Pharmaceutical compositions of anti-HER2 antibody immunoagonist conjugates and their uses

Abstract

The present disclosure relates to pharmaceutical compositions of antibody-immunoagonist conjugates (AIACs) as a new type of tumor-targeted therapy.

Description

[Technical Field]

[0001] The present disclosure relates to the field of biopharmaceuticals, and in particular to pharmaceutical compositions of antibody-immunoagonist conjugates (AIACs) as a new type of tumor-targeted therapy. [Background technology]

[0002] Targeted delivery of therapeutic agents remains a major challenge in cancer therapy, both in development and clinical trials. Current targeted drug conjugates approved by the FDA are primarily antibody-drug conjugates (ADCs), in which the drug (warhead) is typically 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. HER2 amplification or overexpression occurs in approximately 15–30% of breast cancers and 10–30% of gastric / gastroesophageal cancers. HER2 overexpression has also been confirmed in other cancers, such as ovarian, endometrial, bladder, lung, colon, and head and neck cancers (Iqbal N. et al., Mol Biol Int. 2014:852748). Although the effectiveness 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 remains inherently a more aggressive form of the disease, with a poorer prognosis and worse outcomes than patients with HER2-negative (and HR-positive) disease. Furthermore, treatment results have proven disappointing in other HER2-overexpressing cancers. One of the many reasons for poor outcomes is that patients who receive HER2-targeted therapy develop resistance, a process driven by immune evasion by tumor cells.

[0004] Immunotherapy is a novel cancer treatment modality that has shown great promise. Although immune checkpoint inhibitors, primarily T cell-based therapies, represented by CLTA-4 and PD-1 / L1 monoclonal antibodies, have been approved for various cancer indications, many efforts are also being made to explore other mechanisms of the immune system to combat cancer. Targeting myeloid cells, primarily macrophages and DCs, has emerged as a promising direction. Activating macrophages and DCs with agonists or macrophage checkpoint inhibitors not only improves their phagocytic ability to eliminate tumor cells but also their antigen-presenting capacity, thereby activating adaptive antitumor immunity more robustly.

[0005] TLR7 / 8 are two important pattern recognition receptors located on the endosomal membranes of macrophages, DCs, and monocytes. They naturally sense virus-derived ssRNA and mediate immune cell activation and proinflammatory cytokine release. Numerous studies have demonstrated the antitumor activity of TLR7 / 8 agonists. The TLR7 agonist imiquimod has been approved for the topical treatment of genital warts, superficial basal cell carcinoma, and actinic keratosis. The TLR7 / 8 dual agonist resiquimod has been approved for the treatment of cutaneous T-cell lymphoma. Nevertheless, the side effects induced by systemic administration of TLR7 / 8 agonists have limited their use in a wider range of cancers.

[0006] The present disclosure provides pharmaceutical compositions of HER2-directed antibody-immunoagonist conjugates (AIACs), which are novel agents for tumor-targeted immunotherapy.

[0007] As known in the art, the active pharmaceutical ingredient (API) determines the use of a drug, and the formulation of a pharmaceutical preparation is closely related to the properties of the API. For example, the pH of the preparation can affect the stability of the API, and excipients not only affect the solubility and dissolution of the API, but also have a significant impact on the properties of the API, such as permeability and absorption. 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: [ka] In the formula, 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 of 1 to 5, j is an integer of 1 to 3, d is an integer of 1 or 2, and R 1 and R 2 are hydrogen, -OH, -NH2, and -C, respectively. 1-6 independently selected from alkyl, PL is the payload linked to the B2 portion, Preferably, the PL is resiquimod, [ka] and z is an integer of 1 to 4, preferably 1 to 2; A is for V L and V H wherein V L comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 17, an LCDR2 having the amino acid sequence of SEQ ID NO: 18, and an LCDR3 having the amino acid sequence of SEQ ID NO: 19, Hcomprises an HCDR1 having the amino acid sequence of SEQ ID NO: 20, an HCDR2 having the amino acid sequence of SEQ ID NO: 21, and an HCDR3 having the amino acid sequence of SEQ ID NO: 22, and the antibody is modified by the introduction of a ligase donor substrate recognition sequence. In a preferred embodiment, the antibody comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 23 L and V having the amino acid sequence of SEQ ID NO: 24 H and,

[0009] In one aspect, an antibody-drug conjugate of formula (II-1) and / or (II-2) is provided: [ka] In the formula, 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 of 1 to 5, j is an integer of 1 to 3, d is an integer of 1 or 2, and R 1 and R 2 are hydrogen, -OH, -NH2, and -C, respectively. 1-6 independently selected from alkyl, PL is the payload linked to the B2 portion, Preferably, the PL is resiquimod, [ka] and z is an integer of 1 to 4, preferably 1 to 2; A is 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; The antibody comprises:

[0010] The antibody-immune agonist conjugates (AIACs) of the present disclosure provide a new type of tumor-targeting therapy. In vitro experiments demonstrate that AIACs can induce higher TNFα production compared to naked, unmodified antibodies. In vivo experiments of AIACs demonstrate antitumor effects.

[0011] In another aspect, there is provided a pharmaceutical composition 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 explanation of the drawings]

[0012] [Figure 1] Figure 1 shows TNFα induction activity in human PBMC and NCI N87 co-culture assays for the conjugate AC102-6-1-1 and the corresponding naked unmodified antibody Ab0001 (trastuzumab), as well as the agonist resiquimod. [Figure 2] Figure 1 shows TNFα induction activity in human PBMC and NCI N87 co-culture assays for conjugates AC102-6-1-1, AC102-8-1-1, and their corresponding naked unmodified antibodies Ab0001. [Figure 3] Figure 1 shows the TNFα-inducing activity of AC102-6-1-1 and antibodies in co-cultures of PBMC with either NCI N87 or MDA-MB-468 cells. [Figure 4]1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in PBMC co-culture with HCC1954 cells that highly express HER2. [Figure 5] 1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in PBMC co-cultures with SK-BR-3 cells that highly express HER2. [Figure 6] 1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in a co-culture of PBMC with BT474 cells that highly express HER2. [Figure 7] 1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in PBMC co-cultures with JIMT1 cells, which moderately express HER2. [Figure 8] 1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in PBMC co-cultures with Colo205 cells that express low levels of HER2. [Figure 9] 1 shows the TNFα-inducing activity of AC102-8-1-1 and antibodies in PBMC co-cultures with MDA-MB-468 cells that express low levels of HER2. [Figure 10] 1 shows the INF-γ inducing activity of AC102-6-1-1 and antibodies in co-cultures of PBMC with SK-BR-3 cells. [Figure 11] 1 shows the INF-γ inducing activity of AC102-6-1-1 and antibodies in co-cultures of PBMC with HCC1954 cells. [Figure 12] 1 shows the change in tumor volume over time in SCID Beige mice according to the NCI N87 CDX model, administered with vehicle (PBS pH 6.5), antibody, and 5 mg / kg of conjugates AC102-6-1-1 and AC102-8-1-1. [Figure 13] 1 shows the change in tumor volume over time in SCID Beige mice according to the NCI N87 CDX model administered with 0.5, 1, and 3 mg / kg of AC102-8-1-1. [Figure 14]1 shows the change in tumor volume over time in SCID Beige mice of the JIMT1 CDX model administered with 5 mg / kg of AC102-8-1-1. [Figure 15] 1 shows the change in tumor volume over time in the hHER2-overexpressing MC38 model administered with 3 mg / kg and 10 mg / kg of AC102-6-1-1. [Figure 16] 1 shows the change in tumor volume over time in the hHER2-overexpressing MC38 model administered with 3 mg / kg and 10 mg / kg of AC102-8-1-1. DETAILED DESCRIPTION OF THE INVENTION

[0013] Below, specific embodiments are provided to explain the technical content of the present disclosure. Those skilled in the art can easily understand other advantages and effects of the present disclosure through the contents disclosed herein. The present disclosure can also be implemented or applied through other different specific embodiments. Various modifications and variations can be made by those skilled in the art without departing from the spirit of the present disclosure.

