Pharmaceutical composition of anti-HER2 antibody-immune agonist conjugate and application thereof

Abstract

The invention relates to a pharmaceutical composition with an antibody-immune agonist conjugate (AIAC). The pharmaceutical composition is used as a novel tumor targeting therapy.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a pharmaceutical composition having an antibody-immunoagonist conjugate (AIAC), which is a novel tumor-targeted therapy. Background Art

[0002] Targeted delivery of therapeutic agents remains a major challenge for cancer therapy in both development and clinical settings. Currently, the FDA-approved targeted molecule-drug conjugates are mainly antibody-drug conjugates (ADCs), in which the drug (warhead) is usually a small molecule cytotoxin.

[0003] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family with tyrosine kinase activity. Amplification or overexpression of HER2 occurs in about 15% to 30% of breast cancer and 10% to 30% of gastric / gastroesophageal cancer. HER2 overexpression also occurs in other cancers such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer and head and neck cancer (Iqbal N. et al., Mol Biol Int., 2014: 852748). Although the efficacy of HER2-targeted therapies (such as HER2-directed antibodies or antibody-drug conjugates (ADCs)) has significantly improved the life expectancy of patients with HER2-positive disease, HER2-positive breast cancer is still a more aggressive form of the disease, with a poorer prognosis and less ideal outcomes than patients with HER2-negative (and HR-positive) disease. In addition, studies have shown that the treatment results of such therapies in other HER2-overexpressing cancers are still unsatisfactory. One of the many reasons for poor outcomes is the emergence of drug resistance in patients who are administered HER2-targeted therapies. This process is caused by the immune escape of tumor cells.

[0004] As a new mode of cancer treatment, immunotherapy has demonstrated powerful efficacy. Although immune checkpoint inhibitors represented by CLTA-4 and PD-1 / L1 monoclonal antibodies (which are essentially T cell-based therapies) have been approved for various cancer indications, many people are also committed to exploring other mechanisms of action of the immune system to fight cancer. Targeting myeloid cells (mainly macrophages, DCs) has become a promising direction. Activating macrophages and DCs by agonists or by macrophage checkpoint inhibitors not only enhances their phagocytic ability to clear tumor cells, but also enhances their antigen presentation function, which will more effectively stimulate adaptive anti-tumor immunity.

[0005] TLR7 / 8 are two important pattern recognition receptors, which are located in the endosomal membranes of macrophages, DCs and monocytes. They instinctively sense ssRNA derived from viruses, thereby mediating the activation of immune cells and the release of proinflammatory cytokines. A large number of studies have shown that TLR7 / 8 agonists have anti-tumor activity. Imiquimod, a TLR7 agonist, has been approved for topical application for the treatment of genital warts, superficial basal cell carcinoma and actinic keratosis. Resiquimod, a TLR7 / 8 dual agonist, has been approved for the treatment of cutaneous T-cell lymphoma. However, the side effects induced by systemic administration of TLR7 / 8 agonists limit their use in a wider spectrum of cancers.

[0006] The present invention provides a pharmaceutical composition of a HER2-directed antibody-immunoagonist conjugate (AIAC), which is a new drug for tumor-targeted immunotherapy.

[0007] As is known in the art, the active pharmaceutical ingredient (API) determines the use of the drug, and the formulation of the drug preparation is closely related to the properties of the API. For example, the pH of the preparation may affect the stability of the API, and the excipients not only have an effect on the solubility and dissolution of the API, but also have a significant effect on the characteristics of the API such as permeability and absorbability. Therefore, it is very necessary to develop a formulation for a specific API. Summary of the invention

[0008] In one aspect, an antibody-drug conjugate of formula (II-1) and / or (II-2) is provided:

[0009]

[0010] Where B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is an integer from 1 to 5, j is an integer from 1 to 3, d is an integer from 1 or 2, R 1 and R 2 Each independently selected from hydrogen, -OH, -NH2, -C 1-6 alkyl;

[0011] PL is the payload attached to the B2 part,

[0012] Preferably, PL is resiquimod

[0013] z is an integer from 1 to 4; preferably from 1 to 2;

[0014] A contains V L and V H The antibody, wherein the V L comprising LCDR1 having an amino acid sequence of SEQ ID NO: 17, LCDR2 having an amino acid sequence of SEQ ID NO: 18, and LCDR3 having an amino acid sequence of SEQ ID NO: 19, wherein the V H The invention relates to an antibody comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 20, a HCDR2 having an amino acid sequence of SEQ ID NO: 21, and a HCDR3 having an amino acid sequence of SEQ ID NO: 22; wherein the antibody is modified by introducing a ligase donor substrate recognition sequence. In a preferred embodiment, the antibody comprises a V having an amino acid sequence of SEQ ID NO: 23. L and V having the amino acid sequence of SEQ ID NO: 24 H .

[0015] In one aspect, an antibody-drug conjugate of formula (II-1) and / or (II-2) is provided:

[0016]

[0017]

[0018] Where B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is an integer from 1 to 5, j is an integer from 1 to 3, d is an integer from 1 or 2, R 1 and R 2 Each independently selected from hydrogen, -OH, -NH2, -C 1-6 alkyl;

[0019] PL is the payload attached to the B2 part,

[0020] Preferably, PL is resiquimod

[0021] z is an integer from 1 to 4; preferably from 1 to 2;

[0022] A is an antibody comprising: a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2; or

[0023] A light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4; or

[0024] A light chain having the amino acid sequence of SEQ ID NO: 5 and a heavy chain having the amino acid sequence of SEQ ID NO: 6; or

[0025] A light chain having the amino acid sequence of SEQ ID NO:7 and a heavy chain having the amino acid sequence of SEQ ID NO:8; or

[0026] A light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO: 10; or

[0027] A light chain having the amino acid sequence of SEQ ID NO: 11 and a heavy chain having the amino acid sequence of SEQ ID NO: 12; or

[0028] A light chain having the amino acid sequence of SEQ ID NO: 13 and a heavy chain having the amino acid sequence of SEQ ID NO: 14; or

[0029] A light chain having the amino acid sequence of SEQ ID NO:15 and a heavy chain having the amino acid sequence of SEQ ID NO:16.

[0030] The antibody-immunoagonist conjugate (AIAC) disclosed herein provides a novel tumor-targeted therapy. In vitro experiments have shown that AIAC can induce higher TNFα production than naked unmodified antibodies. In vivo experiments have shown that AIAC has anti-tumor effects.

[0031] In another aspect, a pharmaceutical composition is provided, comprising a prophylactically or therapeutically effective amount of a conjugate of formula (II-1) and / or (II-2), and at least one pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : TNFα inducing activity of the conjugate AC102-6-1-1 and the corresponding naked unmodified antibody Ab0001 (trastuzumab) and the agonist resiquimod in human PBMC and NCI N87 co-culture assays.

[0033] Figure 2: TNFα inducing activity of conjugates AC102-6-1-1, AC102-8-1-1 and their corresponding naked unmodified antibody Ab0001 in human PBMC and NCI N87 co-culture assays.

[0034] Figure 3 : TNFα-inducing activity of AC102-6-1-1 and antibodies in co-cultures of PBMCs with NCI N87 or MDA-MB-468 cells.

[0035] Figure 4 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and HCC1954 cells that highly express HER2.

[0036] Figure 5 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and SK-BR-3 cells that highly express HER2.

[0037] Figure 6 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and BT474 cells that highly express HER2.

[0038] Figure 7 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-cultures of PBMCs with JIMT1 cells that moderately express HER2.

[0039] Figure 8 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs and HER2-low expressing Colo205 cells.

[0040] Fig. 9 : TNFα-inducing activity of AC102-8-1-1 and antibodies in co-culture of PBMCs with HER2-low expressing MDA-MB-468 cells.

[0041] Fig.10 : INF-γ inducing activity of AC102-6-1-1 and antibodies in co-culture of PBMCs and SK-BR-3 cells.

[0042] Fig.11 : INF-γ inducing activity of AC102-6-1-1 and antibodies in co-culture of PBMCs and HCC1954 cells.

[0043] Fig.12: Changes in tumor volume over time in SCID Beige mice of the NCI N87 CDX model after administration of 5 mg / kg of vehicle (PBS pH 6.5), antibody, and conjugates AC102-6-1-1 and AC102-8-1-1, respectively.

[0044] Fig.13 : Changes in tumor volume over time in SCID Beige mice of the NCI N87 CDX model after administration of 0.5 mg / kg, 1 mg / kg and 3 mg / kg AC102-8-1-1, respectively.

[0045] Fig.14 : Changes in tumor volume over time after administration of 5 mg / kg AC102-8-1-1 in SCID Beige mice of the JIMT1 CDX model.

[0046] Fig.15 : Changes in tumor volume over time after administration of 3 mg / kg and 10 mg / kg AC102-6-1-1 in the hHER2-overexpressing MC38 model.

[0047] Fig.16 : Changes in tumor volume over time after administration of 3 mg / kg and 10 mg / kg AC102-8-1-1 in the hHER2-overexpressing MC38 model. DETAILED DESCRIPTION

[0048] Specific embodiments are provided below to illustrate the technical content of this article. Through the content disclosed in the specification, those skilled in the art can easily understand other advantages and effects of this article. This article can also be specifically 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 this article.

[0049] definition

[0050] 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 those technologies generally understood in the art, including variants and equivalent substitutes obvious to those skilled in the art. Although it is believed that the following terms are easily understood by those skilled in the art, it is to better illustrate this article to list the following definitions. When a trade name appears in this article, it refers to the corresponding commodity or its active ingredient. All patents cited herein, published patent applications and publications are incorporated herein by reference.

[0051] When a certain amount, concentration, or other value or parameter is listed in the form of a range, a preferred range, or a preferred upper limit or a preferred lower limit, it should be understood that it is equivalent to specifically disclosing any range formed by any upper limit or preferred value combined with any lower limit or preferred value, regardless of whether the range is explicitly listed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 2 to 20" means that i is any integer from 2 to 20, for example, i can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar expressions should also be understood in a similar manner.

[0052] Unless otherwise specified herein, singular forms such as "a / an" and "the / said" 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.

[0053] When used in conjunction with a numerical variable, the terms "about" and "approximately" generally mean that the value of the variable and all values ​​of the variable are within experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% of the specified value, or a wider range.

[0054] The expression "comprising" or similar expressions "including", "containing" and "having" are open ended and do not exclude additional unlisted elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, as well as other elements, steps or ingredients that are optionally present and 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".

[0055] As used herein, concentration "%" means mass volume concentration in g / ml. For example, a 9% sucrose solution means that 9 g of sucrose is dissolved in a solvent to form 100 ml of solution, which means that the solution contains 9 g of sucrose per 100 ml.

[0056] The amount of buffer herein refers to the total amount of buffer pairs in the buffer system constituting buffer.In some embodiments, molar concentration is used as the unit of the amount of buffer, and its numerical value refers to the molar concentration of buffer pairs in the buffer system of buffer.For example, when using the histidine buffer composed of L-Histidine and L-Histidine hydrochloride, the given concentration (for example, 10mM) of histidine buffer is the combined concentration (for example, L-Histidine is 5mM and L-Histidine hydrochloride is 5mM of L-Histidine and L-Histidine hydrochloride; or L-Histidine is 6mM and L-Histidine hydrochloride is 4mM; or L-Histidine is 3.46mM and L-Histidine hydrochloride is 6.54mM etc.).

