L 2 C is a linear or branched alkylene group. 7-34 The alkylene group is an alkylene group, in which one or more -CH2- structures can be optionally replaced by -O-, and the alkylene group is -OR 4 and -NR 5 R 6 It is optionally substituted with one, two, or three substituents selected from. In yet another embodiment, L 2 is -OR 4 and -NR 5 R 6A divalent group is selected from which one, two, or three substituents are optionally substituted, where the divalent group is a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a 1-methylethylene group, a 2-methylethylene group, a 2-methylpropylene group, or a 2-ethylpropylene group.

[0046] In another embodiment, L 2 ha-(C2H4-O) i -C 1-4 It is an alkylene, and i is an integer from 2 to 10. In one embodiment, L 2 is C 1-4 Alkylene-(O-C2H4) i -(C2H4-O) i -" or "-(O-C2H4)" i - represents a structure formed by polymerization of PEG units, where i indicates the number of PEG units. In another embodiment, L 2 ha-(C2H4-O) i -C 1-2 It is an alkylene. In certain embodiments, L 2 ha-(C2H4-O) i In another embodiment, L 2 is C 1-2 Alkylene-(O-C2H4) i -In a particular embodiment, L 2 is -C2H4-(O-C2H4) i - In one embodiment, i is selected from the values ​​2 to 10, 2 to 8, 2 to 6, 2 to 4, or 4 to 6. In a particular embodiment, i is 4.

[0047] In one embodiment, Y and W are either independently absent or selected from cleavable sequence 1, spacer Sp2, and combinations thereof. In a particular embodiment, a is 0 and Y is therefore absent. In another particular embodiment, b is 0 and W is therefore absent. In yet another particular embodiment, neither Y nor W is present. In one embodiment, cleavable sequence 2 comprises an amino acid sequence that is recognizable as an enzyme substrate and is enzymatically cleavable. In a particular embodiment, cleavable sequence 2 is enzymatically cleavable intracellularly, particularly within lysosomes. In another particular embodiment, cleavable sequence 2 is cleavable by a protease, specifically cathepsin. In yet another particular embodiment, cleavable sequence 2 is cleavable by glutaminase. In one embodiment, cleavable sequence 2 is selected from a cathepsin restriction site, a glutaminase restriction site, and combinations thereof. In one embodiment, the cleavable sequence 2 is selected from Phe-Lys, Val-Cit, Val-Lys, GLy-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and combinations thereof.

[0048] In one embodiment, Y and W are either absent or selected from spacer Sp2 independently. In another embodiment, Sp2 is a spacer sequence containing 1 to 10, preferably 1 to 6, more preferably 1 to 4 amino acids. In a particular embodiment, Sp2 is Leu. In another particular embodiment, Sp2 is Gln. In one embodiment, Sp2 is PABC. In yet another embodiment, Y and W are each independently selected from Phe-Lys-PABC, Val-Cit-PABC, and Val-Lys-PABC.

[0049] In one embodiment, the amino acids contained in Y and / or W may be natural or non-natural. In a particular embodiment, Y is amino acid fragment 1. Each amino acid fragment 1 consists of 1 to 30 natural or non-natural amino acids, each independently identical or distinct. Amino acid fragment 1 is selected from cleavable sequences containing 1 to 10 amino acids, spacer sequences containing 1 to 20 amino acids, and combinations thereof. In another particular embodiment, W is amino acid fragment 2. Each amino acid fragment 2 consists of 1 to 30 natural or non-natural amino acids, each independently identical or distinct. Amino acid fragment 2 is selected from cleavable sequences containing 1 to 10 amino acids, spacer sequences containing 1 to 20 amino acids, and combinations thereof.

[0050] The portion containing the recognition sequence of a ligase receptor or donor substrate. In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from natural transpeptidases, non-natural transpeptidases, their variants, and combinations thereof. Non-natural transpeptidase enzymes may, but are not limited to, those obtained by manipulating natural transpeptidases. In a preferred embodiment, the ligase is selected from natural saltases, non-natural saltases, and combinations thereof. Types of natural saltases include saltase A, saltase B, saltase C, saltase D, saltase L. plantarum, etc. (US20110321183A1). The type of ligase corresponds to the ligase recognition sequence and is used to achieve specific conjugation between different molecules or structural fragments. In one embodiment, the recognition sequence of the ligase receptor substrate is selected from oligomeric glycine, oligomeric alanine, and oligomeric glycine / alanine mixtures with a degree of polymerization of 3 to 10. In certain embodiments, the recognition sequence of the ligase receptor substrate is G nHere, G is glycine (Gly) and n is an integer from 3 to 10. In another specific embodiment, the ligase is saltase A derived from Staphylococcus aureus. Accordingly, the ligase recognition sequence may be the typical recognition sequence of the enzyme LPXTG. In yet another specific embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ and the recognition sequence of the ligase receptor substrate is G n Here, X may be any single amino acid, natural or non-natural, and J is an amino acid fragment containing 1 to 10 amino acids, which is absent or optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or non-natural amino acid. In yet another embodiment, J is G m Here, m is an integer from 1 to 10. In yet another specific embodiment, the ligase donor substrate recognition sequence is LPETG. In yet another specific embodiment, the ligase donor substrate recognition sequence is LPETGG. In one embodiment, the ligase is saltase B derived from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN. In another embodiment, the ligase is saltase B derived from Bacillus anthrax, and the corresponding donor substrate recognition sequence may be NPKTG. In yet another embodiment, the ligase is saltase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In yet another embodiment, the ligase is saltase subfamily 5 derived from Streptomyces cericarpa, and the corresponding donor substrate recognition sequence may be LAXTG. In yet another embodiment, the ligase is saltase A derived from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ. The ligase recognition sequence may also be another entirely novel recognition sequence for a transpeptidase optimized by manual screening.

[0051] part containing a reactive group Reactive group for connecting to the payload In one embodiment, B1 or B2 is used for connection to a payload. To connect to the payload, the compound of formula (I') contains a reactive group. In one embodiment, B1 or B2 in the compound of formula (I') is connected to the payload via an amide bond, an ester bond, or an ether bond. In one embodiment, the reactive group in B1 or B2 in formula (I') is independently a reactive group for condensation reactions, nucleophilic addition or electrophilic addition (e.g., a reactive C=O moiety, a reactive C=CC=O moiety, an amino group, an amine group, a hydroxyl group, a thiol group), or a reactive group for substitution reactions (e.g., a leaving group attached to an O, C, N, or S atom). In one embodiment, the reactive group in B1 or B2 is independently selected from carboxyl groups, sulfonic acid groups, phosphoryl groups with a free -OH terminus, active esters, aldehyde groups, isocyanate groups, Michael addition acceptor groups (such as maleimide groups), amino groups, amine groups, hydroxyl groups, thiol groups, pyridyldithiol groups, and haloacetic acid groups. In a specific embodiment, the reactive group in B1 or B2 for connection to the payload is independently selected from amino groups, amine groups, hydroxyl groups, thiol groups, carboxyl groups, and active esters. In another specific embodiment, B1 or B2 is connected to the payload via groups selected from -OH and -COOH, particularly hydroxyl groups of alkyl alcohols or carboxyl groups of alkyl carboxylic acids.

[0052] In one embodiment, the reactive group in B1 or B2 is independently an amino group, an amine group, or a hydroxyl group, which reacts with the corresponding group (e.g., a carboxyl group, a sulfonic acid group, a phosphoryl group with a free -OH terminus, an active ester, an acid chloride, or an isocyanate group) in the payload. In another embodiment, the reactive group in B1 or B2 is independently a carboxyl group, a sulfonic acid group, a phosphoryl group with a free -OH terminus, an active ester, or an isocyanate group, which reacts with the corresponding group (e.g., an amino group, an amine group, or a hydroxyl group) in the payload.

[0053] In one embodiment, the reactive group in B1 or B2 is independently an amino group, a hydroxyl group, or a thiol group, which reacts with the corresponding group (e.g., halogen, hydroxyl group, thiol group, aldehyde group) in the payload. In another embodiment, the reactive group in B1 or B2 is independently a hydroxyl group, which reacts with the corresponding group (e.g., halogen or hydroxyl group) in the payload.

[0054] B1 and B2 In one embodiment, B1 and B2 are independent of each other. 1)-(CH2) k C(O)-, -NR 3 -,-(C2H4-O) j -,-(NH-CR 1 R 2 -C(O)) d -,-(C2H4) g -, -C(O)(CH2) k -,-(O-C2H4) j -,-(C(O)-CR 1 R 2 -NH) d -A combination of one, two or more divalent groups selected, a cleavable sequence 1, and a spacer Sp1, and 2) a terminal group, where, The terminal group is hydrogen or R 7 And, k is an integer between 0 and 20, j is an integer between 1 and 20, d is an integer between 1 and 20, and g is an integer between 1 and 20. Each -(CH2) k C(O)-, -C(O)(CH2) k -and-(C2H4) g - is either unsubstituted independently, or -OH, -NH2, C 1-6 alkyl group, -OC 1-6 Alkyl, -NH-C 1-6 Alkyl, -C 1-6 Alkyl-NH2,-N(C 1-6 Alkyl)-C 1-6 Alkyl, -C(O)-C 1-6 Alkyl, -NHC(O)-C 1-6 Alkyl, -S(=O)2-C 1-6Alkyl and -NHS(=O)2-C 1-6 It is substituted with one or more groups selected from alkyl groups.

[0055] In one embodiment, the terminal group is hydrogen. In one embodiment, R 7 is a hydroxyl group or [ka] That is the case.

[0056] In one embodiment, terminal group R 7 Since represents a structural part that does not appear in the product molecule resulting from the reaction of B1 or B2 with the payload, in the linking unit-payload intermediate (see below), the structural part corresponding to B1 or B2 is one, two or more of the above-mentioned divalent groups.