[0014] definition Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Technical terms used herein refer to those commonly understood in the art, including variations and equivalent substitutions that are obvious to those skilled in the art.Although the following terms are believed to be easily understood by those skilled in the art, the following definitions are provided to better explain this disclosure.When a trade name appears herein, this refers to its corresponding commodity or active ingredient.All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0015] When a particular amount, concentration, or other value or parameter is described in the form of a range, a preferred range, or a preferred upper or lower limit, this should be understood as being equivalent to specifically defining any range formed by combining any upper or preferred value with any lower or preferred value, regardless of whether the range is explicitly stated. Unless otherwise specified, numerical ranges recited herein are intended to include both endpoints of the range and all integers and fractions (decimals) within the range. For example, the phrase "i is an integer from 2 to 20" means that i is any integer from 2 to 20, e.g., i is 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 an analogous manner.

[0016] Unless otherwise stated herein, singular forms such as "a" and "the," include plural forms. "One or more" or "at least one" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0017] The terms "about" and "approximately," when used in conjunction with a numerical variable, generally mean that the value of that variable, and all values ​​of that variable, are within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the stated value, or within a wider range.

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

[0019] As used herein, "%" concentration refers to a mass-volume concentration in g / ml. For example, a 9% sucrose solution refers to dissolving 9 g of sucrose in a solvent to form 100 ml of solution, which means that the solution contains 9 g of sucrose per 100 ml.

[0020] In the present disclosure, the amount of buffering agent refers to the total amount of the buffering agent pair in the buffer system that constitutes the buffer solution. In some embodiments, molar concentration is used as a unit of buffering agent amount, and the numerical value refers to the molar concentration of the buffering agent pair in the buffer system of the buffer solution. For example, when a histidine buffer solution composed of L-histidine and L-histidine hydrochloride is used, a given concentration of the histidine buffer solution (e.g., 10 mM) is the total concentration of L-histidine and L-histidine hydrochloride (e.g., 5 mM L-histidine and 5 mM L-histidine hydrochloride, or 6 mM L-histidine and 4 mM L-histidine hydrochloride, or 3.46 mM L-histidine and 6.54 mM L-histidine hydrochloride, etc.).

[0021] In the solid state, trehalose typically exists in the form of trehalose dihydrate. In some embodiments, trehalose dihydrate can be used for the preparation, and a corresponding amount of another form of trehalose can also be used for the preparation, with the resulting formulation containing the same concentration of trehalose. When the content of trehalose in a formulation using trehalose dihydrate is described in this disclosure, the formulation contains the stated amount of trehalose dihydrate, or a corresponding amount of trehalose or another form of trehalose, or a combination thereof, and vice versa.

[0022] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, etc.). A targeting molecule can deliver a payload to a specific site in vivo through 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 multiple receptors. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins for a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0023] As used herein, the term "antibody" is used broadly and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they possess the desired biological activity. Antibodies can be of any subtype (such as IgG, IgE, IgM, IgD, and IgA) or subclass and can be derived from any suitable species. In some embodiments, antibodies are of human or murine origin. Antibodies can also be fully human, humanized, or chimeric antibodies prepared by recombinant methods.

[0024] A monoclonal antibody is used herein to mean an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a few possible natural variations. Monoclonal antibodies are highly specific for a single antigenic site, multiple antigenic sites, or different epitopes on the same antigen. The word "monoclonal" denotes that the character of the antibody is derived from a substantially homogeneous antibody population and should not be construed as requiring any particular method for producing the antibody.

[0025] An intact or full-length antibody essentially comprises an antigen-binding variable region, as well as a light chain constant region (C L ) and heavy chain constant region (C H ), the latter of which, depending on the antibody subtype, can be C H 1. C H 2. C H 3, and / or C H The antigen-binding variable region (also known as a fragment variable region, Fv fragment) typically comprises a light chain variable region (V L ) and heavy chain variable region (V H ). The constant region can be a constant region comprising a native sequence (such as a constant region comprising a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with a target antigen. The constant region can be recognized by and interacts with the immune system.

[0026] As used herein, "heavy chain constant region (C H The term "antibody heavy chain constant region" includes amino acid sequences derived from an intact antibody or a full-length antibody heavy chain. A polypeptide comprising a heavy chain constant region is H 1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, C H 2 domains, C H 3 domains, C H For example, an antigen-binding polypeptide for use in the present disclosure may comprise at least one of the C4 domains, or a variant or fragment thereof. H A polypeptide chain containing one domain; CH 1 domain, at least a portion of the hinge domain, and C H A polypeptide chain containing two domains; C H 1 domain and C H A polypeptide chain containing three domains; C H 1 domain, at least a portion of the hinge domain, and C H a polypeptide chain containing three domains; or C H 1 domain, at least a portion of the hinge domain, C H 2 domain, and C H It may comprise a polypeptide chain comprising three domains.

[0027] As used herein, "C L " refers to the constant region of the light chain.

[0028] The subunit structures and three-dimensional configurations of the constant regions of various antibody classes are well known. H The term "domain" includes the amino-terminal variable domain of an antibody heavy chain and is used interchangeably with "C H The term "C domain" includes the first (most amino-terminal) constant region domain of an antibody heavy chain. H 1 domain is V H domain and amino-terminal to the hinge region of the antibody heavy chain molecule.

[0029] As used herein, "V L " refers to the variable region of the light chain.

[0030] An antibody fragment may comprise a portion of an intact antibody, preferably the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), V H and C H Examples of fragments include Fd fragments, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs). Fab fragments are antibody fragments obtained by papain digestion of full-length immunoglobulins, or fragments with the same structure produced, for example, by recombinant expression. Fab fragments consist of a light chain (VL and C L ), and another chain, wherein the other chain comprises a variable domain of the heavy chain (V H ) and the heavy chain constant region domain (C H 1). F(ab')2 fragments are antibody fragments obtained by pepsin digestion of immunoglobulins at pH 4.0-4.5, or fragments of the same structure produced, for example, by recombinant expression. F(ab')2 fragments essentially contain two Fab fragments, with each heavy chain containing a few additional amino acids, including a cysteine ​​that forms a disulfide bond connecting the two fragments. Fab' fragments are fragments containing half of an F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may contain multiple chains connected to each other, for example, via disulfide bonds and / or via peptide linkage units. Examples of antibody fragments include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments can include any antibody fragment that, when inserted into an antibody framework (eg, by substitution of the corresponding region), can result in an antibody that immunospecifically binds to an antigen.

[0031] Where there is more than one definition of a term used and / or accepted within the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983), and Chothia et al., J. MoI. Biol. 196:901-917 (1987), both of which are incorporated herein by reference in their entireties. The definitions of CDRs according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. However, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0032] As used herein, "HCDR" means a complementarity determining region of a heavy chain.

[0033] As used herein, "LCDR" means light chain complementarity determining region.

