Anti-HER2 antibody-immune agonist conjugates and uses thereof

Abstract

The present disclosure relates to linking unit molecules of targeting molecule-drug conjugates, and the corresponding conjugates, their preparation and uses, in particular to anti-HER2 antibody-immunoagonist conjugates (AIACs) as a novel type of cancer therapy.

Description

[Technical Field]

[0001] The present disclosure relates to the field of biopharmaceuticals, and in particular to linking units of targeting molecule-immune agonist conjugates, and the corresponding conjugates, their preparation processes and uses. [Background technology]

[0002] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. HER2 amplification or overexpression occurs in approximately 15-30% of breast cancers and approximately 10-30% of gastric / gastroesophageal cancers. HER2 overexpression has also been observed in other cancers, such as ovarian, endometrial, bladder, lung, colon, and head and neck cancers (Iqbal N. et al., Mol Biol Int. 2014:852748). While the efficacy of HER2-targeted therapies, such as HER2-directed antibodies or antibody-drug conjugates (ADCs), has significantly extended the life expectancy of patients with HER2-positive disease, HER2-positive breast cancer remains an aggressive cancer with a worse prognosis and poorer outcomes than patients with HER2-negative (and HR-positive) disease. Furthermore, treatment efficacy has been disappointing in other HER2-overexpressing cancers. Resistance to HER2-targeted therapies is one of the many causes of poor outcomes in patients, and immune escape by tumor cells contributes to this process.

[0003] Immunotherapy is a novel cancer treatment modality that has demonstrated strong efficacy. Immune checkpoint inhibitors, primarily CLTA-4 and PD-1 / L1 monoclonal antibodies, are primarily T cell-based therapies and have been approved for various cancer indications. However, numerous efforts are underway to explore other immune system mechanisms to combat cancer. Targeting myeloid cells, primarily macrophages and DCs, has become a promising direction. Activation of macrophages and DCs by agonists or macrophage checkpoint inhibitors enhances their antigen-presenting function as well as their phagocytic ability to eliminate tumor cells, thereby eliciting stronger adaptive antitumor immunity.

[0004] The present invention provides HER2-directed antibody-immunoagonist conjugates (AIACs), which are novel agents for tumor-targeted immunotherapy. Summary of the Invention

[0005] In one embodiment, a compound of formula (I-1) or (I-2) [ka] In the formula, B2 is -(CH2) k (CO)-NH-(C2H4-O) j where k is an integer from 1 to 5 and j is an integer from 1 to 3; PL is a payload linked to the B2 portion,

[0006] Preferably, the PL is resiquimod [ka] The compound is provided as follows:

[0007] In another embodiment, an antibody drug conjugate of formula (II-1) or (II-2): [ka] In the formula, B2 is -(CH2) k (CO)-NH-(C2H4-O) j wherein k is an integer from 1 to 5 and j is an integer from 1 to 3; PL is a payload linked to the B2 portion,

[0008] Preferably, the PL is resiquimod [ka] and z is an integer of 1 to 4, preferably 1 to 2; A is an antibody comprising 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.

[0009] The antibody-immune agonist conjugates (AIACs) of the present invention provide a novel type of tumor-targeted therapy. In vitro studies have shown that the AIACs can induce higher TNFα production than naked, unmodified antibodies. In vivo studies have shown that the AIACs have antitumor effects. [Brief explanation of the drawings]

[0010] [Figure 1] Illustrative examples of compounds of formula (I'). [Figure 2] Illustrative examples of compounds of formula (II'). [Figure 3] Illustrative examples of compounds of formula (III'). [Figure 4] TNFα-inducing activity of conjugates AC102-5-1-1, AC102-6-1-1, and their corresponding naked unmodified antibody Ab0001 (Trastuzumab) and agonist resiquimod in human PBMC-NCI N87 co-cultures. [Figure 5] TNFα-inducing activity of AC102-6-1-1 and antibodies in co-cultures of PBMC with either NCI N87 or MDA-MB-468 cells. [Figure 6] TNFα-inducing activity of AC102-9-1-1, AC102-12-1-1, AC102-10-1-1, AC102-13-1-1, and antibodies in cocultures of PBMCs and NCI N87 cells. [Figure 7] 7 and 8, arrows below the X-axis indicate the time points of administration. 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 test conjugate (AC102-5-1-1 or AC102-6-1-1). [Figure 8]7 and 8, the arrows below the X-axis indicate the time points of administration. Changes in tumor volume over time in the hHER2-overexpressing MC38 model administered AC102-6-1-1 at 3 mg / kg and 10 mg / kg, and the antibody at 10 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following, specific embodiments are presented to explain the technical content of the present disclosure. Those skilled in the art can easily understand other advantages and effects of the present disclosure from the contents disclosed in this specification. The present 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 the present 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 techniques used herein refer to techniques commonly understood in the art and include variations and equivalent substitutions that are obvious to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, their definitions are provided below to better explain the present disclosure. Product names mentioned herein refer to the corresponding products or their active ingredients. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0013] It should be understood that the description of a particular amount, concentration, or other value or parameter as a range, preferred range, or preferred upper or lower limit is equivalent to specifically disclosing any range combining any upper or preferred value with any lower or preferred value, whether expressly stated or not. Unless otherwise specified, numerical ranges recited herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the phrase "i is an integer from 2 to 20" means that i is any integer from 2 to 20, e.g., i can 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 manner.

