Pharmaceutical composition of antibody-conjugated drug

Abstract

The invention discloses a pharmaceutical composition of an antibody coupling medicine, and relates to the field of biological pharmacy.

Description

Pharmaceutical compositions of antibody-drug conjugates Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a pharmaceutical composition of an antibody-drug conjugate. Background Art

[0002] HER2 has been found to be overexpressed in several cancer types, including breast and gastric cancer, and has been shown to be a promising target for cancer treatment. A variety of HER2-targeted therapies have been approved, including HER2 tyrosine kinase inhibitors (lapatinib, tucatinib), therapeutic HER2 antibodies (Herceptin, Perjeta), and HER2 antibody-drug conjugates (ADCs) (Kadcyla, Enhertu). These therapeutic drugs have significantly improved the survival rates of patients with HER2-positive breast and gastric cancer. In particular, Enhertu has shown good efficacy not only in patients with high HER2 expression, but also in patients with medium / low HER2 expression, which may benefit more patients with HER2-expressing cancers. Although Enhertu has significant efficacy, it causes interstitial lung disease in more than 10%, which limits its use in some patients.

[0003] Enhertu and other commercially available ADCs and most ADCs in clinical trials are prepared by chemical conjugation, using a sulfosuccinimide structure (sulfosuccinimide linkage) to couple small molecule drugs to targeted antibodies or proteins. The sulfosuccinimide structure is formed by the reaction of a sulfhydryl group and a maleimide. However, the sulfosuccinimide linkage is not stable. In the organism, reverse Michael addition or exchange of sulfhydryl groups with other sulfhydryl groups causes cytotoxins to fall off the ADC, which can lead to off-target toxicity, reduce safety and limit clinical application.

[0004] Summary of the Invention

[0005] To address the aforementioned issues with the prior art, the inventors of the present invention developed a novel ADC. During the research and development process, the inventors further discovered that the stability of this ADC is affected by the formulation. Therefore, the inventors developed a pharmaceutical composition based on this ADC, which offers the advantages of high stability, long-term storage, and protection against toxicity caused by ADC degradation. This composition can be used as a pharmaceutical formulation for this ADC.

[0006] In a first aspect, the present invention provides an antibody-drug conjugate (also referred to herein as an "antibody-drug conjugate," "conjugate," or "ADC") having the structure of Formula (III):

[0007] in,

[0008] Q is hydrogen or L 2 ―L 1 ―B―P;

[0009] M is hydrogen or LKa-L 2 ―L 1 ―B―P;

[0010] Each LKa is independently selected

[0011] opSu is or a mixture thereof;

[0012] Each B is independently absent or a combination of 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene and -(CO)-; preferably, B is -NH-CH2-U- or absent or -NH-CH2-U-(CH2) g -(CO)-; g is any integer from 1 to 6, preferably 1;

[0013] Provided that Q and M are not simultaneously hydrogen;

[0014] P is with part B or L 1 partially connected loads;

[0015] A is with G n Partially linked targeting molecule; G is glycine;

[0016] L 1 A cleavable sequence 1 (Cleavable sequence 1) comprising an amino acid sequence that can be cleaved by an enzyme, wherein the cleavable sequence 1 comprises 1-10 amino acids;

[0017] L 2 is a key; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0018] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10; or substituted with -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10, -(PEG) j -R 11 There is a carbonyl group connected between them;

[0019] -(PEG) i -and-(PEG) j - are PEG fragments containing a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end. 1-10 alkylene;

[0020] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Halogenated alkyl, C 4-10 cycloalkylene;

[0021] R 11 It is C 1-10 alkyl;

[0022] n is any integer from 2 to 20;

[0023] d is 0 or any integer from 1 to 6;

[0024] Each i is independently an integer of 1-100, preferably 1-20; preferably, each i is independently an integer of 1-12; more preferably 2-8; especially 4;

[0025] Each j is independently an integer of 1-100, preferably 1-20; preferably, each j is independently an integer of 1-12; more preferably, an integer of 8-12 or 8-13; especially 8, 9, 12 or 13.

[0026] z is an integer from 1 to 20.

[0027] In a second aspect, the present invention provides a pharmaceutical composition comprising an active ingredient, a buffer, a stabilizer, and optionally a surfactant, wherein the active ingredient comprises the conjugate of formula (III) as described above.

[0028] In some embodiments, the buffer is selected from acetate buffer, histidine buffer, citrate buffer, and phosphate buffer. In some embodiments, the buffer is acetate buffer or histidine buffer. In some embodiments, the buffer is histidine buffer. In some embodiments, the concentration of the buffer is 10-50 mM; preferably, the pH of the buffer is 5.0-6.5, or 5.5-6.4.

[0029] In some embodiments, the stabilizer is selected from sucrose and trehalose, preferably sucrose. In some embodiments, the concentration of the stabilizer is 1-20% (w:v).

[0030] In some embodiments, the pH of the pharmaceutical composition is 5.0-6.5, preferably 5.2-6.0, more preferably about 5.5.

[0031] In some embodiments, the surfactant is polysorbate; preferably, the concentration of the surfactant is 0.01-10 mg / ml.

[0032] In some embodiments, a pharmaceutical formulation comprising an active ingredient of formula (III) comprises:

[0033] 20 mM acetate buffer, pH 5.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or

[0034] 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 20 mg / ml active ingredient; or

[0035] 20 mM histidine buffer, pH 6.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or

[0036] 20 mM citrate buffer, pH 6.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or

[0037] 20 mM phosphate buffer, pH 7.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or

[0038] 20 mM histidine buffer at pH 5.5, 8.1% (w:v) trehalose (or 9% trehalose dihydrate), 0.2 mg / ml polysorbate 20, 20 mg / ml active ingredient; or

[0039] 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 0.2 mg / ml polysorbate 20, 20 mg / ml active ingredient; or

[0040] 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 0.2 mg / ml polysorbate 80, 20 mg / ml active ingredient;

[0041] The pharmaceutical preparation is a powder preparation or a liquid preparation.

[0042] In a third aspect, the present invention provides use of the pharmaceutical composition of the present invention in preparing a medicament for treating a disease; wherein the disease is a tumor or an autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 shows the efficacy of the conjugates and corresponding cargoes in the SK-BR-3 HER2-high expressing cell line.

[0044] FIG2 shows the efficacy of the conjugate and the corresponding cargo in the NCI-N87 HER2-high expressing cell line.

[0045] Figure 3 shows the efficacy of the conjugate in the CFPAC-1 HER2-low expressing cell line.

[0046] Figure 4 shows the efficacy of the conjugate in the NCI-H2110 HER2-low expressing cell line.

[0047] Figure 5 shows the efficacy of the conjugate in the MDA-MB-468 HER2 negative cell line.

[0048] Figure 6 shows the mean tumor volume changes over time in SCID Beige mice with the JIMT1 CDX model administered 5 mg / kg of LC1184(8), LC1184(4), LC301-2-1(2), LC302-2-1(4), LC302-2-4(4).

[0049] Figure 7 shows the mean tumor volume changes over time in BALB / c nude mice with the Capan-1 CDX model, which were administered 5 mg / kg LC1184(8), LC1184(4), LC301-2-1(2), LC302-2-1(4), LC302-2-4(4).

[0050] Figure 8 shows the ex vivo serum stability of the conjugates after incubation in pooled human serum for 0, 24, 48 and 96 hours. DETAILED DESCRIPTION

[0051] The following specific embodiments are provided to illustrate the technical content of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention through the contents disclosed in the specification. The present invention can also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present invention.

[0052] definition

[0053] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The technology used herein refers to the technology generally understood in the art, including variations and equivalent substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are readily understood by those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding commercial product or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0054] When a certain amount, concentration or other numerical value or parameter is set forth in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it should be understood to be equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is clearly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include all integers and fractions (decimals) within the endpoints and ranges of the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar expressions such as j, k and g should also be understood in a similar manner.

[0055] As used herein, "%" in relation to a component refers to weight-to-volume (w / v) percentages, where weight may be expressed in grams and volume may be expressed in milliliters. For example, a solution containing 1% stabilizer means that 1 gram of stabilizer is present in 100 milliliters of the solution, or that the stabilizer content is 0.01 g / ml.

[0056] The amount of a buffer in the present invention refers to the total amount of the buffer pair in the buffer system comprising the buffer. In some embodiments, molar concentration is used as the unit of the amount of a buffer (or buffer), and the numerical value refers to the molar concentration of the buffer pair in the buffer system of the buffer (or buffer). For example, when a histidine buffer composed of histidine and histidine hydrochloride is used as the buffer, a given concentration of histidine salt buffer (e.g., 20 mM) is the combined concentration of histidine and histidine hydrochloride (e.g., 15 mM histidine and 5 mM histidine hydrochloride; or 3.6 mM histidine and 16.4 mM histidine hydrochloride).

[0057] In a solid state, trehalose is typically present as trehalose dihydrate. Therefore, in some embodiments, when a formulation is prepared using trehalose dihydrate, a corresponding amount of trehalose in another form can also be used to prepare the formulation, and the resulting formulation contains the same concentration of trehalose. When trehalose dihydrate is used herein to describe the trehalose content in a formulation, the formulation can contain the stated amount of trehalose dihydrate, a corresponding amount of trehalose or another form of trehalose, or a combination thereof, and vice versa.

[0058] Unless the context clearly dictates otherwise, singular forms such as "a", "an", and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0059] The terms "about" and "approximately" when used with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% or more of the stated value.

[0060] The term "stoichiometric ratio" refers to the mixing of various substances in a certain weight ratio. For example, in the present invention, the active ingredient is mixed with the filler, binder and lubricant in a specified weight ratio.

[0061] The term "optional" or "optionally" means that the subsequently described event may occur but does not necessarily occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0062] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0063] The term "targeting molecule" refers to a molecule that has an affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). Targeting molecules can deliver a payload to a specific site in the body through targeted delivery. Targeting molecules can recognize one or more targets. A specific target is defined by the target it recognizes. For example, a receptor-targeting targeting molecule can deliver a cytotoxin to a site containing a large number of receptors. 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.

[0064] As used herein, the term "antibody" is used in a broad manner and particularly includes complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody can be of any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and can be derived from any suitable species. In some embodiments, the antibody is human or mouse. The antibody can also be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by a recombinant method.