[0034] Antibodies according to the present disclosure can be prepared using techniques well known in the art, such as the following techniques or a combination thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques well known in the art. For example, a genetically engineered recombinant antibody (or antibody mimic) can be expressed in a suitable culture system (e.g., E. coli or mammalian cells). The modification of an antibody can mean, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0035] HER2 means human epidermal growth factor receptor 2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this disclosure, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0036] As used herein, the term "targeted molecule drug conjugate" refers to a "conjugate." Examples of conjugates include, but are not limited to, antibody drug conjugates.

[0037] As used herein, the expression "concentration of an AIAC" has the same meaning as "concentration of a protein of an AIAC," and they can be used interchangeably.

[0038] "Low molecular weight compounds" refers to molecules having a size comparable to that of organic molecules commonly used in medicine. This term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of a low molecular weight compound can be, for example, about 100 to about 2,000 Da, about 200 to about 1,000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0039] By immune agonist is meant an agonist capable of inducing or enhancing an immune response against 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, including but not limited to agonists of TLR7 and / or TLR8 and / or TLR9 (e.g., imiquimod, resiquimod, 852A, and VTX-2337), and STING agonists (e.g., ADU-S100 and MK-1454), are well known in the art.

[0040] A linking unit refers to a functional group that covalently links two or more moieties in a compound or material. For example, a linking unit can serve to covalently link a targeting molecule and / or an adjuvant moiety of a payload.

[0041] A spacer is a structure that is located between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, where the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that functions as a spacer, and examples thereof include, but are not limited to, a single amino acid such as Leu or Gln, a sequence containing multiple amino acids, for example, a sequence containing two amino acids such as GA, or a sequence such as GGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGGS. Other examples of spacers include self-immolative spacers, such as PABC (p-benzyloxycarbonyl).

[0042] The term "alkyl" means a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is attached to the rest of the molecule through a single bond. An alkyl group can contain 1 to 20 carbon atoms, and is represented by C1-C 20 It refers to an alkyl group, such as a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl group, a C3 alkyl group, a C4 alkyl group, or a C3-C6 alkyl group. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof, but are not limited to these.

[0043] A divalent radical refers to a group obtained from the corresponding monovalent radical by removing a hydrogen atom from a carbon atom that has a free valence electron. A divalent radical has two points of attachment to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group that is either straight-chain or branched. Examples of alkylene groups include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexylene (-CH 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, and the like.

[0044] As used herein, when a group combines with another group, the bond between these groups may be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination may be bonded to the rest of the molecule through any suitable atom in the structure, preferably through the specified chemical bond. For example, C 1-4 When depicting a combination of alkylene with one of the groups including -CH2-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-, C 1-4 Alkylene is a linear bond to the above groups, e.g., C 1-4 Alkylene-CH2-, C 1-4 Alkylene-NH-, C 1-4 Alkylene-(CO)-, C 1-4 Alkylene-NH(CO)-, C 1-4 Alkylene -(CO)NH-, -CH2-C 1-4 Alkylene, -NH-C 1-4 Alkylene, -(CO)-C 1-4 Alkylene, -NH(CO)-C 1-4 Alkylene, -(CO)NH-C 1-4 An alkylene can be formed, and the resulting divalent structure can be further attached to other parts of the molecule.

[0045] The term "acidic buffer" refers to a buffer with a pH of <7, for example, a buffer with a pH of 4.0-6.0, pH of 4.0, pH of 4.5, pH of 5.0, pH of 5.5 or pH of 6.0.

[0046] "Stabilizer" means a chemical product that can increase the stability of a solution, colloid, solid, mixture, etc., and has the functions of slowing down reactions, maintaining chemical balance, reducing surface tension, and preventing light, thermal, or oxidative degradation; for example, the stabilizer may be sucrose, trehalose dihydrate, sorbitol, and combinations thereof.

[0047] As used herein, "surfactant" refers to a substance that, when added in small amounts, can significantly change the interfacial state of a solution system. Examples of surfactants include, but are not limited to, Tween 20 (PS 20), Tween 80 (PS 80), Span 20 (SP 20), and Span 80 (SP 80). In some embodiments, surfactants can be used as stabilizers.

[0048] As used herein, "Tm" means melting temperature, which indicates the temperature at which a protein begins to denature.

[0049] As used herein, "Tagg" refers to the aggregation temperature, which refers to the temperature at which a protein begins to aggregate.

[0050] Compound of formula (I') In one aspect, a pharmaceutical composition comprising an AIAC is provided, wherein the AIAC comprises a compound of formula (I') (formula (I'-1) or formula (I'-2) or a mixture thereof), [ka] During the ceremony, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -R 3 , or -(CH2) k (CO)-(NH-CR 1 R 2 -(CO)) d -R 3 and R 1 are hydrogen, -OH, -NH2, and -C 1-6 alkyl, R 2 are hydrogen, -OH, -NH2, and -C 1-6 alkyl, R 3 is a group that can be eliminated upon reaction with a group in the payload, Each k is independently an integer of 1 to 5; j is an integer of 1 to 3; d is 1 or 2.

[0051] In one embodiment, R 1 and R 2 are each independently hydrogen or C 1-6 In a preferred embodiment, R 1 and R 2 are each independently hydrogen or C 1-6 In a more preferred embodiment, R 1 and R 2 are both hydrogen.

[0052] In one embodiment, B2 in formulas (I'-1) and (I'-2) are the same.

[0053] In one embodiment, k is 2. In one embodiment, j is 1.

[0054] In one embodiment, d is 1.

[0055] In one embodiment, the terminal group R 3 is hydrogen. In one embodiment, R 3 is hydroxy or [ka] is.

[0056] In one embodiment, the terminal group R 3 represents a portion of the structure of B2 that does not appear in the product molecule obtained by reaction of B2 with a payload; therefore, in the linking unit-payload intermediate (see below), the structural portion corresponding to B2 is one of the above divalent groups or a combination of two or more divalent groups.

[0057] Thiosuccinimides are unstable under physiological conditions and prone to reverse Michael addition, resulting in cleavage at the conjugation site. Furthermore, when other thiol compounds are present in the system, thiosuccinimides may also undergo thiol exchange with other thiol compounds. Both of these reactions cause a reduction in payload and lead to toxic side effects. In the present disclosure, the open-ring succinimide structure [ka] does not undergo retro-Michael addition or thiol exchange, and therefore the product is more stable. Methods for the ring-opening reaction can be found in WO2015165413A1.

[0058] Specific embodiments of compounds of formula (I') In one embodiment, in the compound of formula (I'), B2 is -(CH2) k (CO)-NH-(C2H4-O) j -H, k is 2, j is 1, and the structure of the linking unit is a mixture of the following two structures (linking unit LN102-6): [ka]

[0059] 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 where k is 2, d is 1, and 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): [ka]

[0060] R 3is a group that can be eliminated upon reaction with a group in the payload, and in one embodiment, R 3 is hydroxy or [ka] is.

[0061] Two or more R's x (where x is 1, 2, 3, 4, 5, 6, 7, etc.), then each R x In some embodiments, "x" in a molecule may be represented with or without an additional single apostrophe (') or multiple apostrophes ('', ''', ''', etc.), e.g., R, R 1’ , R 1’’ , R 1’’’ , R 2’ , R 2’’ , R 2’’’ etc. R 3 Other R x should be understood analogously.

[0062] Compound of formula (I') as a linking unit In one embodiment, the reactive group contained in B2 can be used to covalently conjugate with a payload containing another reactive group, thereby rendering the compound of formula (I') bear a payload.