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

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

[0016] The term "stoichiometric ratio" refers to the blending of various materials in specific amounts by weight. For example, in this disclosure, active ingredients are mixed with fillers, binders, and lubricants in specified weight ratios.

[0017] The terms "optional" or "optionally" mean that the subsequently described event may, but does not necessarily, occur, and the phrase includes cases where the event or circumstance occurs or does not occur.

[0018] The expression "comprises" or similar expressions such as "comprising," "containing," and "having" are open-ended and do not exclude additional, unrecited elements, steps, or ingredients. The expression "consisting of" excludes any element, step, or ingredient not expressly recited. The expression "consisting essentially of" means limiting the scope to the specified elements, steps, or ingredients, and any optionally present elements, steps, or ingredients that do not materially affect the essential and novel characteristics of the claimed subject matter. The expression "comprising" should be understood to encompass the expressions "consisting essentially of" and "consisting of."

[0019] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, etc.). A targeting molecule can deliver a payload to a specific site in the body by targeted delivery. A targeting molecule can recognize one or more targets. A 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 said receptor. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins for a given antigen, antibody mimics, 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, so long as they possess the desired biological activity. The antibody may be of any subtype (such as IgG, IgE, IgM, IgD, and IgA) or subclass and may be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may be fully human, humanized, or a chimeric antibody prepared by recombinant methods.

[0021] Monoclonal antibody is used herein to refer to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a few possible natural variations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" indicates that the properties of the antibody are derived from a substantially homogeneous antibody population and should not be construed as requiring any particular method for producing the antibody.

[0022] An intact or full-length antibody essentially consists of an antigen-binding variable region, a light chain constant region (C L ) and heavy chain constant region (C H ), and the heavy chain constant region (C H ) depending on the antibody subtype, C H 1. C H 2. C H 3 and C H 4. The antigen-binding variable region (also known as a fragment variable region, Fv fragment) typically comprises a light chain variable region (V L ) and heavy chain variable region (V H The constant region may be a constant region having a native sequence (such as a constant region having a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with a target antigen. The constant region can be recognized by the immune system and interacts with it.

[0023] An antibody fragment may contain a portion of an intact antibody, preferably the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), V, H and C H These fragments include Fd fragments, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs). Fab fragments are antibody fragments obtained by papain digestion of full-length immunoglobulins, or fragments with the same structure as those produced by, for example, recombinant expression. Fab fragments are composed of a light chain (V L and C L ) and another chain, wherein the other chain comprises a heavy chain (VH ) and the variable domains of the heavy chain (C H The F(ab')2 fragment contains the constant region domain of the F(ab')2 antibody fragment obtained by pepsin digestion of immunoglobulins at pH 4.0 to 4.5, or a fragment having the same structure as that produced by, for example, recombinant expression. The F(ab')2 fragment essentially contains two Fab fragments, with each heavy chain containing a few additional amino acids, including a cysteine ​​that forms a disulfide bond connecting the two fragments. The Fab' fragment contains half of the F(ab')2 fragment (one heavy chain and one light chain). The antibody fragment 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 typically at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework (eg, by substitution of the corresponding region), can result in an antibody that immunospecifically binds to an antigen.

[0024] Antibodies according to the present disclosure can be prepared using techniques known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination thereof, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed in an appropriate culture system (e.g., E. coli or mammalian cells). Such engineering can include, for example, introducing ligase-specific recognition sequences into the termini.

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

[0027] A small molecule compound refers to a molecule of a size comparable to the organic molecules commonly used in pharmaceuticals. The term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low-molecular-weight peptides such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc., or their derivatives. Typically, the molecular weight of the small molecule compound may be, for example, about 100 to about 2,000 Da, about 200 to about 1,000 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.

[0028] An immune agonist is an agonist that can induce or enhance an immune response against a tumor, such as through activation of immune cells, including but not limited to DCs, B cells, macrophages, NK cells, and T cells. Non-limiting examples of immune agonists are known in the art, such as TLR agonists, including but not limited to TLR7 and / or TLR8 and / or TLR9 agonists (e.g., imiquimod, resiquimod, 852A, and VTX-2337), and STING agonists (e.g., ADU-S100 and MK-1454).

[0029] A linking unit refers to a functional group that covalently links two or more moieties in a compound or material. For example, the linking unit can serve to covalently link a targeting molecule and / or an adjuvant moiety of a payload.

[0030] A spacer is a structure located between different structural modules that can spatially separate the structural modules. 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 acids and non-amino acid structures. 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 functions as a spacer, and examples include, but are not limited to, a single amino acid such as Leu or Gln, a sequence containing multiple amino acids, a sequence containing two amino acids such as GA, or sequences such as GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGGS. Other examples of spacers include self-immolative spacers such as PABC (p-benzyloxycarbonyl).