[0065] As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. A monoclonal antibody is highly specific for a single antigenic site or for different epitopes or multiple antigenic sites on the same antigen. The term "monoclonal" refers to the characteristic of an antibody as being derived from a substantially homogeneous antibody population and should not be construed as requiring some specific method for producing the antibody.

[0066] Intact antibodies or full-length antibodies essentially comprise an antigen-binding variable region and a light chain constant region (CL) and a heavy chain constant region (CH), which may include CH1, CH2, CH3, and CH4, depending on the subtype of the antibody. The antigen-binding variable region (also referred to as a fragment variable region, an Fv fragment) typically comprises a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region with a native sequence (e.g., a constant region with a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized by the immune system and interacts with it.

[0067] Antibody fragments may comprise a portion of an intact antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting a full-length immunoglobulin with papain, or fragments with the same structure produced, for example, by recombinant expression. Fab fragments comprise a light chain (comprising VL and CL) and another chain, wherein the other chain comprises the variable region (VH) of the heavy chain and one constant region (CH1) of the heavy chain. F(ab')2 fragments are antibody fragments obtained by digesting an immunoglobulin with pepsin at pH 4.0-4.5, or fragments with the same structure produced, for example, by recombinant expression. F(ab')2 fragments essentially comprise two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including cysteine ​​that forms a disulfide bond connecting the two fragments. Fab' fragments are fragments comprising half of a F(ab')2 fragment (a heavy chain and a light chain). The antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically comprise at least or about 50 amino acids, typically at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), can obtain an antibody that immunospecifically binds to an antigen.

[0068] The antibodies of the present invention can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology, phage display technology, synthetic technology, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed in a suitable culture system (e.g., Escherichia coli (E. Coli) or mammalian cells). The engineering can refer, for example, to the introduction of a ligase-specific recognition sequence at its terminus.

[0069] HER2 refers to human epidermal growth factor receptor-2, which 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.

[0070] As used herein, the term "antibody-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0071] Small molecule compounds refer to molecules of a size comparable to organic molecules commonly used in pharmaceuticals. The term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but does encompass low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, small molecule compounds have a molecular weight of, 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.

[0072] Cytotoxins refer to substances that inhibit or prevent the expression activity, cell function, and / or cause cell damage in cells. Cytotoxins commonly used in ADCs are more toxic than chemotherapy drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and regulation of apoptosis. Drugs targeting the microtubule cytoskeleton can, for example, be microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins. Drugs targeting DNA can, for example, be drugs that directly damage DNA structure or topoisomerase inhibitors. Examples of drugs that directly damage DNA structure include, but are not limited to, DNA double strand breakers, DNA alkylating agents, and DNA intercalators. DNA double strand breakers can, for example, be enediyne antibiotics, including, but not limited to, daneimycin, esperamicin, neocarzinostatin, and uncialamycin. DNA alkylating agents can be, for example, DNA bis-alkylators (i.e., DNA cross linkers) or DNA mono-alkylators. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepines. (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), the structures of which are described below), camptothecins, and anthracyclines. Drugs targeting RNA may, for example, be drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport may, for example, be mitotic kinesin inhibitors, including, but not limited to, kinesin spindle (KSP) inhibitors.

[0073] "Spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function, nor does it limit whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-immolative spacer is a covalent component that causes two chemical bonds to be cleaved sequentially after activation of a protective moiety in a precursor: the protective moiety (e.g., a cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order. Examples of self-immolative spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0074] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups can have 1 to 20 carbon atoms and refer to "C1-C 20"alkyl", such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. 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 free radical refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of the corresponding monovalent free radical. A divalent free radical has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon group. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc.

[0075] As used herein, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be connected to other parts of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more groups selected from -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 The heterocyclic group and the divalent group of -(CO)- are combined to form a combination, and two or more divalent groups can form a linear connection with each other, such as -CR 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-C 1-10 Alkylene-(CO)-, -CR 1 R2 -CR 1 R 2’ -(CO)-、-CR 1 R 2 -CR 1' R 2' -CR 1” R 2” -(CO)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0076] As used herein, the expressions "antibody-drug conjugate" and "antibody-drug conjugate" have the same meaning.

[0077] Compounds of formula (I)

[0078] In a first aspect, the present invention provides a compound of formula (I):

[0079] in,

[0080] W is hydrogen or LKb;

[0081] Y is hydrogen or LKa-LKb;

[0082] Provided that W and Y are not both hydrogen;

[0083] Each LKa is independently selected

[0084] opSu is or a mixture thereof;

[0085] Each LKb is independently selected from L 2 ―L 1 ―B;

[0086] Each B is independently a terminal group R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0087] R 10 It is hydrogen or a group that can leave when reacting with a group in the load;

[0088] L 1A cleavable sequence 1 comprising an amino acid sequence cleavable by an enzyme, wherein the cleavable sequence 1 comprises 1-10 amino acids;

[0089] L 2 is a key; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0090] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10; or substituted with -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10, -(PEG) j -R 11 There is a carbonyl group connected between them;

[0091] -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene;

[0092] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R8 、R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Halogenated alkyl, C 4-10 cycloalkylene;

[0093] R 11 It is C 1-10 alkyl;

[0094] n is any integer from 2 to 20;

[0095] d is 0 or any integer from 1 to 6;

[0096] Each i is independently an integer of 1-100, preferably 1-20; preferably, each i is independently an integer of 1-12; more preferably 2-8; especially 4;

[0097] Each j is independently an integer of 1-100, preferably 1-20; preferably, each j is independently an integer of 1-12; more preferably 8-12 or 13; especially 8, 9, 12 or 13.

[0098] In one embodiment, L 2 Selected from: -(CH2) p -(CH2)2(CO)-, p is 0 or an integer from 1 to 5; b is an integer from 1 to 10.

[0099] In one embodiment, the above L 2 The carbonyl group and L in the structure 1 The other linker site is connected to opSu.

[0100] In one embodiment, p is 0-3; preferably 3.

[0101] Ld2 and each Ld1 are independently selected from a bond, or

[0102] Each i, j and k is independently selected from an integer from 1-100.

[0103] In one embodiment, each i, j, and k is independently selected from an integer from 1 to 20. In one embodiment, each i, j, and k is independently selected from an integer from 1 to 12.

[0104] In one embodiment, each i is independently selected from an integer from 2 to 8; particularly 4.

[0105] In one embodiment, each j is independently selected from an integer of 8-12 or 8-13; particularly 8, 9, 12 or 13.

[0106] In one embodiment, each k is independently selected from an integer of 1-7; in particular 1 or 3 or 5.

[0107] In one embodiment, Ld2 and each Ld1 are independently selected from a bond; or a C with an amino group and a carbonyl group at both ends. 1-20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (represented as -(PEG) i -), or one or more natural amino acids, each of which is independently unsubstituted or substituted with a PEG fragment of a certain length (denoted as -(PEG) j -)replace.

[0108] In one embodiment, -(PEG) i -Contains -(O-C2H4) i -or-(C2H4-O) i -, and optionally appended with C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O) j -, and optionally appended with C at one end 1-10 In a very specific embodiment, -(PEG) i - contains -C2H4-(O-C2H4) i -or-(C2H4-O) i -C2H4-.

[0109] It should be understood that when there are two or more Ld1, B, L 2 or L 1 When constructing, select each Ld1, B, L independently. 2 or L 1 When there are two or more R x (x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.), each R x In some embodiments, "x" in the molecule is represented with or without an additional single prime (') or multiple primes (e.g., ", "', "", etc.), e.g., R, R 1’ 、R 1” 、R 1”’ 、R 2’ 、R 2” 、R 2”’ etc., where each R with or without an additional single or multiple apostrophes x , are independently selected. Other R x , such as R 3 、R4 、R 5 、R 6 、R 7 、R 8 、R 9 and "Ld1", "B", "L 2 ” and “L 1 ", should be understood in a similar manner. In some embodiments, "i" in the molecule is represented with or without an additional number, such as i1, i2, i3, i4, etc., wherein the number does not represent any order but is only used to distinguish "i". And each "i" with or without an additional number is independently selected.

[0110] In one embodiment, the cleavable sequence 1 is selected from GLy-GLy-Phe-GLy, Phe-Lys, Val-Cit, Val-Lys, GLy-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Ala-Ala-Ala and combinations thereof; the preferred cleavable sequence 1 is GLy-GLy-Phe-Gly.

[0111] In one embodiment, W is hydrogen.

[0112] In one embodiment, R 11 It is C 1-6 Alkyl, preferably methyl.

[0113] In one embodiment, n is an integer from 2 to 5, in particular 3.

[0114] In one embodiment, d is 0, or any integer from 1 to 4; preferably 0, 1, 2 or 3.

[0115] Sulfosuccinimide is unstable under physiological conditions and is prone to reverse Michael addition, resulting in cleavage at the conjugation site. In addition, when another sulfhydryl compound is present in the system, sulfosuccinimide can also undergo sulfhydryl exchange with another sulfhydryl compound. Both reactions will cause the load to fall off and produce toxic side effects. In the present invention, when applied to the linker, the ring-opened succinimide structure no longer undergoes reverse Michael addition or sulfhydryl exchange, so the product is more stable. The method of the ring-opening reaction can be found in WO2015165413A1.

[0116] Compounds containing a ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain high purity and defined components, regardless of the efficiency of the succinimide ring-opening reaction.

[0117] The portion containing the recognition sequence for the ligase acceptor or donor substrate

[0118] In one embodiment, G of the compound of formula (I)n The moiety is a recognition sequence for a ligase acceptor substrate, which facilitates the enzymatic coupling of the compound of formula (I) with the targeting molecule under ligase catalysis. The targeting molecule is optionally modified and comprises a corresponding recognition sequence for a ligase donor substrate.

[0119] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of: natural transpeptidase, non-natural transpeptidase, its variant and combination thereof. Non-natural transpeptidase can be but is not limited to those obtained by engineering natural transpeptidase. In a preferred embodiment, the ligase is selected from the group consisting of: natural Sortase enzyme, non-natural Sortase enzyme and combination thereof. The type of natural Sortase enzyme includes Sortase A, Sortase B, Sortase C, Sortase D, Sortase L.plantarum etc. (US20110321183A1). The type of ligase corresponds to the ligase recognition sequence, thereby being used to realize the specific conjugation between different molecules or structural fragments.