[0063] In another embodiment, the ligase recognition sequence GGG (where G is glycine) contained in formula (I') can be used in complex formation with the corresponding ligase recognition sequence LPETGG (SEQ ID NO: 28) by a ligase.

[0064] Thus, the compounds of formula (I') can be used as linking units that can be attached to a targeting molecule (such as an antibody or an antigen-binding fragment thereof) and / or a payload.

[0065] One skilled in the art can synthesize the linking units by conventional solid or solution phase methods.

[0066] Compounds of formula (I') with a payload The reactive group contained in B2 is covalently conjugated to a payload containing another reactive group to provide a compound of Formula (I') bearing the payload.

[0067] In yet another aspect, a pharmaceutical composition comprising an AIAC is provided, wherein the AIAC comprises a compound having the structure of formula (II'-1) or (II'-2), or a mixture thereof: [ka] During the ceremony, PL is a payload linked to the B2 moiety of the compound of formula (I').

[0068] payload In the present disclosure, the payload can be selected from small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics, and proteins. In one embodiment, the payload is selected from small molecule compounds, nucleic acid molecules, and tracer molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from cytotoxins and fragments thereof. In a more preferred embodiment, the payload is selected from immune agonists and fragments thereof.

[0069] In one embodiment, the immune agonist is selected from a TLR agonist, such as a TLR agonist (e.g., 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.

[0070] In one embodiment, the immune agonist is resiquimod: [ka]

[0071] In one embodiment, the linking unit and the payload are connected via the reactive groups defined above using any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc.

[0072] In one embodiment, the payload is an immune agonist and the antibody-immune agonist conjugate (numbered LPx) is one of the compounds shown in the table below. [Table 1]

[0073] Preparation of compounds of formula (I') bearing a payload In one embodiment, the linking unit and the payload are connected via the reactive groups defined above using any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc.

[0074] Compound of formula (III') In one aspect, there is provided a compound of formula (III'): [ka] wherein B2 is as defined in formula (I').

[0075] In one embodiment, the compound of formula (III') can be used to prepare a compound of formula (I') bearing a payload by the following route: [ka]

[0076] The conversion of a compound of Formula (III') bearing a payload to a compound of Formula (I') bearing a payload can be carried out using any method known in the art or as described herein, for example, by performing a single-step or multi-step synthesis to obtain a structural fragment. [ka] can be introduced into the maleimide ring of a compound of Formula (III') bearing a payload, and the resulting molecule containing a succinimide moiety can then undergo a ring-opening reaction to open the succinimide ring and obtain a compound of Formula (I') bearing a payload (i.e., a compound of Formula (II')). In one embodiment, LU102 is introduced into a compound of Formula (III') bearing a payload by reaction of the maleimide group contained in compound of Formula (III') with a thiol group of LU102.

[0077] Conjugates and their preparation In yet another aspect, there is provided a conjugate having the structure of formula (II-1) and / or (II-2): [ka] During the ceremony, PL is a payload linked to the B2 moiety of the compound of formula (I'), z is an integer of 1 to 4, preferably 1 to 2; A is the target molecule.

[0078] In one embodiment, the payload is an immune agonist, as defined above, hi one embodiment, the conjugate is an antibody-immune agonist conjugate.

[0079] target molecule In one embodiment, the target molecule is an antibody, or an antigen-binding fragment thereof.

[0080] In some embodiments of the present disclosure, the target recognized by the targeting molecule (such as an antibody or antigen-binding fragment thereof) is ErbB2 / HER2.

[0081] In one embodiment, the target molecule is an anti-human HER2 antibody or an antigen-binding fragment thereof. Examples of anti-human HER2 antibodies include, but are not limited to, trastuzumab. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2 and is approved for the treatment of HER2-positive breast cancer and gastric cancer.

[0082] In a preferred embodiment, the anti-human HER2 antibody is one or more selected from recombinant anti-HER2 antibodies based on trastuzumab.

[0083] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment, and an antibody mimetic. In one embodiment, the antibody mimetic is selected from an scFv, a minibody, a diabody, and a nanobody. For conjugation with a compound of Formula (I'), Formula (II'-1), or Formula (II'-2), the targeting molecule of the present disclosure can include a modifying moiety for binding to a compound of Formula (I'), Formula (II'-1), or Formula (II'-2). The introduction position of such a modifying moiety is not limited; for example, when the targeting molecule is an antibody, the introduction position can be, but is not limited to, the C-terminus and / or N-terminus of the heavy chain and / or light chain of the antibody.

[0084] In one embodiment, the target molecule of the present disclosure is an antibody or antigen-binding fragment thereof, which may include a terminal modification. Terminal modification refers to a modification at the C-terminus and / or N-terminus of the heavy and / or light chain of the antibody, which may include, for example, a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp1 containing 2 to 10 amino acids, wherein the antibody, Sp1, and ligase recognition sequence are contiguously linked. In a specific embodiment, Sp1 is a spacer sequence selected from GA, GGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), and GGGSGGGGSGGGGS (SEQ ID NO: 27), particularly GA.

[0085] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof is of three types: wild-type (LC), C-terminally modified light chain (LCCT), which is modified by direct introduction of the ligase recognition sequence LPETGG, and C-terminally modified light chain (LCCT), which is modified by introduction of a short peptide spacer and, in addition, the ligase donor substrate recognition sequence LPETGG. LThe heavy chain of the antibody or antigen-binding fragment thereof includes three types: wild-type (HC), C-terminally modified heavy chain (HCCT) modified by direct introduction of the ligase recognition sequence LPETGG, and C-terminally modified heavy chain (HCCT) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPETGG. L When z in the compounds of formula (II-1) and / or formula (II-2) is 1 or 2, eight preferred antibody molecules can be formed by the combination of the heavy chain and light chain, and for these, please refer to the amino acid sequence listing.

[0086] In one embodiment, the target molecule of the present disclosure is V L and V H wherein V L comprises an LCDR1 having the amino acid sequence (RASQDVNTAVA) of SEQ ID NO: 17, an LCDR2 having the amino acid sequence (SASFLYS) of SEQ ID NO: 18, and an LCDR3 having the amino acid sequence (QQHYTTPPT) of SEQ ID NO: 19, H comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 20 (DTYIH), an HCDR2 having the amino acid sequence of SEQ ID NO: 21 (RIYPTNGYTRYADSVKG), and an HCDR3 having the amino acid sequence of SEQ ID NO: 22 (WGGDGFYAMDY). In one embodiment, the antibody is modified by the introduction of a ligase donor substrate recognition sequence. In one embodiment, the antibody comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 23 (DTYIH), an HCDR2 having the amino acid sequence of SEQ ID NO: 21 (RIYPTNGYTRYADSVKG), and an HCDR3 having the amino acid sequence of SEQ ID NO: 22 (WGGDGFYAMDY). In one embodiment, the antibody is modified by the introduction of a ligase donor substrate recognition sequence. In one embodiment, the antibody comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 23 (WGGDGFYAMDY). L and V having the amino acid sequence of SEQ ID NO: 24 H and,

[0087] In one embodiment, the sequences of the CDRs and variable domains are defined according to the Kabat numbering system. In one embodiment, the target molecule of the present disclosure 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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. In one embodiment, the target molecule of the present disclosure is an antibody comprising 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.

[0088] The conjugates of the present disclosure can further comprise a payload, which is as described above.