[0031] The term "alkyl group" means a linear or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is connected to the rest of the molecule by a single bond. An alkyl group can contain 1 to 20 carbon atoms, i.e., C1-C 20It may be alkyl, such as a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl group, a C3 alkyl group, a C4 alkyl group, or a C3-C6 alkyl group. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof. A divalent radical refers to a group obtained by removing a hydrogen atom from the carbon atom bearing the free valence electron of the corresponding monovalent radical. A divalent radical has two linking points that connect it 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 group (-CH 12 -), 1-methylethylene group (-CH(CH3)CH2-), 2-methylethylene group (-CH2CH(CH3)-), methylpropylene group, ethylpropylene group, and the like.

[0032] As used herein, when one group is combined with another group, the linkage of the groups may be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination may be linked to the rest of the molecule by any suitable atom within the structure, preferably by a designated chemical bond. For example, C 1-4When describing an alkylene group in combination with one of the groups containing -CH2-, -NH-, -(CO)-, -NH(CO)-, or -(CO)NH-, C 1-4 The alkylene group can form a linear linkage with the above groups, for example, C 1-4 Alkylene-CH2-, C 1-4 Alkylene-NH-, C 1-4 Alkylene-(CO)-, C 1-4 Alkylene-NH(CO)-, C 1-4 Alkylene -(CO)NH-, -CH2-C 1-4 Alkylene, -NH-C 1-4 Alkylene, -(CO)-C 1-4 Alkylene, -NH(CO)-C 1-4 Alkylene, -(CO)NH-C 1-4 An alkylene can be formed, and the resulting divalent structure can be further linked to other parts of the molecule.

[0033] Compound of formula (I') In one embodiment, a compound of formula (I') (formula (I'-1) or formula (I'-2) or a mixture thereof) [ka] During the ceremony, B2 is -(CH2) k1 (CO)-NH-(C2H4-O) j -(CH2) k2 (CO)-Lys-R 3 , -(CH2) k (CO)-NH-(C2H4-O) j -H, or -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 and R 1 Ha-C 1-6 is an alkyl group, R 2 is hydrogen and -C 1-6 alkyl groups, R 3is a group that can be released when reacting with a group in the payload, wherein k, k1, and k2 each independently represent an integer of 1 to 5; j represents an integer of 1 to 3; and d represents 1 or 2.

[0034] In one embodiment, B2 in formulas (I'-1) and (I'-2) are the same.

[0035] In one embodiment, d is 1.

[0036] In one embodiment, the terminal group is hydrogen. 3 is a hydroxy group or [ka] is.

[0037] In one embodiment, the terminal group R 3 represents a structural moiety that does not appear in the product molecule resulting from the reaction of B2 with the payload, so that in the linking unit-payload intermediate (see below), the structural moiety corresponding to B2 is one or a combination of two or more of the above divalent groups.

[0038] Thiosuccinimides are unstable under physiological conditions and tend to reverse the Michael addition, causing cleavage at the conjugation site. Thiosuccinimides can also undergo transthiolation with other thiol compounds if other thiol compounds are present in the system. Both reactions result in payload loss and toxic side effects.

[0039] In the present disclosure, the ring-opened succinimide structure [ka] The product is more stable because it does not undergo retro-Michael addition or thiol exchange. For ring-opening reaction methods, see WO2015165413A1.

[0040] Specific embodiments of compounds of formula (I') In one embodiment, in the compound of formula (I'), B2 is -(CH2) k1 (CO)-NH-(C2H4-O) j -(CH2) k2 (CO)-(Lys-OH), k1 is 5, j is 3, k2 is 1, Lys is connected to the remainder of B2 via its α-amino group, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-5). [ka]

[0041] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-NH-(C2H4-O) j -H, k is 2, j is 1, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-6). [ka]

[0042] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 where k is 2, d is 1, and R 1 is an (S)-methyl group, and R 2 is hydrogen, and the structure of the linking unit is a mixture of the following two (linking unit LN102-9). [ka]

[0043] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2-(CO)) d -R 3 where k is 2, d is 1, and R 1 is an (R)-methyl group, and R 2 is hydrogen, and the structure of the linking unit is a mixture of the following two (linking unit LN102-10). [ka]

[0044] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 where k is 2, d is 2, and R 1 and R 2 is a methyl group, and R 1’ is an (S)-methyl group, and R 2’ is hydrogen, and the structure of the linking unit is a mixture of the following two (linking unit LN102-12). [ka]

[0045] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 where k is 2, d is 2, and R 1 and R 2 is a methyl group, and R 1’ is an (R)-methyl group, and R 2’ is hydrogen, and the structure of the linking unit is a mixture of the following two (linking unit LN102-13). [ka]

[0046] In one embodiment, the compound of formula (I') is one of the compounds shown in FIG.

[0047] Two or more -(CH2) k It should be understood that when a C(O)-group is present, each k value is independently selected. In some embodiments, "k" in a molecule may or may not have an additional number, e.g., k1, k2, k3, etc., where the number does not indicate order but merely serves to distinguish the "k." Other subscripts g, j, d should be understood similarly.