[0057] In one embodiment, each - (CH2) k C(O)-, -C(O)(CH2) k -and-(C2H4) g - is either independently unsubstituted or -OH, C 1-6 alkyl group, -OC 1-6 Alkyl, -N(C 1-6 Alkyl)-C 1-6 Alkyl, -C(O)-C 1-6 Alkyl, -NHC(O)-C 1-6 Alkyl, -S(=O)2-C 1-6 Alkyl and -NHS(=O)2-C 1-6 Substituted with one or more groups selected from alkyl groups, more preferably -C 1-6 Alkyl or -NHC(O)-C 1-6 Substituted with one or more groups selected from alkyl groups. In another embodiment, each -(CH2) k C(O)-, -C(O)(CH2) k -and-(C2H4) g - is either independently unsubstituted, or -C(O)-NH2, -NH2, -C 1-6 Alkyl-NH2, -NH-C 1-6Substituted with one or more groups selected from alkyl groups, preferably -C(O)-NH2, -C 1-6 Alkyl-NH2, -NH-C 1-6 It is substituted with one or more groups selected from alkyl groups.

[0058] In one embodiment, k is selected from the values ​​of 0, 1, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3, preferably 0, 1, or 2, particularly 1 or 2. In one embodiment, j is selected from the values ​​of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, particularly 1, 3, or 4. In one embodiment, d is selected from the values ​​of 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2. In a particular embodiment, d is 1 or 2. In a particular embodiment, d is 1. In one embodiment, g is selected from the values ​​of 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4.

[0059] Two or more -(CH2) molecules within the molecule k It should be understood that when a C(O)- group is present, the value of each k is selected independently. In some embodiments, the "k"s in the molecule may or may not have an additional number, e.g., k1, k2, k3, etc., where the number does not indicate order but is simply to distinguish the "k"s. Other subscripts such as g, j, d should be understood similarly.

[0060] Two or more R x If (x is 1, 2, 3, 4, 5, 6, 7, etc.), then each R x It should be understood that these are selected independently. In some embodiments, the "x"s in the molecule may or may not have an additional apostrophe (') or apostrophe ('', ''', '''', etc.), e.g., R, R 1’ , R 1’’ , R 1’’’ , R 2’ , R 2’’ , R 2’’’ Etc. R 3 , R 4 , R5 , R 6 , R 7 Other R's, etc. x 's' should be understood similarly.

[0061] In another embodiment, the cleavable sequence 1 includes an enzyme cleavage site selected from a protease-sensitive oligomeric peptide, a cathepsin cleavage site, a glutaminase cleavage site, and a combination thereof. In a particular embodiment, the cleavable sequence 1 is selected from Phe-Lys, Val-Cit, and Val-Lys.

[0062] In another embodiment, Sp1 is a spacer sequence containing 1 to 10, preferably 1 to 6, and more preferably 1 to 4 amino acids. In one embodiment, Sp1 is PABC.

[0063] In one embodiment, B1 and B2 are each optionally derivatized lysine. In another embodiment, the derivatization of lysine is 1) amidation of the carboxyl group, and the resulting amide NH2 is C 1-6 1) the amidation which is optionally substituted with an alkyl group, and 2) the linkage of a carboxyl group and / or an amino group to an amino acid fragment containing 1 to 10 amino acids or a nucleotide fragment containing 1 to 10 nucleotides, wherein the amino acid fragment is preferably glycan.

[0064] In one embodiment, when a terminal group is combined with a divalent group, the following divalent groups each form the following structural moieties: -(CH2) k C(O)- is -(CH2) k Forms C(O)-OH, -NR 3 -ha-NHR 3 or R 3 Forms HN-, -(C2H4-O) j -ha-(C2H4-O) j -H forms, -(NH-CR 1 R 2 -C(O)) d - is - (NH-CR 1 R2 -C(O)) d Forms an OH group, -(C2H4) g -ha-(C2H4) g -H, -(C2H4) g -OH,H-(C2H4) g -or HO-(C2H4) g - forms -C(O)(CH2) k - is HO-C(O)(CH2) k -forms -(O-C2H4) j - is H-(O-C2H4) j - forms -(C(O)-CR 1 R 2 -NH) d - is HO-(C(O)-CR 1 R 2 -NH) d - forms, where -(CH2) k C(O)-, -C(O)(CH2) k -and-(C2H4) g Each of these is either independently non-substitutable or substituted by one or more of the groups defined above.

[0065] In one embodiment, B2 is -(CH2) k C(O)-OH, -(NH-CR 1 R 2 -C(O)) d -OH, -NH-(C2H4-O) j -H, -Val-Cit-PABC, -(Lys-NH2), -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H, -(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -H, -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O)j -H, -(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-(Lys-OH), -Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-Val-Cit-PABC and -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - Selected from OH

[0066] In one embodiment, B2 is -(CH2) k C(O)-OH, -(NH-CR 1 R 2 -C(O)) d -OH, -NH-(C2H4-O) j -H, -Val-Cit-PABC, -(Lys-NH2), -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H, -(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -H, -(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-(Lys-OH), -Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-Val-Cit-PABC and -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - Selected from OH

[0067] In one embodiment, B2 is -(CH2) k C(O)-OH, -(NH-CR 1 R 2 -C(O)) d -OH, -NH-(C2H4-O) j -H, -Val-Cit-PABC, -(Lys-NH2), -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H, -(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -H, -(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-(Lys-OH), -Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -OH and -(CH2) k It is selected from C(O)-Val-Cit-PABC.

[0068] In one embodiment, B2 is -(Lys-NH2). In one embodiment, B1 is (Lys-NH2)-.

[0069] In one embodiment, B2 is -(CH2) k C(O)-OH, -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H, -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j-H, -(CH2) k C(O)-Val-Cit-PABC and -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - Selected from OH

[0070] In one embodiment, B2 is -(CH2) k C(O)-OH, -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H, -(CH2) k C(O)-Val-Cit-PABC and -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - Selected from OH

[0071] In one embodiment, B2 is -(CH2) k C(O)-OH, -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -OH,-(CH2) k C(O)-NH-(C2H4-O) j -H and -(CH2) k It is selected from C(O)-Val-Cit-PABC.

[0072] In one embodiment, B1 is HO-C(O)(CH2) k -, HO-(C(O)-CR 1 R 2 -NH) d -, H-(O-C2H4) j -NH-, (Lys-NH2)-, HO-(C(O)-CR 1 R 2 -NH) d -C(O)(CH2) k-, H-(O-C2H4) j -NH-C(O)(CH2) k -, H-(O-C2H4) j -NH-(C(O)-CR 1 R 2 -NH) d -, H-(O-C2H4) j -NH-(C(O)-CR 1 R 2 -NH) d C(O)(CH2) k -, (Lys-OH)-C(O)(CH2) k1 -(O-C2H4) j -NH-C(O)(CH2) k2 -, HO-(C(O)-CR 1 R 2 -NH) d -Val-Cit-PABC-, and HO-(C(O)-CR 1 R 2 -NH) d -Val-Cit-PABC-C(O)(CH2) k1 - will be selected from.

[0073] In one embodiment, B1 is HO-C(O)(CH2) k -, HO-(C(O)-CR 1 R 2 -NH) d -, H-(O-C2H4) j -NH-, (Lys-NH2)-, HO-(C(O)-CR 1 R 2 -NH) d -C(O)(CH2) k -, H-(O-C2H4) j -NH-C(O)(CH2) k -, H-(O-C2H4) j -NH-(C(O)-CR 1 R 2 -NH) d -, (Lys-OH)-C(O)(CH2) k1 -(O-C2H4) j -NH-C(O)(CH2) k2 -, HO-(C(O)-CR 1 R 2-NH) d -Val-Cit-PABC-, and HO-(C(O)-CR 1 R 2 -NH) d -Val-Cit-PABC-C(O)(CH2) k1 - will be selected from.

[0074] In one embodiment, R 1 and R 2 Both are hydrogen or both are -C 1-6 They are alkyl, preferably both hydrogen or both -C 1-3 Alkyl, more preferably both hydrogen or both -C 1-2 It is alkyl, and in particular both are hydrogen or both are methyl groups. In one embodiment, R 3 is hydrogen or -C 1-6 It is alkyl, preferably hydrogen or -C 1-2 It is alkyl, and especially hydrogen.

[0075] A and Lm In one embodiment, A1 and A2 in formula (I') are each independently residues obtained from reactive groups selected from amino compounds, thiol compounds, pyridyldithiol compounds, and isocyanates. In another embodiment, A1 and A2 are each independently moieties conjugated to Lm1 or Lm2 via reactive groups selected from amino groups, thiol groups, pyridyldithio groups, and isocyanate groups. In a particular embodiment, A1 and A2 are each independently selected from optionally derivatized amino acids, preferably optionally derivatized cysteine ​​or lysine.

[0076] In another specific embodiment, A1 and A2 are each independently selected from optionally derivatized cysteine. In a preferred embodiment, the derivatization of cysteine ​​is 1) amidation of the carboxyl group, wherein the resulting amide NH2 is C 1-6The amidation is optionally substituted with an alkyl group; the acylation of an amino group; and the linkage of a carboxyl group and / or an amino group to an amino acid fragment containing 1 to 10 amino acids or a nucleotide fragment containing 1 to 10 nucleotides, wherein the amino acid fragment is preferably glycine. In certain embodiments, cysteine ​​derivatization refers to the amidation or linkage of the carboxyl group of cysteine ​​to glycine.

[0077] In one embodiment, A2 is [ka] Here, x is selected from hydrogen, OH, NH2, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides, and is preferably NH2. In one embodiment, A1 is [ka] Here, x is selected from hydrogen, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides, preferably NH2. In one embodiment, amino group acylation is defined as the C of the amino group of cysteine. 1-6 This refers to substitution with an alkylcarbonyl group.

[0078] In one embodiment, the linkage between the A portion and the Lm portion may be formed such that the thiol group in the cysteine ​​structure reacts with the maleimide group contained in Lm to produce a thiosuccinimide structure.

[0079] In certain embodiments, the thiol group in the cysteine ​​structure is connected to the maleimide group by Michael addition.

[0080] Thiosuccinimide is unstable under physiological conditions and tends to reverse Michael addition, causing cleavage at the conjugation site. Additionally, if another thiol compound is present in the system, thiosuccinimide may undergo thiol exchange with the other thiol compound. Both reactions result in a reduced payload and toxic side effects. In this disclosure, ring-opening of succinimide is carried out by a ring-opening reaction after the Michael addition step. After ring-opening, succinimide no longer undergoes reverse Michael addition or thiol exchange, making the product more stable. For the method of the ring-opening reaction, please refer to WO2015165413A1.