[0089] Specific Embodiments of the Conjugate 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 complex is as follows (formula AC102-6): [ka]

[0090] 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 where k is 2, d is 1, and R 1 and R 2 is hydrogen and the structure of the complex is as follows (formula AC102-8): [ka]

[0091] Preparation of the complex The conjugates of the present disclosure can be prepared by any method known in the art. In some embodiments, the conjugates are prepared by ligase-catalyzed site-specific conjugation of a target molecule with a compound of formula (I') having a payload, wherein the target molecule is modified with a ligase recognition sequence, such as a ligase donor substrate recognition sequence. This method includes Step A and Step B.

[0092] Step A. Preparation of the Linking Unit-Payload Intermediate In a preferred embodiment, B2 in the compound of formula (I') is covalently linked via a reactive group to a payload containing a corresponding reactive group, where each reactive group is as defined above.

[0093] The linking unit-payload intermediates prepared using the compounds of formula (I') of the present disclosure have defined structures, defined compositions, and high purity, so that when conjugation reactions with antibodies are carried out, fewer or no impurities are introduced. When such intermediates are used for ligase-catalyzed site-specific conjugation with modified antibodies containing ligase recognition sequences, homogeneous ADCs with highly controllable quality are obtained.

[0094] Step B. Linking a targeting molecule to a payload-bearing compound of formula (I') The target molecule of the present disclosure can be conjugated with a compound of Formula (I') bearing a payload (i.e., a compound of Formula (II')) by any method known in the art. For example, a site-specific conjugation technique catalyzed by a ligase is applied, and the target molecule and the compound of Formula (I') bearing a payload are linked to each other via a ligase-specific recognition sequence in the substrate. In one embodiment, the target molecule is an antibody having a recognition sequence-based terminal modification introduced at the C-terminus of the light chain and / or heavy chain, and the target molecule is conjugated with a compound of Formula (II') under the catalysis of a wild-type or optimized recombinant ligase, or any combination thereof, and under suitable catalytic reaction conditions.

[0095] In a specific embodiment, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme: [ka]

[0096] The triangle and pentagon each represent either a portion of an antibody or a portion of a compound of formula (II'). Each N is as defined above. G is the corresponding recognition sequence of the acceptor substrate. n When complexed with , the peptide bond upstream of the glycine in the LPETGG sequence is cleaved by sortase A, and the resulting intermediate is G n The resulting amino acid sequence is LPETG n (SEQ ID NO: 30). Sequence G n and LPETGG are as defined above.

[0097] Table of specific complexes In one embodiment, the payload is an immunoagonist. In one embodiment, the antibody-immunoagonist conjugate is as shown in the table below: [Table 2]

[0098] Pharmaceutical compositions and medicines Another aspect of the present disclosure is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a conjugate of the present disclosure and at least one pharmaceutically acceptable carrier.

[0099] The pharmaceutical composition of the present disclosure can be administered by any method as long as it achieves the effect of preventing, alleviating, preventing, or curing symptoms in humans or animals. For example, various suitable dosage forms can be prepared according to the administration route, particularly injections such as lyophilized powder for injection, injections, or sterile powder for injection.

[0100] The term "pharmaceutically acceptable" means, within the scope of ordinary medical judgment, that when in contact with the tissues of a patient, it does not produce excessive toxicity, irritation, or allergic reaction, and has a reasonable ratio of benefits to drawbacks, and is effective for its intended use.

[0101] In one embodiment, a pharmaceutical composition of the present disclosure has an antibody-drug ratio (DAR) that is an integer or non-integer between 1 and 4, such as about 1 to 4, about 1 to 3.5, about 1 to 3, about 1 to 2.5, and preferably about 1 to 2. In one embodiment, a pharmaceutical composition of the present disclosure has a DAR of about 1.5 to about 2, preferably about 1.6 to about 2, and more preferably about 1.7 to about 2.

[0102] In one embodiment, the pharmaceutical composition comprises an AIAC, a buffering agent, and one or more stabilizers.

[0103] 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.

[0104] In some embodiments, the buffer concentration is about 10-40 mM. In preferred embodiments, the buffer concentration is about 10 mM, about 14 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 26 mM, about 30 mM, about 33 mM, about 37 mM, or about 40 mM. In preferred embodiments, the buffer concentration is about 20 mM. In some embodiments, the buffer concentration is about 15-25 mM. In a preferred embodiment, the buffer concentration is about 20 mM. In one embodiment, the buffer in the pharmaceutical composition comprises about 15-25 mM of one or more of sodium acetate, sodium citrate, sodium phosphate, histidine, Tris, and glycine. In some embodiments, the buffer concentration is about 15-25 mM. In some embodiments, the buffer comprises about 15-25 Mm citrate buffer, about 15-25 Mm phosphate buffer, about 15-25 Mm histidine buffer, and about 15-25 Mm acetate buffer.

[0105] 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.

[0106] In one embodiment, the buffer in the pharmaceutical composition comprises about 15-25 Mm of one or more of sodium acetate, sodium citrate, and histidine.

[0107] In one embodiment, the buffer in the pharmaceutical composition is about 15-25 mM histidine, hi some embodiments, the buffer is about 15-25 mM, e.g., about 15 mM, about 16.5 mM, about 17 mM, about 18.3 mM, about 19 mM, about 21 mM, about 22 mM, about 23 mM, or about 25 mM histidine buffer.

[0108] In one embodiment, the buffering agent in the pharmaceutical composition is about 20 Mm histidine. In some embodiments, the buffering agent in the pharmaceutical composition is about 20 Mm histidine buffering agent.

[0109] In one embodiment, the pH of the buffer in the pharmaceutical composition is about 4.0 to 6.0, for example, 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.

[0110] In one embodiment, the pH of the buffer in the pharmaceutical composition is about 5.5.

[0111] In one embodiment, the stabilizer in the pharmaceutical composition comprises one or more of sucrose, trehalose dihydrate, trehalose, and sorbitol.

[0112] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 5-15% (w / v) of one or more 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).

[0113] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 9% (W / V) of one or more of sucrose, trehalose dihydrate, trehalose, and sorbitol.

[0114] In one embodiment, the stabilizer in the pharmaceutical composition comprises sucrose.

[0115] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 5-15% (w / v) sucrose, hi 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%.

[0116] In one embodiment, the stabilizer in the pharmaceutical composition comprises about 9% (W / V) sucrose.

[0117] In some embodiments, the pharmaceutical composition further comprises 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 0.05-0.5 mg / ml. In one embodiment, the surfactant in the pharmaceutical composition comprises about 0.05-0.5 mg / ml of PS 20. In some embodiments, the concentration of PS 20 is about 0.05 mg / ml, about 0.1 mg / ml, about 0.15 mg / ml, about 0.2 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.37 mg / ml, about 0.4 mg / ml, about 0.48 mg / ml, or about 0.5 mg / ml.

[0118] In one embodiment, the surfactant in the pharmaceutical composition comprises 0.2 mg / ml PS 20. In another embodiment, the surfactant in the pharmaceutical composition comprises 0.4 mg / ml PS 20.

[0119] In some embodiments, the pharmaceutical composition comprises about 10-40 mM buffer (pH 4.0-6.0), about 5-15% (W / V) stabilizer, and about 0.05-0.5 mg / ml surfactant. In some embodiments, the pharmaceutical composition comprises about 15-25 mM buffer (pH 4.0-6.0), about 5-15% (W / V) stabilizer, and about 0.05-0.5 mg / ml surfactant. In one embodiment, the pharmaceutical composition comprises 10-40 mM histidine buffer (pH 4.0-6.0), 5-15% (W / V) sucrose, and 0.05-0.5 mg / ml PS 20. In one embodiment, the pharmaceutical composition comprises 15 to 25 mM histidine buffer (pH 4.0 to 6.0), 5 to 15% (W / V) sucrose, and 0.05 to 0.5 mg / ml PS 20.