[0048] Two or more R x (x is 1, 2, 3, 4, 5, 6, 7, etc.), each R x It should be understood that "x" is independently selected. In some embodiments, the "x" in a 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 Other R x should be understood in the same way.

[0049] Compound of formula (I') as a linking unit In one embodiment, the reactive group contained in B2 can be used to covalently conjugate a payload containing another reactive group such that the compound of formula (I') bears the payload.

[0050] In another embodiment, the ligase recognition sequence GGG (where G is glycine) contained in formula (I') can be used for ligase-mediated conjugation with the corresponding ligase recognition sequence LPETGG.

[0051] Thus, the compounds of formula (I') can be used as linking units that can be attached to a targeting molecule (eg, an antibody or an antigen-binding fragment thereof) and / or a payload.

[0052] Those skilled in the art can synthesize the linking units by conventional solid or solution phase methods.

[0053] Payload-bearing formula (I') compounds A reactive group contained in B2 is covalently conjugated to a payload containing another reactive group to provide a payload-bearing compound of formula (I').

[0054] In another embodiment, a compound having the structure of formula (II'-1) or (II'-2): [ka] During the ceremony, The PL provides a compound of formula (I') wherein said compound is a payload linked to the B2 moiety of said compound.

[0055] payload In the present disclosure, the payload may be selected from small molecule compounds, nucleic acids and their analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics, 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 cytotoxins and fragments thereof. In a more preferred embodiment, the payload is selected from immune agonists and fragments thereof.

[0056] In one embodiment, the immune agonist is selected from a TLR agonist, such as a TLR agonist (e.g., a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist), and a STING agonist. In one embodiment, the immune agonist is selected from a TLR agonist.

[0057] In one embodiment, the immune agonist is resiquimod. [ka]

[0058] In one embodiment, the linking unit and the payload are connected via the reactive group defined above by any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc.

[0059] 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 below and in FIG.

[0060] [Table 1]

[0061] Preparation of payload-bearing compounds of formula (I') In one embodiment, the linking unit and the payload are connected via the reactive group defined above by any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc.

[0062] Compound of formula (III') In one embodiment, a compound of formula (III') [ka] wherein B2 is as defined in formula (I').

[0063] In one embodiment, the compound of formula (III') can be used to prepare a payload-bearing compound of formula (I') by the following route: [ka]

[0064] The conversion of a payload-bearing compound of formula (III') to a payload-bearing compound of formula (I') can be carried out by any method known in the art or as described herein, for example, by carrying out a single-step or multi-step synthesis to obtain the structural fragment [ka] can be introduced into the maleimide ring in the payload-bearing compound of Formula (III'), and the resulting molecule containing the succinimide moiety can undergo a ring-opening reaction to open the succinimide ring to obtain the payload-bearing compound of Formula (I') (i.e., the compound of Formula (II')). In one embodiment, LU102 is introduced into the payload-bearing compound of Formula (III') by reaction of the maleimide group contained in the compound of Formula (III') with the thiol group of LU102.

[0065] Conjugates and their preparation In another embodiment, a conjugate having the structure of formula (IV'-1) or (IV'-2): [ka] During the ceremony, PL is a payload linked to the B2 moiety of the compound of formula (I'), A is a targeting molecule linked to the D1 or D2 moiety of the compound of formula (I'); wherein z is 1 or 2.

[0066] In one embodiment, the payload is an immune agonist as defined above, hi one embodiment, the conjugate is an antibody-immune agonist conjugate.

[0067] targeting molecule In one embodiment, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0068] In some embodiments of the present disclosure, the target recognized by the targeting molecule (e.g., an antibody or antigen-binding fragment thereof) is 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, SLC44A 4 / AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, nectin4, 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, and FLT3.

[0069] In one embodiment, the targeting molecule is an anti-human HER2 antibody or an antigen-binding fragment thereof. Examples of anti-human HER2 antibodies include, but are not limited to, trastuzumab. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2 and is approved for the treatment of Her2-positive breast cancer and gastric cancer.

[0070] In a preferred embodiment, the anti-human HER2 antibody is one or more selected from trastuzumab-based engineered anti-HER2 antibodies.

[0071] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment, and an antibody mimetic. In one embodiment, the antibody mimetic is selected from an scFv, a minibody, a diabody, and a nanobody. For conjugation with a compound of formula (I'), the targeting molecule of the present disclosure may include a modifying moiety for connecting to the compound of formula (I'). The introduction position of such a modifying moiety is not limited. For example, when the targeting molecule is an antibody, the introduction position may be, but is not limited to, the C-terminus or N-terminus of the heavy or light chain of the antibody.

[0072] In one embodiment, the targeting molecule of the present disclosure is an antibody or antigen-binding fragment thereof and may include a terminal modification. Terminal modification refers to a modification at the C-terminus or N-terminus of the antibody heavy or light chain, for example, including a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp1 containing 2 to 10 amino acids, wherein the antibody, Sp2, and the ligase recognition sequence are sequentially linked. In a specific embodiment, Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGG, particularly GA.