[0081] Regardless of the efficiency of the succinimide ring-opening reaction, the ring-opened compound of formula (I') can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain a payload-supported formula (I') compound with high purity and the specified composition.

[0082] In one embodiment, Lm1 and Lm2 are independently [ka] It is a mixture of. In one embodiment, Lm1 is [ka] or a mixture thereof. In one embodiment, Lm2 is [ka] Or a mixture thereof.

[0083] Specific Embodiments of Compound (I') 1. Compounds of formula (I') in which A and Lm exist p is 1, D1 is G n G is glycine, and A2 is the reaction between the thiol group and Lm2. [ka] The linking unit of formula (I'-1) and the structure of the compound of formula (I'-1) are as shown in formula (I'-1-1) below. [ka] In the formula, n is an integer between 3 and 10, and Lm2 is [ka] or a mixture thereof, x is selected from hydrogen, OH, NH2, amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides. Y, Lk, and W are defined in equation (I'), respectively.

[0084] In a preferred embodiment, in formula (I'-1-1), x is selected from OH, NH2, and Gly, and is particularly NH2.

[0085] In a particular embodiment, in formula (I'-1-1), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the connecting unit is as follows (connecting unit LN102).

[0086] [ka]

[0087] In one embodiment, B2 in the connecting unit LN102 is selected from the table below.

[0088] [Table 1]

[0089] In one embodiment, B2 in the connecting unit LN102 is selected from the table below.

[0090] [Table 2]

[0091] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-R 7 Therefore, k is 2, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-1).

[0092] [ka]

[0093] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-R 7 Therefore, k is 5, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-2).

[0094] [ka]

[0095] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) kThe structure is C(O)-Val-Cit-PABC, k is 5, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-3).

[0096] [ka]

[0097] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -R 7 And k is 5, d is 1, R 1 and R 2 It is hydrogen, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-4).

[0098] [ka]

[0099] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-NH-(C2H4-O) j -H is 2, k is 1, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-6).

[0100] [ka]

[0101] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -H is , k is 2, d is 1, j is 1, R 1 and R 2 The group is a methyl group, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-7).

[0102] [ka]

[0103] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is 2, d is 1, R 1 and R 2 It is hydrogen, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-8).

[0104] [ka]

[0105] In one embodiment, in the connecting unit LN102, Lm2 is [ka] or a mixture thereof, where B2 is -(CH2)k C(O)-(NH-CR 1 R 2 -C(O)) d -R 7 And k is 2, d is 2, R 1 and R 2 R is a methyl group, 1’ and R 2’ It is hydrogen, and the structure of the connecting unit is a mixture of the following two structures (connecting unit LN102-11).

[0106] [ka]

[0107] In a particular embodiment, in formula (I'-1-1), a is 0, b is 0, n=3, B2 is -Cys-NH2, and the structure of the connecting unit is as follows (connecting unit LN105).

[0108] [ka]

[0109] In a particular embodiment, in formula (I'-1-1), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The relationship is -C2H4-, i=4, x is OH, and the structure of the connecting unit is as follows (connecting unit LN106).

[0110] [ka]

[0111] In a particular embodiment, in formula (I'-1-1), a is 1, b is 0, Y is L, L is leucine (Leu), n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the connecting unit is as follows (connecting unit LN107).

[0112] [ka]

[0113] In yet another specific embodiment, in formula (I'-1-1), a is 1, b is 0, Y is Q, Q is glutamine (Gln), n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the connecting unit is as follows (connecting unit LN108).

[0114] [ka]

[0115] In a particular embodiment, in formula (I'-1-1), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 -C5H 10-The structure of the connecting unit is as follows (connecting unit LN109).

[0116] [ka]

[0117] In yet another specific embodiment, in formula (I'-1-1), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 is one -NR 1 R 2 -C5H substituted with a group 10 - is a base, R 1 is hydrogen, R 2 x is -C(O)CH3, x is NH2, and the structure of the connecting unit is as follows (connecting unit LN110).

[0118] [ka]

[0119] D2 is LPXTG, and A1 is the reaction between the thiol group and Lm2. [ka] In this case, the linking unit of formula (I'-2) and the structure of the compound of formula (I'-2) are as shown in formula (I'-2-1) below. [ka] In the formula, x is selected from hydrogen, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides, and Lm1 is [ka] or a mixture thereof, Y, Lk, and W are defined in equation (I'), respectively.

[0120] In one embodiment, x is hydrogen.

[0121] 2. Compounds of formula (I') in which A and Lm are absent.

[0122] p is 0, D1 is G n The linking unit of formula (I'-1), where G is glycine, and the structure of the compound of formula (I'-1) are as shown in formula (I'-1-2) below. [ka] In the formula, n is an integer between 3 and 10. Y, Lk, W, a, and b are defined in equation (I'), respectively.

[0123] In one embodiment, in equation (I'-1-2), a is 0, b is 0, n=3, B2 is -(Lys-NH2), and the structure of the connecting unit is as follows (connecting unit LN201).

[0124] [ka] In the formula, n is an integer between 3 and 10.

[0125] In one embodiment, the compound of formula (I') is one of the compounds shown in Figure 1.

[0126] Compound of formula (I') as a linking unit In one embodiment, a reactive group contained in B1 or B2 can be used to covalently conjugate with a payload containing another reactive group so that the compound of formula (I') supports the payload.

[0127] In another embodiment, the ligase recognition sequence contained in D1 or D2 can be used for ligase conjugation with the corresponding ligase recognition sequence. As a result, the compound of formula (I') can be linked to a molecule containing a ligase recognition sequence, where the ligase recognition sequence contained in the molecule is a ligase donor / receptor substrate recognition sequence corresponding to the ligase recognition sequence contained in D1 or D2.

[0128] In one embodiment, the molecule includes a recognition sequence for a ligase donor substrate, and accordingly, D1 or D2 is independently a recognition sequence for a ligase receptor substrate. In another embodiment, the molecule includes a recognition sequence for a ligase receptor substrate, and accordingly, D1 or D2 is independently a recognition sequence for a ligase donor substrate.

[0129] Therefore, the compound of formula (I') can be used as a linking unit that can be linked to a targeting molecule (e.g., an antibody or its antigen-binding fragment) and / or a payload. The linking unit may contain a ligase-recognition sequence for conjugation of the linking unit and the targeting molecule. The linking unit may further contain a reactive group for co-conjugation with the payload.

[0130] Depending on the type of terminal modification of the target molecule to be conjugated, the ligase recognition sequence included in the conjugation unit is either a ligase receptor substrate recognition sequence or a ligase donor substrate recognition sequence. The recognition sequence corresponds to the ligase being employed.

[0131] Depending on the type of reactive group in the payload to be conjugated, the reactive groups included in the linking unit belong to a type that allows the condensation reaction to proceed.

[0132] The linking unit can influence the properties of the drug conjugate formed therein. For example, the linking unit can optionally be used to provide appropriate hydrophilicity, and it can optionally contain cleavage sites to achieve an appropriate release profile for the payload.

[0133] In alternative embodiments, the linking unit further comprises one or more non-enzymatic cleavage sites, each independently located at any suitable position. In one embodiment, the non-enzymatic cleavage site may be a pH-sensitive hydrazone. In another embodiment, the non-enzymatic cleavage site may be a reducing agent-sensitive disulfide bond. In yet another alternative embodiment, the linking unit further comprises one or more enzymatic cleavage sites, each independently located at any suitable position other than Y and W. In one embodiment, the enzymatic cleavage sites are selected from protease-sensitive oligomeric peptides, cathepsin cleavage sites, glutaminase cleavage sites, and combinations thereof.

[0134] In yet another alternative embodiment, to improve the DAR of the targeted molecule-drug conjugate, the linking unit may further include branched structural fragments. The backbone of this branched structure is formed of multifunctional molecules according to a specific linking pattern, and the number and structure of the branches may be made to correspond to a desired number of payloads. Each branch may include the structure of the linear linking unit described above.

[0135] Those skilled in the art can synthesize the connecting units using conventional solid-phase or liquid-phase methods.

[0136] Payload-supported (I') compounds The reactive group contained in B1 or B2 is covalently conjugated with a payload containing another reactive group to provide a payload-supported (I') compound.

[0137] In yet another embodiment, a compound having the structure of formula (II') (formula (II'-1) or formula (II'-2)), (Compound of formula (I')) - PL t (II'-1) PL t —(Compound of formula (I')) Compound (II'-2) During the ceremony, PL is a payload attached to the B1 or B2 portion of the compound of formula (I'), The present invention provides the compound in which t is an integer from 1 to 20. t represents the number of PL(s) linked to the compound of formula (I').

[0138] In one embodiment, t is an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0139] In one embodiment, t is 1, and the compounds of formula (II'-1) and (II'-2) have the structure of formula (II'-1-1) or formula (II'-2-1), respectively. D1-Y a ―Lk―W b ―A2 p ―Lm2 p ―B2―PL (II'-1-1) PL-B1-Lm1 p ―A1 p ―W b ―Lk―Y a ―D2 (II'-2-1) In the formula, A1, A2, Lm1, Lm2, B1, B2, D1, D2, Y, Lk, W, a, b, and p are as defined above.

[0140] In another embodiment, t is 2 to 20, and the structure of the compound of formula (II') is as shown in any of the following formulas (II'-3) to (II'-6): D1-Y a ―Lk―W b ―A2 p ―Lm2 p ―(B2―PL) t (II'-1-2) D1-Y a ―Lk―W b ―(A2 p ―Lm2 p ―B2―PL) t (II'-1-3) D1―(Y a ―Lk―W b ―A2 p ―Lm2 p ―B2―PL) t (II'-1-4) (PL-B1) t ―Lm1 p ―A1 p ―W b ―Lk―Y a ―D2 (II'-2-2) (PL-B1-Lm1 p ―A1 p ) t ―W b ―Lk―Y a ―D2 (II'-2-3) (PL-B1-Lm1 p ―A1 p ―W b ―Lk―Y a ) t ―D2 (II'-2-4) In the formula, A1, A2, Lm1, Lm2, B1, B2, D1, D2, Y, Lk, W, a, b, and p are defined as in formula (II'-1) or formula (II'-2), respectively.