[0120] In some embodiments, the ligase is a Sortase enzyme selected from the group consisting of Sortase A, Sortase B, Sortase C, Sortase D, and Sortase L. plantarum. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligoglycine, oligoalanine, and an oligoglycine / alanine mixture with a degree of polymerization of 3-10. In a particular embodiment, the recognition sequence of the ligase acceptor substrate is G n , wherein G is glycine (Gly) and n is an integer of 2-10.

[0121] In another specific embodiment, the ligase is Sortase A from Staphylococcus aureus. Therefore, the ligase recognition sequence can be the typical recognition sequence LPXTG of the enzyme. In another specific embodiment, the ligase donor substrate recognition sequence is LPXTGJ and the ligase acceptor substrate recognition sequence is G n , wherein X can be any natural or non-natural single amino acid; J is absent or is an amino acid fragment comprising 1-10 amino acids, which may optionally be labeled. In one embodiment, J is absent. In another embodiment, J is an amino acid fragment comprising 1-10 amino acids, wherein each amino acid is independently any natural or non-natural amino acid. In another embodiment, J is G m, wherein m is an integer from 1 to 10. In another specific embodiment, the ligase donor substrate recognition sequence is LPETG. In another specific embodiment, the ligase donor substrate recognition sequence is LPETGG.

[0122] In one embodiment, the ligase is Sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN. In another embodiment, the ligase is Sortase B from Bacilus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG.

[0123] In another embodiment, the ligase is Sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence can be LPXTGJ, wherein J is as defined above. In another embodiment, the ligase is Sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence can be LAXTG; wherein X can be any natural or unnatural single amino acid.

[0124] In another embodiment, the ligase is Sortase A from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ.

[0125] The ligase recognition sequence may also be other new transpeptidase recognition sequences that are artificially screened and optimized.

[0126] Contains active group part

[0127] Active groups for connecting with loads

[0128] In one embodiment, B is a terminal group R 10 , and L 1In the cleavable sequence 1 is connected to the load. In this case, B is not present in the resulting molecule that the cleavable sequence 1 is connected to the load. In one embodiment, B is used to be connected to the load. In order to be connected to the load, the compound of formula (I) comprises an active group. In one embodiment, B in the compound of formula (I) is connected to the load via an amide bond, an ester bond, or an ether bond. In one embodiment, the active group in the B of formula (I) is independently an active group (such as an active C=O part, an active C=CC=O part, an amino group, an amine group, a hydroxyl group, or a sulfhydryl group) for condensation reaction, nucleophilic addition, or electrophilic addition, or an active group (such as a leaving group connected to an O, C, N, or S atom) for substitution reaction. In one embodiment, the active group in B is independently selected from carboxyl, active ester, aldehyde, amino, amine group, hydroxyl group, and sulfhydryl group. In a specific embodiment, the active group for connecting the load in B is independently selected from amino, amine group, hydroxyl group, sulfhydryl group, carboxyl group, and active ester.

[0129] In one embodiment, the reactive groups in B are independently amino, amine, or hydroxyl groups, which react with corresponding groups in the support (e.g., carboxyl, sulfonic acid, phosphoryl with a free -OH terminal end, active ester, acid chloride, or isocyanate). In another embodiment, the reactive groups in B are independently carboxyl or active ester groups, which react with corresponding groups in the support (e.g., amino, amine, or hydroxyl groups).

[0130] In one embodiment, the reactive groups in B are independently amino, hydroxyl, or thiol, which react with corresponding groups in the support (e.g., halogen, hydroxyl, aldehyde). In another embodiment, the reactive groups in B are independently hydroxyl, which react with corresponding groups in the support (e.g., halogen or hydroxyl).

[0131] In one embodiment, each B is independently R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene and -(CO)-; and 3) terminal group R 10 .

[0132] In one embodiment, Sp1 is selected from PABC, acetal, heteroacetal, and combinations thereof. In one embodiment, Sp1 is acetal, heteroacetal, or PABC. In one embodiment, heteroacetal is selected from N,O-heteroacetal. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is attached to cleavable sequence 1. U is O, S, or NH, preferably O or S. In one embodiment, U is O, S, or N, preferably O or S.

[0133] In one embodiment, B is R 10 Or -NH-CH2-UR 10 Or -NH-CH2-U-(CH2) g -(CO)-R 10 .

[0134] In one embodiment, R 10 is hydrogen, hydroxyl; or In one embodiment, R 10 In one embodiment, R 10 is hydroxyl or

[0135] In one embodiment, R 10 represents a structural portion that will not appear in the product molecule produced by the reaction of B with the load.

[0136] Specific embodiments of the compound of formula (I)

[0137] In one embodiment, W is hydrogen, each Lka is In one embodiment, the compound of formula (I) has the structure of formula (I-1)

[0138] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (I-1) is as follows:

[0139] In one embodiment, d is 0, Ld2 is In one embodiment, the compound of formula (I-1) is as follows:

[0140] In one embodiment, d is 1, 2 or 3, Ld2 and each Ld1 are independently selected from In one embodiment, the compound of formula (I-1) is as follows:

[0141] In one embodiment, Ld2 is d is 0. In one embodiment, the compound of formula (I-1) is as follows:

[0142] In one embodiment, d is 1, 2 or 3, and Ld2 is and each Ld1 is independently selected from In one embodiment, the compound of formula (I-1) is as follows:

[0143] In one embodiment, n is 3, L 2 Yes - (CH2) p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CH2) g -(CO)-R 10 , U is O, and g is 1. In one embodiment, the linker LA301-1 has the following structure:

[0144] In one embodiment, linker LA301-2 has the following structure:

[0145] In one embodiment, linker LA301-3 has the following structure:

[0146] In one embodiment, linker LA301-4 has the following structure:

[0147] In one embodiment, linker LA301-5 has the following structure:

[0148] In one embodiment, linker LA302-1 has the following structure:

[0149] In one embodiment, linker LA302-2 has the following structure:

[0150] In one embodiment, linker LA302-3 has the following structure:

[0151] In one embodiment, linker LA302-4 has the following structure:

[0152] In one embodiment, i is 4, g is 1, R 11 It's methyl.

[0153] In one embodiment, the linker LA301-2 is as follows (linkers LA301-2-1 to LA301-2-3):

[0154] In one embodiment, i is 4, j is 8, g is 1, R 11 In one embodiment, the linker LA302-2 is as follows (linkers LA302-2-1 to LA302-2-3):

[0155] In one embodiment, i is 4, j is 12, g is 1, R 11 In one embodiment, the linker LA302-2 is as follows (linkers LA302-2-4 to LA302-2-6):

[0156] Among them, opSu is or a mixture thereof.

[0157] Compound of formula (I) with a load

[0158] The active group contained in B is covalently conjugated with a load containing another active group to obtain a compound of formula (I) carrying the load.

[0159] In another aspect, the present invention provides a compound having the structure of formula (II)

[0160] in

[0161] Q is hydrogen or LKb-P;

[0162] M is hydrogen or LKa-LKb―P;

[0163] Provided that Q and M are not simultaneously hydrogen;

[0164] P is the same as the B part or L of the compound of formula (I) 1 partially connected loads;

[0165] n, d, Ld1, Ld2, LKa and LKb are as defined in formula (I).

[0166] As defined above, each LKb in the compound of formula (I) is independently selected from L 2 ―L 1 ―B; each B is independently a terminal group R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen or a group that can leave when reacting with a group in the support. In one embodiment, R 10 represents a structural portion that will not appear in the product molecule produced by the reaction of B with the load.

[0167] In one embodiment, P is linked to the B portion of the compound of formula (I) to form the compound of formula (II). As described above, R does not appear in the B-P structure of the compound of formula (II). 10 .

[0168] It should be understood that when B in the compound of formula (I) is a terminal group R 10 When R 10 Therefore, B in the B-P structure of the compound of formula (II) does not exist.

[0169] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein P is the B part or L of the compound of formula (I) 1 Partially attached payload; each B is independently a terminal group R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen or a group that can leave when reacting with a group in the load; R 10 represents a structural portion that will not appear in the product molecule produced by the reaction of B with the load.

[0170] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent or a combination of the following 1) and 2): 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 In a preferred embodiment, M is hydrogen or LKa-L 2 ―L1 ―B―P; wherein each B is independently absent, or is -NH-CH2-U-, or is -NH-CH2-U-(CH2) g -(CO)-; g is any integer from 1 to 6, preferably 1. In another embodiment, M is LKa-L 2 ―L 1 ―B―P. In one embodiment, LKa-L 2 ―L 1 In one embodiment, LKa-L 2 ―L 1 B in —B—P is a combination of 1) and 2) as follows: 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: —CR 1 R 2 -、C 1-10 Alkylene and -(CO)-. In one embodiment, LKa-L 2 ―L 1 B in ―B―P is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-; g is any integer from 1 to 6, preferably 1. U is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is connected to the support through an amide bond, an ester bond or an ether bond.

[0171] As mentioned above, when B in the compound of formula (I) is a terminal group R 10 , B in the B-P structure of the compound of formula (II) does not exist accordingly. 1 The cleavable sequence 1 in the formula (II) is connected to the cargo to form a compound of formula (II), wherein B is not present in the molecule obtained by connecting the cleavable sequence 1 to the cargo. Therefore, in one embodiment, P is the same as L of the compound of formula (I). 1 Thus, in one embodiment, M is LKa-L 2 ―L 1 ―B―P and B does not exist, M can also be expressed as LKa-L 2 ―L 1 ―P.

[0172] load

[0173] In the present invention, the cargo can be selected from the group consisting of small molecule compounds, nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics, and proteins. In one embodiment, the cargo is selected from the group consisting of small molecule compounds, nucleic acid molecules, and tracer molecules. In a preferred embodiment, the cargo is selected from the group consisting of small molecule compounds. In a more preferred embodiment, the cargo is selected from the group consisting of cytotoxins and fragments thereof.