[0073] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof is selected from the group consisting of wild-type (LC), C-terminally modified light chains (LCCT) modified by direct introduction of the ligase recognition sequence LPETGG, and C-terminally modified light chains (LCCT) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPETGG. L The heavy chain of the antibody or antigen-binding fragment thereof includes three types: wild type (HC), C-terminal modified heavy chain (HCCT) modified by direct introduction of the ligase recognition sequence LPETGG, and C-terminal modified heavy chain (HCCT) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPETGG. L As shown in the amino acid sequence table, when z in the compound of formula (IV') is 1 or 2, the combination of the heavy chain and light chain can form eight preferred antibody molecules.

[0074] The conjugates of the present disclosure may further comprise a payload, which is as described above.

[0075] Specific Embodiments of the Conjugates In one embodiment, in formula (IV'), B2 is -(CH2) k1 (CO)-NH-(C2H4-O) j -(CH2) k2 (CO)-(Lys-OH)-, k1 is 5, j is 3, k2 is 1, and Lys is connected to the remainder of B2 via the α-amino group, giving the conjugate structure (Formula AC102-5): [ka]

[0076] In one embodiment, in formula (IV'), B2 is -(CH2) k (CO)-NH-(C2H4-O) j -, k is 2, j is 1, and the structure of the conjugate is as follows (Formula AC102-6): [ka]

[0077] In one embodiment, in formula (IV'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -, k is 2, d is 1, and R 1 is an (S)-methyl group, and R 2 is hydrogen, and the structure of the conjugate is as follows (Formula AC102-9): [ka]

[0078] In one embodiment, in formula (IV'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -, k is 2, d is 1, and R 1 is an (R)-methyl group, and R 2 is hydrogen, and the structure of the conjugate is as follows (Formula AC102-10): [ka]

[0079] In one embodiment, in formula (IV'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -, k is 2, d is 2, and R 1 and R 2 is a methyl group, and R 1’ is an (S)-methyl group, and R 2’ is hydrogen, and the structure of the conjugate is as follows (Formula AC102-12): [ka]

[0080] In one embodiment, in formula (IV'), B2 is -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -, k is 2, d is 2, and R 1 and R 2 is a methyl group, and R 1’ is an (R)-methyl group, and R 2’ is hydrogen, and the structure of the conjugate is as follows (Formula AC102-13): [ka]

[0081] Preparation of conjugates The conjugate of the present disclosure can be prepared by any known method in the art. In some embodiments, the conjugate is prepared by ligase-catalyzed site-specific conjugation of a targeting molecule with a payload-bearing compound of formula (I'), wherein the targeting molecule is modified with a ligase recognition sequence. The method includes steps A and B.

[0082] Step A, Preparation of Linker Unit-Payload Intermediate In a preferred embodiment, B2 in the compound of formula (I') is covalently linked via a reactive group to a payload containing a corresponding reactive group, wherein each of the reactive groups is as defined above.

[0083] Linking unit-payload intermediates prepared using compounds of formula (I') of the present disclosure have well-defined structures, well-defined compositions, and high purity, resulting in fewer or no impurities being introduced during 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 homogeneous and highly controllable quality.

[0084] Step B, Linking a Targeting Molecule to a Payload-Bearing Formula (I') Compound The targeting molecule of the present disclosure can be conjugated to a payload-bearing compound of Formula (I') (i.e., a compound of Formula (II')) by any method known in the art. For example, a ligase-catalyzed site-specific conjugation technique is applied to link the targeting molecule and the payload-bearing compound of Formula (I') to each other via a ligase-specific recognition sequence in the substrate. In one embodiment, the targeting molecule is an antibody having terminal modifications based on the recognition sequence introduced into the C-terminus of the light chain and / or heavy chain, and the targeting molecule is conjugated to a compound of Formula (II') under appropriate catalytic reaction conditions under the catalysis of a wild-type or optimally engineered ligase, or any combination thereof.

[0085] In certain embodiments, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme: [ka]

[0086] The triangle and pentagon represent either a portion of an antibody or a portion of a compound of formula (II'), respectively. n is as defined above. G, the corresponding recognition sequence of the receptor substrate, n When conjugated with , the peptide bond upstream of the glycine in the LPETGG sequence is cleaved by sortase A, and the resulting intermediate is G n The resulting amino acid sequence is LPETG n The array G n and LPETGG is as defined above.

[0087] Table of specific conjugates In one embodiment, the payload is an immune agonist. In one embodiment, the antibody-immune agonist conjugate is as shown in the table below and in FIG.

[0088] [Table 2]

[0089] Pharmaceutical compositions and pharmaceutical preparations Another object of the present disclosure is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a conjugate of the present disclosure and at least one pharmaceutically acceptable carrier.

[0090] The pharmaceutical composition of the present disclosure may be administered in any manner as long as it can achieve the effect of preventing, alleviating, preventing, or curing symptoms in humans or animals. For example, depending on the administration route, various appropriate dosage forms, particularly injections such as lyophilized powder for injection, injection drug, or sterile powder for injection, can be prepared.

[0091] The term "pharmaceutically acceptable" means, within the scope of ordinary medical judgment, that when in contact with the tissues of a patient, it does not produce undue toxicity, irritation, or allergic response, etc., has a reasonable benefit / disadvantage ratio, and is effective for its intended use.