[0141] payload In this disclosure, the payload may be selected from small molecule compounds, nucleic acids and their analogues, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptide mimes, and proteins. In one embodiment, the payload is selected from small molecule compounds, nucleic acid molecules, and tracer molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from cytotoxics and their fragments. In a more preferred embodiment, the payload is selected from immunoagonists and their fragments.

[0142] In one embodiment, the immunoagonist is selected from TLR agonists and STING agonists, preferably TLR agonists (e.g., TLR7 agonist, TLR8 agonist, TLR7 / 8 agonist) and STING agonists.

[0143] In one embodiment, the immunoagonist is selected from imidazoquinolines. In one embodiment, the immunoagonist has the structure of formula i, [ka] During the ceremony, Each R 9 They are independently hydrogen, halogen, and C 1-7 Alkyl-OC(O)-C 1-7 Alkyl, C 1-7 Alkyl-OC(O)-C 2-7 Selected from alkenyls and 5- to 7-membered heterocycles, R 10 and R 11 Each of them independently consists of hydrogen and C 1-7 Selected from alkyl groups, R 12 C 1-7 Alkyl and C 1-7 Alkoxy-C 1-7 Selected from alkyl groups, R 13 C is optionally substituted with substituents selected from -OH and -NH2. 1-7 Selected from alkyl groups, u is 1, 2, 3, or 4.

[0144] In one embodiment, the immunoagonist is selected from compounds i-1 to i-5. [ka]

[0145] In one embodiment, the immunoagonist is selected from 9H-purine. In one embodiment, the immunoagonist has the structure of formula ii, [ka] During the ceremony, L 4 It is selected from -CH2-, -NH-, -O-, and -C(O)-. R 14 C 1-7 Alkyl alkyl group, C1-7 Alkoxy groups and C 1-7 Alkyl-OC(O)-C 1-7 Selected from alkyl groups, R 15 and R 16 Each of them independently consists of hydrogen and C 1-7 Selected from alkyl groups, R 17 -NH2, -OH, C 1-7 Alkyl alkyl group, C 1-7 Alkoxy groups and -NH-C 1-7 Selected from alkyl groups, R 18 The aryl group and the heteroaryl group are selected from -CH2-aryl and -CH2-heteroaryl, and each of these groups is independently -C(O)OH or [ka] It is optionally substituted by substituents selected from the following.

[0146] In one embodiment, the immunoagonist is selected from compounds ii-1 and ii-2. [ka]

[0147] In one embodiment, the immunoagonist is selected from 5H-pyrrolo[3,2-d]pyrimidine. In one embodiment, the immunoagonist has the structure of formula iii, [ka] During the ceremony, R 19 -OH, -NH2, C 1-7 Alkyl alkyl group, C 1-7 Alkoxy groups and -NH-C 1-7 Selected from alkyl groups, R 20 The aryl group is selected from -CH2-aryl groups, and the aryl group is -OH, C 1-7 Alkoxy and -C 1-7It is optionally substituted with two substituents selected from alkyl-piperidinyl.

[0148] In one embodiment, the immunoagonist is compound iii-1. [ka]

[0149] In one embodiment, the immunoagonist is selected from 3H-benzo[b]azepines. In one embodiment, the immunoagonist has the structure of formula iv, [ka] During the ceremony, L 5 These are selected from -CH2-, -NH-, -C(O)-, -NHC(O)-, and -C(O)NH-. R 21 teeth, [ka] Selected from, B is a heteroaryl ring, R 22 is hydrogen and C 1-7 Selected from alkyl groups, R 23 and R 24 Each of them independently consists of hydrogen and C 1-7 Selected from alkyl groups, L 6 It is selected from -CH2- and -C(O)-, R 25 is -N(C 1-7 Alkyl)(C 1-7 Selected from alkyl groups, R 26 , R 27 and R 28 Each of them independently consists of hydrogen and C 1-7 Selected from alkyl groups, v is 1, 2, or 3.

[0150] In one embodiment, the immunoagonist is compound iv-1. [ka]

[0151] Preparation of payload-supported (I') compounds In one embodiment, the linking unit and the payload are linked via the reactive groups defined above by any reaction known in the art, including but not limited to condensation reactions, nucleophilic additions, electrophilic additions, and the like.

[0152] In one embodiment, the payload is an immune agonist, and the antibody-immune agonist conjugate (numbered LPx) is one of the compounds shown in the table and Figures 2a and 2b below.

[0153] [Table 3]

[0154] Compound of formula (III') In one embodiment, a compound of formula (III') (formula (III'-1) or formula (III'-2)), [ka] The present invention provides the compound wherein B1 and B2 are as defined in formula (I').

[0155] In one embodiment, the compound of formula (III') can be used to prepare the payload-supported compound of formula (I') by the following route.

[0156] [ka]

[0157] The conversion from payload-supported (III') compounds to payload-supported (I') compounds can be carried out by any known method in the art or as described herein. For example, the structural fragment "D1-Y" can be synthesized in a single-step or multi-step process. a ―Lk―W b ―A2 p " or "A1 p ―W b ―Lk―Y a "D1-Y" can be introduced into the maleimide ring of the payload-supported (III') compound, and the resulting molecule containing the succinimide moiety can undergo a ring-opening reaction to open the succinimide ring and obtain the payload-supported (I') compound (i.e., the (II') compound). In one embodiment, "D1-Y" a ―Lk―W b ―A2 p " or "A1 p ―W b ―Lk―Y a "-D2" is introduced into the payload-supported compound of formula (III') by the reaction of a maleimide group contained in the compound of formula (III') with a thiol group or an amino group, and the thiol group or amino group is "D1-Y a ―Lk―W b ―A2 p " or "A1 p ―W b ―Lk―Y a —This is part of the "D2" construction block. In one embodiment, the thiol group is contained in optionally derivatized cystine. In one embodiment, the amino group is contained in optionally derivatized lysine.

[0158] In one embodiment, the compound of formula (III') is one of the compounds shown in Figure 3.

[0159] Conjugates and their preparation Furthermore, since the payload-supported compound (I') having a portion containing a ligase recognition sequence can be conjugated with another molecule containing a ligase recognition sequence, it can be used, for example, in the preparation of targeted molecule-drug conjugates such as antibody-drug conjugates. Accordingly, in yet another embodiment, a conjugate comprising a compound of formula (I'), a targeted molecule, and a payload is provided.

[0160] Specific composition of the conjugate In yet another embodiment, a conjugate having the structure of formula (IV') (formula (IV'-1) or formula (IV'-2)), A—((compound of formula (I'))—PL t ) z (IV'-1) ((Compound of formula (I')) - PL t ) z ―A (IV'-2) During the ceremony, PL is a payload attached to the A1 or A2 portion of the compound of formula (I'), A is a targeting molecule linked to the D1 or D2 portion of the compound in formula (I'), z is an integer between 1 and 20. The present invention provides the conjugate in which t is an integer between 1 and 20. t represents the number of PL(s) linked to the compound of formula (I').

[0161] In one embodiment, the payload is an immune agonist as defined above. In one embodiment, the conjugate is an antibody-immune agonist conjugate.

[0162] In one embodiment, the ligase recognition sequence represented by D1 or D2 in the compound of formula (I') corresponds to the ligase recognition sequence in the targeting molecule to be conjugated with it, thereby achieving site-directed conjugation between the targeting molecule and the compound of formula (I'). If the terminal modification of the targeting molecule to be conjugated is a terminal modification based on the recognition sequence of a ligase donor substrate, then D1 or D2 is independently the recognition sequence of a ligase receptor substrate. Alternatively, if the terminal modification of the targeting molecule to be conjugated is a terminal modification based on the recognition sequence of a ligase receptor substrate, then D1 or D2 is independently the recognition sequence of a ligase donor substrate.

[0163] In one embodiment, z is an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0164] In one embodiment, t is an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0165] In one embodiment, t is 1, and the conjugate of formula (IV') has the structure of formula (IV'-1-1) or formula (IV'-2-1) below, A―(D1―Y a ―Lk―W b ―A2 p ―Lm2 p ―B2―PL) z (IV'-1-1) (PL-B1-Lm1 p ―A1 p ―W b ―Lk―Y a ―D2) z ―A (IV'-2-1) In the formula, PL, A1, A2, D1, D2, Y, W, Lk, a, b, and p are as defined above.

[0166] In another embodiment, t is 2 to 20, and the conjugate of formula (IV') has the following structures: (IV'-1-2), (IV'-1-3), (IV'-1-4), (IV'-2-2), (IV'-2-3), and (IV'-2-4). A―(D1―Y a ―Lk―W b ―A2 p ―Lm2 p ―(B2―PL) t ) z (IV'-1-2) A―(D1―Y a ―Lk―W b ―(A2 p ―Lm2 p ―B2―PL) t ) z (IV'-1-3) A―(D1―(Y a ―Lk―W b ―A2 p ―Lm2 p ―B2―PL) t ) z (IV'-1-4) ((PL-B1) t ―Lm1 p ―A1 p ―W b ―Lk―Y a ―D2) z ―A (IV'-2-2) ((PL―B1―Lm1 p ―A1 p ) t ―W b ―Lk―Y a ―D2) z ―A (IV'-2-3) ((PL―B1―Lm1 p ―A1 p ―W b ―Lk―Y a ) t ―D2) z ―A (IV'-2-4) In the formula, PL, A1, A2, D1, D2, Y, W, Lk, a, b, p, and z are defined as in formula (IV'-1-1) or formula (IV'-2-1), respectively.

[0167] targeting molecule In one embodiment, the targeting molecule is an antibody or its antigen-binding fragment.

[0168] In some embodiments of this disclosure, the targets recognized by the targeting molecule (e.g., an antibody or its antigen-binding fragment) are CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII, SLC44A4 / AGS-5, etc. This includes, but is not limited to, sothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, cadherin6, FGFR2, FGFR3, CA6, CanAg, integrinαV, TDGF1, ephrinA4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2.

[0169] In one embodiment, the targeting molecule is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include, but are not limited to, pertuzumab and trastuzumab. Pertuzumab binds to the second extracellular domain (ECD2) of HER2 and is approved for the treatment of HER2-positive breast cancer. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2 and is approved for the treatment of HER2-positive breast cancer and gastric cancer.

[0170] In a preferred embodiment, the anti-human HER2 antibody is one or more selected from trastuzumab-based engineered anti-HER2 antibodies.