[0174] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10, and vinblastine. 10) and its analogs, halichondrin B, eribulin, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors such as camptothecins and their derivatives, mitoxantrone, and mitoguanidine. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitroglycerin hydrochloride, melphalan, mechlorethamine, methambucil, phenylephrine, prednimustine, trofosfamide, and uracil. In another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ranimustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines. In a preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carboquinone, metodepa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, aclarubicin.In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, carminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zoloft, and daunorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, baculozolin A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, and 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of dimethylformamide, methotrexate, pteropterin, trimetrexate, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamidine, and thioguanine. In another embodiment, the cytotoxin is selected from the group consisting of pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenal agents. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In another embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, and pyrrolobenzodiazepine (PBD). In a specific embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, and maytansinoids.

[0175] In a particular embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f.

[0176] In a particular embodiment, the cytotoxin is a maytansinoid, such as DM1. It should be noted that when a cytotoxin comprising a sulfhydryl moiety is used, the sulfhydryl moiety can react with a maleimide moiety to form a sulfosuccinimide, such as a maytansinoid, such as DM1, and the cytotoxin can be directly linked via the sulfosuccinimide. In this case, it will be understood that in some embodiments, the cargo and the sulfhydryl moiety together constitute the cytotoxin, and therefore in this case, the cargo represents the remainder of the cytotoxin molecule except the sulfhydryl moiety.

[0177] In a particular embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0178] The carrier contains an active group that can react with the active group in the compound of formula (I) and thus covalently conjugate the carrier to the compound of formula (I). Compounds without active groups require appropriate derivatization to obtain the carrier.

[0179] In a particular embodiment, the cytotoxin is a compound of formula (i)

[0180] Where g is any integer from 1 to 6;

[0181] In one embodiment, g is any integer from 1 to 3, preferably 1.

[0182] In one embodiment, the cytotoxin is selected from the following compounds 1-14; wherein the wavy bond represents the site of attachment to the compound of formula (I).

[0183] In some embodiments, the loading material is selected from DX8951f (Compound 9), DXd-(1) (Compound 10) and DXd-(2) (Compound 14), preferably DX8951f or DXd-(1), more preferably DXd-(1).

[0184] Preparation of compounds of formula (I) with loads

[0185] In one embodiment, the linker unit and the load are linked via the reactive group as defined above using any reaction known in the art, including but not limited to condensation reaction, nucleophilic addition, electrophilic addition, and the like.

[0186] In one embodiment, the cargo is a cytotoxin. In one embodiment, the linker unit-cargo intermediate (numbered LBx) is shown in the following table. *: For all listed linkers, n is 3.

[0187] Conjugates and their preparation

[0188] In addition, the compound of formula (I) with a load having a portion comprising a ligase recognition sequence can be conjugated to other molecules comprising a ligase recognition sequence, thereby being used, for example, in the preparation of antibody-drug conjugates, such as antibody-drug conjugates. Therefore, in another aspect, the present invention provides a conjugate comprising a compound of formula (I), a targeting molecule and a load.

[0189] In another aspect, the present invention provides a conjugate having the structure of formula (III):

[0190] in,

[0191] n, d, Ld1 and Ld2 are as defined in formula (I);

[0192] Q is hydrogen or LKb-P;

[0193] M is hydrogen or LKa-LKb―P;

[0194] Provided that Q and M are not simultaneously hydrogen;

[0195] P is the same as the B part or L of the compound of formula (I) 1 partially connected loads;

[0196] A is G of the compound of formula (I) n Partially linked targeting molecule; G is glycine;

[0197] z is an integer from 1 to 20.

[0198] In one embodiment, LKa and LKb are as defined in formula (I).

[0199] As defined above, in one embodiment, G of the compound of formula (I) n The moiety is a recognition sequence for a ligase acceptor or donor substrate, which promotes the enzymatic coupling of the compound of formula (I) with the targeting molecule under the catalysis of the ligase. The targeting molecule is optionally modified and comprises a corresponding recognition sequence for a ligase acceptor or donor substrate.

[0200] It should be understood that the targeting molecule A and the G of the compound of formula (I) n When the moieties are conjugated under the catalysis of the ligase, the ligase acceptor substrate recognition sequence and the ligase donor substrate recognition sequence react to obtain the corresponding product sequence.

[0201] In one embodiment, the targeting molecule A comprises a ligase donor substrate recognition sequence LPXTGJ, wherein X and J are as defined above. n During conjugation, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by SortaseA, and the resulting intermediate is linked to G n A new peptide bond is generated at the free N-terminus. The resulting amino acid sequence is LPXTG n . Sequence G n and LPXTGJ are as defined above.

[0202] In one embodiment, P is in the presence of moiety B or L of a compound of formula (I). 1 Partial connection, A and G of the compound of formula (I) n The moieties are connected to form a compound of formula (III).

[0203] As mentioned above, R does not appear in the B-P structure of the compound of formula (III). 10 As mentioned above, when B in the compound of formula (I) is a terminal group R 10 , B in the B-P structure of the compound of formula (III) does not exist accordingly.

[0204] As mentioned above, A-G of the compound of formula (III) n In the structure, A optionally comprises a ligase acceptor substrate recognition sequence and a ligase donor substrate recognition sequence to react and obtain a corresponding product sequence.

[0205] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein P is the B part or L of the compound of formula (I)1 Partially attached payload; each B is independently a terminal group R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen or a group that can leave when reacting with a group in the load; R 10 represents a structural portion that will not appear in the product molecule produced by the reaction of B with the load.

[0206] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent or a combination of the following 1) and 2): 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 In a preferred embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or is -NH-CH2-U-, or is -NH-CH2-U-(CH2) g -(CO)-; g is any integer from 1 to 6, preferably 1. In another embodiment, M is LKa-L 2 ―L 1 ―B―P. In one embodiment, LKa-L 2 ―L 1 In one embodiment, LKa-L 2 ―L 1 B in —B—P is a combination of 1) and 2) as follows: 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: —CR 1 R 2 -、C 1-10 Alkylene and -(CO)-. In one embodiment, LKa-L 2 ―L 1 B in ―B―P is -NH-CH2-U- or -NH-CH2-U-(CH2)g -(CO)-. U is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is connected to the load via an amide bond, an ester bond or an ether bond. In one embodiment, M is LKa-L 2 ―L 1 ―B―P and B does not exist, M can also be expressed as LKa-L 2 ―L 1 ―P.

[0207] Targeting molecules

[0208] In one embodiment, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0209] 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.

[0210] In a preferred embodiment, the anti-human HER2 antibody is one or more engineered anti-HER2 antibodies selected from trastuzumab-based anti-HER2 antibodies.

[0211] 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 a scFv, a mini antibody, a bispecific antibody, and a nanobody. For conjugation with a compound of formula (I), the targeting molecule of the present invention may comprise a modified portion to connect the G in the compound of formula (I). n The position of introduction of such modified moieties is not limited. For example, when the targeting molecule is an antibody, the position of introduction thereof can be, but is not limited to, the C-terminus or N-terminus of the heavy chain or light chain of the antibody.

[0212] In an alternative embodiment, for use with G in the compound of formula (I) n The conjugated modifying moiety can be introduced at a non-terminal position of the heavy or light chain of the antibody using, for example, chemical modification methods.

[0213] In one embodiment, the targeting molecule of the present invention is an antibody or an antigen-binding fragment thereof, 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, which, for example, include a ligase recognition sequence. In another embodiment, the terminal modifications may further include a spacer Sp2 of 2-100 amino acids, wherein the antibody, Sp2, and ligase recognition sequence are sequentially connected. In a preferred embodiment, Sp2 is a spacer sequence comprising 2-20 amino acids. In a particular embodiment, Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, particularly GA.

[0214] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof includes three types: wild type (LC); C-terminal modified light chain (LCCT) modified by directly introducing the ligase recognition sequence LPXTG; and C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer and a 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-terminal modified heavy chain (HCCT) modified by directly introducing the ligase recognition sequence LPXTG; and C-terminal modified heavy chain (HCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. L ). X can be any natural or non-natural single amino acid. When z in the compound of formula (III) is 1 or 2, the above heavy and light chains can be combined to form 8 preferred antibody molecules, as shown in the amino acid sequence table.

[0215] The conjugate of the present invention may further comprise a loading substance, which is as described above.

[0216] Specific embodiments of the conjugate

[0217] In one embodiment, Q is hydrogen, each Lka is In one embodiment, the compound of formula (III) has the structure of formula (III-1):

[0218] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (III-1) is as follows:

[0219] In one embodiment, d is 0, Ld2 is In one embodiment, the compound of formula (III-1) is as follows:

[0220] In one embodiment, d is 1, 2 or 3, Ld2 and each Ld1 are independently selected from In one embodiment, the compound of formula (III-1) is as follows:

[0221] In one embodiment, Ld2 is d is 0. In one embodiment, the compound of formula (III-1) is as follows:

[0222] In one embodiment, d is 1, 2 or 3, and Ld2 is and each Ld1 is independently selected from In one embodiment, the compound of formula (III-1) is as follows:

[0223] In one embodiment, z is 1-4; in one embodiment, z is 2 or 4; in one embodiment, z is 2; in one embodiment, in conjugates LC301-1, LC301-2, LC302-1, z is 2 or 4. In one embodiment, in conjugates LC301-3, LC301-4, LC301-5, LC302-2, LC302-3, and LC302-4, z is 2.

[0224] In one embodiment, B in the compound of formula (I) is a terminal group R 10 , and L 1 The cleavable sequence 1 in the formula is connected to the load to form a compound of formula (II), wherein B is not present in the molecule obtained by connecting the cleavable sequence 1 to the load. In this case, it can also be understood that M is LKa-L 2 ―L 1 ―B―P and B does not exist. In this case, M can also be expressed as LKa-L 2 ―L 1 In one embodiment, n is 3, L 2 Yes - (CH2) p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-U- or absent or -NH-CH2-U-(CH2) g -(CO)-, U is O, g is 1. In one embodiment, the conjugate LC301-1 has the following structure:

[0225] In one embodiment, the conjugate LC301-2 has the following structure:

[0226] In one embodiment, the conjugate LC301-3 has the following structure:

[0227] In one embodiment, the conjugate LC301-4 has the following structure:

[0228] In one embodiment, the conjugate LC301-5 has the following structure:

[0229] In one embodiment, the conjugate LC302-1 has the following structure:

[0230] In one embodiment, the conjugate LC302-2 has the following structure:

[0231] In one embodiment, the conjugate LC302-3 has the following structure:

[0232] In one embodiment, the conjugate LC302-4 has the following structure:

[0233] In one embodiment, i is 4, g is 1, R 11 It's methyl.