[0057] When trehalose is in solid state, it is usually present in the form of trehalose dihydrate. In some embodiments, trehalose dihydrate can be used in preparations, and corresponding amounts of other forms of trehalose can also be used in preparations, and the preparations obtained contain the same concentration of trehalose. When trehalose dihydrate is used to describe the trehalose content in a preparation herein, the preparation contains the amount of trehalose dihydrate or a corresponding amount of trehalose or other forms of trehalose or a combination thereof, and vice versa.

[0058] 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, etc.). A targeting molecule can deliver a payload to a specific site in the body by targeted delivery. A targeting molecule can recognize one or more targets. A specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a payload to a site containing a large 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, and the like.

[0059] As used herein, the term "antibody" is used in a broad sense, and specifically 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 have any subtype (such as 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.

[0060] Monoclonal antibody is used herein to refer to antibodies obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a small amount of possible natural mutations. Monoclonal antibodies are highly specific to a single antigenic site, multiple antigenic sites or different epitopes of the same antigen. The term "monoclonal" refers to the characteristic of an antibody derived from a substantially homogeneous antibody population, and should not be construed as requiring some ad hoc methods to produce the antibody.

[0061] An intact antibody or full-length antibody essentially comprises an antigen-binding variable region and a light chain constant region (C L ) and the heavy chain constant region (C H ), the heavy chain constant region may include C H 1. C H 2. C H 3 and / or C H 4. This depends on the subtype of the antibody. The antigen-binding variable region (also called variable region fragment, Fv fragment) usually contains the light chain variable region (V L ) and the heavy chain variable region (V H ). The constant region can be a constant region with a native sequence (such as a constant region with a human native sequence), or an amino acid sequence variant thereof. The variable region recognizes and interacts with a target antigen. The constant region can be recognized and interacted with by the immune system.

[0062] As used herein, the term "heavy chain constant region (C H )" comprises an amino acid sequence derived from a complete antibody or a full-length antibody heavy chain. The polypeptide containing the heavy chain constant region comprises at least one of the following: C H 1 domain, hinge (e.g., upper, middle and / or lower hinge region) domain, C H 2 domains, C H 3 domains, C H 4 domains or variants or fragments thereof. For example, the antigen-binding polypeptide used herein may comprise: H 1 domain polypeptide chain; containing C H 1 domain, at least a portion of the hinge domain and C H 2-domain polypeptide chain; containing C H 1 domain and C H 3-domain polypeptide chain; containing C H 1 domain, at least a portion of the hinge domain and C H 3 domain polypeptide chain; or containing C H 1 domain, at least a portion of the hinge domain, C H 2 domains and C H 3-domain polypeptide chain.

[0063] As used herein, “CL ” refers to the constant region of the light chain.

[0064] The subunit structures and three-dimensional configurations of the constant regions of the various antibody classes are well known. H The term "C H The "1 domain" includes the first (amino-terminal) constant region domain of the antibody heavy chain. H 1 domain and V H The domains are adjacent and amino terminal to the hinge region of the antibody heavy chain molecule.

[0065] As used herein, “V L ” refers to the variable region of the light chain.

[0066] Antibody fragments may include a portion of an intact antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, V H and C H 1 domain, and an isolated complementarity determining region (CDR). The Fab fragment is an antibody fragment obtained by digesting the full-length immunoglobulin with papain, or a fragment with the same structure produced, for example, by recombinant expression. The Fab fragment contains the light chain (containing the V L and C L ) and another chain, wherein the other chain comprises the variable domain of the heavy chain (V H ) and the constant region domain of the heavy chain (C H 1). F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0 to 4.5, or fragments having the same structure produced by, for example, recombinant expression. F(ab')2 fragments essentially contain two Fab fragments, wherein each heavy chain portion contains a small amount of additional amino acids, including cysteine ​​that forms a disulfide bond connecting the two fragments. Fab' fragments (one heavy chain and one light chain) are fragments that contain half of a F(ab')2 fragment. Antibody fragments may contain, for example, multiple chains joined together via disulfide bonds and / or via peptide linking units. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that is capable of producing an antibody that immunospecifically binds to an antigen when inserted into an antibody framework region (e.g., by replacing the corresponding region).

[0067] In the case where there are two or more definitions of a term used and / or accepted in the art, unless there is a clear statement to the contrary, the definition of the term as used herein is intended to include all such meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of both heavy chain polypeptides and light chain polypeptides. This particular region has been described in Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J.MoI.Biol.196:901-917 (1987), which are incorporated herein by reference in their entirety. The definition of CDR according to Kabat and Chothia includes overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition of CDRs involving antibodies or variants thereof is intended to fall within the scope of the term as defined and used herein. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues comprise a particular CDR based on the variable region amino acid sequence of a given antibody.

[0068] As used herein, "HCDR" refers to the heavy chain complementarity determining region.

[0069] As used herein, "LCDR" refers to the complementarity determining region of the light chain.

[0070] Antibodies according to the present invention can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed by a suitable culture system (e.g., E. coli or mammalian cells). The engineering of antibodies can refer, for example, to the introduction of a ligase-specific recognition sequence at its terminus.

[0071] HER2 refers to human epidermal growth factor receptor 2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family.Herein, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0072] As used herein, the term "targeting molecule-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0073] As used herein, the expression "AIAC concentration" has the same meaning as "AIAC protein concentration", and they can be used interchangeably.

[0074] Small molecule compounds refer to molecules of comparable size to organic molecules commonly used in medicine. The term does not encompass biomacromolecules (e.g., proteins, nucleic acids, etc.), but encompasses low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, and the like. Typically, the molecular weight of a small molecule compound can be, for example, about 100Da to about 2000Da, about 200Da to about 1000Da, about 200Da to about 900Da, about 200Da to about 800Da, about 200Da to about 700Da, about 200Da to about 600Da, about 200Da to about 500Da.

[0075] An immune agonist refers to an agonist that can induce or enhance an immune response to a tumor, such as by activating immune cells, including but not limited to DCs, B cells, macrophages, NK cells, and T cells. Non-limiting examples of immune agonists such as TLR agonists include but are not limited to agonists of TLR7 and / or TLR8 and / or TLR9 known in the art (e.g., imiquimod, resiquimod, 852A, and VTX-2337) and STING agonists (e.g., ADU-S100 and MK-1454).

[0076] A linking unit refers to a functional group that covalently bonds two or more moieties in a compound or substance. For example, a linking unit can be used to covalently link a targeting molecule and / or a derivative group of a payload.

[0077] The spacer is a structure that is located between different structural modules and can separate these structural modules in space. The definition of the spacer is not limited by whether it has a certain function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, amino acid derivatives or analogs. "Spacer sequence" refers to an amino acid sequence used as a spacer, and examples thereof include, but are not limited to, single amino acids such as Leu, Gln, etc.; sequences containing multiple amino acids, such as sequences containing two amino acids such as GA, etc., or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. Other examples of spacers include, for example, self-resection spacers, such as PABC (p-benzyloxycarbonyl), and the like.

[0078] 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. The alkyl group can contain 1 to 20 carbon atoms and is related to C1-C 20Alkyl groups, such as C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C3-C6 alkyl groups. Non-limiting examples of alkyl groups 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 the like, or isomers thereof. A divalent free radical refers to a group obtained from a corresponding monovalent free radical by removing a hydrogen atom from a carbon atom with free valence electrons. A divalent free radical has two attachment sites connected to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a straight or branched saturated divalent hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H8-), styryl (-CH2-), styryl (-CH4-), styryl (-CH6-), styryl (-CH8-), styryl (-CH8-), styryl (-CH2-), styryl (-CH6-), styryl (-CH8-), styryl (-CH8-), styryl (-CH2-), styryl (-CH2-), styryl (-CH6-), styryl (-CH8 ... 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, and the like.

[0079] As used herein, when a group is combined with another group, the connection of these groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by this combination can be connected to other parts of the molecule via any suitable atom in the structure, preferably via a specified chemical bond. For example, when describing C 1-4 When an alkylene group is combined with one of the groups including -CH2-, -NH-, -(CO)-, -NH(CO)-, -(CO)NH-, the C 1-4 The alkylene group can form a linear connection with the above groups, such as C 1-4 Alkylene -CH2-, C 1-4 Alkylene-NH-, C 1-4 Alkylene-(CO)-, C 1-4 Alkylene -NH(CO)-, C 1-4 Alkylene -(CO)NH-, -CH2-C 1-4 Alkylene, -NH-C 1-4 Alkylene, -(CO)-C 1-4 Alkylene, -NH(CO)-C 1-4 Alkylene, -(CO)NH-C1-4 The resulting divalent structure can be further linked to other parts of the molecule.

[0080] The term "acidic buffer" refers to a buffer having a pH < 7, for example, a buffer having a pH of 4.0 to 6.0, pH = 4.0, pH = 4.5, pH = 5.0, pH = 5.5 or pH = 6.0.

[0081] "Stabilizer" refers to a chemical that can increase the stability of solutions, colloids, solids, mixtures, etc. and has the functions of delaying reactions, maintaining chemical equilibrium, reducing surface tension, and preventing light, thermal or oxidative decomposition. For example, the stabilizer can be sucrose, trehalose dihydrate, sorbitol, and combinations thereof.

[0082] As used herein, "surfactant" refers to a substance that can significantly change the interfacial state of a solution system by adding a small amount. Examples of surfactants include, but are not limited to, Tween 20 (PS20), Tween 80 (PS 80), Spandex 20 (SP20), Spandex 80 (SP 80). In some embodiments, the surfactant can be used as a stabilizer.

[0083] As used herein, "Tm" refers to the melting temperature, which indicates the temperature at which a protein begins to denature.

[0084] As used herein, "Tagg" refers to aggregation temperature, which indicates the temperature at which a protein begins to aggregate.

[0085] Compound of formula (I')

[0086] In one aspect, a pharmaceutical composition comprising AIAC is provided, wherein the AIAC comprises a compound of formula (I') (formula (I'-1) or formula (I'-2) or a mixture thereof):

[0087]

[0088] in,

[0089] B2 is –(CH2) k (CO)-NH-(C2H4-O) j -R 3 or –(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 ;

[0090] R 1 Selected from hydrogen, -OH, -NH2 and -C 1-6 alkyl;

[0091] R 2 Selected from hydrogen, -OH, -NH2 and -C 1-6 alkyl;

[0092] R 3 is a group that can be dissociated when reacting with a group in the payload;

[0093] k is an integer of 1 to 5, j is an integer of 1 to 3, and d is 1 or 2, each independently.

[0094] In one embodiment, R 1 and R 2 are independently hydrogen or C 1-6 In a preferred embodiment, R 1 and R 2 are independently hydrogen or C 1-6 In a more preferred embodiment, R 1 and R 2 All are hydrogen.

[0095] In one embodiment, B2 in formula (I'-1) and (I'-2) are the same.

[0096] In one embodiment, k is 2. In one embodiment, j is 1.

[0097] In one embodiment, d is 1.

[0098] In one embodiment, the terminal group R 3 In one embodiment, R 3 Hydroxyl or

[0099] In one embodiment, the terminal group R 3 represents a structural moiety that will not appear in the product molecule obtained by the reaction of B2 with the payload, so that in the linker unit-payload intermediate (see below), the structural moiety corresponding to B2 is one of the divalent groups or a combination of two or more divalent groups.

[0100] Thiosuccinimide is unstable under physiological conditions and is prone to reverse Michael addition, resulting in cleavage at the coupling site. In addition, when another thiol compound is present in the system, thiosuccinimide can also undergo thiol exchange with other thiol compounds. Both reactions can cause the payload to fall off and lead to toxic side effects. In this paper, the ring-opened succinimide structure No reverse Michael addition or thiol exchange is performed, so the product is more stable. The method of the ring-opening reaction can be found in WO2015165413A1.