[0120] In one embodiment, the pharmaceutical composition comprises about 20 mM histidine buffer (pH about 5.5), about 9% (W / V) sucrose, and about 0.2 mg / ml PS 20.

[0121] In one embodiment, the concentration of the AIAC protein in the pharmaceutical composition is about 18-110 mg / ml, e.g., 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 concentration of the AIAC protein in the pharmaceutical composition is about 62.2 mg / ml. In another preferred embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 82.2 mg / ml. In another preferred embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 100.4 mg / ml.

[0122] In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 18 mg / ml to 66 mg / ml, e.g., 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 protein concentration of the AIAC in the pharmaceutical composition is about 18 mg / ml to 66 mg / ml, e.g., 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.

[0123] In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 20 mg / ml to 60 mg / ml. In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 20 mg / ml to 60 mg / ml.

[0124] In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 20 mg / ml.In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 20 mg / ml.

[0125] In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 40 mg / ml.In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 40 mg / ml.

[0126] In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 60 mg / ml.In one embodiment, the protein concentration of the AIAC in the pharmaceutical composition is about 60 mg / ml.

[0127] 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.

[0128] 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.

[0129] In some embodiments, the AIAC has the 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.

[0130] Treatment and Use The conjugates of the present disclosure are useful for treating tumors and / or autoimmune diseases. Tumors amenable to conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors that can be recognized by the target molecule in the conjugate and be susceptible to the immune cell activating activity of the agonist in the conjugate.

[0131] Thus, in yet another aspect, there is also provided the use of a conjugate of the present disclosure or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for preventing, alleviating, or treating a disease, disorder, or condition selected from a tumor or an autoimmune disease.

[0132] In another aspect, there is provided a conjugate of the present disclosure or a pharmaceutical composition of the present disclosure for use in the prevention, alleviation, or treatment of a tumor or an autoimmune disease.

[0133] In a further aspect, there is provided a method for preventing, alleviating, or treating a tumor or an autoimmune disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0134] In a preferred embodiment, the conjugate of the present disclosure formed by conjugating an anti-human HER2 antibody with a payload can specifically bind to HER2 on the surface of tumor cells and selectively kill tumor cells expressing HER2. Another preferred embodiment provides a use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for preventing, alleviating, or treating a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0135] The dose of the conjugate administered to a subject can be adjusted to a considerable extent. The dose can vary according to the particular route of administration, the needs of the subject, and can be subject to the judgment of a health care professional.

[0136] Beneficial effects The present disclosure utilizes a linking unit with a unique structure and catalyzes the conjugation of an antibody (i.e., an anti-HER2 antibody) and an agonist using a ligase. The conjugates of the present disclosure have good homogeneity, high activity, and high selectivity. In particular, the intracellular metabolites exhibit significantly reduced cell proliferation toxicity against cells that express low levels or no target antigen. Furthermore, the toxicity of the linking unit-agonist intermediate is much lower than that of the free agonist, thus reducing the adverse impact on the drug manufacturing process, which is advantageous for industrial production. Furthermore, the formulations of the present disclosure ensure that the ADCs and AIACs of the present disclosure have good physicochemical and biological properties.

[0137] The composite of the present disclosure achieves at least one of the following technical effects: (1) High inhibitory activity against target cells or strong killing effect against target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., good stability in plasma, adequate half-life and duration of action). (4) Good safety (low toxicity to normal cells or tissues other than the target, and / or fewer side effects, wider therapeutic window), etc.

[0138] The drug can prevent the patient's resistance to HER2-targeted therapy and activate bone marrow cells to enhance the innate and adaptive immune response, which can overcome the low response rate of current HER2-directed therapy. And the formulation of the present disclosure can ensure at least one or more of the above technical effects of the AIAC of the present disclosure.

[0139] Example Preparation Examples In order to more clearly illustrate the objectives and technical solutions, the present disclosure will be further described below with reference to specific examples. It should be understood that the examples are not intended to limit the scope of the present disclosure. Specific experimental methods not mentioned in the following examples were carried out according to conventional experimental methods.

[0140] Equipment, materials and reagents Unless otherwise stated, the instruments and reagents used in the examples are commercially available. Reagents can be used directly without further purification. MS: Thermo Fisher Q Exactive Plus, Water 2795-Quattro micro triple quadrupole mass spectrometer HPLC:Waters 2695,Agilent 1100,Agilent 1200 Semi-preparative HPLC: Lisure HP plus 50D Flow cytometry: CytoFLEX S HIC-HPLC: Butyl-HIC; Mobile phase A: 25 mM PB, 2 M (NH4)2SO4, pH 7.0; Mobile phase B: 25 mM PB, pH 7.0; Flow rate: 0.8 ml / min; Collection time: 25 min; Injection volume: 20 μg; Column temperature: 25°C; Detection wavelength: 280 nm; Sample chamber temperature: 8°C. SEC-HPLC: Column: TSK-gel G3000 SWXL, TOSOH 7.8 mm ID x 300 mm, 5 μm; Mobile phase: 0.2 M KH2PO4, 0.25 M KCl, pH 6.2; Flow rate: 0.5 ml / min; Collection time: 30 min; Injection volume: 50 μl; Column temperature: 25 °C; Detection wavelength: 280 nm; Sample tray temperature: 8 °C. CHO cells were obtained from Thermo Fisher Scientific. pcDNA 3.3 was obtained from Life Technology. HEK293F cells were obtained from Prejin. PEIMAX transfection reagent was obtained from Polyscience. MabSelect Sure ProA was obtained from GE. Capto S ImpAct was obtained from GE. Rink-Amide-MBHA-resin and dichloro resin were obtained from Nankai synthesis. HCC1954 cells were obtained from ATCC catalog number CRL-2338. SK-BR-3 cells were obtained from ATCC catalog number HTB-30. BT474 cells were obtained from ATCC catalog number HTB-20. JIMT1 cells were obtained from DSMZ catalog number ACC589. Colo205 cells were obtained from ATCC catalog number CRL-222. MC38Hher2 mouse colorectal carcinoma cells were obtained from Biocytogen. NUGC4 human gastric cancer cells were obtained from JCRB catalog number JCRB0834. NCI-N87 cells (ATCC catalog number CRL-5822); MDA-MB-468 were obtained from ATCC catalog number HTB-132.

[0141] Example 1 Construction of antibody expression vector, antibody expression, purification, and identification 1.1 Modified anti-human HER2 antibody Ab0001-LCCT L -HC production Antibody Ab0001-LCCT L An expression plasmid for Ab0001-HC (light chain: SEQ ID NO: 1, heavy chain: SEQ ID NO: 2) was constructed as follows: L The sequence of -HC: GALPETGG (SEQ ID NO: 29) was introduced into the C-terminus of the light chain based on the amino acid sequence of trastuzumab, where LPETGG is the recognition sequence for the ligase donor substrate and GA is the spacer sequence. The plasmid was transfected into CHO cells, a cell population was established, and a highly expressing cell population was screened. This was cultured in a 5-10 L reactor based on the culture process of trastuzumab, and the supernatant was collected.