[0234] In one embodiment, the conjugate LC301-2 is as follows (conjugate LC301-2-1):

[0235] In one embodiment, i is 4, j is 8, g is 1, R 11 In one embodiment, the conjugate LC302-2 is as follows (conjugate LC302-2-1):

[0236] In one embodiment, i is 4, j is 12, g is 1, R 11 In one embodiment, the conjugate LC302-2 is as follows (conjugate LC302-2-4):

[0237] Preparation of conjugates

[0238] The conjugates of the present invention can be prepared by any method known in the art. In some embodiments, the conjugates are prepared by site-specific conjugation of a targeting molecule and a compound of formula (I) with a payload under ligase catalysis, wherein the targeting molecule is modified with a ligase recognition sequence. The method comprises steps A and B.

[0239] Step A. Preparation of Linker-Loader Intermediate

[0240] In a preferred embodiment, B in the compound of formula (I) is covalently linked to a carrier containing another reactive group via a reactive group.

[0241] The linker-load intermediates prepared using the compounds of formula (I) of the present invention have a well-defined structure, composition, and high purity, resulting in minimal or no introduction of impurities during the coupling reaction with antibodies. When such intermediates are used for site-specific conjugation with modified antibodies containing a ligase recognition sequence under ligase catalysis, homogeneous ADCs with highly controllable quality are obtained.

[0242] Step B. Connecting the targeting molecule to the compound of formula (I) with a payload

[0243] The targeting molecule of the present invention can be conjugated to the compound of formula (I) (ie, the compound of formula (II)) carrying a load by any method known in the art.

[0244] The targeting molecule and the compound of formula (I) with a load are connected to each other through the ligase-specific recognition sequence of the substrate. The recognition sequence depends on the specific ligase used. In one embodiment, the targeting molecule is an antibody that introduces a terminal modification based on the recognition sequence at the C-terminus of the light chain and / or heavy chain, and is catalyzed by a wild-type or optimized engineered ligase or any combination thereof, and under suitable catalytic reaction conditions, the targeting molecule is conjugated to the compound of formula (II).

[0245] In a specific embodiment, the ligase is Sortase A, and the conjugation reaction can be represented by the following scheme:

[0246] The triangle represents a portion of the antibody; and the pentagon represents a portion of the compound of formula (II). n, X and J are as defined above. n During conjugation, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by Sortase A, and the resulting intermediate is linked to G n A new peptide bond is generated at the free N-terminus. The resulting amino acid sequence is LPXTG n . Sequence G n and LPXTGJ are as defined above.

[0247] Metabolism of conjugates in physiological environments

[0248] When part or all of the linkers in tumor cells are cleaved, the anti-tumor compound is partially released to exhibit the anti-tumor effect of the anti-tumor compound. Since the linker is cleaved at the site of connection with the drug, the anti-tumor compound is released in its inherent structure to exhibit its inherent anti-tumor effect.

[0249] In one embodiment, the cleavable sequence 1 (eg, GGFG) is cleavable by a lysosomal enzyme (eg, cathepsin B and / or cathepsin L).

[0250] In one embodiment, Sp1 comprises a self-immolative spacer. In one embodiment, Sp1 comprises PABC, acetal, or heteroacetal. In one embodiment, L 1 is GGFG. In one embodiment, the linker comprises -GGFG-NH-CH2-O-. In one embodiment, -GGFG-NH-CH2-O- represents a combination of a restriction enzyme site and a self-immolative spacer, which will be cleaved in the cell and release the target molecule (e.g., a drug).

[0251] Specific Conjugate Table

[0252] In one embodiment, the cargo is a cytotoxin or a fragment thereof. In one embodiment, the antibody is a modified trastuzumab, preferably Ab0001-LCCT L -HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) or Ab0001-LCCT L -HCCT L (Light chain: SEQ ID NO: 3, heavy chain: SEQ ID NO: 4). Ab0001-LCCT L -HC and Ab0001-LCCT L -HCCT L The sequences of the two antibodies are based on the amino acid sequence of Ab0001 (trastuzumab), with the GALPETGG sequence introduced at the C-terminus of the light chain (Ab0001-LCCT L -HC) or by introducing GALPETGG at the C-terminus of the light and heavy chains (Ab0001-LCCT L -HCCT L ), wherein LPETGG is the recognition sequence of the ligase donor substrate and GA is the spacer sequence. L -HCCT L In the present invention, the lysine at the C-terminus of the heavy chain of Ab0001 can be maintained as in SEQ ID NO: 4, or can be deleted before the introduction of GALPETGG (the resulting sequence is not shown in the sequence listing).

[0253] In a preferred embodiment, in the conjugate of the present invention, the payload (represented by P in formula (III)) is a compound represented by formula (i) or compound 1-14, preferably compound 10. In a preferred embodiment, in the conjugate of the present invention, the linker-payload (the portion other than A in formula (III)) has a structure selected from LB301-1-1, LB301-2-1, LB301-3-1, LB301-4-1, LB301-5-1, LB302-1-1, LB302-1-4, LB302-2-1, LB302-2-4, LB302-3-1, LB302-3-4, LB302-4-1 and LB302-4-4 as described above. In a preferred embodiment, in the conjugate of the present invention, the targeting molecule (represented by A in formula (III)) is selected from Ab0001-LCCT L -HC, Ab0001-LCCT L -HCCT L .

[0254] In a more preferred embodiment, the conjugates of the present invention are shown in the following table: (Nomenclature of ADC: the numbers in brackets indicate the number of cargo (drug) molecules intended to be attached to the antibody).

[0255] Pharmaceutical compositions and pharmaceutical preparations

[0256] The pharmaceutical composition of the present invention comprises an active ingredient, a buffer, a stabilizer, and an optional surfactant, wherein the active ingredient comprises the conjugate of formula (III) described above. Depending on actual needs and without hindering the technical effects of the present invention, at least one other pharmaceutically acceptable carrier other than the above may also be included.

[0257] To improve storage stability and reduce degradation of the active ingredient, the buffer is preferably selected from acetate buffer, histidine buffer, citrate buffer, and phosphate buffer; more preferably, the buffer is acetate buffer or histidine buffer, and even more preferably, a histidine buffer. The molar concentration of the buffer can be 10-50 mM, for example, about 15 mM, 16 mM, 18 mM, 19 mM, 20 mM, 21 mM, 23 mM, 24 mM, or 25 mM.

[0258] The stabilizer used in the formulation of the present invention may include a sugar. To improve storage stability and reduce degradation of the active ingredient, the sugar is preferably selected from sucrose and trehalose (or trehalose dihydrate); more preferably, the sugar is sucrose. The concentration of the sugar may be 1-20% (w:v), for example, approximately 6%, 7%, 8%, 8.1%, 9%, 10%, or 12% (w:v).

[0259] In order to improve storage stability and reduce degradation of active ingredients, preferably, the pH value of the pharmaceutical composition is weakly acidic to neutral, for example, 5.0-6.5, preferably 5.2-6.0, more preferably about 5.5.

[0260] In order to reduce the surface tension of the solution, increase the solubility of the protein, and simultaneously suppress the aggregation of protein molecules under mechanical stress, the pharmaceutical composition of the present invention can also contain a surfactant. Those skilled in the art can select the surfactant according to actual needs. The surfactant can be a polysorbate (Tween), for example, polysorbate 20 or polysorbate 80. The concentration of the surfactant can be 0.01-10 mg / ml, for example, 0.1-1 mg / ml, for example, about 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, or 1 mg / ml.

[0261] The pharmaceutical composition of the present invention can be administered in any manner, as long as it achieves the effect of preventing, alleviating or treating symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration, in particular injections such as lyophilized powder injections, injections or sterile injection powders. The pharmaceutical composition of the present invention may contain water. Those skilled in the art can judge based on their common sense that in some dosage forms (such as injections), the pharmaceutical composition of the present invention contains water; while in other dosage forms (such as lyophilized powder injections), although they are in a water-containing form during preparation and use, they are stored in an anhydrous form during storage.

[0262] The term "pharmaceutically acceptable" means that it does not produce undue toxicity, irritation or allergic reaction when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages to disadvantages, and is effective for the intended use.

[0263] The term pharmaceutically acceptable carrier refers to any 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 substances that allow the composition to approach physiological conditions, such as pH adjusters and buffers, toxicity modifiers, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0264] In one embodiment, the drug / antibody ratio (DAR) of the pharmaceutical composition of the invention is an integer or non-integer of 1-20, such as about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2. In a particular embodiment, the conjugate of the invention has a DAR of about 2, about 4, about 6, or about 8.

[0265] Treatment methods and uses

[0266] The pharmaceutical compositions of the present invention can be used to treat tumors and / or autoimmune diseases. Tumors susceptible to conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors. These tumor cells can be recognized by the targeting molecules in the conjugate and, in turn, killed by the cargo / cytotoxin in the conjugate.

[0267] Therefore, in another aspect, the present invention also provides use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating, improving or alleviating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0268] In another aspect, the present invention provides the pharmaceutical composition of the present invention for use in treating tumors or autoimmune diseases.

[0269] In a further aspect, the present invention provides a method for treating tumors or autoimmune diseases, comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.

[0270] In a preferred embodiment, the conjugate formed by linking an anti-human HER2 antibody provided herein with a small molecule cytotoxin can specifically bind to HER2 on the surface of tumor cells, selectively killing HER2-expressing tumor cells. In another preferred embodiment, the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating, ameliorating, or alleviating a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0271] The amount of active ingredient (i.e., conjugate) in the pharmaceutical composition administered to a subject can be adjusted to a considerable extent. The amount can vary depending on the specific route of administration and the needs of the subject, and can be determined by a healthcare professional. Beneficial effects

[0272] The antibody-drug conjugate of the present invention uses a specially designed linker-load, is more stable, and can achieve good therapeutic effects at a lower DAR, thereby reducing side effects and improving the therapeutic index.

[0273] The present invention utilizes a linker unit with a unique structure and uses a ligase to catalyze the conjugation of a targeting molecule with a cargo. The conjugates of the present invention exhibit good homogeneity, high activity, and high selectivity. Furthermore, the toxicity of the linker-carrier intermediate is much lower than that of the free cargo, making the drug manufacturing process less hazardous and facilitating industrial production.