[0101] Specific embodiments of the compound of formula (I')

[0102] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-NH-(C2H4-O) j -H, k is 2, j is 1, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-6):

[0103]

[0104] In one embodiment, in the compound of formula (I'), B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -R 3 , k is 2, d is 1, R 1 and R 2 is hydrogen, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-8):

[0105]

[0106] R 3 is a group that can be detached when reacting with a group in the payload; in one embodiment, R 3 Hydroxyl or

[0107] It should be understood that when there are two or more R x (x is 1, 2, 3, 4, 5, 6, 7, etc.), each R x In some embodiments, "x" in the molecule is represented with or without an additional prime (') or multiple primes (such as ", "', "", etc.), for example, R, R 1 '、R 1 ”、R 1 ”'、R 2 '、R 2 ”、R 2 ”' etc. Other R x Such as R 3 It should be understood in a similar way.

[0108] Compounds of formula (I') as linking units

[0109] In one embodiment, the reactive group contained in B2 can be used for covalent coupling with a payload containing another reactive group, so that the compound of formula (I') carries the payload.

[0110] In another embodiment, the ligase recognition sequence GGG (G is glycine) contained in formula (I') can be used for coupling with the corresponding ligase recognition sequence LPETGG (SEQ ID NO: 28) by a ligase.

[0111] Therefore, the compound of formula (I') can be used as a linker unit that can be linked to a targeting molecule (such as an antibody or an antigen-binding fragment thereof) and / or a payload.

[0112] Those skilled in the art can synthesize the linking unit by conventional solid phase or liquid phase methods.

[0113] Compounds of formula (I') carrying a payload

[0114] The reactive group contained in B2 is covalently coupled to a payload containing another reactive group to obtain a compound of formula (I') carrying the payload.

[0115] In yet another aspect, a pharmaceutical composition comprising AIAC is provided, wherein the AIAC comprises a compound having a structure of formula (II'-1) or (II'-2) or a mixture thereof,

[0116]

[0117] in

[0118] PL is the payload attached to the B2 moiety of the compound of formula (I').

[0119] Payload

[0120] In this article, the payload can be selected from small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptide mimetics and proteins. In one embodiment, the payload is selected from small molecule compounds, nucleic acid molecules and tracer molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from cytotoxins and fragments thereof. In a more preferred embodiment, the payload is selected from immune agonists and fragments thereof.

[0121] In one embodiment, the immune agonist is selected from a TLR agonist, such as a TLR agonist (eg, a TLR 7 agonist, a TLR 8 agonist, a TLR 7 / 8 agonist) and a STING agonist. In one embodiment, the immune agonist is selected from a TLR agonist.

[0122] In one embodiment, the immune agonist is resiquimod:

[0123]

[0124] In one embodiment, the linker unit and the payload are attached via the reactive groups as defined above using any reaction known in the art including but not limited to condensation reactions, nucleophilic additions, electrophilic additions, and the like.

[0125] In one embodiment, the payload is an immunoagonist and the antibody-immunoagonist conjugate (coded as LPx) is one of the compounds shown in the following table.

[0126] Compound of formula (II') Connection unit PL (immune agonist) LP102-6-1 LN102-6 Recimotte LP102-8-1 LN102-8 Recimotte

[0127] Preparation of compounds of formula (I') carrying a payload

[0128] In one embodiment, the linker unit and the payload are attached via the reactive groups as defined above using any reaction known in the art including but not limited to condensation reactions, nucleophilic additions, electrophilic additions, and the like.

[0129] Compound of formula (III')

[0130] In one aspect, a compound of formula (III') is provided:

[0131]

[0132] wherein B2 is as defined in formula (I').

[0133] In one embodiment, the compound of formula (III') can be used to prepare the compound of formula (I') carrying a payload by the following route:

[0134]

[0135] The compounds of formula (III') carrying the payload can be converted to compounds of formula (I') carrying the payload using methods known in the art or as described herein. For example, one or more steps can be performed to convert the structural fragments (LU102) is introduced into the maleimide ring of the compound of formula (III') carrying the payload, and then the resulting molecule containing the succinimide moiety can undergo a ring-opening reaction to open the succinimide ring and obtain the compound of formula (I') carrying the payload (i.e., the compound of formula (II')). In one embodiment, LU102 is introduced into the compound of formula (III') carrying the payload by reacting the maleimide group in the compound of formula (III') with the thiol group of LU102.

[0136] Conjugates and their preparation

[0137] In another aspect, a conjugate having the structure of formula (II-1) and / or (II-2) is provided.

[0138]

[0139] in,

[0140] PL is a payload attached to the B2 moiety of the compound of formula (I');

[0141] z is an integer from 1 to 4; preferably from 1 to 2;

[0142] A is a targeting molecule.

[0143] In one embodiment, the payload is an immunoagonist as defined above. In one embodiment, the conjugate is an antibody-immunoagonist conjugate.

[0144] Targeting molecules

[0145] In one embodiment, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0146] In some embodiments herein, the target recognized by the targeting molecule (such as an antibody or antigen-binding fragment thereof) is ErbB2 / HER2.

[0147] In one embodiment, the targeting molecule is an anti-human HER2 antibody or an antigen-binding fragment thereof. Examples of anti-human HER2 antibodies include, but are not limited to, trastuzumab. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2, and the antibody is approved for the treatment of HER2-positive breast cancer and gastric cancer.

[0148] In a preferred embodiment, the anti-human HER2 antibody is one or more of an engineered anti-HER2 antibody selected from trastuzumab.

[0149] 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 scFv, a mini antibody, a double antibody, and a nano antibody. In order to couple with a compound of formula (I'), formula (II'-1) or formula (II'-2), the targeting molecule herein may include a modified portion to be connected with a compound of formula (I'), formula (II'-1) or formula (II'-2). The introduction position of such a modified portion is not limited, for example, when the targeting molecule is an antibody, its introduction position may be, but is not limited to, located at the C-terminus and / or N-terminus of the antibody heavy chain and / or light chain.

[0150] In one embodiment, the targeting molecule herein is an antibody or an antigen-binding fragment thereof, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus and / or N-terminus of the antibody heavy chain and / or light chain, which, for example, include a ligase recognition sequence. In another embodiment, the terminal modifications may also include a spacer Sp1 containing 2 to 10 amino acids, wherein the antibody, Sp1, and ligase recognition sequences are sequentially connected. In a specific embodiment, Sp1 is a spacer sequence selected from GA, GGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), GGGGSGGGGSGGGGS (SEQ ID NO: 27), especially GA.

[0151] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof includes three types: wild type (LC); C-terminally modified light chain (LCCT), which is modified by directly introducing the ligase recognition sequence LPETGG; and C-terminally modified light chain (LCCT L ), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPETGG. The heavy chain of the antibody or its antigen-binding fragment includes 3 types: wild type (HC); C-terminally modified heavy chain (HCCT), which is modified by directly introducing a ligase recognition sequence LPETGG; and C-terminally modified heavy chain (HCCT). L ), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPETGG. When z in the compound of formula (II-1) and / or formula (II-2) is 1 or 2, the combination of the above heavy chain and light chain can form 8 preferred antibody molecules, see the amino acid sequence table.

[0152] In one embodiment, the targeting molecule herein is a L and V H The antibody, wherein the V L comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 17 (RASQDVNTAVA), a LCDR2 having an amino acid sequence of SEQ ID NO: 18 (SASFLYS), and a LCDR3 having an amino acid sequence of SEQ ID NO: 19 (QQHYTTPPT), wherein V HThe antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 20 (DTYIH), a HCDR2 having an amino acid sequence of SEQ ID NO: 21 (RIYPTNGYTRYADSVKG), and a HCDR3 having an amino acid sequence of SEQ ID NO: 22 (WGGDGFYAMDY). In one embodiment, the antibody is modified by introducing a ligase donor substrate recognition sequence. In one embodiment, the antibody comprises a V having an amino acid sequence of SEQ ID NO: 23. L and V having the amino acid sequence of SEQ ID NO: 24 H .

[0153] In one embodiment, the sequences of the CDRs and variable domains are defined according to the Kabat numbering system. In one embodiment, the targeting molecule herein is an antibody comprising the following items: a light chain having an amino acid sequence of SEQ ID NO: 1 and a heavy chain having an amino acid sequence of SEQ ID NO: 2. In one embodiment, the targeting molecule herein is an antibody comprising the following items: a light chain having an amino acid sequence of SEQ ID NO: 3 and a heavy chain having an amino acid sequence of SEQ ID NO: 4. In one embodiment, the targeting molecule herein is an antibody comprising the following items: a light chain having an amino acid sequence of SEQ ID NO: 5 and a heavy chain having an amino acid sequence of SEQ ID NO: 6. In one embodiment, the targeting molecule herein is an antibody comprising the following items: a light chain having an amino acid sequence of SEQ ID NO: 7 and a heavy chain having an amino acid sequence of SEQ ID NO: 8. In one embodiment, the targeting molecule herein is an antibody comprising the following items: a light chain having an amino acid sequence of SEQ ID NO: 9 and a heavy chain having an amino acid sequence of SEQ ID NO: 10. In one embodiment, the targeting molecule herein is an antibody comprising: a light chain having an amino acid sequence of SEQ ID NO: 11 and a heavy chain having an amino acid sequence of SEQ ID NO: 12. In one embodiment, the targeting molecule herein is an antibody comprising: a light chain having an amino acid sequence of SEQ ID NO: 13 and a heavy chain having an amino acid sequence of SEQ ID NO: 14. In one embodiment, the targeting molecule herein is an antibody comprising: a light chain having an amino acid sequence of SEQ ID NO: 15 and a heavy chain having an amino acid sequence of SEQ ID NO: 16.

[0154] The conjugates herein may also comprise a payload. The payload is as described above.

[0155] Specific embodiments of the conjugate

[0156] In one embodiment, in formula (II-1) and / or formula (II-2), B2 is -(CH2) k (CO)-NH-(C2H4-O) j -, k is 2, j is 1, and the structure of the conjugate (Formula AC102-6) is as follows:

[0157]

[0158] In one embodiment, in formula (II-1) and / or formula (II-2), B2 is -(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is 2, d is 1, R 1 and R 2 is hydrogen, and the structure of the conjugate (Formula AC102-8) is as follows:

[0159]

[0160] Preparation of conjugates

[0161] The conjugate herein can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by coupling a targeting molecule with a compound of formula (I') carrying a payload by a site-specific coupling method catalyzed by a ligase, wherein the targeting molecule is modified by a ligase recognition sequence (such as a ligase donor substrate recognition sequence). The method comprises steps A and B.

[0162] Step A. Preparation of Linker-Payload Intermediates

[0163] In a preferred embodiment, B2 in the compound of formula (I') is covalently linked via a reactive group to a payload containing a corresponding reactive group, each of which is defined above.

[0164] The linker-payload intermediate prepared using the compound of formula (I') herein has a well-defined structure, well-defined composition and high purity, so when coupled with an antibody, less impurities or no other impurities are introduced. When this intermediate is used for ligase-catalyzed site-specific coupling with a modified antibody containing a ligase recognition sequence, a homogeneous ADC with highly controllable quality is obtained.