[0142] 1.2 Antibody Ab0001-LCCT L -HC purification Ab0001-LCCT was purified using a standard process using a combination of MabSelect affinity chromatography and Sepharose S cation exchange chromatography. L Purification of -HC was performed and the purified product was dissolved in the original trastuzumab drug buffer (5 mM histidine-HCl, 2% trehalose, 0.009% polysorbate 20, pH 6.0) and frozen in small aliquots.

[0143] 1.3 Antibody Ab0001-LCCT L -HC quality control The antibody Ab0001-LCCT purified above L The purity of -HC is 98.5% by SDS-PAGE, the content of high molecular weight polymers in the sample is less than 0.4% by SEC-HPLC, and the endotoxin content is less than 0.098 EU / mg.

[0144] 1.4 Preparation of Other Modified Anti-Human Antibodies Following a similar method, terminal modifications based on ligase recognition sequences were introduced into the C-terminus of the light chain and / or heavy chain of trastuzumab, respectively, to obtain modified antibodies.

[0145] Modified anti-human HER2 antibodies based on Ab0001 (trastuzumab) are listed in Table 1. LPETGG in the terminal modification sequence is the recognition sequence for the ligase donor substrate, and GA is the spacer sequence. [Table 3]

[0146] CDR sequences of modified anti-human HER2 antibodies based on Ab0001 (trastuzumab), V L , and V H are listed in Table 2. [Table 4]

[0147] Example 2 Preparation of Intermediates 2.1 Preparation of Linking Units The connecting unit is [ka] Represents.

[0148] Linker unit fragment LU102 was synthesized by conventional solid-phase polypeptide synthesis using Rink-amide-MBHA-resin or dichloro-resin. Fmoc was used to protect the amino acids in the linker unit. Conjugation reagents were selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the resin was cleaved using trifluoroacetic acid. The product was purified by HPLC, lyophilized, and stored for use. The linker unit fragments are listed in the table below. [Table 5]

[0149] The linking unit fragments in the table above were reacted with a linking unit fragment containing a maleimide structure or its derivative, followed by a ring-opening reaction using the method described in WO2015165413A1 to obtain linking units LN102-6 and LN102-8, whose structures are shown above. The linking unit fragments are listed in the table below. [Table 6] In the formula, Mal is [ka] When fragment 1 and fragment 2 react to form a linking unit, the maleimide ring of fragment 2 opens, forming a ring-open succinimide structure. [ka] Form.

[0150] 2.2 Preparation of Linker Unit-Agonist Intermediate 2.2.1 Preparation of Linker Unit-Agonist Intermediate LP102-6-1 [ka]

[0151] Step 1: Resiquimod (25.0 g, 79.5 mmol) was dissolved in MeCN (500 mL) and treated with Trt-Cl (33.25 g, 119.3 mmol) followed by TEA (20.12 mL, 20.12 mmol). The reaction was refluxed for 2-3 h (TLC). The reaction mixture was concentrated in vacuo. The mixture was then treated with AcOEt (700 mL) and HO (400 mL), stirred for 30 min, and separated. The organic phase was concentrated in vacuo to 300 mL and treated with n-heptane (400 mL). The mixture was then stirred for 20 min. After filtration, the cake was washed with EtOH / HO (1:1, 200 mL) and filtered. The cake was dried in vacuo to give the target compound HX20031-a as a white solid (43.9 g, 99.1%).

[0152] Step 2: Compound (HX20031-a) (40.02 g, 72.9 mmol) was dissolved in DMF (200 mL) and cooled to 0-10 °C. NaH (60%, 3.74 g, 93.4 mmol) was added in a batch. The suspension was stirred vigorously at 0-10 °C for 1 h, then warmed to 20-30 °C and stirred for 1 h. The mixture was then cooled to 0-10 °C and treated with compound 1186g (20.88 g, 93.4 mmol) in one portion. The mixture was stirred at room temperature overnight and then slowly treated with a mixture of 10% NaH2PO4 (1 L) and AcOEt (1 L). The reaction mixture was stirred for 3 h and filtered. The organic layer was concentrated and purified by silica gel column chromatography (n-heptane → n-heptane / AcOEt=10:1 → n-heptane / AcOEt=4:1) to obtain the target compound HX20031-b (24.89 g, 46.9%).

[0153] Step 3: Compound HX20031-b (10 g, 14.3 mmol) was treated with a mixture of TFA (40 mL) and HO (80 mL). The reaction mixture was stirred at room temperature for 24 h. The mixture was then poured into MTBE (400 mL) and stirred for 2 h. After filtration, the cake was washed with MTBE (200 mL) and filtered. The cake was dried in vacuo to give the target compound HX20031-c as a white solid (8.51 g, 100%).

[0154] Step 4: Compound HX20031-c (6.0 g, 10.2 mmol) was dissolved in DMF (50 mL) and treated with DIPEA (3.5 mL, 20.4 mmol) and N-succinimidyl 3-maleimidopropionate (3.27 g, 12.3 mmol). The reaction was maintained at room temperature for 3 hours (HPLC), after which the mixture was used directly in the next step.

[0155] Steps 5-6: The mixture from Step 4 was treated with a solution of fragment HX18041 (LU102) (5.5 g, 15.3 mmol) and HO (50 mL). The mixture was allowed to react at 0-40°C for 0.5-20 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 allowed to proceed at 0-40°C for 0.2-20 hours. After completion of the reaction, the product was purified by semi-preparative / preparative HPLC and lyophilized to give the linked unit-agonist LP102-6-1 (3.3 g, 30% over three steps). MS m / z 1065.6 [M+H] + . [Table 7]

[0156] Example 3 Preparation of target molecule-drug conjugates Each linking unit-agonist intermediate was conjugated to an antibody in a site-specific manner by a ligase, thereby forming an AIAC. The method for the conjugation reaction can be found in WO2015165413A1. The resulting AIACs are listed in the table below: [Table 8]

[0157] Efficacy Example 1 In vitro evaluation of antibody-immune agonist conjugates Isolation of human peripheral mononuclear cells Human peripheral mononuclear cells were isolated from healthy blood donors using SepMate 50 and Lymphoprep (Stem Cell Technologies). Viable cells were counted and the cell concentration was adjusted to 1.25 × 10 in RPMI 1640 medium containing 10% FBS. 6 Tumor cells were detached with trypsin and collected. Viable cells were counted and the cell concentration was adjusted to 2.5 × 10 / ml in RPMI 1640 medium containing 10% FBS. 5 / ml. 12.5 × 10 4 human PBMCs, and 2.5 x 10 4 After adding tumor cells (PBMC:tumor cells = 5:1), antibodies or conjugates were added at the indicated concentrations. After incubating the cell mixture with the drug for 18 hours, cell-free supernatants were collected for human TNFα ELISA.

[0158] To evaluate the activity of HER2-targeted immunoconjugates, human PBMCs and NCI-N87 human gastric cancer cells were cocultured at a 5:1 ratio, and the antibody or test immunoconjugate (AC102-6-1-1 or AC102-8-1-1) was added at the indicated concentrations. AC102-6-1-1 induced higher TNFα production than the antibody Ab0001, and the effective concentration of AC102-6-1-1 was much lower than that of the payload resiquimod (Figure 1). AC102-8-1-1 induced higher levels of TNFα than Ab0001, which were similar to those of AC102-6-1-1 (Figure 2). The activity of AC102-6-1-1 was not observed in cocultures of human PBMCs and MDA-MB-468 HER2-negative cells, indicating that the activity of AC102-6-1-1 is highly dependent on the expression of HER2 in the target tumor cells (Figure 3). Given this data, the activity of the immunoconjugate was tested in cocultures of human PBMCs with cancer cells with various HER2 expression levels, including HCC1954 (Figure 4), SK-BR-3 (Figure 5), BT474 (Figure 6), JIMT1 (Figure 7), Colo205 (Figure 8), and MDA-MB-468 (Figure 9). These data indicated that AC102-6-1-1 could induce TNFα only in cocultures of PBMCs and HER2-high tumor cells.