[0274] The conjugates of the present invention achieve at least one of the following technical effects:

[0275] (1) It has high inhibitory activity on target cells or has a strong killing effect on target cells.

[0276] (2) Excellent physicochemical properties (e.g., solubility, physical and / or chemical stability).

[0277] (3) Excellent pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action).

[0278] (4) Excellent safety (lower toxicity and / or fewer side effects to non-target normal cells or tissues, wider therapeutic window), etc.

[0279] (5) Highly modular design, enabling easy assembly of multiple drugs.

[0280] The preparation of the present invention is stable and can achieve long-term stable storage of the drug.

[0281] Example

[0282] Preparation Example

[0283] In order to more clearly illustrate the purpose and technical solutions of the present invention, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The specific experimental methods not mentioned in the following examples are all carried out according to conventional experimental methods.

[0284] Instruments, materials and reagents

[0285] Unless otherwise specified, the instruments and reagents used in the examples are commercially available and can be used directly without further purification.

[0286] MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer

[0287] HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0288] Semi-preparative HPLC: Lisure HP plus 50D

[0289] Flow cytometer CytoFLEX S

[0290] 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; acquisition time: 25 min; injection volume: 20 μg; column temperature: 25°C; detection wavelength: 280 nm; sample chamber temperature: 8°C.

[0291] SEC-HPLC: Chromatographic 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; injection volume: 50μg; column temperature: 25℃; detection wavelength: 280nm; sample tray temperature: 8℃.

[0292] CHO cells were 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; Rink-amide-MBHA-resin and dichlororesin were obtained from Nankai synthesis; HCC1954 was obtained from ATCC Cat#CRL-2338; SK-BR-3 was obtained from ATCC Cat#HTB-30; BT-474 was obtained from ATCC Cat#HTB-20; NCI-N87 (ATCC Cat#CRL-5822); MCF7 was obtained from ATCC Cat#HTB-22; MDA-MB-231 was obtained from ATCC Cat#HTB-26; MDA-MB-468 was obtained from ATCC Cat#HTB-132; CFPAC-1 was obtained from ATCC CAT#CRL-1918; NCI-H2110 was obtained from ATCC CAT#CRL-5924; JIMT-1 was obtained from Wuxi Apptech; Capan-1 was obtained from ATCC CAT#CRL-1573; the antibody trastuzumab was prepared according to the known sequence; the optimized recombinant enzyme Sortase A from Staphylococcus aureus was prepared in E. coli.

[0293] Example 1 Construction of antibody expression vector, antibody expression, purification and identification

[0294] 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L -HC production

[0295] Antibody Ab0001-LCCT L -HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) expression plasmid was constructed as follows. Antibody Ab0001-LCCT L -HC sequence: Based on the amino acid sequence of trastuzumab, 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 establish cell colonies and screen for high-expressing cells. Cultures were performed similarly to the trastuzumab culture process in a 5-10 L reactor, and the supernatant was collected.

[0296] 1.2 Antibody Ab0001-LCCT L Purification of -HC

[0297] Ab0001-LCCT L -HC was purified using a standard process combining MabSelect affinity chromatography and Sepharose S cation exchange chromatography, and the purified product was dissolved in native trastuzumab drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) and frozen in small aliquots.

[0298] 1.3 Antibody Ab0001-LCCT L -HC quality control

[0299] The antibody Ab0001-LCCT purified by SDS-PAGE L The purity of -HC is 98.5%; SEC-HPLC detected that the content of high molecular weight polymers in the sample was less than 0.4%; and the endotoxin content was less than 0.098EU / mg.

[0300] 1.4 Preparation of other modified anti-human antibodies

[0301] According to a similar method, terminal modifications based on ligase recognition sequences were introduced into the C-termini of the trastuzumab light chain and / or heavy chain to obtain modified antibodies.

[0302] The modified anti-human HER2 antibodies based on Ab0001 (trastuzumab) are listed in Table 1. LPETGG in the terminal modification sequence is the recognition sequence of the ligase donor substrate, and GA is a spacer sequence.

[0303] Table 1 Modified anti-human HER2 antibodies *: “-” indicates no terminal modification

[0304] Example 2 Preparation of intermediate

[0305] 2.1 Preparation of intermediate Mc-GGFG-Dxd (RF1184)

[0306] The intermediate Mc-GGFG-Dxd (RF1184) is commercially available or prepared according to the procedure described in EP 2907824. This compound is used to prepare the linker-load intermediate (compound of formula (IV)) and is also used to directly link (optionally modified) antibodies to prepare reference ADCs.

[0307] 2.2 Preparation of linker-load intermediate LB301-2-1

[0308] Step 1: Preparation of linker HX18041

[0309] HX18041 was synthesized using conventional solid-phase peptide synthesis using Rink-amide-MBHA resin. Fmoc was used to protect the amino acids in the linker. 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 and lyophilized for future use. Theoretical molecular weight: 538.24, measured value: [M+H] + =539.2.

[0310] Step 2: Preparation of linker-load intermediate LB301-2-1

[0311] HX18041 and the intermediate MC-GGFG-Dxd (molar ratio ~1.2:1) were weighed and dissolved in water and DMF, respectively. The mixture was thoroughly mixed and reacted at 0-40°C for 0.5-20 hours. After the reaction was complete, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued at 0-40°C for another 0.2-20 hours. After the reaction was complete, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the linker-load compound LB301-2-1. Theoretical mass: 1589.65, measured value: [(M+2H) / 2] + =796.6.

[0312] 2.3 Preparation of linker-load intermediate LB302-2-1

[0313] Step 1: Preparation of linker HX20113

[0314] HX20113 was synthesized using conventional solid-phase peptide synthesis using Rink-amide-MBHA resin. Fmoc was used to protect the amino acids in the linker. 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 and lyophilized for future use. Theoretical mass: 1207.59, measured value: [MH] - =1206.7.

[0315] Step 2: Preparation of linker-load intermediate LB302-2-1

[0316] HX20113 and the intermediate MC-GGFG-Dxd (molar ratio ~1:2) were weighed and dissolved in water and DMF, respectively. The mixture was thoroughly mixed and reacted at 0-40°C for 0.5-30 hours. After the reaction was complete, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued 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 linker-load intermediate LB302-2-1. Theoretical mass: 3310.41, measured value: [(M+3H) / 3] + =1104.5.

[0317] 2.4 Preparation of linker-load intermediate LB302-2-4

[0318] Step 1: Preparation of linker HX20111

[0319] HX20111 was synthesized using conventional solid-phase peptide synthesis using Rink-amide-MBHA resin. Fmoc was used to protect the amino acids in the linker. 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 and lyophilized for future use. Theoretical mass: 1383.70, measured value: [MH] - =1382.6.

[0320] Step 2: Preparation of linker-load intermediate LB302-2-4

[0321] HX20111 and the intermediate MC-GGFG-Dxd (molar ratio ~1:2) were weighed and dissolved in water and DMF, respectively. The mixture was thoroughly mixed and reacted at 0-40°C for 0.5-30 hours. After the reaction was complete, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued 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 linker-load intermediate LB302-2-4. Theoretical mass: 3486.52, measured value: [(M+3H) / 3] + =1163.3.

[0322] Example 3 Preparation of Antibody-Drug Conjugates

[0323] 3.1 The linker-load intermediates were conjugated to the antibody in a site-specific manner using a ligase to form an ADC. The conjugation reaction method can be found in WO2015165413A1. The resulting ADCs are shown in Table 2 below:

[0324] Table 2 Generated ADC

[0325] 3.2 Reference ADCs LC1184 (8) and LC1184 (4) were prepared by directly linking the intermediate RF1184 to an (optionally modified) antibody (Cys conjugation, i.e., conjugation via a maleimide structure and a sulfhydryl group of a cysteine). Methods for conjugation reactions are known in the art. LC1184 (8) incorporates eight RF1184s via reduced interchain cysteines. LC1184 (4) incorporates four RF1184s via reduced interchain cysteines.

[0326] Example 4 Effect of HER2-targeted conjugates on cell proliferation

[0327] Cytotoxicity assays were performed using HER2-high expressing cancer cells SK-BR-3 (Figure 1) and NCI-N87 (Figure 2), HER2-low expressing cancer cells CFPAC-1 (Figure 3) and NCI-H2110 (Figure 4), and HER2-negative cell line MDA-MB-468 (Figure 5) to analyze the effects of the conjugates on tumor cell proliferation. Test samples included conjugates LC302-2-1 (4), LC302-2-4 (4) and small molecule compound 10 (structure as described above). Briefly, 3000 to 5000 cells were plated in 96-well plates and the cells were allowed to adhere after growing overnight. The cells were treated with different concentrations of the designated drugs for 120 hours. By Luminescent Cell Viability Assay was used to examine cell viability and calculate the percentage of cell viability.

[0328] In HER2-high-expressing SK-BR-3 and NCI-N87, the conjugates LC302-2-1(4) and LC302-2-4(4) showed similar efficacy. The IC values ​​of LC302-2-1(4) and LC302-2-4(4) were 50 The values ​​were lower than those of the small molecule loading compound Dxd. In HER2 low-expressing and negative cells, the conjugates LC302-2-1 (4) and LC302-2-4 (4) showed the least efficacy. The specific results are shown in Table 3.

[0329] Table 3. Test results of the effects of HER2-targeted conjugates on cell proliferation N / A indicates weak inhibitory effect, and - indicates not tested.

[0330] Example 5 In vivo evaluation of conjugates

[0331] For in vivo antitumor efficacy studies, 5 × 10 6 JIMT-1 human breast cancer cells (HER2 culture medium). After 7 days, when the average tumor volume reached 142mm 3 Tumor-bearing mice were assigned and LC1184(8) and five other different conjugates were administered intravenously at 5 mg / kg. Tumor volume was measured twice a week with a caliper. LC1184(8) showed better efficacy than LC1184(4), indicating that a higher DAR will lead to better efficacy when using the same loading material. LC302-2-1(4) and LC302-2-4(4) were more effective than LC1184(8). LC301-2-1(4) showed efficacy comparable to LC1184(8), while LC301-2-1(2) showed efficacy comparable to LC1184(4) ( FIG6 ). The conjugates of the present invention achieved good efficacy at lower DARs.