[0165] Step B. Attaching the targeting molecule to the compound of formula (I') carrying the payload

[0166] The targeting molecule herein can be coupled to a compound of formula (I') carrying a payload (i.e., a compound of formula (II')) by any method known in the art. For example, a site-specific coupling technique catalyzed by a ligase is used, and the targeting molecule and the compound of formula (I') carrying a payload are connected to each other via a ligase-specific recognition sequence of a substrate. In one embodiment, the targeting molecule is an antibody having a terminal modification based on a recognition sequence introduced at the C-terminus of a light chain and / or a heavy chain, and the targeting molecule is coupled to a compound of formula (II') under the catalysis of a wild-type or optimized engineered ligase or any combination thereof and under suitable catalytic reaction conditions.

[0167] In a specific embodiment, the ligase is sortase A, and the coupling reaction can be represented by the following scheme:

[0168]

[0169] The triangle and pentagon represent either of the following: a portion of an antibody or a portion of a compound of formula (II'). n is as defined above. n When coupled to α-Glycine (which is the corresponding recognition sequence of the acceptor substrate), the upstream peptide bond of the glycine in the LPETGG sequence is cleaved by sortase A, and the resulting intermediate reacts with G n The free N-terminus of 1,2-dihydropyrimidine ... n (SEQ ID NO:30). Sequence G n and LPETGG are as defined above.

[0170] Specific conjugate table

[0171] In one embodiment, the payload is an immunostimulant. In one embodiment, the antibody-immunostimulant conjugate is shown in the following table:

[0172] AIAC Mode PL (immune agonist) A (targeting molecule) AC102-6-1-1 AC102-6 Recimotte <![CDATA[Ab0001-LCCT L -HC]]> AC102-8-1-1 AC102-8 Recimotte <![CDATA[Ab0001-LCCT L -HC]]>

[0173] Pharmaceutical compositions and pharmaceutical preparations

[0174] Another aspect of the present invention provides a pharmaceutical composition comprising a preventively or therapeutically effective amount of the conjugate described herein, and at least one pharmaceutically acceptable carrier.

[0175] The pharmaceutical composition herein can be administered in any manner, as long as it achieves the effect of preventing, alleviating or curing the symptoms of humans or animals. For example, various suitable dosage forms, especially injections, such as lyophilized powder for injection, injection or sterile powder for injection can be prepared according to the administration route.

[0176] The term "pharmaceutically acceptable" means that when in contact with patient tissues, it does not produce undue toxicity, irritation or allergic response, etc., has a reasonable benefit-risk ratio, and is effective for the intended use within the scope of normal medical judgment.

[0177] In one embodiment, the pharmaceutical composition herein has a drug-to-antibody ratio (DAR) of an integer or non-integer of 1 to 4, such as about 1 to 4, about 1 to 3.5, about 1 to 3, about 1 to 2.5, preferably about 1 to 2. In one embodiment, the pharmaceutical composition herein has a DAR of about 1.5 to about 2, preferably about 1.6 to about 2, more preferably about 1.7 to about 2.

[0178] In one embodiment, the pharmaceutical composition comprises AIAC, a buffer, and one or more stabilizers.

[0179] In one embodiment, the buffer in the pharmaceutical composition comprises one or more of sodium acetate, sodium citrate, sodium phosphate, histidine, Tris and glycine. In some embodiments, the buffer is selected from citrate buffer, phosphate buffer, histidine buffer and acetate buffer. In some embodiments, the citrate buffer comprises citric acid and sodium citrate, the histidine buffer comprises L-histidine and L-histidine hydrochloride, and the acetate buffer comprises acetic acid and sodium acetate. In another embodiment, the buffer comprises a succinate buffer. In one embodiment, the succinate buffer comprises succinic acid and sodium succinate.

[0180] In some embodiments, the concentration of the buffer is about 10mM to 40mM. In a preferred embodiment, the concentration of the buffer is about 10mM, about 14mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 26mM, about 30mM, about 33mM, about 37mM or about 40mM. In a preferred embodiment, the concentration of the buffer is about 20mM. In some embodiments, the concentration of the buffer is about 15mM to 25mM. In a preferred embodiment, the concentration of the buffer is about 20mM. In one embodiment, the buffer in the pharmaceutical composition comprises one or more of the following: about 15mM to 25mM sodium acetate, sodium citrate, sodium phosphate, histidine, Tris and glycine. In some embodiments, the concentration of the buffer is about 15mM to 25mM. In some embodiments, the buffer comprises about 15 mM to 25 mM citrate buffer, about 15 mM to 25 mM phosphate buffer, about 15 mM to 25 mM histidine buffer, or about 15 mM to 25 mM acetate buffer.

[0181] In one embodiment, the buffer in the pharmaceutical composition comprises one or more of sodium acetate, sodium citrate, and histidine. In some embodiments, the buffer comprises one or more of a citrate buffer, a histidine buffer, and an acetate buffer. In some embodiments, the buffer is a citrate buffer, a histidine buffer, or an acetate buffer.

[0182] In one embodiment, the buffer in the pharmaceutical composition comprises about 15 mM to 25 mM of one or more of sodium acetate, sodium citrate, and histidine.

[0183] In one embodiment, the buffer in the pharmaceutical composition comprises about 15mM to 25mM histidine. In some embodiments, the buffer is about 15mM to 25mM histidine buffer, such as about 15mM, about 16.5mM, about 17mM, about 18.3mM, about 19mM, about 21mM, about 22mM, about 23mM or about 25mM.

[0184] In one embodiment, the buffer in the pharmaceutical composition comprises about 20 mM histidine. In some embodiments, the buffer in the pharmaceutical composition is about 20 mM histidine buffer.

[0185] In one embodiment, the buffer pH in the pharmaceutical composition is about 4.0 to 6.0, such as about 4.0, about 4.4, about 4.8, about 5.0, about 5.2, about 5.4, about 5.7, or about 6.0.

[0186] In one embodiment, the pH of the buffer in the pharmaceutical composition is about 5.5.

[0187] In one embodiment, the stabilizer in the pharmaceutical composition comprises one or more of the following: sucrose, trehalose dihydrate, trehalose, and sorbitol.

[0188] In one embodiment, the stabilizer in the pharmaceutical composition comprises one or more of the following: about 5% to 15% (W / V) of sucrose, trehalose dihydrate, trehalose, and sorbitol. In some embodiments, the concentration of the stabilizer is about 5%, 5.8%, 6.5%, 7.3%, 7.9%, 8.2%, 8.5%, 9%, 9.9%, 10%, 11%, 12%, 13%, 14% or 15% (W / V).

[0189] In one embodiment, the stabilizer in the pharmaceutical composition comprises one or more of the following: about 9% (W / V) sucrose, trehalose dihydrate, trehalose, and sorbitol.

[0190] In one embodiment, the stabilizer in the pharmaceutical composition comprises sucrose.

[0191] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 5% to 15% (W / V) sucrose. In some embodiments, the concentration of sucrose is about 5%, 5.8%, 6.5%, 7.3%, 7.9%, 8.2%, 8.5%, 9%, 9.9%, 10%, 11%, 12%, 13%, 14% or 15%.

[0192] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 9% (W / V) sucrose.

[0193] In some embodiments, the drug also includes a surfactant. In some embodiments, the surfactant is Tween 20 (PS20) and / or Tween 80 (PS80). In some embodiments, the concentration of the surfactant is about 0.05mg / ml to 0.5mg / ml. In one embodiment, the surfactant in the pharmaceutical composition includes about 0.05mg / ml to 0.5mg / mlPS20. In some embodiments, the concentration of PS20 is about 0.05mg / ml, about 0.1mg / ml, about 0.15mg / ml, about 0.2mg / ml, about 0.24mg / ml, about 0.25mg / ml, about 0.3mg / ml, about 0.37mg / ml, about 0.4mg / ml, about 0.48mg / ml or about 0.5mg / ml.

[0194] In one embodiment, the surfactant in the pharmaceutical composition comprises 0.2 mg / ml PS20. In another embodiment, the surfactant in the pharmaceutical composition comprises 0.4 mg / ml PS20.

[0195] In some embodiments, the pharmaceutical composition comprises: about 10mM to 40mM buffer, pH 4.0 to 6.0; about 5% to 15% (W / V) stabilizer; about 0.05mg / ml to 0.5mg / ml surfactant. In some embodiments, the pharmaceutical composition comprises: about 15mM to 25mM buffer, pH 4.0 to 6.0; about 5% to 15% (W / V) stabilizer; about 0.05mg / ml to 0.5mg / ml surfactant. In one embodiment, the pharmaceutical composition comprises 10mM to 40mM histidine buffer, pH 4.0 to 6.0; 5% to 15% (W / V) sucrose; 0.05mg / ml to 0.5mg / ml PS20. In one embodiment, the pharmaceutical composition comprises 15 mM to 25 mM histidine buffer, pH 4.0 to 6.0; 5% to 15% (W / V) sucrose; 0.05 mg / ml to 0.5 mg / ml PS20.

[0196] In one embodiment, the pharmaceutical composition comprises about 20 mM histidine buffer, pH about 5.5; about 9% (W / V) sucrose; about 0.2 mg / ml PS20.

[0197] In one embodiment, the AIAC protein concentration in the pharmaceutical composition is about 18 mg / ml to 110 mg / ml, such as about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 24 mg / ml, about 27 mg / ml, about 30 mg / ml, about 37 mg / ml, about 40 mg / ml, about 47 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 62 mg / ml, about 66 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 82 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, about 105 mg / ml or about 110 mg / ml. In a preferred embodiment, the AIAC protein concentration in the pharmaceutical composition is about 62.2 mg / ml. In another preferred embodiment, the AIAC protein concentration in the pharmaceutical composition is about 82.2 mg / ml. In another preferred embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 100.4 mg / ml.

[0198] In one embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 18 mg / ml to 66 mg / ml, such as about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 24 mg / ml, about 27 mg / ml, about 30 mg / ml, about 37 mg / ml, about 40 mg / ml, about 47 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml or about 66 mg / ml. In one embodiment, the concentration of AIAC in the pharmaceutical composition is about 18 mg / ml to 66 mg / ml, such as about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 24 mg / ml, about 27 mg / ml, about 30 mg / ml, about 37 mg / ml, about 40 mg / ml, about 47 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml or about 66 mg / ml.

[0199] In one embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 20 mg / ml to 60 mg / ml. In one embodiment, the concentration of AIAC in the pharmaceutical composition is about 20 mg / ml to 60 mg / ml.

[0200] In one embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 20 mg / ml. In one embodiment, the concentration of AIAC in the pharmaceutical composition is about 20 mg / ml.

[0201] In one embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 40 mg / ml. In one embodiment, the concentration of AIAC in the pharmaceutical composition is about 40 mg / ml.

[0202] In one embodiment, the concentration of AIAC protein in the pharmaceutical composition is about 60 mg / ml. In one embodiment, the concentration of AIAC in the pharmaceutical composition is about 60 mg / ml.

[0203] In some embodiments, the formulation comprises: 20 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.5), 9% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 40 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.5), 9% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 60 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.5), 5% sucrose, and 0.2 mg / ml Tween 20.

[0204] In some embodiments, the formulation comprises: 62.2 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.65), 7.5% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 82.2 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.68), 7.5% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 100.4 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.69), 7.5% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 62.2 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.65), 9% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 82.2 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.68), 9% sucrose, and 0.2 mg / ml Tween 20. In some embodiments, the formulation comprises: 100.4 mg / ml AIAC protein, 20 mM histidine buffer (pH 5.69), 9% sucrose, and 0.2 mg / ml Tween 20.

[0205] In some embodiments, the AIAC has a structure of formula (II-1), (II-2) or a mixture thereof. In some embodiments, the AIAC is AC102-6-1-1. In some embodiments, the AIAC is AC102-8-1-1.