[0092] 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. Pharmaceutically acceptable carriers also include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, toxicity adjusters, and the like, as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0093] In one embodiment, the pharmaceutical composition of the present disclosure has a drug-antibody ratio (DAR) that is an integer or non-integer 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, or 1 to 2.5, and preferably 1 to 2. In one embodiment, the pharmaceutical composition of the present disclosure has a DAR of about 1.4 to about 2, preferably about 1.5 to about 2, and more preferably about 1.55 to about 1.95. In one embodiment, the pharmaceutical composition of the present disclosure has a DAR of about 1.6 to about 1.8.

[0094] Treatment Methods and Uses The conjugates of the present disclosure are useful for treating tumors and / or autoimmune diseases. Tumors amenable to conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors, and those that can be recognized by the targeting molecule in the conjugate and be susceptible to the immune cell activating activity of the agonist in the conjugate.

[0095] Accordingly, in another aspect, there is further provided a use of a conjugate of the present disclosure or a pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0096] In another aspect, there is provided a use of a conjugate of the present disclosure or a pharmaceutical composition of the present disclosure for treating a tumor or an autoimmune disease.

[0097] In a further aspect, there is provided a method of treating a tumor or an autoimmune disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate of the present disclosure or a pharmaceutical composition of the present disclosure.

[0098] In a preferred embodiment, the conjugate of the present disclosure formed by conjugating an anti-human HER2 antibody with a payload specifically binds to HER2 on the surface of tumor cells and can selectively kill HER2-expressing tumor cells. In another preferred embodiment, there is provided use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating a disease, disorder, or symptom selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder, or symptom is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0099] The dose of the conjugate administered to a subject can be varied within a wide range, and may vary depending on the particular route of administration and the needs of the subject, and may be subject to the judgment of a medical professional.

[0100] Beneficial effects The present disclosure utilizes a linking unit with a unique structure and catalyzes the conjugation of an anti-HER2 antibody and an agonist using a ligase. The conjugates of the present disclosure have high homogeneity, high activity, and high selectivity. In particular, they significantly reduce cell proliferation toxicity in cells with low or no expression of the target antigen. Furthermore, the toxicity of the linking unit-agonist intermediate is much lower than that of the free agonist, resulting in fewer adverse effects during the drug manufacturing process and favorable for industrial production.

[0101] The conjugates of the present disclosure achieve at least one of the following technical effects: (1) High inhibitory activity against target cells or a strong 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, adequate half-life and duration of action). (4) High safety (low toxicity to non-target normal cells or tissues, and / or few side effects, wide therapeutic window), etc.

[0102] The drug can prevent patient resistance to HER2-targeted therapy, activate bone marrow cells, and enhance innate and adaptive immune responses, overcoming the low response rate of current HER2-directed therapy.

[0103] Example Preparation example In order to more clearly explain the objectives and technical solutions of the invention, the present 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 present disclosure. Specific experimental methods not described in the following examples are carried out according to conventional experimental methods.

[0104] Apparatus, materials and reagents Unless otherwise stated, the instruments and reagents used in the examples are commercially available. 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: 25 mM PB, 2 M (NH4)2SO4, pH 7.0, mobile phase B: 25 mM PB, pH 7.0, flow rate: 0.8 ml / min, collection time: 25 min, sample injection amount: 20 μg, column temperature: 25°C, detection wavelength: 280 nm, sample chamber temperature: 8°C. SEC-HPLC: Column: TSK-gel G3000 SWXL, TOSOH 7.8 mm ID × 300 mm, 5 μm, Mobile phase: 0.2 M KH2PO4, 0.25 M KCl, pH 6.2, Flow rate: 0.5 ml / min, Collection time: 30 min, Sample injection volume: 50 μl, Column temperature: 25 °C, Detection wavelength: 280 nm, Sample tray temperature: 8 °C. CHO cells were obtained from Thermo Fisher Scientific. pcDNA3.3 was obtained from Life Technology. HEK293F cells were obtained from Prejin. PEIMAX transfection reagent was obtained from Polyscience. MabSelect Sure ProA was obtained from GE. Capto S ImpAct was obtained from GE. Rink Amide MBHA resin and dichloro resin were obtained from Nankai synthesis. HCC1954 cells were obtained as ATCC CAT# CRL-2338. SK-BR-3 cells were 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. MC38hHER2 mouse colorectal carcinoma cells were obtained from Biocytogen. NUGC4 human gastric cancer cells were obtained as JCRB CAT# JCRB0834. NCI-N87 cells (ATCC CAT# CRL-5822) and MDA-MB-468 cells were obtained as ATCC CAT# HTB-132.

[0105] 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 antibody Ab0001-LCCT-HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) is constructed as follows: LThe -HC sequence 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, and a cell population for high expression was constructed and screened. The cell population was cultured in a 5-10 L reactor based on the trastuzumab culture process, and the supernatant was collected.

[0106] 1.2 Antibody Ab0001-LCCT L -HC purification Ab0001-LCCT L Purification of -HC was performed using a standard process using a combination of MabSelect affinity chromatography and Sepharose S cation exchange chromatography, and 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 aliquots.