[0171] In one embodiment, the targeting molecule is one or more selected from anti-human TROP2 antibodies or their antigen-binding fragments. In a particular embodiment, the anti-human TROP2 antibody is one or more selected from optionally modified anti-TROP2 antibodies based on Ab0064.

[0172] In one embodiment, the targeting molecule is one or more selected from anti-human CLDN18.2 antibodies or their antigen-binding fragments. In a particular embodiment, the anti-human CLDN18.2 antibody is one or more selected from optionally modified anti-CLDN18.2 antibodies based on Ab0098.

[0173] In preferred embodiments, the anti-human HER2, TROP2, or CLDN18.2 antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetic bodies. In one embodiment, the antibody mimetic body is selected from scFv, minibody, diabody, and nanobody. For conjugation with the compound of formula (I'), the targeting molecule of this disclosure may include a modification moiety that connects to D1 or D2 in the compound of formula (I'). The location of such a modification moiety is not limited; for example, if the targeting molecule is an antibody, its location may be at the C-terminus or N-terminus of the heavy or light chain of the antibody, but is not limited thereto.

[0174] In an alternative embodiment, the modification moiety for conjugation with D1 or D2 in the compound of formula (I') may be introduced to the non-terminal position of the antibody's heavy or light chain, for example, by a chemical modification method.

[0175] In one embodiment, the targeted molecule of the present disclosure is an antibody or its antigen-binding fragment, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus or N-terminus of the heavy or light chain of the antibody, for example, including a ligase recognition sequence. In another embodiment, the terminal modifications may further include a spacer Sp3 containing 2 to 100 amino acids, where the antibody, Sp3, and ligase recognition sequence are sequentially linked. In a preferred embodiment, Sp3 is a spacer sequence containing 2 to 20 amino acids. In a particular embodiment, Sp3 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGGS, particularly GA.

[0176] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment comprises a wild-type (LC), a C-terminally modified light chain (LCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and a C-terminally modified light chain (LCCT) modified by introduction of a short-chain peptide spacer and the ligase donor substrate recognition sequence LPXTG. L The heavy chain of the antibody or its antigen-binding fragment includes three types: wild-type (HC), C-terminally modified heavy chain (HCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and C-terminally modified heavy chain (HCCT) modified by introduction of a short-chain peptide spacer and the ligase donor substrate recognition sequence LPXTG. L ) and three other types. X may be any natural or non-natural single amino acid. As shown in the amino acid sequence table, when z in the compound of formula (IV') is 1 or 2, the above heavy chain and light chain combinations can form eight preferred antibody molecules.

[0177] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment comprises a wild-type (LC), an N-terminally modified light chain (LCNT) modified by direct introduction of the ligase recognition sequence GGG, and an N-terminally modified light chain (LCNT) modified by introduction of a short-chain peptide spacer and the ligase receptor substrate recognition sequence GGG. LThe heavy chain of the antibody or its antigen-binding fragment includes three types: wild-type (HC), N-terminally modified heavy chain (HCNT) modified by direct introduction of the ligase recognition sequence GGG, and N-terminally modified heavy chain (HCNT) modified by introduction of a short-chain peptide spacer and the ligase receptor substrate recognition sequence GGG. L This includes three types: ) and .

[0178] The conjugate of this disclosure may further include a payload, which is as described above.

[0179] Specific implementations of the conjugate 1. Compounds of formula (IV') in which A and Lm exist p is 1, D1 is G n G is glycine, and A2 is the reaction between the thiol group and Lm2. [ka] The structure of the conjugate of formula (IV'-1-1) and the compound of formula (IV'-1-1) is as shown in the following formula (referred to as formula IV'-1-1-1 or formula II), [ka] In the formula, n is an integer between 3 and 10, and Lm2 is [ka] or a mixture thereof, x is selected from hydrogen, OH, NH2, amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides. Y, Lk, and W are defined as shown in equation (IV'), respectively.

[0180] In a preferred embodiment, in formula (II), x is selected from OH, NH2, and Gly, and is particularly NH2.

[0181] In one embodiment, in equation (II), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the compound of formula (II) is as shown in the following formula (II-1). [ka]

[0182] In one embodiment, B2 in formula (II-1) is selected from the table below.

[0183] [Table 4]

[0184] In one embodiment, B2 in formula (II-1) is selected from the table below.

[0185] [Table 5]

[0186] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k The compound is C(O)-, k is 2, and the structure of the conjugate is as follows (Equation AC102-1). [ka]

[0187] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k The compound is C(O)-, k is 5, and the structure of the conjugate is as follows (Equation AC102-2). [ka]

[0188] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k The structure is C(O)-Val-Cit-PABC-, where k is 5, and the structure of the conjugate is as follows (Equation AC102-3).

[0189] [ka]

[0190] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -, k is 5, d is 1, R 1 and R 2 is hydrogen, and the structure of the conjugate is as follows (Equation AC102-4). [ka]

[0191] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -, k is 2, d is 1, j is 1, R 1 and R 2 The group is a methyl group, and the structure of the conjugate is as follows (formula AC102-7). [ka]

[0192] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is 2, d is 1, R 1 and R 2 is hydrogen, and the structure of the conjugate is as follows (Equation AC102-8). [ka]

[0193] In one embodiment, in formula (II-1), Lm2 is [ka] or a mixture thereof, where B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is 2, d is 2, R 1 and R 2 R is a methyl group,1’ and R 2’ is hydrogen, and the structure of the conjugate is as follows (Equation AC102-11). [ka]

[0194] In a particular embodiment, in equation (II), a is 0, b is 0, n=3, B2 is -(Cys-NH2)-, and the structure of the conjugate is as follows (equation AC105). [ka]

[0195] In a particular embodiment, in formula (II), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The structure is -C2H4-, i=4, x is OH, and the structure of the conjugate is as follows (Equation AC106). [ka]

[0196] In a particular embodiment, in formula (II), a is 1, b is 0, Y is L, L is leucine (Leu), n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the conjugate is as follows (Equation AC107). [ka]

[0197] In yet another specific embodiment, in formula (II), a is 1, b is 0, Y is Q, Q is glutamine (Gln), n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, x is NH2, and the structure of the conjugate is as follows (Equation AC108). [ka]

[0198] In a particular embodiment, in formula (II), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 -C5H 10 -The structure of the conjugate is as follows (Equation AC109). [ka]

[0199] In yet another specific embodiment, in equation (II), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 is one -NR 1 R 2 -C5H substituted with a group 10 - is a base, R 1 is hydrogen, R 2x is -C(O)CH3, x is NH2, and the structure of the conjugate is as follows (Equation AC110). [ka]

[0200] D2 is LPXTG, and A1 is the reaction between the thiol group and Lm2. [ka] In this case, the structure of the conjugate of formula (IV'-2) and the compound of formula (IV'-2-1) are as shown in the following formula (IV'-2-1-1), [ka] In the formula, x is selected from hydrogen, an amino acid fragment containing 1 to 10 amino acids, and a nucleotide fragment containing 1 to 10 nucleotides, and Lm1 is [ka] or a mixture thereof, Y, Lk, and W are defined as shown in equation (IV'), respectively.

[0201] In one embodiment, x is hydrogen.

[0202] 2. Compounds of formula (IV') in which A and Lm are absent p is 0, D1 is G n The linking unit of formula (IV'-1-1), where G is glycine, and the structure of the compound of formula (IV'-1-1) are as shown in the following formula (IV'-1-1-2), [ka] In the formula, n is an integer between 3 and 10. Y, Lk, W, a, and b are defined as shown in equation (IV').

[0203] In one embodiment, in equation (IV'-1-1-2), a is 0, b is 0, n=3, and Lk is L 1 -L 2 -L 3 And L 1 is -NH-, L 3 is -C(O)- and L 2 ha-(C2H4-O) i The compound is -C2H4-, i=4, B2 is -(Lys-NH2)-, and the structure of the conjugate is as follows (Equation AC201). [ka]

[0204] Preparation of Conjugates The conjugates of this disclosure may be prepared by any known method of the art. In some embodiments, the conjugate is prepared by ligase-catalyzed site-specific conjugation of a targeting molecule and a payload-supported (I') compound, wherein the targeting molecule is modified by a ligase-recognition sequence. The method comprises steps A and B.

[0205] Step A: Preparation of the connecting unit-payload intermediate In a preferred embodiment, B1 or B2 in the compound of formula (I') are independently covalently linked to a payload containing the corresponding reactive group via a reactive group, where the reactive group is as defined above.

[0206] The ligation unit-payload intermediates prepared using the compound of formula (I') of this disclosure have a specified structure, specified composition and high purity, so that fewer impurities are introduced or no other impurities are introduced when they are used in conjugation reactions with antibodies. When such intermediates are used in ligase-catalyzed site-specific conjugation with modified antibodies containing ligase recognition sequences, the resulting ADCs have a homogeneous and highly controllable quality.

[0207] Step B: Linking the target molecule to the payload-supported (I') compound. The targeted molecules of this disclosure can be conjugated with payload-supported (I') compounds (i.e., compounds of formula (II')) by any known method of the art. For example, ligase-catalyzed site-specific conjugation techniques are applied to link the targeted molecule and the payload-supported (I') compound to each other by a ligase-specific recognition sequence of the substrate. The recognition sequence is determined by the specific ligase employed. In one embodiment, the targeted molecule is an antibody having a recognition sequence-based terminal modification introduced at the C-terminus of the light chain and / or heavy chain, and the targeted molecule is conjugated with a compound of formula (II') under appropriate catalytic reaction conditions under the catalytic action of a wild-type or optimally engineered ligase or any combination thereof.

[0208] In a specific embodiment, the ligase is saltase A, and the conjugation reaction can be represented by the following scheme. [ka]

[0209] The triangle and pentagon represent either a part of the antibody or a part of the compound of formula (II'), and an interchangeable position, respectively. n, X, and J are as defined above. G is the corresponding recognition sequence of the receptor substrate. n When conjugated, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by saltase A, and the resulting intermediate is G n It is linked to the free N-terminus to form a new peptide bond. The resulting amino acid sequence is LPXTG n This is the case. Array G n And LPXTGJ is as defined above.