[0206] Treatments and uses

[0207] The conjugates herein can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to conjugate treatment include those characterized by specific tumor-associated antigens or cell surface receptors, and these tumors will be recognized by the targeting molecule in the conjugate and may be affected by the immune cell activation activity of the agonist in the conjugate.

[0208] Therefore, in yet another aspect, there is also provided use of the conjugate herein or the pharmaceutical composition herein in the manufacture of a medicament for preventing, alleviating or treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0209] In another aspect, provided is the conjugate or pharmaceutical composition for preventing, alleviating or treating a tumor or an autoimmune disease.

[0210] In yet another aspect, provided is a method for preventing, alleviating or treating tumors or autoimmune diseases, the method comprising administering an effective amount of the conjugate or pharmaceutical composition described herein to an individual in need thereof.

[0211] In a preferred embodiment, the conjugate herein formed by coupling anti-human HER2 antibodies and payloads can specifically bind to HER2 on the surface of tumor cells and selectively kill tumor cells expressing HER2. In another preferred embodiment, there is provided the use of the conjugate herein or the pharmaceutical composition herein in the manufacture of a drug for preventing, alleviating or treating a disease, disorder or condition, the disease, disorder or condition being selected from HER2 positive tumors. In a more preferred embodiment, the disease, disorder or condition is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial carcinoma, and the like.

[0212] The dosage of the conjugate administered to a subject can be adjusted to a considerable degree. The dosage may vary depending on the specific route of administration and the needs of the subject, and can be determined by a healthcare professional.

[0213] Beneficial Effects

[0214] This article utilizes a connecting unit with a unique structure and uses a ligase to catalyze the coupling of an antibody (i.e., an anti-HER2 antibody) and an agonist. The conjugate herein has good uniformity, high activity, and high selectivity. Specifically, intracellular metabolites show significantly reduced cell proliferation toxicity to cells that low-express or do not express the target antigen. In addition, the toxicity of the connecting unit-agonist intermediate is much lower than that of the free agonist, so the harmfulness of the drug manufacturing process is less, which is conducive to industrial production. And the preparation herein ensures that the ADC and AIAC herein have good physicochemical properties and biological properties.

[0215] The conjugate herein achieves at least one of the following technical effects:

[0216] High inhibitory activity on target cells, or strong killing effect on target cells.

[0217] Good physicochemical properties (e.g. solubility, physical and / or chemical stability).

[0218] Good pharmacokinetic properties (e.g., good stability in plasma, adequate half-life and duration of action).

[0219] Good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider therapeutic window), etc.

[0220] The drug can prevent patients from developing resistance to HER2-targeted therapy and activate myeloid cells, thereby enhancing innate immune responses and adaptive immune responses. The drug can overcome the low response rate of current HER2-targeted therapy. And the preparation herein can ensure that the AIAC herein achieves at least one or more of the above-mentioned technical effects.

[0221] Example

[0222] Preparation Example

[0223] In order to more clearly illustrate the purpose and technical solutions, the present invention is further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of this invention. The specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0224] Instruments, materials, and reagents

[0225] Unless otherwise specified, the instruments and reagents used in the examples are commercially available and the reagents were used directly without further purification.

[0226] MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer

[0227] HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0228] Semi-preparative HPLC: Lisure HP plus 50D

[0229] Flow cytometry: CytoFLEX S

[0230] HIC-HPLC: Butyl-HIC; mobile phase A: 25mM PB, 2M (NH4)2SO4, pH 7.0; mobile phase B: 25mM PB, pH 7.0; flow rate: 0.8ml / min; acquisition time: 25min; injection volume: 20μg; column temperature: 25℃; detection wavelength: 280nm; sample chamber temperature: 8℃.

[0231] SEC-HPLC: column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID×300mm, 5μm; mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2; flow rate: 0.5ml / min; acquisition time: 30 minutes; injection volume: 50μl; column temperature: 25℃; detection wavelength: 280nm; sample plate temperature: 8℃.

[0232] CHO was obtained from Thermo Fisher Scientific; pcDNA3.3 was obtained from Life Technology; HEK293F was obtained from Prejin; PEIMAX transfection reagent was obtained from Polyscience; MabSelect Sure ProA was obtained from GE; Capto SImpAct 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; BT474 cells were obtained from ATCC CAT#HTB-20; JIMT1 cells were obtained from DSMZ CAT#ACC589; Colo205 cells were obtained from ATCC CAT#CRL-222; MC38Hher2 mouse colorectal cancer cells were obtained from Biocytogen; NUGC4 human gastric cancer cells were obtained from JCRB CAT#JCRB0834; NCI-N87 cells (ATCC CAT#CRL-5822); MDA-MB-468 was obtained from ATCC CAT#HTB-132.

[0233] Example 1 Construction of antibody expression vector, antibody expression, purification and identification

[0234] 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L -HC production

[0235] Antibody Ab0001-LCCT L The expression plasmid of -HC (light chain SEQ ID NO: 1, heavy chain SEQ ID NO: 2) was constructed as follows. Antibody Ab0001-LCCT L -HC sequence: Based on the amino acid sequence of trastuzumab, GALPETGG (SEQ ID NO: 29) was introduced at the C-terminus of the light chain, where LPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence. The plasmid was transfected into CHO cells, a cell population was established and a high-expressing cell population was screened, and these cells were cultured in a 5L to 10L reactor with reference to the culture method of trastuzumab, and the supernatant was collected.

[0236] 1.2 Antibody Ab0001-LCCT L Purification of -HC

[0237] Ab0001-LCCT L Purification of -HC was performed according to standard methods using a combination of MabSelect affinity chromatography and Sepharose S cation exchange chromatography, and the purified product was dissolved in original trastuzumab drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) and frozen in small aliquots.

[0238] 1.3 Antibody Ab0001-LCCT L -HC quality control

[0239] The purified antibody Ab0001-LCCT was detected by SDS-PAGE. L -HC has a purity of 98.5%; the content of high molecular weight polymers in the sample is less than 0.4% as determined by SEC-HPLC; and the endotoxin content is less than 0.098EU / mg.

[0240] 1.4 Preparation of other modified anti-human antibodies

[0241] According to a similar method, terminal modifications based on the ligase recognition sequence were introduced into the C-terminus of the trastuzumab light chain and / or heavy chain, respectively, to obtain modified antibodies.

[0242] The anti-human HER2 antibodies modified with 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.

[0243] Table 1 Modified anti-human HER2 antibodies

[0244] sequence Sequence introduced at the end <![CDATA[Ab0001-LCCT L -HC light chain]]> SEQ ID NO:1 GALPETGG <![CDATA[Ab0001-LCCT L -HC heavy chain]]> SEQ ID NO:2 -* Ab0001-LC-HCCT light chain SEQ ID NO:3 - Ab0001-LC-HCCT heavy chain SEQ ID NO:4 LPETGG <![CDATA[Ab0001-LC-HCCT L Light Chain]]> SEQ ID NO:5 - <![CDATA[Ab0001-LC-HCCT L Heavy Chain]]> SEQ ID NO:6 GALPETGG Ab0001-LCCT-HC light chain SEQ ID NO:7 LPETGG Ab0001-LCCT-HC Heavy Chain SEQ ID NO:8 - Ab0001-LCCT-HCCT light chain SEQ ID NO:9 LPETGG Ab0001-LCCT-HCCT heavy chain SEQ ID NO:10 LPETGG <![CDATA[Ab0001-LCCT-HCCT L Light Chain]]> SEQ ID NO:11 LPETGG <![CDATA[Ab0001-LCCT-HCCT L Heavy Chain]]> SEQ ID NO:12 GALPETGG <![CDATA[Ab0001-LCCT L -HCCT light chain]]> SEQ ID NO:13 GALPETGG <![CDATA[Ab0001-LCCT L -HCCT heavy chain]]> SEQ ID NO:14 LPETGG <![CDATA[Ab0001-LCCT L -HCCT L Light Chain]]> SEQ ID NO:15 GALPETGG <![CDATA[Ab0001-LCCT L -HCCT L Heavy Chain]]> SEQ ID NO:16 GALPETGG

[0245] *: “-” indicates no terminal modification.

[0246] CDR sequences, V L and V H Listed in Table 2.

[0247] Table 2 CDR, V L and V H sequence

[0248] sequence LCDR1 SEQ ID NO:17 LCDR2 SEQ ID NO:18 LCDR3 SEQ ID NO:19 HCDR1 SEQ ID NO:20 HCDR2 SEQ ID NO:21 HCDR3 SEQ ID NO:22 <![CDATA[V L ]]> SEQ ID NO:23 <![CDATA[V H ]]> SEQ ID NO:24

[0249] Example 2 Preparation of intermediate

[0250] 2.1 Preparation of the connection unit

[0251] The connection unit is composed of

[0252]

[0253] The linker fragment LU102 was synthesized by conventional solid phase peptide synthesis using Rink-amide-MBHA-resin or dichlororesin. Fmoc was used to protect the amino acids in the linker. The coupling reagent was selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the resin was cleaved using trifluoroacetic acid. The product was purified by HPLC, lyophilized and stored for later use.

[0254] The linker fragments are listed in the table below.

[0255]

[0256] The linker unit fragments in the above table are reacted with the linker unit fragments containing a maleimide structure or a derivative thereof, and then subjected to a ring-opening reaction using the method described in WO2015165413A1 to obtain linker units LN102-6 and LN102-8. Their structures are shown above. The linker unit fragments are listed in the following table:

[0257]

[0258] Among them, Mal has the structure When fragment 1 and fragment 2 react to form a linker unit, the maleimide ring of fragment 2 opens to form an open-ring succinimide structure.

[0259] 2.2 Preparation of Linker-Agonist Intermediates

[0260] 2.2.1 Preparation of the linked unit agonist intermediate LP102-6-1

[0261]

[0262] Step 1: Resimod (25.0 g, 79.5 mmol) was dissolved in MeCN (500 mL) and treated with Trt-Cl (33.25 g, 119.3 mmol), followed by TEA (20.12 mL, 20.12 mmol). The reactant was refluxed for 2 to 3 hours (TLC). The reaction mixture was concentrated in vacuo. The mixture was then treated with AcOEt (700 mL) and H2O (400 mL), stirred for 30 minutes and separated. The organic phase was concentrated in vacuo to 300 mL and treated with n-heptane (400 mL). The mixture was then stirred for 20 minutes. After filtration, the filter cake was washed with EtOH / H2O (1:1, 200 mL) and filtered. The filter cake was dried in vacuo to obtain the target compound HX20031-a as a white solid (43.9 g, 99.1%).

[0263] Step 2: Compound (HX20031-a) (40.02g, 72.9mmol) is dissolved in DMF (200mL), and is cooled to 0 ℃ to 10 ℃. NaH (60%, 3.74g, 93.4mmol) is added in batches. The suspension is stirred vigorously at 0 ℃ to 10 ℃ for 1 hour, then warmed to 20 ℃ to 30 ℃ and stirred for 1 hour. Then the mixture is cooled to 0 ℃ to 10 ℃, and treated once with 1186g (20.88g, 93.4mmol) compound. The mixture is stirred at room temperature overnight, and then slowly treated with a mixture of 10% NaH2PO4 (1L) and AcOEt (1L). The reaction mixture is stirred for 3 hours and filtered. The organic layer was concentrated and purified by silica gel column chromatography (elution gradient: n-heptane→n-heptane / AcOEt=10:1→n-heptane / AcOEt=4:1) to obtain the target compound HX20031-b (24.89 g, 46.9%).