[0107] 1.3 Antibody Ab0001-LCCT L -HC quality control The purified antibody Ab0001-LCCT L The purity of -HC is 98.5% by SDS-PAGE, the high molecular weight polymer content of the sample is less than 0.4% by SEC-HPLC, and the endotoxin content is less than 0.098 EU / mg.

[0108] 1.4 Preparation of Other Modified Anti-Human Antibodies Using a similar method, terminal modifications based on ligase recognition sequences were introduced into the C-terminus of the light chain and / or heavy chain of trastuzumab, respectively, to obtain modified antibodies.

[0109] Modified anti-human HER2 antibodies based on Ab0001 (trastuzumab) are shown in Table 1. In the terminal modification sequence, LPETGG is a recognition sequence for the ligase donor substrate, and GA is a spacer sequence.

[0110] [Table 3]

[0111] Example 2 Preparation of intermediates 2.1 Preparation of linking units Linking unit where A and Lm are present [ka]

[0112] Linking unit fragment LU102 containing moiety A of formula (I') was synthesized by conventional solid-phase polypeptide synthesis using Rink amide MBHA resin or dichloro resin. Fmoc was used to protect the amino acid and amino groups of the Lk structure in the linking unit. The conjugation reagent was selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was cleaved using trifluoroacetic acid. The product was purified by HPLC, lyophilized, and stored for use. The linking unit fragment is shown in the table below.

[0113] [Table 4]

[0114] The linking unit fragments in the above table were reacted with linking unit fragments containing a maleimide structure or a derivative thereof, followed by a ring-opening reaction using the method described in WO2015165413A1 to obtain linking units LN102-5, LN102-6, LN102-9, LN102-10, LN102-12, and LN102-13, whose structures are as shown above. In the table below, [Table 5]

[0115] 2.2 Preparation of Linker Unit-Agonist Intermediate 2.2.1 Preparation of Linker Unit-Agonist Intermediate LP102-6-1 [ka]

[0116] In step 1, resiquimod (25.0 g, 79.5 mmol) was dissolved in MeCN (500 mL) and treated with Trt-Cl (33.25 g, 119.3 mmol), followed by TEA (20.12 mL, 20.12 mmol). The reaction was refluxed for 2–3 h (TLC). The reaction mixture was concentrated in vacuo. The mixture was then treated with AcOEt (700 mL) and HO (400 mL), stirred for 30 min, and separated. The organic phase was concentrated in vacuo to 300 mL and treated with n-heptane (400 mL). The mixture was then stirred for 20 min. After filtration, the cake was triturated with EtOH / HO (1:1, 200 mL) and filtered. The cake was dried in vacuo to give the target compound HX20031-a (43.9 g, 99.1%) as a white solid.

[0117] In step 2, compound (HX20031-a) (40.02 g, 72.9 mmol) was dissolved in DMF (200 mL) and cooled to 0–10 °C. NaH (60%, 3.74 g, 93.4 mmol) was added in a batchwise manner. The suspension was vigorously stirred at 0–10 °C for 1 h, then warmed to 20–30 °C and stirred for 1 h. The mixture was then cooled to 0–10 °C and treated in one portion with compound 1186g (20.88 g, 93.4 mmol). The mixture was stirred overnight at room temperature and then slowly treated with a mixture of 10% NaH2PO4 (1 L) and AcOEt (1 L). The reaction mixture was stirred for 3 h and filtered. The organic layer was concentrated and purified by silica gel column chromatography (n-heptane → n-heptane / AcOEt = 10:1 → n-heptane / AcOEt = 4:1) to obtain the target compound HX20031-b (24.89 g, 46.9%).

[0118] In step 3, compound (HX20031-b) (10 g, 14.3 mmol) was treated with a mixture of TFA (40 mL) and HO (80 mL). The reaction mixture was stirred at room temperature for 24 h. The mixture was then poured into MTBE (400 mL) and stirred for 2 h. After filtration, the cake was washed with MTBE (200 mL) and filtered. The cake was dried in vacuo to give the target compound HX20031-c (8.51 g, 100%) as a white solid.

[0119] In step 4, compound HX20031-c (6.0 g, 10.2 mmol) was dissolved in DMF (50 mL) and treated with DIPEA (3.5 mL, 20.4 mmol) and N-succinimidyl 3-maleimidopropionate (3.27 g, 12.3 mmol). The reaction was maintained at room temperature for 3 h (HPLC), and then the mixture was used directly in the next step.

[0120] In steps 5-6, the mixture from step 4 was treated with a solution of linking unit LU102 (5.5 g, 15.3 mmol) and HO (50 mL). The mixture was allowed to react for 0.5-20 h at 0-40 °C. The reaction mixture was then mixed with an appropriate amount of Tris base solution or other solution to promote the ring-opening reaction, and the reaction was allowed to proceed for 0.2-20 h at 0-40 °C. After completion of the reaction, the product was purified by semi-preparative / preparative HPLC and lyophilized to give linking unit-agonist LP102-6-1 (3.3 g, 30% over three steps). MS m / z 1065.6 [M+H] + .