[0210] Table of specific conjugates In one embodiment, the payload is an immune agonist. In one embodiment, the antibody is modified trastuzumab, preferably Ab0001-LCCT.L -HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2). Ab0001-LCCT L -The HC sequence is based on the amino acid sequence of Ab0001 (trastuzumab), with GALPETGG introduced at the C-terminus of the light chain, where LPETGG is the ligase donor substrate recognition sequence and GA is the spacer sequence. In one embodiment, the antibody-immunoagonist conjugate is as shown in the table and Figures 4a and 4b below.

[0211] [Table 6]

[0212] Pharmaceutical compositions and pharmaceutical preparations Another object of this disclosure is to provide a pharmaceutical composition comprising a prophylactic or therapeutically effective amount of the conjugate of this disclosure and at least one pharmaceutically acceptable carrier.

[0213] The pharmaceutical compositions of this disclosure may be administered in any manner as long as they achieve an effect of preventing, alleviating, preventing or curing symptoms in humans or animals. For example, various suitable dosage forms, particularly injectable preparations such as lyophilized powders for injection, injectable drugs, or sterile powders for injection, can be prepared depending on the route of administration.

[0214] The term "pharmaceutically acceptable" means that, within the bounds of normal medical judgment, contact with a patient's tissue does not cause undue toxicity, irritation, or allergic reactions, has a reasonable benefit-to-defect ratio, and is effective for its intended purpose.

[0215] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline solutions. Pharmacochemically acceptable carriers also include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, toxicity modifiers, and sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0216] In one embodiment, the pharmaceutical composition of the present disclosure has an integer or non-integer drug-antibody ratio (DAR) of 1 to 20, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, and preferably 1 to 2. In one embodiment, the pharmaceutical composition of the present disclosure has a DAR of about 1.5 to about 2, preferably about 1.6 to about 2, and more preferably about 1.7 to about 2.

[0217] Treatment methods and use The conjugates of this disclosure are useful for the treatment of tumors and / or autoimmune diseases. Tumors susceptible to conjugate therapy include tumors characterized by specific tumor-associated antigens or cell surface receptors, and tumors that are recognized by targeted molecules in the conjugate and may be affected by the immune cell activation activity of agonists in the conjugate.

[0218] Accordingly, in yet another embodiment, the use of the conjugates or pharmaceutical compositions of the Disclosure is further provided for manufacturing agents for treating diseases, disorders or symptoms selected from tumors or autoimmune diseases.

[0219] In another embodiment, the use of the conjugates or pharmaceutical compositions of the Disclosure is provided for the treatment of tumors or autoimmune diseases.

[0220] In a further embodiment, the present invention provides a method for treating a tumor or autoimmune disease, comprising administering an effective amount of the conjugate or pharmaceutical composition of the present disclosure to an individual in need.

[0221] In a preferred embodiment, the conjugate of the present disclosure, formed by the conjugation of an anti-human HER2 antibody and a payload, can specifically bind to HER2 on the surface of tumor cells and selectively kill HER2-expressing tumor cells. In another preferred embodiment, the use of the conjugate of the present disclosure or a pharmaceutical composition of the present disclosure is provided for the production of a drug for treating a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial carcinoma, etc.

[0222] In a preferred embodiment, the conjugate of the present disclosure, formed by the conjugation of an anti-human TROP2 antibody and a payload, can specifically bind to TROP2 on the surface of tumor cells and selectively kill TROP2-expressing tumor cells. In another preferred embodiment, the use of the conjugate of the present disclosure or a pharmaceutical composition of the present disclosure is provided for the production of a drug for treating a disease, disorder, or condition selected from TROP2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from breast cancer, urothelial carcinoma, lung cancer, liver cancer, endometrial cancer, head and neck cancer, ovarian cancer, and the like.

[0223] In a preferred embodiment, the conjugate of the present disclosure, formed by the conjugation of an anti-human CLDN18.2 antibody and a payload, can specifically bind to CLDN18.2 on the surface of tumor cells and selectively kill CLDN18.2-expressing tumor cells. In another preferred embodiment, the use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure is provided for the manufacture of a drug for treating a disease, disorder, or condition selected from CLDN18.2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from gastric cancer or pancreatic cancer, etc.

[0224] The dose of the conjugate administered to the subject can be adjusted within a considerable range. This dose may vary depending on the specific route of administration and the needs of the subject, and may also be subject to the judgment of a healthcare professional.

[0225] Beneficial effects This disclosure utilizes a uniquely structured linking unit to catalyze the conjugation of a target molecule and an agonist using a ligase. The conjugate of this disclosure exhibits high homogeneity, high activity, and high selectivity. In particular, the intracellular metabolite significantly reduces cytoproliferative toxicity in cells with low or no expression of the target antigen. Furthermore, the toxicity of the linking unit-agonist intermediate is far lower than that of the free agonist, resulting in fewer adverse effects from the drug manufacturing process and making it advantageous for industrial production.

[0226] The conjugate of this disclosure achieves at least one of the following technical effects: (1) High inhibitory activity against target cells, or a potent cell-killing effect against target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., high stability in plasma, appropriate half-life and duration of action). (4) High safety (low toxicity to non-target normal cells or tissues, and / or few side effects, and a wide therapeutic window), etc.

[0227] Examples Preparation example To more clearly explain the purpose of the invention and the technical solutions, the disclosure will be further described below with specific examples. It should be understood that these examples are not intended to limit the scope of the disclosure. Specific experimental methods not described in the following examples will be carried out according to conventional experimental methods.

[0228] Instruments, materials, and reagents Unless otherwise specified, the equipment and reagents used in the examples are commercially available. The reagents can be used directly without further purification. MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer HPLC: Waters2695, Agilent1100, Agilent1200 Semi-preparative HPLC: Lisure HP plus 50D Flow cytometry: CytoFLEX S HIC-HPLC: Butyl-HIC, Mobile phase A: 25mM PB, 2M (NH4)2SO4, pH 7.0, Mobile phase B: 25mM PB, pH 7.0, Flow rate: 0.8 ml / min, Acquisition time: 25 min, Sample injection volume: 20 μg, Column temperature: 25°C, Detection wavelength: 280 nm, Sample chamber temperature: 8°C. SEC-HPLC: Column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID × 300mm, 5μm; Mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2; Flow rate: 0.5ml / min; Acquisition time: 30min; Sample injection volume: 50μl; Column temperature: 25℃; Detection wavelength: 280nm; Sample tray temperature: 8℃. CHO was obtained from Thermo Fisher Scientific. pcDNA3.3 was obtained from Life Technology. HEK293F was obtained from Prejin. PEIMAX transfection reagent was obtained from Polyscience. MabSelect Sure ProA was obtained from GE. Capto S ImpAct was obtained from GE. Linkamide MBHA resin and dichloro resin were obtained from Nankai Synthesis. HCC1954 was obtained as ATCC CAT# CRL-2338. SK-BR-3 was obtained as ATCC CAT# HTB-30. BT474 cells were obtained as ATCC CAT# HTB-20. JIMT1 cells were obtained as DSMZ CAT# ACC589. Colo205 cells were obtained as ATCC CAT# CRL-222. NCI-N87-Claudin18.2 human gastric cancer cells were obtained from KYinno Biotechnology Co., Ltd. Claudin 18.2-negative NCI-N87 parental cells were obtained as ATCC CAT# CRL-5822. MC38hHER2 mouse colorectal cancer cells were obtained from Biocytogen. NUGC4 human gastric cancer cells were obtained as JCRB CAT# JCRB0834. NCI-N87 cells (ATCC CAT# CRL-5822). MDA-MB-468 was obtained as ATCC CAT# HTB-132.

[0229] Example 1: Construction of antibody expression vector, antibody expression, purification, and identification. 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L - HC production Antibody Ab0001-LCCT L The expression plasmid for -HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) is constructed as follows: Antibody Ab0001-LCCT LThe sequence of -HC was based on the amino acid sequence of trastuzumab, and GALPETGG was introduced at the C-terminus of the light chain, where LPETGG is the recognition sequence for the ligase donor substrate and GA is the spacer sequence. The plasmid was transfected into CHO cells to construct and screen cell populations for high expression. These cell populations were cultured in a 5-10 L reactor following the trastuzumab culture process, and the supernatant was collected.

[0230] 1.2 Antibody Ab0001-LCCT L - Purification of HC Ab0001-LCCT L - The purification of HC was performed using a standard process combining MabSelect affinity chromatography and Sepharose S cation exchange chromatography. The purified product was dissolved in the original trastuzumab drug buffer (5 mM histidine HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) and frozen in small batches.

[0231] 1.3 Antibody Ab0001-LCCT L -HC Quality Control The purified antibody Ab0001-LCCT L -The purity of HC was 98.5% by SDS-PAGE, the high molecular weight polymer content of the sample was less than 0.4% by SEC-HPLC, and the endotoxin content was less than 0.098 EU / mg.

[0232] 1.4 Preparation of other modified anti-human antibodies Following a similar method, terminal modifications based on ligase recognition sequences were introduced into the C-terminuses of the light and / or heavy chains of trastuzumab, respectively, to obtain modified antibodies. Table 1 shows modified anti-human HER2 antibodies based on Ab0001 (trastuzumab). In the terminal modification sequence, LPETGG is the recognition sequence for the ligase donor substrate, and GA is the spacer sequence.

[0233] [Table 7]

[0234] Modified anti-human TROP2 antibody Ab0064-LCCT L -HC is shown in Table 2. In the terminal modification sequence, LPETGG is the ligase donor substrate recognition sequence, and GA is the spacer sequence.

[0235] [Table 8]

[0236] Modified anti-human CLDN18.2 antibodies are shown in Table 3. In the terminal modification sequence, LPETGG is the recognition sequence for the ligase donor substrate, and GA is the spacer sequence.

[0237] [Table 9]

[0238] Example 2 Preparation of the intermediate 2.1 Preparation of the connecting unit 2.1.1 Connecting Units with A and Lm Linked unit fragments LU102 to LU110 containing part A of formula (I') were synthesized by conventional solid-phase polypeptide synthesis using link amide MBHA resin or dichloro resin. Fmoc was used to protect the amino acids and amino groups of the Lk structure within the linked unit. The conjugation reagent was selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was cleaved with trifluoroacetic acid. The product was purified by HPLC, lyophilized, and stored for use. The linked unit fragments are shown in the table and Figure 5 below.