[0264] Step 3: Compound HX20031-b (10 g, 14.3 mmol) was treated with a mixture of TFA (40 mL) and H2O (80 mL). The reaction mixture was stirred at room temperature for 24 hours. The mixture was then poured into MTBE (400 mL) and stirred for 2 hours. After filtration, the filter cake was washed with MTBE (200 mL) and filtered. The filter cake was vacuum dried to obtain the target compound HX20031-c as a white solid (8.51 g, 100%).

[0265] Step 4: Compound HX20031-c (6.0 g, 10.2 mmol) was dissolved in DMF (50 mL) and treated with DIPEA (3.5 mL, 20.4 mmol) and N-succinimidyl 3-maleimido propionate (3.27 g, 12.3 mmol). The reaction was allowed to stand at room temperature for 3 hours (HPLC), and the mixture was then used directly in the next step.

[0266] Step 5 to Step 6: The mixture from Step 4 was treated with a solution of fragment HX18041 (LU102) (5.5 g, 15.3 mmol) and H2O (50 mL). The mixture was reacted at 0°C to 40°C for 0.5 to 20 hours. The reaction mixture was then mixed with an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction, and the reaction was continued at 0°C to 40°C for 0.2 to 20 hours. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and lyophilized to give the linker-agonist LP102-6-1 (3.3 g, 30% yield for three steps). MS m / z 1065.6 [M+H] + .

[0267]

[0268] Example 3 Preparation of Targeting Molecule-Drug Conjugates

[0269] The linker-agonist intermediate is coupled to the antibody in a site-specific manner by a ligase to form an AIAC. The method of the coupling reaction can be found in WO2015165413A1. The resulting AIAC is listed in the following table:

[0270] AIAC Linker-agonist intermediate A (targeting molecule) DAR AC102-6-1-1 LP102-6-1 <![CDATA[Ab0001-LCCT L -HC]]> 1.74 AC102-8-1-1 LP102-8-1 <![CDATA[Ab0001-LCCT L -HC]]> 1.79

[0271] Effect Example 1 In vitro evaluation of antibody immunoagonist conjugates

[0272] Isolation of human peripheral blood mononuclear cells

[0273] Human peripheral blood mononuclear cells were isolated from healthy blood donors using SepMate 50 and Lymphoprep (Stem Cell Technologies). The viable cells were counted and the cell concentration was adjusted to 1.25 × 10 6 Tumor cells were separated by trypsin and collected. The viable cells were counted and the cell concentration was adjusted to 2.5×10 in RPMI1640 medium containing 10% FBS. 5 / ml. 4 Individual PBMC and 2.5 × 104 Tumor cells (PBMC: tumor cells = 5:1) were added to the wells of a 96-well plate, and then antibodies or conjugates were added at the indicated concentrations. The cell mixture was incubated with the drug for 18 hours, and then the cell-free supernatant was collected for human TNFα ELISA.

[0274] To evaluate the activity of HER2-targeted immunoconjugates, human PBMCs and NCI-N87 human gastric cancer cells were co-cultured at a 5:1 ratio and the indicated concentrations of antibodies or test immunoconjugates (AC102-6-1-1 or AC102-8-1-1) were added. AC102-6-1-1 induced higher TNFα production compared to antibody Ab0001, and the effective concentration of AC102-6-1-1 was much lower than that of the payload resiquimod ( Figure 1 AC102-8-1-1 induced higher levels of TNFα than Ab0001, which was similar to AC102-6-1-1 ( Figure 2 No activity of AC102-6-1-1 was observed in co-cultures of human PBMCs and MDA-MB-468 HER2-negative cells, indicating that the activity of AC102-6-1-1 is highly dependent on HER2 expression on target tumor cells ( Figure 3 Based on this data, the activity of the immunoconjugate was tested in co-cultures of human PBMCs and other cancer cells with different HER2 expression levels, including HCC1954 ( Figure 4 )、SK-BR-3( Figure 5 )、BT474( Figure 6 )、JIMT1( Figure 7 )、Colo205( Figure 8 )、MDA-MB-468( Fig. 9 ). The data showed that AC102-8-1-1 was only able to induce TNFα in co-cultures of PBMCs and tumor cells that highly expressed HER2.

[0275] Effect Example 2 In vitro evaluation of antibody immunoagonist conjugates

[0276] To evaluate the activity of HER2-targeted immunoconjugates, human PBMCs and SK-BR-3 ( Fig.10 ) or HCC1954( Fig.11) Human breast cancer cells were co-cultured at a ratio of 5:1, and the immunoconjugate (AC102-6-1-1) and antibody (Ab0001) at the indicated concentrations were added. The cells were incubated with the drug for 18 hours, and then the cell-free supernatant was collected for detection of human IFNγ by ELISA. The separation and experimental setup of human PBMCs were similar to those in Example 1. AC102-6-1-1 induced a higher IFNγ production than antibody Ab0001, indicating that the former has the potential to activate T cell responses.

[0277] Effect Example 3 In vivo evaluation of antibody immunoagonist conjugates

[0278] For in vivo antitumor efficacy studies, 1×10 7 NCI-N87 human gastric cancer cells were subcutaneously inoculated into the right flank of SCIDBeige mice. After 6 days, when the tumor volume reached an average of 173 mm 3 At 1 hr, tumor-bearing mice were divided into groups and given 5 mg / kg of Ab0001 or the test immunoconjugate (AC102-6-1-1 or AC102-8-1-1) intravenously. Tumor volumes were measured twice a week with a caliper. The antitumor activity of Ab0001 antibody itself was very limited. AC102-6-1-1 and AC102-8-1-1 almost cured the tumors at the end ( Fig.12 In a separate study, AC102-8-1-1 demonstrated dose-dependent activity at 0.5 mg / kg, 1 mg / kg, and 3 mg / kg ( Fig.13 ).

[0279] 5×10 6 JIMT1 human breast cancer cells were subcutaneously inoculated into the right flank of SCID Beige mice to generate a xenograft model. After 9 days, when the tumor volume reached an average of 149 mm 3 When 5 mg / kg AC102-8-1-1 was intravenously administered to tumor-bearing mice, tumor growth was significantly inhibited ( Fig.14 ).

[0280] 5×10 5 MC38 hHER2 mouse colorectal cancer cells overexpressing human HER2 were subcutaneously inoculated into the right flank of C57BL / 6 mice. After 8 days, when the tumor volume reached an average of 90 mm 3 At 10 mg / kg, Ab0001 did not show significant antitumor activity. 3 mg / kg and 10 mg / kg AC102-6-1-1 dose-dependently inhibited tumor growth ( Fig.15In a similar setting, both 3 mg / kg and 10 mg / kg AC102-8-1-1 induced complete tumor regression in all (100%) mice ( Fig.16 ).

[0281] Examples of Formulations Containing AIAC

[0282] Examples of pH

[0283] Preliminary selection of the stable pH range of the formulation

[0284] I.1 Experimental Materials

[0285] Sample: AC102-8-1-1; Batch No. 20200815;

[0286]

[0287] z is an integer from 1 to 4;

[0288] A is modified trastuzumab.

[0289] I.2 Experimental methods

[0290] Five groups of formulation buffers with different pH values ​​(pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0) were prepared, and the five groups of formulation buffers were as follows:

[0291]

[0292]

[0293] AC102-8-1-1 samples were dialyzed with 5 buffers and the protein concentration was adjusted to about 20 mg / ml. The stability of AC102-8-1-1 in different pH buffers was tested at 2°C to 8°C, 25°C and 40°C to determine the stable pH range.

[0294] The sample preparation procedure is as follows:

[0295] Thaw samples.

[0296] The sample was loaded into dialysis tubing.

[0297] The sample was dialyzed against 100 times the sample volume of buffer at room temperature for 2 hours.

[0298] Change the dialysis buffer and dialyze overnight at 2°C to 8°C twice.

[0299] The protein concentration was tested and adjusted to 20 mg / ml.

[0300] The samples were sterile filtered through a 0.22 μm filter.

[0301] Fill, label and test samples for stability.

[0302] The stability test conditions and times are shown in the table below:

[0303]

[0304] Test items: purity (reduced CE-SDS method, non-reduced CE-SDS method), DAR value (HIC-HPLC), free drug (RP-HPLC).

[0305] Detection Methods

[0306] Purity (reduced CE-SDS)

[0307] The test sample was diluted to about 10 mg protein per 1 mL with ultrapure water as the test solution. 85 μL sample buffer (4 mL pH 6.2 citric acid-phosphate buffer plus 1 mL 10% SDS, water was added to make up to 25 mL) and 5 μL β-mercaptoethanol were added to 10 μL test solution. The solution was vortexed and mixed thoroughly. The test solution was then heated at 70 ° C for 5 minutes, cooled to room temperature, centrifuged at 13,000 rpm for 10 minutes before loading, and 85 μL supernatant was injected into a sample vial for analysis. The loading voltage was 5 KV, the loading time was 20 seconds, the separation voltage was 15 KV, the separation time was 35 minutes, and the detector wavelength was 220 nm. The sum of the percentages of the corrected peak areas of the light chain and the heavy chain is the sample purity.

[0308] Purity (non-reduced CE-SDS)

[0309] The test sample was diluted to about 10 mg / mL with ultrapure water as the test solution. 85 μL sample buffer (4 mL pH 6.2 citric acid-phosphate buffer plus 1 mL 10% SDS, water was added to 25 mL) and 5 μL 500 mM iodoacetamide were added to 10 μL test solution. The solution was vortexed and mixed thoroughly. The test solution was then heated at 70°C for 5 minutes, cooled to room temperature, centrifuged at 13,000 rpm for 10 minutes before loading, and the supernatant was injected into a sample vial for analysis. The loading voltage was 5 KV, the loading time was 20 seconds, the separation voltage was 15 KV, the separation time was 35 minutes, and the detector wavelength was 220 nm. The percentage of the corrected peak area of ​​the monomer peak is the sample purity.

[0310] DAR value (HIC)

[0311] DAR value analysis was performed by hydrophobic chromatography. The analytical column was TSK gel Butyl NPR (4.6 mm × 10 cm, particle size 2.5 μm); 25 mmol / L phosphate buffer + 2 mol / L ammonium sulfate solution (pH 7.0) was used as mobile phase A. Mobile phase B consisted of 25 mmol / L phosphate solution (pH 7.0) and isopropanol mixed in a volume ratio of 70:30. The flow rate was 0.3 mL / min, the column temperature was 30 ° C, and the detection wavelength was 280 nm. The test sample was diluted with pure water to a solution containing about 5 mg per 1 mL as the test solution. The injection volume was 5 μL. The elution gradient was performed according to the table below.

[0312]

[0313] The area percentages of DAR0, DAR1, and DAR2 were calculated according to the area normalization method: DAR=DAR1%+DAR2%*2.

[0314] Free drug (RP-HPLC)

[0315] Reverse phase high performance liquid chromatography was used for free drug detection. The analytical column was ACQUITY PREMIER C18AX, 2.1 mm × 150 mm and had a particle size of 1.7 μm. A 10 mmol / L aqueous ammonium acetate solution was used as mobile phase A, and an acetonitrile solution was used as mobile phase B. The flow rate was 0.3 mL / min, the column temperature was 40 ° C, and the detection wavelength was 248 nm. 400 μL of acetone was added to 200 μL of the test sample, and the solution was mixed evenly. The solution was centrifuged at 13,000 rpm for 15 minutes, and the supernatant was taken as the test solution. Take an appropriate amount of reference material to prepare a linker-agonist reference material solution containing 0.267 μg to 5.3 μg per 1 mL and an agonist reference material solution containing 0.067 μg to 1.3 μg per 1 mL. The injection volume is 3 μl. And operate the reference solution in the same way. Perform an elution gradient according to the table below.