[0332] 5x10 6 Capan-1 human pancreatic cancer cells (HER2 low expression) were used to prepare a transplanted tumor model. After 8 days, when the average tumor volume reached 178 mm 3 Tumor-bearing mice were intravenously administered LC1184(8) and five other different conjugates at 5 mg / kg. Tumor volume was measured twice weekly with a caliper. LC1184(8) was more potent than LC1184(4). LC302-2-1(4), LC302-2-4(4), and LC301-2-1(4) showed comparable potency to LC1184(8), while LC301-2-1(2) showed comparable potency to LC1184(4) ( FIG7 ).

[0333] Example 6 In vitro serum stability of the conjugate

[0334] For in vitro serum stability studies, conjugates LC302-2-1(4), LC302-2-4(4), and LC1184(8) were inoculated in pooled human serum at 37°C. At 0, 24, 48, and 96 hours, the conjugates were captured with antigen, deglycosylated with glycosidase, and cleaved with acid. The supernatant was collected and centrifuged, and then analyzed by high-resolution LC-MS to determine the DAR.

[0335] LC302-2-1(4) and LC302-2-4(4) were very stable after 96 hours of incubation in serum. The DAR of LC302-2-1(4) and LC302-2-4(4) did not decrease significantly after 96 hours of incubation. However, the DAR of LC1184(8) decreased from 7.8 to 2.6 after 96 hours of incubation (Figure 8). The stability of the linker indicates that more cargo is delivered to the targeted tumor, while off-target free cargo release is reduced, thereby improving the therapeutic index.

[0336] Example 7 Preparation Study

[0337] 1. Detection method

[0338] 1.1. Appearance and visible foreign matter (visual inspection)

[0339] Visual inspection is used to inspect the appearance of the product. Ensure the light intensity of the clarity meter is maintained between 1000 and 1500 lx. Hold the sample at eye level and gently shake or invert it to avoid introducing air bubbles. Visual inspection is performed against both a black and white background. Results are recorded for both appearance and visible foreign matter.

[0340] 1.2. Protein concentration (UV method)

[0341] The protein concentration was detected using a UV-visible spectrophotometer (dual wavelength method). Wash the cuvette three times with ultrapure water and then add 400 μL of ultrapure water to the cuvette, click to measure, and use ultrapure water for blank correction. After mixing the test sample, dilute it with water for injection (the dilution factor each time should not exceed 10 times) to a final concentration of approximately 0.5 mg / mL. Each sample needs to be prepared in parallel for 3 times, mixed and set aside. Use a UV spectrophotometer to measure the absorbance (A280 and A360) of the diluted sample at 280 nm and 360 nm. The measured values ​​of the three parallel samples are recorded as A2801, A3601, A2802, A3602, A2803, and A3603, respectively. Calculate the A280 and A360 of each test sample according to the following formula.

[0342] A280=(A2801+A2802+A2803) / 3; A360=(A3601+A3602+A3603) / 3

[0343] The A calculated above 280 and A 360 Substitute the following formula (1) and formula (2) to calculate C mAb , C mAb Substituting into formula (3) we can get the protein concentration C of the test sample; 280 =ε mAb-280 *L*C mAb +εLinker-Drug-280*L*C Linker-Drug (1) A 360 =εLinker-Drug-360*L*C linker-Drug (2) C=C mAb *N (3)

[0344] The extinction coefficient of naked antibody at 280nm is 1.47 (g / L) -1 cm -1 , Linker-Drug extinction coefficient at 280nm is 2.39 (g / L) -1 cm -1 , the extinction coefficient at 360nm is 8.73 (g / L) -1 cm -1 . N is the dilution multiple.

[0345] Purity (SEC-HPLC)

[0346] Sample purity was determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The column was a TSKgel G3000SWXL, 7.8 × 300 mm, gel-free column; the mobile phase consisted of 0.2 mol / L potassium dihydrogen phosphate-0.25 mol / L potassium chloride solution, pH 6.2. The sample was diluted with ultrapure water to a concentration of approximately 2 mg per mL. A 50 μL sample solution was injected into the HPLC instrument. The column temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the injector temperature was 4°C, and the analysis time was 30 minutes. The relative percentages of aggregates, monomers, and fragments were calculated using the area normalization method.

[0347] 1.4. Charge variants (CEX-HPLC)

[0348] The charge heterogeneity of the samples was determined by cation exchange high performance liquid chromatography (CEX-HPLC). The chromatographic column was a ProPac WCX-10 (4×250 mm) cation exchange column. The mobile phase consisted of A: 100% water, B: 200 mM NaCl, C: 100 mM sodium dihydrogen phosphate, and D: 100 mM sodium dihydrogen phosphate. The sample was diluted with ultrapure water to approximately 2 mg per 1 mL to prepare the sample solution. 15 μL of the sample solution was injected into the liquid chromatograph and gradient eluted according to the table below. The column temperature was 45°C, the flow rate was 0.8 mL / min, and the detection wavelength was 214 nm. The relative percentages of the acidic component, main peak, and basic component were calculated using the area normalization method.

[0349] 1.5. Purity (reduced CE-SDS)

[0350] Sample purity was determined using the reduced CE-SDS method. The sample solution was diluted to a concentration of 1.0 mg per mL in sample buffer (1% SDS-0.04 mmol / L sodium dihydrogen phosphate, pH 6.5). 95 μL of the sample solution and 5 μL of β-mercaptoethanol were vortexed and mixed. The sample solution was heated in a 70°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 12,000 rpm for 5 minutes before loading. Separation was performed using uncoated capillaries (AB Sciex, capillary inner diameter: 50 μm, length: 30.2 cm, effective separation length: 20 cm). The injection voltage was -5 kV, the injection time was 20 seconds, the separation voltage was -15 kV, the analysis time was 35 minutes, the column temperature was 25°C, and the sample chamber temperature was 15°C. The area normalization method was used to calculate the purity of LC, LC-Drug, and HC, and the sum of the three was the sample purity.

[0351] 1.6.DAR value (HIC)

[0352] DAR values ​​were determined using hydrophobic chromatography (HIC) using a MabPac HIC-20 column (4.6 × 100 mm, 5 μm particle size). Mobile phase A consisted of 50 mM PB + 1 M sodium sulfate, pH 7.0 ± 0.05; mobile phase B consisted of 50 mM PB (phosphate buffer), pH 7.0 ± 0.05. The sample solution was diluted with pure water to approximately 5 mg per mL. A 10 μL aliquot of the sample solution was injected into the liquid chromatograph. Chromatographic parameters were: column temperature 30°C, sample chamber temperature 5 ± 3°C, flow rate 1.0 mL / min, injection volume 10 μL, detection wavelength 280 nm, elution time 30 minutes, and the elution gradient is shown in the table below. The percentages of the DAR0, DAR2, and DAR4 peaks were calculated using area normalization: DAR value = DAR2% × 2 + DAR4% × 4.

[0353] 1.7. Free drug (RP-HPLC)

[0354] Free drug was detected by reverse-phase high-performance liquid chromatography (RP-HPLC) using an Agilent Poroshell 120 EC-C18 column, 4.6 × 100 mm, with a particle size of 2.7 μm. Mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and mobile phase B consisted of 0.1% TFA in acetonitrile. 100 μL of the test sample was added to 200 μL of acetone and mixed thoroughly. Centrifuged at 13,000 rpm for 5 minutes, the supernatant was used as the test solution. An appropriate amount of small molecule sample was prepared to prepare a reference solution containing 0.133 μg to 1.33 μg per 1 mL. 7 μL of the test solution was injected into the liquid chromatograph, and the reference solution was processed in the same manner. The flow rate was 1.0 mL / min, the column temperature was 40°C, the detection wavelength was 261 nm, and the injector temperature was 4°C. Gradient elution was performed according to the table below. The free small molecule content in the sample was calculated using the external standard method.

[0355] 1.8.Binding Activity (ELISA)

[0356] Determined by enzyme-linked immunosorbent assay (ELISA). Add the antigen (Human Her2 protein) to the enzyme-labeled plate, coat it at 2-8°C overnight, and then block it with blocking solution. After washing the plate with washing solution, add the reference sample and test sample solution respectively, and incubate with shaking at 25°C for 1 hour; after washing the plate with washing solution, add the horseradish peroxidase-labeled mouse anti-human IgG-Fc antibody solution to the enzyme-labeled plate, and incubate with shaking at 25°C for 1 hour; after washing the plate with washing solution, add TMB for color development, and color development in the dark for 10 minutes; after terminating the reaction with stop solution, measure the absorbance at a wavelength of 450nm. Use the four-parameter logarithmic regression (4PL) model to fit the EC values ​​of the reference sample and the test sample respectively. 50 . Through the reference product EC 50 / Test Sample EC 50 Calculate the relative activity of the test article.

[0357] 2. Research content

[0358] 2.1 Experimental steps

[0359] 2.1.1 Preparation of replacement fluid

[0360] Prepare 2 L of the corresponding replacement fluid according to Table 4. Soak the dialysis bag in buffer solution, seal one end with a dialysis clamp, carefully transfer the LC302-2-4(4) sample (Lot: 20210409) to the dialysis bag, and clamp the bag mouth. Place it in a beaker filled with buffer solution and let it stand at room temperature for dialysis. After 4 hours of dialysis, transfer the dialysis bag to new dialysis solution and dialyze it at 2-8°C overnight. Dialyze it twice at 2-8°C overnight. During each dialysis solution change, control the liquid system to be replaced: the replacement fluid volume is not higher than 1:100. After dialysis is completed, measure the protein concentration and dilute it to the target concentration of 20 mg / ml. Note: The trehalose in the examples is trehalose crystals (trehalose dihydrate).

[0361] Table 4 Prescription ingredients

[0362] 2.1.2 Preparation of 20 mg / ml Polysorbate 20 / 80 Stock Solution

[0363] Before sterilizing and filtration, prepare fresh polysorbate stock solution and add the corresponding volume of 2% (20 mg / ml) polysorbate 20 or polysorbate 80 stock solution according to the prescription design scheme and the volume of the stock solution.

[0364] 2.1.3 Sterilization, filtration and filling

[0365] The product was aseptically filled into 2R vials (0.5 ml / vial) in a clean bench, with 25 vials per group. After stoppering, capping, labeling, and stability testing, the vials were sampled and tested.