[0121] [Table 6]

[0122] Example 3 Preparation of targeting molecule-drug conjugates The linking unit-agonist intermediates were each site-specifically conjugated to the antibody using a ligase to form an AIAC. The conjugation reaction method is described in WO2015165413A1. The resulting AIACs are shown in the table below.

[0123] [Table 7]

[0124] Efficacy Example 1: In vitro evaluation of antibody-immunoagonist conjugates Isolation of human peripheral mononuclear cells Human peripheral mononuclear cells were isolated from healthy blood donors using SepMate50 and Lymphoprep (Stem Cell Technologies). Viable cells were counted and the cell concentration was adjusted to 1.25 x 10 in RPMI 1640 medium containing 10% FBS. 6 Tumor cells were detached with trypsin and collected. The cells were counted and adjusted to a cell concentration of 2.5 x 10 / ml in RPMI 1640 medium containing 10% FBS. 5 / ml. 12.5x10 4 of human PBMCs and 2.5x10 4 Tumor cells (PBMC:tumor cells = 5:1) were added to wells of a 96-well plate, followed by the addition of antibody or conjugate at the indicated concentration. The cell mixture was incubated with the drug for 18 hours, and then the cell-free supernatant was collected for human TNFα ELISA.

[0125] To evaluate the activity of HER2-targeting immunoconjugates, human PBMCs and NCI N87 human gastric cancer cells were cocultured at a 5:1 ratio, and the antibody or test immunoconjugate (AC102-6-1-1 or AC102-5-1-1) was added at the indicated concentrations. AC102-6-1-1 induced higher TNFα production than the antibody Ab0001, and the effective concentration of AC102-6-1-1 was significantly lower than that of the payload, resiquimod. The other immunoconjugate, AC102-5-1-1, did not show significantly different activity compared to Ab0001 (Figure 4). No activity of AC102-6-1-1 was observed in cocultures of human PBMCs and MDA-MB-468 HER2-negative cells, indicating that AC102-6-1-1 activity is highly dependent on HER2 expression on the target tumor cells (Figure 5).

[0126] In a similar experimental setting, the in vitro activity of several other conjugates was evaluated (Figure 6).

[0127] Efficacy Example 2: In vivo evaluation of antibody-immunoagonist conjugates 1x10 for in vivo antitumor efficacy studies 7 NCI N87 human gastric cancer cells were inoculated subcutaneously into the right flank of SCID beige mice. Six days later, tumor volumes averaged 173 mm. 3 When tumor volume reached 100 μg / kg, tumor-bearing mice were randomly assigned to receive 5 mg / kg of Ab0001 or the test immunoconjugate (AC102-5-1-1 or AC102-6-1-1) intravenously. Tumor volume was measured twice weekly with a caliper. The antibody itself, Ab0001, showed very limited antitumor activity. AC102-5-1-1 and AC102-6-1-1 ultimately cured the tumor (Figure 7).

[0128] MC38 hHER2 5x10 overexpressing mouse colorectal cancer cells 5 Human HER2 was subcutaneously inoculated into the right flank of C57BL / 6 mice. After 8 days, the tumor volume was an average of 90 mm 3 When tumor growth reached 100 mg / kg, tumor-bearing mice were divided and intravenously administered Ab0001 or AC102-6-1-1. Ab0001 at 10 mg / kg showed no significant antitumor activity. AC102-6-1-1 at 3 mg / kg and 10 mg / kg dose-dependently inhibited tumor growth (Figure 8).

[0129] [Sequence table] SEQ ID NO: 1: Ab0001-LCCT L -HC light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG SEQ ID NO: 2: Ab0001-LCCT L -HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3: Ab0001-LC-HCCT Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 4: Ab0001-LC-HCCT heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG sequence number 5: Ab0001-LC-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 6: Ab0001-LC-HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG SEQ ID NO: 7: Ab0001-LCCT-HC Light Chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID NO: 8: Ab0001-LCCT-HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 9: Ab0oo1-LCCT-HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG Sequence number 10: Ab0001-LCCT-HCCT heavy chain: It should be noted that there is a small error in the Chinese text where "配列番号" should be "序列番号". The above translation is adjusted accordingly.EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG sequence number 11: Ab0001-LCCT-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG sequence number 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: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG

Claims

1. A compound selected from the following structures: 【Chemistry 1】

2. An antibody-drug conjugate of formula (II-1) or (II-2): 【Chemistry 2】 In the formula, B2 is -(CH 2 ) k (CO)-NH-(C 2 H4-O)j-, where k is an integer from 1 to 5 and j is an integer from 1 to 3; PL is the payload linked to the B2 portion, PL is Resiquimod 【Transformation 3】 and z is an integer from 1 to 4, A is an antibody comprising 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.

3. 3. The antibody drug conjugate of claim 2, wherein k is 2.

4. 4. The antibody drug conjugate of claim 3, wherein j is 1.

5. selected from the following structures: 【Chemistry 4】 z is an integer from 1 to 4, 5. The antibody drug conjugate of claim 2, wherein A is an antibody comprising 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.

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