[0316]

[0317] The content of free drug was calculated by the external standard method.

[0318] I.3 Results

[0319] The results of the purity test are shown below:

[0320] Results of non-reduced CE-SDS purity (%) test:

[0321]

[0322]

[0323] Note: "N / A" means no value was detected. Results of reduced CE-SDS purity (%) test:

[0324]

[0325] The results of DAR value detection are as follows:

[0326]

[0327] Results of free drug testing:

[0328]

[0329]

[0330] I.4 Conclusion

[0331] The results of reduced CE-SDS purity analysis, non-reduced CE-SDS purity analysis, DAR value detection (HIC-HPLC) and free drug (RP-HPLC) analysis showed that at pH 7.0, 8.0 and 9.0, the molecular stability of AIAC was poor; the protein was more easily degraded; the small molecules coupled to the antibody were more easily detached; and the DAR value decreased significantly. Therefore, AIAC is not suitable for long-term storage at pH 7.0 to 9.0.

[0332] The optimal pH range of AIAC was further screened in the range of pH 4.0 to pH 6.0.

[0333] II.1 Experimental Materials

[0334] Sample: AC102-8-1-1; Batch number 20200815.

[0335] II.2 Experimental methods

[0336] Four groups of formulation buffers with different pH values ​​(pH values ​​of 4.0, 5.0, 5.5, and 6.0, respectively) were prepared, and the four groups of formulation buffers were as follows:

[0337] serial number Recipe ID Buffer excipient pH 1 A4Su 20 mM sodium acetate 9% (w / v) sucrose 4.0 2 C5Su 20 mM sodium citrate 9% (w / v) sucrose 5.0 3 H5.5Tr 20mM L-histidine 9% (w / v) Trehalose dihydrate 5.5 4 H6Su 20mM L-histidine 9% (w / v) sucrose 6.0

[0338] AC102-8-1-1 samples were dialyzed with 4 buffers for buffer exchange and the protein concentration was adjusted to about 20 mg / ml. The stable pH range was determined by testing the stability of AC102-8-1-1 in different pH buffers at 2°C to 8°C, 25°C and 40°C. The sample preparation procedure was the same as in Example I.2:

[0339] The stability test conditions and times are shown in the table below:

[0340]

[0341] Test items: purity (SE-HPLC), charge variant (CEX-HPLC), T m / T agg .

[0342] Detection Methods

[0343] Purity (SE-HPLC)

[0344] The sample purity was checked by size exclusion high performance liquid chromatography (SE-HPLC). The analytical column was TSKgelG3000SWXL, 7.8×300 mm; the mobile phase was 0.2 mol / L potassium dihydrogen phosphate-0.25 mol / L potassium chloride solution, pH 6.2; the test sample was diluted to about 2 mg per 1 mL with ultrapure water as the test solution, and 50 μL of the test solution was injected into the high performance liquid chromatograph. The column temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, and the elution time was 30 minutes. The percentages of aggregates, monomers, and fragment components were calculated using area normalization.

[0345] Charge variants (CEX-HPLC)

[0346] Charge heterogeneity was detected by cation exchange high performance liquid chromatography (CEX-HPLC). The chromatographic column was a weak cation exchange column (Propac WCX-10, 4×250 mm); 10 mM phosphate buffer (5 mM NaH2PO4·2H2O+5 mM Na2HPO4·12H2O) was used as phase A, 10 mM phosphate buffer (5 mM NaH2PO4·2H2O+5 mM Na2HPO4·12H2O) and 200 mM sodium chloride were used as phase B, the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm. The test sample was diluted to about 2 mg per 1 mL with ultrapure water as the test solution. 50 μL of the test solution was injected into the liquid chromatograph and the elution gradient was performed according to the table below.

[0347]

[0348] The percentages of the reported major, acidic, and basic components were calculated based on the area normalization method.

[0349] T m / T agg

[0350] T using the Uncle analysis system (Unchained Labs) m and T agg Analysis. The analysis parameters are as follows:

[0351] SLS settings: Experiment: Example 1; Start temperature (°C): 25; Incubation time (seconds): 180; Slope (°C / min): 0.7; Plate hold time (seconds): 45; End point temperature (°C): 95.

[0352] DLS settings: Initial acquisition: Yes; Final acquisition: Yes; Number of acquisitions: 4; Acquisition time (seconds): 5; Attenuator control: Auto; Laser control: Auto.

[0353] II.3 Results

[0354] Purity (%) (SEC-HPLC) test results:

[0355]

[0356] Results of charge variant (%) (CEX-HPLC) analysis:

[0357]

[0358] Results of protein thermal stability analysis:

[0359]

[0360] Note: “ND” means no value was detected; T agg 266 refers to the measurement of SLS (static light scattering) at 266 nm, which is more sensitive and suitable for detecting smaller particles; T agg 473 refers to measuring SLS at 473 nm, which is more sensitive and suitable for detecting larger particles; Tm1 is related to the C H 2 dissociation, and Tm2 is related to the C H 3Dissociation related.

[0361] II.4 Conclusion

[0362] At pH 4.0 and 5.0, more fragments were produced and the purity decreased rapidly. The results of protein thermal stability analysis also showed that AIAC molecules have lower T at low pH. m and T agg value, and it is easier to aggregate. When the pH is 6.0, the acid component rises faster. Therefore, the optimal pH finally screened out is 5.5.

[0363] Filler selection

[0364] 2.1 Experimental Purpose

[0365] Screen out fillers (stabilizers) that stabilize AIAC.

[0366] 2.2 Experimental Materials

[0367] Sample: AC102-8-1-1; Batch number 20200815.

[0368] 2.3 Experimental methods

[0369] Protein stabilizers commonly used in biological products include sucrose, trehalose and sorbitol. In this embodiment, three different formulations were prepared (the pH values ​​of the three formulations were all set to 5.5, and the three formulations contained 9% sucrose, 9% trehalose and 5% sorbitol, respectively), and the three formulations were as follows:

[0370] serial number Buffer excipient Protein concentration 1 20 mM L-His pH 5.5 9% (w / v) sucrose 20mg / mL 2 20 mM L-His pH 5.5 9% (w / v) Trehalose dihydrate 20mg / mL 3 20 mM L-His pH 5.5 5% (w / v) sorbitol 20mg / mL

[0371] The AC102-8-1-1 sample was dialyzed with 3 groups of buffers for buffer exchange, and the protein concentration was adjusted to about 20 mg / ml. The stability of AC102-8-1-1 in different formulations was tested to screen out suitable stabilizers. The sample preparation procedure was the same as in Example 1.2:

[0372] Detection Methods

[0373] Binding activity (ELISA)

[0374] Binding activity was determined by enzyme-linked immunosorbent assay (ELISA). The antigen (human HER2 protein) was added to a microtiter plate, coated overnight at 2°C to 8°C, and blocked with a blocking solution. After washing the plate with a washing solution, the reference solution and the test solution were added respectively, and incubated at 25°C for 1 hour; after washing the plate with a washing solution, a mouse anti-human IgG-Fc antibody solution labeled with horseradish peroxidase was added to the ELISA plate, and incubated with shaking at 25°C for 1 hour; after washing the plate with a washing solution, TMB was added for color development, and the color was developed in the dark for 10 minutes; after terminating the reaction with a stop solution, the absorbance was measured at a wavelength of 450nm. A four-parameter logistic regression (4PL) model was used to fit the EC values ​​of the reference product and the test product, respectively. 50 . By reference product EC 50 / Test Article EC 50 The relative activity of the test articles was calculated.

[0375] 2.4 Results

[0376] Results of the purity (SEC-HPLC) test (% aggregates):

[0377]

[0378] Results of relative binding activity assay:

[0379] serial number T0 2-8℃ 12W 1 100％ 98％ 2 105％ 97％ 3 108％ 103％

[0380] 2.5 Conclusion

[0381] Summarizing the SEC and binding activity results, sucrose is superior to trehalose and sorbitol as a bulking agent (stabilizer).

[0382] Study on the effect of surfactant

[0383] 3.1 Experimental Purpose

[0384] The effect of surfactants on the stability of AIAC formulations was studied.

[0385] 3.2 Experimental Materials

[0386] Sample: AC102-8-1-1; Batch number 20200815.

[0387] Experimental methods

[0388] Groups 1 to 3 were prepared without Tween 20, and Groups 4 to 7 contained 0.2 mg / mL Tween 20. The buffer, pH value, and protein concentration of the 7 groups of formulations remained unchanged. The formulations of the 7 groups of formulations are as follows:

[0389]

[0390] Note: “Met” means methionine.

[0391] The AC102-8-1-1 sample was dialyzed with 7 groups of buffers for buffer exchange, and the protein concentration was adjusted to about 20 mg / ml. The protective effect of surfactant (Tween 20, i.e., PS20) on AIAC protein was determined by testing the stability of AC102-8-1-1 in different formulations. The sample preparation procedure was the same as in Example 1.2.

[0392] Test parameters: Visible foreign matter inspection (visual method) (F / T is the abbreviation of freeze / thaw).

[0393] 3.4 Results

[0394]

[0395] √: Colorless clear liquid. No visible particles.

[0396] 1: White precipitate was detected.

[0397] 2: Smaller visible particles are present.

[0398] 3: 1 white fiber (foreign particle) was detected.

[0399] 3.5 Conclusion

[0400] Tween 20 can effectively inhibit the aggregation and precipitation of AIAC molecules during freeze-thaw and freezing.

[0401] Summarizing all previous screening results, the formulations of AIAC were identified as:

[0402] 20 mM His, pH 5.5 (buffer),

[0403] 9% (W / V) sucrose (stabilizer),

[0404] 0.2 mg / mL PS20 (stabilizer and surfactant).

[0405] AIAC preparation concentration

[0406] 4.1 Experimental Purpose

[0407] After determining the formulation composition, the appropriate protein concentration range is further selected.

[0408] 4.2 Experimental Materials

[0409] Sample: AC102-6-1-1; Batch No. 20210114;

[0410]

[0411] z is an integer from 1 to 4;

[0412] A is modified trastuzumab.

[0413] Experimental methods

[0414] As shown in the table below, the protein concentrations of Groups 1 to 3 are 20 mg / mL, 40 mg / mL, and 60 mg / mL, respectively. The other formulation components are the same. The formulations of the three formulations are as follows:

[0415]

[0416] AC102-6-1-1 samples were dialyzed for buffer exchange with three groups of buffers, and the protein concentrations were adjusted to about 20 mg / mL, 40 mg / mL, and 60 mg / mL, respectively. The appropriate protein concentration range was determined by testing the stability of AC102-6-1-1 at different protein concentrations. The sample preparation procedure was the same as in Example 1.2.

[0417] The test conditions and sampling time points are as follows (F / T is the abbreviation for freeze / thaw):

[0418] Storage conditions T0 2 cycles 5 cycles 3 days 1 week 2 weeks 4 weeks 8 weeks 12 weeks -20℃ x x 2-8℃ x x 25℃ x x x x oscillation x x F / T x x -40℃ x -80℃ x

[0419] Test parameters: visible particle test (visual method), SEC-HPLC purity test, non-reduced CE-SDS purity test, reduced CE-SDS purity test, DAR value test, free drug test. The test method is similar to the above method.

[0420] 4.4 Results

[0421] Visible particle detection results:

[0422]

[0423] Note: √: Basically free of visible particles.