[0239] [Table 10]

[0240] The linking unit fragments in the table above were reacted with linking unit fragments containing a maleimide structure or a derivative thereof, and then a ring-opening reaction was carried out using the method described in WO2015165413A1 to obtain linking units LN102-1-1, LN102-2-1, LN102-3-1, LN102-4-1, LN102-7-1, LN102-8-1, and LN102-11-1. Their structures are as shown above. In the table below, [Table 11]

[0241] 2.1.2 Connecting units without A and Lm Link amide MBHA resin was used to synthesize the LN201 linkage unit by conventional solid-phase polypeptide synthesis. Fmoc was used to protect the amino acids in the linkage unit. The conjugation reagent was selected from HOBt, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was cleaved with trifluoroacetic acid. The product was purified by HPLC, lyophilized, and stored for use. Theoretical molecular weight: 785.9, measured values: [M+H] + = 786.8.

[0242] 2.2 Preparation of linking unit-agonist intermediate 2.2.1 Preparation of linking unit-agonist intermediate LP102-1-4 [ka]

[0243] In Step 1, 4-chloro-3-nitroquinoline (6.25 g, 30.0 mmol) was dissolved in DCM (100 mL), treated with BocNH(CH2)4NH2 (5.76 g, 30.6 mmol), and then with TEA (8.0 mL, 61.8 mmol). After maintaining the reaction at room temperature for 24 hours, the mixture was washed with H2O (80 mL) and brine (50 mL), dried over MgSO4, and concentrated under vacuum. The target compound HX20099-a was obtained as a yellow solid (10.0 g, 92.5%). MS m / z 361.5 [M+H] + .

[0265] In step 2, the nitro compound (HX20099-a) (3.0 g, 8.33 mmol) was dissolved in THF (100 mL) and water (80 mL). Zinc (13.54 g, 208.3 mmol) was added in one step, followed by NH4Cl (13.4 g, 250.0 mmol). The suspension was vigorously stirred at room temperature for 1 hour (TLC). After filtration, the cake was washed with THF (20 mL x 2). NaCl was added to the filtrate until the aqueous phase was saturated. The liquid phase was collected and the THF layer was separated. The aqueous layer was extracted with THF / EA (50 mL / 50 mL). The organic layers were combined, dried over MgSO4, and concentrated to obtain the residue for the next step (HX20099-b) (3.1 g, >100%). MS m / z 331.5 [M+H] + .

[0244] In step 3, the amine compound (HX20099-b) (660 mg, crude, <2.0 mmol) and triethyl orthovalerate (816 mg, 4.0 mmol) were suspended in toluene (50 mL) and heated to 110°C. Pyridine HCl (23.2 mg, 0.2 mmol) was then added. The reaction was heated for 4 hours. The mixture was maintained at room temperature for 48 hours (TLC). The liquid was decanted, and the remaining solid / residue was stirred with toluene (10 mL x 2), mixed with the liquid, and concentrated. The residue was dissolved in DCM and purified by silica gel column chromatography (methanol in DCM, 0-10-20%, 30 g column) to obtain the target compound HX20099-c (300 mg, 37.8% in 2 steps). MS m / z 397.5 [M+H] + .

[0245] In step 4, compound HX20099-c (200 mg, 0.51 mmol) was dissolved in DCM (8 mL) and treated with mCPBA (261.5 mg, 1.52 mmol). The reaction was maintained at room temperature for 4 hours. The mixture was washed with saturated NaHCO3 solution (15 mL x 3), dried, and concentrated to obtain the crude product HX20099-d for the next step. MS m / z 413.5 [M+H] + .

[0246] In step 5, compound HX20099-d (200 mg, <0.48 mmol) was dissolved in dichloroethane (15 mL) in a pressure tube, treated with concentrated ammonium hydroxide (28%, 0.24 mL), and the temperature was brought to 0°C. To this mixture, after cooling, tosyl chloride (104 mg, 0.54 mmol) was gradually added over 3 minutes. Concentrated ammonium hydroxide (0.12 mL) was added, and the tube was sealed. The tube was heated at 80°C for 4 hours (TLC). After cooling, the mixture was diluted with DCM (25 mL), washed with water (30 mL), dried, and purified by silica gel column chromatography to obtain the target compound (HX20099-e) (150 mg, 76%). MS m / z 412.5 [M+H] + .

[0247] In step 6, compound HX20099-e (150 mg, 0.36 mmol) was treated with TFA / DCM (2 mL / 6 mL) at room temperature for 2 hours (HPLC). Next, the reaction was dried under vacuum, and the residue was purified by semi-preparative / preparative HPLC and lyophilized to obtain compound i-3 (74 mg, 66%). MS m / z 312.5 [M+H] + .

[0270] In step 7, compound i-3 (62 mg, 0.2 mmol) was dissolved in DMF (5 mL) and treated with DIPEA (66 μL, 0.4 mmol) and N-succinimidyl 3-maleimide propionate (63.9 mg, 0.24 mmol). The reaction was maintained at room temperature for 3 hours (HPLC), and the mixture was used directly in the next step.

[0248] In steps 8-9, the mixture from step 7 was treated with a solution of ligation unit LU102 (161 mg, 0.3 mmol) and H2O (5 mL). The mixture was reacted at 0-40°C for 0.5-20 hours. The reaction mixture was then mixed with an appropriate amount of Tris base solution or another solution to promote ring-opening, and the reaction was allowed to proceed at 0-40°C for 0.2-20 hours. After the reaction was complete, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the ligation unit-agonist LP102-1-4 (60 mg, 30% in 3 steps). MS m / z 1019.5 [M+H] + .

[0249] 2.2.2 Preparation of the Linking Unit-Agonist LP201-1-1 [ka]

[0250] Step 1: Preparation of Fmoc-LP201-1-1 The purchased agonist ii-1 was weighed and dissolved in DMF. Then, DIEA or Et3N and a conjugation reagent selected from HOBt, HOAt / DIC, DCC, EDCI, or HATU were added. The mixture was stirred for 10-120 minutes. Next, a solution of the coupling unit LN201 and DMF was added to the reaction mixture, and the reaction was carried out at 0-40°C for 0.5-20 hours. After the reaction was complete, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain Fmoc-LP201-1-1. Theoretical molecular weight: 1127.2, measured value: [M+H] + = 1127.7.

[0251] Step 2: Preparation of the linking unit-agonist LP201-1-1 Fmoc-LP201-1-1 was weighed and dissolved in piperidine / DMF (v / v=1:4). The mixture was then reacted at 0–40°C for 0.5–20 hours. After the reaction was complete, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain LP201-1-1. Theoretical molecular weight: 905.0, measured values: [M+H] + = 905.6.

[0252] [Table 12]

[0253] Example 3: Preparation of Targeted Molecule-Drug Conjugates AIACs were formed by site-specific conjugation of the linking unit-agonist intermediate to the antibody using ligase. The conjugation reaction method can be found in WO2015165413A1. The obtained AIACs are shown in the table below.

[0254] [Table 13]

[0255] Example 1: Evaluation of antibody-immune agonist conjugates in vitro Isolation of human peripheral mononuclear cells Human peripheral mononuclear cells were isolated from healthy blood donors using SepMate50 and Lymphoprep (Stem Cell Technologies). Live cells were counted and the cell concentration was increased to 1.25 x 10⁶ in RPMI1640 medium containing 10% FBS. 6 The concentration was adjusted to / ml. Tumor cells were detached with trypsin and collected. Live cells were counted and the cell concentration was increased to 2.5 x 10⁶ in RPMI1640 medium containing 10% FBS. 5 Adjusted to / ml. 12.5x10 4 Human PBMC and 2.5x10 4 Tumor cells (PBMC:tumor cells = 5:1) were added to the wells of a 96-well plate, followed by the addition of antibody or conjugate at the specified concentration. The cell mixture was incubated with the drug for 18 hours, and then the cell-free supernatant was collected for human TNFα ELISA.

[0256] To evaluate the activity of HER2-targeted immune conjugates, human PBMCs and NCI N87 human gastric cancer cells were co-cultured in a 5:1 ratio, and the antibody or test immune conjugate (AC102-6-1-1 or AC102-8-1-1) was added at specified concentrations. AC102-6-1-1 induced higher TNFα production than antibody Ab0001, and the effective concentration of AC102-6-1-1 was much lower than the payload reximod (Figure 6). AC102-8-1-1, similar to AC102-6-1-1, induced higher levels of TNFα than Ab0001 (Figure 7). The activity of AC102-6-1-1 was not observed in co-culture of human PBMCs and MDA-MB-468 HER2-negative cells, indicating that the activity of AC102-6-1-1 is highly dependent on HER2 expression on target tumor cells (Figure 8). Considering this data, the activity of the immune conjugate was tested in co-cultures of human PBMCs with other cancer cells with different HER2 expression levels, including HCC1954 (Figure 9), SK-BR-3 (Figure 10), BT474 (Figure 11), JIMT1 (Figure 12), Colo205 (Figure 13), and MDA-MB-468 (Figure 14). The data indicated that AC102-8-1-1 can induce TNFα only in co-cultures of PBMCs with HER2-high tumor cells.

[0257] In a similar experimental setup, the in vitro activity of several other conjugates was evaluated (Figures 15-17). AC102-2-1-1 and AC102-3-1-1 have the same agonist payload but differ in their ligation units and induced different levels of TNFα production (Figure 15).

[0258] Example 2: Evaluation of antibody-immune agonist conjugates in vitro To evaluate the activity of TROP2-targeted immune conjugates, human PBMCs and NCI-N87 human gastric cancer cells were co-cultured in a 5:1 ratio, and either the test immune conjugate (AC102-8-2-1 or AC102-6-2-1) or naked, unmodified anti-TROP2 antibody was added at specified concentrations. Isolation of human PBMCs and experimental setup were the same as in Effect Example 1. AC102-8-2-1 and AC102-6-2-1 induced higher TNFα production than antibody Ab0064 (light chain: SEQ ID NO: 17, heavy chain: SEQ ID NO: 18) (Figure 18).