[0366] 2.2 Experimental Results

[0367] (1) Appearance and visible foreign matter

[0368] According to the results in Table 5, all samples were colorless, clear liquids with no visible foreign matter at T0. Prescription FD-01 produced a small amount of fine, visible particles after five freeze-thaw cycles at -20°C. Prescription FD-04 produced a small amount of fine, visible particles after four weeks of storage at -20°C. Prescription FD-05 produced a small amount of fine, visible particles after five freeze-thaw cycles at -20°C or seven days of storage at 40°C. Prescriptions FD-02, 03, 06, 07, and 08 showed no visible foreign matter under all storage conditions.

[0369] Table 5 Appearance and visible foreign matter detection results Note: FVP: colorless, clear liquid with no visible foreign matter; 1: fine particles are produced

[0370] (2) SEC-HPLC purity results

[0371] According to the SEC aggregate content results in Table 6, under accelerated and high temperature conditions, the proportion of aggregates in formulations FD-04 and 05 increased slightly, while the proportion of aggregates in formulations FD-01, 03, 06, 07, and 08 did not increase significantly. After being placed at 2-8°C and -20°C for 12 weeks, no significant increase in aggregates was observed in any of the formulations.

[0372] Table 6 SEC-HPLC aggregate content (%) detection results

[0373] (3) Charge isomers (CEX-HPLC)

[0374] According to the CEX-HPLC purity results in Tables 7-9, the proportions of acidic components in formulations FD-03, 04, and 05 increased to varying degrees after accelerated and high-temperature storage, while there was no significant change in the acidic components of formulations FD-01, 02, and FD-06, 07, and 08. The proportions of basic components in formulations FD-01, 02, 03, and FD-06, 07, and 08 all increased significantly, while there was no significant change in formulation FD-04. The proportion of basic components in formulation FD-5 decreased slightly. All samples showed a decrease in the purity of the main peak, with the decrease being slower in formulations FD-01, 02, 06, 07, and 08, and more significant in formulation FD-05. After 12 weeks of storage at both 2-8°C and -20°C, no sample showed a significant decrease in purity.

[0375] Table 7 CEX-HPLC acidic peak (%) detection results

[0376] Table 8 CEX-HPLC main peak (%) detection results

[0377] Table 9 CEX-HPLC basic peak (%) detection results

[0378] (4) Reduced CE-SDS purity

[0379] According to the reduced CE-SDS purity results in Table 10, the purity of formulations FD-04 and 05 decreased under accelerated and high-temperature storage conditions. The purity of formulation FD-08 also decreased slightly after 7 days of high-temperature storage. Significant differences were observed among the other groups. All formulations showed no significant changes in purity after 12 weeks of storage at both 2-8°C and -20°C.

[0380] Table 10 Reduced CE-SDS purity (%) test results

[0381] (5)DAR value

[0382] According to Table 11, the DAR values ​​of different prescriptions under different inspection conditions and time points did not change significantly.

[0383] Table 11 DAR value detection results

[0384] (6) Free drugs

[0385] Tables 12 and 13 show the results of free drug testing under eight different conditions and time points. The results show that low formulation pH (e.g., 5.5 and below) promotes Dxd shedding. Higher pH (e.g., 6.5-7.0) accelerates LB302-2-4 shedding. At 2-8°C and -20°C, and at a moderate pH (e.g., 5.5), free drug shedding was not significant, regardless of the presence of a surfactant, meeting stringent quality control requirements.

[0386] Table 12 Free drug (LB302-2-4) test results

[0387] Table 13 Free drug (Dxd) detection results

[0388] (7) Binding activity (ELISA)

[0389] According to the combined activity results in Table 14, none of the formulations showed a decrease in activity after 7 days at high temperature and 12 weeks at accelerated and long-term conditions.

[0390] Table 14 Binding activity detection results

[0391] 3. Conclusion

[0392] According to the results of visible foreign matter detection, each prescription has a certain stability, among which prescriptions FD-02, 03, 06, 07, and 08 have better stability. According to the SEC aggregate detection results and comprehensive analysis of other analysis results (such as free drug data), better stability can be achieved when the buffer system is an acetic acid and histidine buffer system, and a histidine buffer system is more preferred. From the CEX acid-base peak results, the purity of the main peaks of the five prescriptions FD-01, 02, 06, 07, and 08 is better maintained. From the free drug detection results, low pH conditions will accelerate the shedding of Dxd, and high pH conditions will promote the shedding of LB302-2-4, but when the pH is moderate (for example, around 5.5), even without the addition of a surfactant, the shedding of the free drug can be suppressed.

[0393]

[0394]

Claims

1. A pharmaceutical composition comprising an active ingredient, a buffer, a stabilizer, and optionally a surfactant, in, The active ingredient includes a conjugate having the structure of formula (III): in, n is any integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10; d is 0 or any integer selected from 1, 2, 3, 4, 5, 6; Each Ld1 and Ld2 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10; or substituted with -(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids with a degree of polymerization of 2-10, -(PEG) j -R 11 There is a carbonyl group connected between them; -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene; Q is hydrogen or LKb-P; M is hydrogen or LKa-LKb-P; provided that Q and M are not both hydrogen; Each LKa is independently selected opSu is or a mixture thereof; Each LKb is independently selected from L 2 ―L 1 ―B; Each B is independently absent or a combination of 1) a self-immolative spacer Sp1; and 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene and -(CO)-; or each B is independently a terminal group R 10 or a combination of 1), 2), or 3) below: 1) a self-immolative spacer Sp1; 2) a bond, or a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 It is hydrogen or a group that can leave when reacting with a group in the load; Sp1 is selected from PABC, acetal, heteroacetal and combinations thereof; preferably, Sp1 is acetal, heteroacetal or PABC; preferably, heteroacetal is selected from N, O-heteroacetal; preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-; wherein -O- or -NH- is cleavable Sequence 1 is connected, U is O, S or NH, preferably O or S; Preferably, B is -NH-CH2-U- or absent or -NH-CH2-U-(CH2) g -(CO)-; wherein U is absent, or is O, S or NH, preferably O or S; L 1 is a cleavable sequence 1 comprising an amino acid sequence cleavable by an enzyme, wherein the cleavable sequence 1 comprises 1-10 amino acids; preferably, the cleavable sequence 1 is selected from the group consisting of GLy-GLy-Phe-GLy, Phe-Lys, Val-Cit, Val-Lys, GLy-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Ala-Ala-Ala, and combinations thereof; L 2 is a key; or C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Halogenated alkyl, C 4-10 cycloalkylene; R 11 It is C 1-10 alkyl; Each i is independently an integer of 1-100, preferably 1-20; preferably, each i is independently an integer of 1-12; more preferably 2-8; especially 4; Each j is independently an integer of 1-100, preferably 1-20; preferably, each j is independently an integer of 1-12; more preferably an integer of 8-12 or 8-13; especially 8, 9, 12 or 13; P is with part B or L 1 A partially connected payload selected from the following compounds 1-14, A is a targeting molecule, which is an antibody or an antigen-binding fragment thereof; preferably, the antibody or antigen-binding fragment comprises a ligase donor recognition sequence and is covalently coupled to (Gly) in formula (III) via a ligase. n Preferably, the antibody or antigen-binding fragment is covalently coupled to a -Gly-Gly-Gly- portion; preferably, the heavy chain and / or light chain termini of the antibody or antigen-binding fragment are connected to a ligase donor substrate recognition sequence; Preferably, the antibody is an anti-human HER2 antibody; preferably, the C-terminus of the light chain of the anti-HER2 antibody is linked to the amino acid sequence GALPETGG, or the C-termini of both the light chain and the heavy chain of the anti-HER2 antibody are linked to the amino acid sequence GALPETGG; z is an integer from 1 to 20.

2. The pharmaceutical composition of claim 1, wherein The conjugate has the structure of the following formula (III-1):

3. The pharmaceutical composition according to claim 1 or 2, wherein The conjugate has the following structure: Preferably, z is 1-4; preferably 2; Each i, i1, i2, i3, i4 is independently an integer of 1-100, preferably 1-20; preferably, each i, i1, i2, i3, i4 is independently an integer of 1-12; more preferably 2-8; especially 4; Each j is independently an integer of 1-100, preferably 1-20; preferably, each j is independently an integer of 1-12; more preferably an integer of 8-12 or 8-13; especially 8, 9, 12 or 13; Preferably, n is 3, L 2 Yes - (CH2) p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-U- or absent or -NH-CH2-U-(CH2) g -(CO)-, U is O, g is 1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the loading substance is selected from:

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the conjugate is selected from: Each g is independently an integer of 1-6, preferably 1-3; more preferably 1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the buffering agent forms a buffer selected from acetate buffer, histidine buffer, citrate buffer, and phosphate buffer; preferably, the buffering agent is acetate buffer or histidine buffer, further more preferably a histidine buffer; preferably, the concentration of the buffer is 10-50 mM; preferably, the pH value of the buffer is 5.0-6.5, or 5.5-6.

4.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the stabilizer is selected from sucrose and trehalose, preferably sucrose; preferably, the concentration of the stabilizer is 1-20% (w:v).

8. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pH value of the pharmaceutical composition is 5.0-6.5, preferably 5.2-6.0, more preferably about 5.

5.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the surfactant is a polysorbate; preferably, the concentration of the surfactant is 0.01-10 mg / ml.

10. A pharmaceutical preparation comprising an active ingredient of formula (III), comprising: 20 mM acetate buffer, pH 5.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 20 mg / ml active ingredient; or 20 mM histidine buffer, pH 6.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or 20 mM citrate buffer, pH 6.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or 20 mM phosphate buffer, pH 7.0, 9% (w:v) sucrose, 20 mg / ml active ingredient; or 20 mM histidine buffer, pH 5.5, 8.1% (w:v) trehalose, 0.2 mg / ml polysorbate 20, 20 mg / ml active ingredient; or 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 0.2 mg / ml polysorbate 20, 20 mg / ml active ingredient; or 20 mM histidine buffer, pH 5.5, 9% (w:v) sucrose, 0.2 mg / ml polysorbate 80, 20 mg / ml active ingredient; The pharmaceutical preparation is a powder preparation or a liquid preparation.

11. Use of the pharmaceutical composition according to any one of claims 1 to 10 in the preparation of a medicament for treating a disease; wherein the disease is a tumor or an autoimmune disease; preferably a HER2-positive tumor; Preferably, the HER2-positive tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer and urothelial cancer.

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