[0424] Results of SEC-HPLC aggregation percentage (%) detection:

[0425]

[0426]

[0427] Note: “ / ” indicates that no value was detected.

[0428] Results of non-reduced CE-SDS purity (%) test:

[0429]

[0430]

[0431] Results of reduced CE-SDS purity (%) test:

[0432]

[0433]

[0434] Results of the DAR value test:

[0435]

[0436]

[0437] Results of free drug detection (linker-payload, ppm):

[0438]

[0439]

[0440] Results of free drug testing (agonist, ppm):

[0441]

[0442]

[0443] 4.5 Conclusion

[0444] The results of visible particles, SEC, reduced and non-reduced CE-SDS detection, DAR value detection, and free drug detection all showed that AC102-6-1-1 in the preparation had good stability in the concentration range of 20 mg / mL to 60 mg / mL.

[0445] High protein concentration of AIAC

[0446] 5.1 Experimental Purpose

[0447] Further development of high protein concentration formulations and validation of their stability.

[0448] 5.2 Experimental Materials

[0449] Sample:AC102-6-1-1.

[0450] Experimental methods

[0451] The formula for the high protein concentration formulation is as follows:

[0452] serial number Buffer Stabilizer Surfactants Protein concentration 1 20 mM L-histidine pH 5.65 7.5% (w / v) sucrose 0.2mg / ml PS20 62.2mg / ml 2 20mM L-histidine pH 5.68 7.5% (w / v) sucrose 0.2mg / ml PS20 82.2mg / ml 3 20mM L-histidine pH 5.69 7.5% (w / v) sucrose 0.2mg / ml PS20 100.4mg / ml 4 20 mM L-histidine pH 5.65 9% (w / v) sucrose 0.2mg / ml PS20 62.2mg / ml 5 20 mM L-histidine pH 5.68 9% (w / v) sucrose 0.2mg / ml PS20 82.2mg / ml 6 20mM L-histidine pH 5.69 9% (w / v) sucrose 0.2mg / ml PS20 100.4mg / ml

[0453] AC102-6-1-1 samples were dialyzed with 6 buffers and the protein concentrations were adjusted to 62.2 mg / ml, 82.2 mg / ml and 100.4 mg / ml, respectively. The feasibility of high protein concentration formulations was verified by testing the stability of AC102-6-1-1 at high protein concentrations. The sample preparation procedure was the same as in Example 1.2.

[0454] The test conditions and sampling time points are as follows (F / T is the abbreviation for freeze / thaw):

[0455]

[0456] Detection parameters: visible particle inspection (visual method), SEC-HPLC purity detection, non-reducing CE-SDS purity detection.

[0457] 5.4 Results

[0458] Visible particle detection results:

[0459]

[0460]

[0461] Note: “CL” means clear liquid; “SY” means light yellow.

[0462]

[0463] Note: √: Basically free of visible particles.

[0464] Results of SEC-HPLC purity (%) test:

[0465]

[0466] Note: "Mono" means monomer; "Aggr" means aggregate; "Frac" means fragment. Results of non-reducing CE-SDS purity (%) test:

[0467]

[0468] 5.5 Conclusion

[0469] The results of visible particle test, SEC-HPLC purity test and non-reduced CE-SDS purity test showed that AC102-6-1-1 had good stability in high protein concentration formulations (such as 62.2 mg / ml, 82.2 mg / ml and 100.4 mg / ml). Therefore, in the formulation of the present invention, AIAC can be used in high concentration.

[0470] Although the specific embodiments of this invention have been described above, it should be understood that they are for illustrative purposes only, and the scope of this invention is limited by the appended claims. Those skilled in the art may make various changes or modifications to the embodiments without departing from the essence and scope of this invention, and such changes and modifications fall within the scope of this invention.

[0471] Sequence Listing

[0472] SEQ ID No. 1: Ab0001-LCCT L -HC light chain:

[0473] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0474] SEQ ID No. 2: Ab0001-LCCT L-HC heavy chain:

[0475] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0476] SEQ ID No.3: Ab0001-LC-HCCT light chain:

[0477] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0478] SEQ ID No.4: Ab0001-LC-HCCT heavy chain:

[0479] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGGSEQ ID No.5: Ab0001-LC-HCCT L Light chain:

[0480] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0481] SEQ ID No.6: Ab0001-LC-HCCT L Heavy chain:

[0482] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG

[0483] SEQ ID No.7: Ab0001-LCCT-HC Light Chain:

[0484] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG

[0485] SEQ ID No.8: Ab0001-LCCT-HC Heavy Chain:

[0486] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0487] SEQ ID No.9: Ab0001-LCCT-HCCT Light Chain:

[0488] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG

[0489] SEQ ID No.10: Ab0001-LCCT-HCCT Heavy Chain:

[0490] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID No.11: Ab0001-LCCT-HCCT L Light chain:

[0491] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG

[0492] SEQ ID No.12: Ab0001-LCCT-HCCT L Heavy chain:

[0493] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG

[0494] SEQ ID No.13: Ab0001-LCCT L -HCCT light chain:

[0495] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0496] SEQ ID No.14: Ab0001-LCCT L -HCCT heavy chain:

[0497] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGGSEQ ID No.15: Ab0001-LCCT L -HCCT L Light chain:

[0498] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0499] SEQ ID No.16: Ab0001-LCCT L -HCCT L Heavy chain:

[0500] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG

[0501] SEQ ID No.17:LCDR1:RASQDVNTAVA

[0502] SEQ ID No.18:LCDR2:SASFLYS

[0503] SEQ ID No.19:LCDR3:QQHYTTPPT

[0504] SEQ ID No.20:HCDR1:DTYIH

[0505] SEQ ID No.21:HCDR2:RIYPTNGYTRYADSVKG

[0506] SEQ ID No.22:HCDR3:WGGDGFYAMDY

[0507] SEQ ID No.23:V L :

[0508] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV

[0509] SEQ ID No.24:V H :

[0510] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

Claims

1. A pharmaceutical composition comprising: Antibody-immunoagonist conjugates (AIACs), Buffer, and Stabilizers; The AIAC has a structure of formula (II-1), (II-2) or a mixture thereof: Where B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is an integer from 1 to 5, j is an integer from 1 to 3, d is an integer from 1 or 2, R 1 and R 2 Each independently selected from hydrogen, -OH, -NH2, -C 1-6 alkyl; PL is the payload attached to the B2 part, Preferably, PL is resiquimod z is an integer from 1 to 4; preferably from 1 to 2; A contains V L and V H Antibodies Wherein V L comprising LCDR1 having the amino acid sequence of SEQ ID NO: 17, LCDR2 having the amino acid sequence of SEQ ID NO: 18, and LCDR3 having the amino acid sequence of SEQ ID NO: 19, Wherein V H comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 20, a HCDR2 having the amino acid sequence of SEQ ID NO: 21, and a HCDR3 having the amino acid sequence of SEQ ID NO: 22; wherein the antibody is modified by introducing a ligase donor substrate recognition sequence.

2. The pharmaceutical composition according to claim 1, wherein the antibody comprises a V having an amino acid sequence of SEQ ID NO:

23. L and V having the amino acid sequence of SEQ ID NO: 24 H .

3. A pharmaceutical composition comprising: Antibody-immunoagonist conjugates (AIACs), Acid buffers, and Stabilizers; The AIAC has a structure of formula (II-1), (II-2) or a mixture thereof: Where B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -, k is an integer from 1 to 5, j is an integer from 1 to 3, d is an integer from 1 or 2, R 1 and R 2 Each independently selected from hydrogen, -OH, -NH2, -C 1-6 alkyl; PL is the payload attached to the B2 part, Preferably, PL is resiquimod z is an integer from 1 to 4; preferably from 1 to 2; A is an antibody comprising the following A light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO: 2; or A light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 4; or A light chain having the amino acid sequence of SEQ ID NO: 5 and a heavy chain having the amino acid sequence of SEQ ID NO: 6; or A light chain having the amino acid sequence of SEQ ID NO:7 and a heavy chain having the amino acid sequence of SEQ ID NO:8; or A light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO: 10; or A light chain having the amino acid sequence of SEQ ID NO: 11 and a heavy chain having the amino acid sequence of SEQ ID NO: 12; or A light chain having the amino acid sequence of SEQ ID NO: 13 and a heavy chain having the amino acid sequence of SEQ ID NO: 14; or A light chain having the amino acid sequence of SEQ ID NO:15 and a heavy chain having the amino acid sequence of SEQ ID NO:

16.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein R 1 and R 2 are independently hydrogen or C 1-6 Alkyl; preferably, R 1 and R 2 are independently hydrogen or C 1-6 More preferably, R 1 and R 2 All are hydrogen.

5. The pharmaceutical composition according to claim 4, wherein k is 2, and / or j is 1, and / or d is 1.

6. The pharmaceutical composition according to claim 4, wherein the AIAC is selected from the following structures or mixtures thereof: z is an integer from 1 to 4; preferably from 1 to 2; A contains V L and V H The antibody, wherein the V L comprising LCDR1 having an amino acid sequence of SEQ ID NO: 17, LCDR2 having an amino acid sequence of SEQ ID NO: 18, and LCDR3 having an amino acid sequence of SEQ ID NO: 19, wherein the V H comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 20, a HCDR2 having the amino acid sequence of SEQ ID NO: 21, and a HCDR3 having the amino acid sequence of SEQ ID NO: 22; wherein the antibody is modified by introducing a ligase donor substrate recognition sequence; or A is an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 1 and a heavy chain having the amino acid sequence of SEQ ID NO:

2.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the conjugate of the AIAC has a drug-antibody ratio (DAR) of an integer or non-integer from 1 to 4, i.e., 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, 1 to 2, 1.5 to 2, 1.6 to 2 or 1.7 to 2.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein The acidic buffer comprises one or more of sodium acetate, sodium citrate, and histidine; preferably histidine; or The acidic buffer is selected from the group consisting of a citrate buffer, a phosphate buffer, a histidine buffer and an acetate buffer.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the concentration of the buffer is 10 mM to 40 mM; or the concentration of the buffer is 15 mM to 25 mM; preferably 20 mM.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pH of the pharmaceutical composition is 4.0 to 6.0; preferably the pH is 5.

5.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the stabilizer comprises one or more of sucrose, trehalose dihydrate, trehalose and sorbitol; preferably sucrose.

12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the stabilizer comprises one or more of the following: 5% to 15% (W / V) sucrose, trehalose dihydrate, trehalose and sorbitol; preferably 9% (W / V) sucrose, trehalose dihydrate, trehalose and sorbitol; more preferably 9% (W / V) sucrose.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition comprises a surfactant.

14. The pharmaceutical composition according to claim 13, wherein the surfactant comprises 0.05 mg / ml to 0.5 mg / ml Tween 20 or Tween 80; preferably 0.2 mg / ml Tween 20.

15. A pharmaceutical composition according to any one of claims 1 to 14, comprising: 10 mM to 40 mM histidine buffer, pH 4.0 to 6.0; 5% to 15% (W / V) sucrose; 0.05mg / ml to 0.5mg / ml Tween 20; or 15 mM to 25 mM histidine buffer, pH 4.0 to 6.0; 5% to 15% (W / V) sucrose; 0.05mg / ml to 0.5mg / ml Tween 20.

16. A pharmaceutical composition according to any one of claims 1 to 15, comprising: 20 mM histidine buffer, pH 5.5; 9% (W / V) sucrose; 0.2mg / ml Tween 20.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the AIAC protein concentration is 18 mg / ml to 110 mg / ml; or the AIAC protein concentration is 18 mg / ml to 66 mg / ml.

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