[0259] Example 3: Evaluation of antibody-immune agonist conjugates in vitro Claudin 18.2 can also be used as an example of an AIAC target. To evaluate the activity of claudin 18.2-targeted immune conjugates, NCI-N87-Claudin18.2 human gastric cancer cells overexpressing human claudin 18.2 or parental NCI-N87 cells were co-cultured with human PBMCs in a 1:5 ratio, and either the test immune conjugate (AC102-8-3-1 or AC201-1-3-1) or naked, unmodified anti-claudin 18.2 antibody was added at specified concentrations. Isolation of human PBMCs and experimental setup were the same as in Effect Example 1. The AC102-8-3-1 and AC201-1-3-1 conjugates induced higher TNFα production compared to antibody Ab0098 (Figure 19), but these conjugates induced only very small amounts of TNFα production at very high doses in claudin 18.2-negative NCI-N87 parental cells (Figure 20). Linker-payload intermediate LP201-1-1 and Ab0098-LCCT L -Conjugates prepared using HC are linker-payload intermediates LP102-8-1 and Ab0098-LCCT L Compared to conjugates prepared using -HC, these two payloads exhibited milder activity, consistent with their potency.

[0260] Example 4: Evaluation of antibody-immune agonist conjugates in vitro To evaluate the activity of HER2-targeted immune conjugates, human PBMCs and SK-BR-3 (Figure 21) or HCC1954 (Figure 22) human breast cancer cells were co-cultured in a 5:1 ratio, and the immune conjugate (AC102-6-1-1) and antibody (Ab0001) were added at specified concentrations. After incubating the cells with the drugs for 18 hours, the cell-free supernatant was collected for detection of human IFNγ by ELISA. The isolation of human PBMCs and the experimental setup were the same as in Effect Example 1. AC102-6-1-1 induced higher IFNγ production than the antibody Ab0001, suggesting its potential ability to activate the T cell response.

[0261] Example 5: Evaluation of antibody-immune agonist conjugates in vivo For in vivo antitumor efficacy studies, 1 x 10 7 NCI-N87 human gastric cancer cells were subcutaneously inoculated into the right flank of SCID beige mice. Six days later, the average tumor volume was 173 mm². 3 When tumor-bearing mice reached a certain level, they were randomly assigned and intravenously administered Ab0001 or an experimental immunoconjugate (AC102-6-1-1 or AC102-8-1-1) at 5 mg / kg. Tumor volume was measured twice weekly using calipers. The antibody itself, Ab0001, showed very limited antitumor activity. AC102-6-1-1 and AC102-8-1-1 eventually almost cured the tumors (Figure 23). In another study, AC102-8-1-1 showed dose-dependent activity at 0.5, 1, and 3 mg / kg (Figure 24).

[0262] 5x10 6 JIMT1 human breast cancer cells were subcutaneously inoculated into the right flank of SCID beige mice to generate a xenograft model. After 9 days, the average tumor volume was 149 mm². 3 When the tumor-bearing mice reached a certain stage, AC102-8-1-1 was administered intravenously at a dose of 5 mg / kg. Tumor growth was significantly inhibited (Figure 25).

[0263] MC38 hHER2 5x10⁶ mice overexpressing colorectal cancer cells 5Human HER2 was subcutaneously inoculated into the right flank of C57BL / 6 mice. Eight days later, the average tumor volume was 90 mm². 3 When the target was reached, tumor-bearing mice were sorted and administered intravenously with either Ab0001 or AC102-6-1-1. Ab0001 at 10 mg / kg did not show any significant antitumor activity. AC102-6-1-1 at 3 mg / kg and 10 mg / kg inhibited tumor growth in a dose-dependent manner (Figure 26). Under similar settings, both AC102-8-1-1 at 3 mg / kg and 10 mg / kg induced complete tumor regression in 100% of mice (Figure 27).

[0264] Example 6: Evaluation of antibody-immune agonist conjugates in vivo To test the in vivo antitumor effect of anti-TROP2 AIAC, 1x10 7 NCI-N87 human gastric cancer cells were subcutaneously inoculated into the right flank of SCID beige mice. The average tumor volume was 182 mm². 3 When tumor-bearing mice reached a certain stage, they were randomly assigned to receive intravenous administration of either a vehicle or an experimental immunoconjugate (AC102-6-2-1, AC102-8-2-1, or AC201-1-2-1) at a dose of 5 mg / kg. Tumor volume was measured twice weekly using calipers. Compared to the vehicle control group, AC102-6-2-1 and AC102-8-2-1 showed stronger antitumor activity (Figure 28).

[0265] A xenograft model was generated by subcutaneously inoculating 10 million MDA-MB-468 human breast cancer cells into the right flank of SCID beige mice. The average tumor volume was 183 mm². 3 When the target was reached, tumor-bearing mice were sorted and administered intravenously with either a vehicle or an experimental immunoconjugate (AC102-8-2-1 or AC201-1-2-1). AC102-8-2-1 and AC201-1-2-1 showed good and comparable antitumor activity at 3 mg / kg. AC102-8-2-1 showed a higher antitumor response at 3 mg / kg than at 0.5 mg / kg, demonstrating a dose-dependent effect (Figure 29).

[0266] Example 7: Evaluation of antibody-immune agonist conjugates in vivo The in vivo antitumor effect of anti-claudin 18.2AIAC was evaluated using the NUGC4 model. A xenograft model was generated by subcutaneously inoculating 1 million NUGC4 human gastric cancer cells into the right flank of BALB / c nude mice. The average tumor volume was 108 mm². 3 When tumor-bearing mice reached a certain stage, they were sorted and administered intravenously at a dose of 5 mg / kg with either the test anti-claudin 18.2 conjugate (AC102-8-3-1) or the corresponding naked, unmodified antibody on days 0 and 14. Tumor volume was measured twice weekly using calipers. The antibody alone, Ab0098 (light chain: SEQ ID NO: 21, heavy chain: SEQ ID NO: 22), showed no antitumor activity, while AC102-8-3-1 showed antitumor activity (Figure 30).

[0267] Sequence List Sequence ID 1: Ab0001-LCCT L -HC Light Chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG Sequence ID 2: Ab0001-LCCT L -HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 3: Ab0001-LC-HCCT Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 4: Ab0001-LC-HCCT Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID NO:5:Ab0001-LC-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:6:Ab0001-LC-HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG Sequence ID 7: Ab0001-LCCT-HC Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG Sequence ID 8: Ab0001-LCCT-HC Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 9: Ab0001-LCCT-HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG Sequence number 10: Ab0001-LCCT-HCCT heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID NO:11:Ab0001-LCCT-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID NO:12:Ab0001-LCCT-HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG Sequence number 13: Ab0001-LCCT L -HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 14: Ab0001-LCCT L -HCCT heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG sequence number 15:Ab0001-LCCT L -HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG sequence number 16:Ab0001-LCCT L -HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG Sequence ID 17: Ab0064 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQGINNYLSWYQQKPGKAPKSLIYRANRLVSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 18: Ab0064 Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGYRFTDYVINWVRQAPGQGLEWMGQIYPGSDTFHYNQKFQGRATLTADKSTNTAYMELSSLRSEDTAVYYCARFFEGLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 19: Ab0064-LCCT L -HC Light Chain DIQMTQSPSSLSASVGDRVTITCKASQGINNYLSWYQQKPGKAPKSLIYRANRLLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG Sequence ID 20: Ab0064-LCCT L -HC heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYRFTDYVINWVRQAPGQGLEWMGQIYPGSDTFHYNQKFQGRATLTADKSTNTAYMELSSLRSEDTAVYYCARFFEGLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 21: Ab0098 Light Chain DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 22: Ab0098 Heavy Chain QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 23: Ab0098-LCCT L -HC Light Chain DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG Sequence ID 24: Ab0098-LCCT L -HC heavy chain QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A compound selected from the following structures. 【Chemistry 13-1】 【Chemistry 13-2】

2. An antibody-drug conjugate selected from the following structures, wherein z is an integer from 1 to 20, and A is a targeting molecule which is an antibody or antigen-binding fragment. 【Chemistry 16-1】 【Chemistry 16-2】

3. One or more targets recognized by the targeting molecule are CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII, SLC44A4 / AGS-5, Mesothelin, CEACAM5, PS An antibody-drug conjugate according to claim 2, selected from MA, TIM1, LY6E, LIV1, Nectin 4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2, FGFR3, CA6, CanAg, Integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2.

4. The antibody-drug conjugate according to claim 2, wherein the antibody is an anti-human HER2 antibody.

5. The antibody-drug conjugate according to claim 4, wherein the anti-human HER2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2, or a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4, or a light chain having the amino acid sequence of SEQ ID NO: 5 and a heavy chain having the amino acid sequence of SEQ ID NO: 6, or a light chain having the amino acid sequence of SEQ ID NO: 7 and a heavy chain having the amino acid sequence of SEQ ID NO: 8, or a light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO: 10, or a light chain having the amino acid sequence of SEQ ID NO: 11 and a heavy chain having the amino acid sequence of SEQ ID NO: 12, or a light chain having the amino acid sequence of SEQ ID NO: 13 and a heavy chain having the amino acid sequence of SEQ ID NO: 14, or a light chain having the amino acid sequence of SEQ ID NO: 15 and a heavy chain having the amino acid sequence of SEQ ID NO:

16.

6. The antibody-drug conjugate according to claim 2, wherein the antibody is an anti-human TROP2 antibody.

7. The antibody-drug conjugate according to claim 6, wherein the anti-human TROP2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 17 and a heavy chain having the amino acid sequence of SEQ ID NO: 18, or a light chain having the amino acid sequence of SEQ ID NO: 19 and a heavy chain having the amino acid sequence of SEQ ID NO:

20.

8. The antibody-drug conjugate according to claim 2, wherein the antibody is an anti-human CLDN18.2 antibody.

9. The antibody-drug conjugate according to claim 8, wherein the anti-human CLDN18.2 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 21 and a heavy chain having the amino acid sequence of SEQ ID NO: 22, or a light chain having the amino acid sequence of SEQ ID NO: 23 and a heavy chain having the amino acid sequence of SEQ ID NO:

24.

10. The antibody-drug conjugate according to any one of claims 2 to 9, wherein the conjugate has a drug-antibody ratio (DAR) of an integer or non-integer of 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, 1 to 2, 1.5 to 2, 1.6 to 2, or 1.7 to 2.

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