[0159] Efficacy Example 2 In vitro evaluation of antibody-immune agonist conjugates To evaluate the activity of the HER2-targeted immunoconjugate, human PBMCs were co-cultured with SK-BR-3 ( FIG. 10 ) or HCC1954 ( FIG. 11 ) human breast cancer cells at a 5:1 ratio, and the immunoconjugate (AC102-6-1-1) and antibody (Ab0001) were added at the indicated concentrations. After incubating the cells with the drug for 18 hours, cell-free supernatants were collected for human IFNγ detection by ELISA. The isolation of human PBMCs and the experimental setup were similar to those in Efficacy Example 1. AC102-6-1-1 induced higher IFNγ production than antibody Ab0001, suggesting its potential ability to activate T cell responses.

[0160] Efficacy Example 3: In vivo evaluation of antibody-immune agonist conjugates 1 × 10 for in vivo antitumor efficacy studies 7 NCI-N87 human gastric cancer cells were subcutaneously inoculated into the right flank of SCID Beige mice. Six days later, tumor volumes averaged 173 mm 3 When tumor-bearing mice reached 0.5 mg / kg, tumor-bearing mice were assigned and intravenously administered Ab0001 or test immunoconjugates (AC102-6-1-1 or AC102-8-1-1) at 5 mg / kg. Tumor volumes were measured twice weekly with calipers. Antibody Ab0001 itself showed very limited antitumor activity. AC102-6-1-1 and AC102-8-1-1 nearly cured tumors at termination (Figure 12). In a separate study, AC102-8-1-1 showed dose-dependent activity at 0.5, 1, and 3 mg / kg (Figure 13).

[0161] 5×10 6 JIMT1 human breast cancer cells were inoculated subcutaneously into the right flank of SCID Beige mice to generate xenograft models. After 9 days, tumor volumes averaged 149 mm 3 When tumor growth reached 100%, AC102-8-1-1 was administered intravenously at 5 mg / kg to tumor-bearing mice. Tumor growth was significantly inhibited (FIG. 14).

[0162] Overexpressing human HER2, 5 × 10 5 MC38 hHER2 murine colorectal cancer cells were inoculated subcutaneously into the right flank of C57BL / 6 mice. After 8 days, tumor volumes averaged 90 mm 3 When the tumor-bearing mice reached 100 mg / kg, tumor-bearing mice were assigned and intravenously administered Ab0001 or AC102-6-1-1. Ab0001 at 10 mg / kg showed no significant antitumor activity. AC102-6-1-1 at 3 mg / kg and 10 mg / kg inhibited tumor growth in a dose-dependent manner (Figure 15). In a similar setting, AC102-8-1-1 at both 3 mg / kg and 10 mg / kg induced complete tumor regression in 100% of mice (Figure 16).

[0163] Examples of formulations containing AIAC 1. pH Example I. Preliminary selection of a stable pH range for the formulation.

[0164] I.1 Experimental materials Sample: AC102-8-1-1; Batch number 20200815; [ka] z is an integer from 1 to 4, A is the modified antibody trastuzumab.

[0165] I.2 Experimental Method Five groups of formulation buffers with different pH (pH values ​​were 5.0, 6.0, 7.0, 8.0, 9.0) were prepared, and the five groups of formulation buffers were as follows: [Table 9]

[0166] The AC102-8-1-1 sample was buffer-exchanged using five buffers by dialysis, and the protein concentration was adjusted to approximately 20 mg / ml. The stable pH range of AC102-8-1-1 was determined by testing its stability in buffers of different pH values ​​at 2-8°C, 25°C, and 40°C.

[0167] The sample preparation procedure was as follows: a) Thaw the sample. b) Load the sample into the dialysis tubing. c) Dialyze against 100 times the sample volume of buffer at room temperature for 2 hours. d) Change the dialysis buffer and dialyze twice overnight at 2-8°C. e) Test protein concentration and adjust to 20mg / ml. f) Sterile filter the sample using a 0.22 μm filter. g) Fill, label and test samples for stability.

[0168] The stability test conditions and times are shown in the table below: [Table 10]

[0169] Test items: Purity (reduced CE-SDS method, non-reduced CE-SDS method), DAR value (HIC-HPLC), free drug (RP-HPLC).

[0170] Detection Method Purity (reduced CE-SDS) To prepare the test solution, the test sample was diluted with ultrapure water to approximately 10 mg of protein per mL. 85 μL of sample buffer (4 mL of pH 6.2 citrate-phosphate buffer + 1 mL of 10% SDS, water added to 25 mL) and 5 μL of β-mercaptoethanol were added to 10 μL of 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, and centrifuged at 13,000 rpm for 10 minutes before loading. 85 μL of 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 sum of the corrected peak areas (%) of the light and heavy chains was used to determine the purity of the sample.

[0171] Purity (non-reduced CE-SDS) To prepare the test solution, the test sample was diluted to approximately 10 mg / mL with ultrapure water. 85 μL of sample buffer (4 mL of pH 6.2 citrate-phosphate buffer + 1 mL of 10% SDS, water added to 25 mL) and 5 μL of 500 mM iodoacetamide were added to 10 μL of the 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, and centrifuged at 13,000 rpm for 10 minutes before loading. The supernatant was injected into a sample vial for analysis. The load voltage was 5 kV, the load time was 20 seconds, the separation voltage was 15 kV, the separation time was 35 minutes, and the detector wavelength was 220 nm. The corrected peak area percentage of the monomer peak was used to determine the purity of the sample.

[0172] DAR value (HIC) The DAR value was analyzed by hydrophobic chromatography. The analytical column was a 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) mixed with isopropanol 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. For the test solution, the test sample was diluted with purified water to a solution containing approximately 5 mg per mL. The injection volume was 5 μL. The elution gradient was performed according to the table below. [Table 11]

[0173] The area percentages (%) of DAR0, DAR1, and DAR2 were calculated according to the area normalization method: DAR = DAR1% + DAR2% * 2.

[0174] Free drug (RP-HPLC) Free drug was detected by reversed-phase high-performance liquid chromatography. The analytical column was an ACQUITY PREMIER C18AX, 2.1 mm x 150 mm, with 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 test sample, and the solution was thoroughly mixed. The solution was centrifuged at 13,000 rpm for 15 minutes, and the supernatant was collected as the test solution. Appropriate amounts of reference substances were prepared: linker-agonist reference substance solutions containing 0.267 μg to 5.3 μg per mL, and agonist reference substance solutions containing 0.067 μg to 1.3 μg per mL. The injection volume was 3 μL. Reference solutions were then prepared in the same manner. The elution gradient was performed according to the table below. [Table 12]

[0175] The content of free drug was calculated by the external standard method.

[0176] I.3 Results The purity test results were as follows:

[0177] Non-reduced CE-SDS Purity (%) Test Results: [Table 13]

[0178] Reduced CE-SDS Purity (%) Test Results: [Table 14]

[0179] DAR value detection results: [Table 15]

[0180] Free drug detection results: [Table 16]

[0181] I.4 Conclusion 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 indicate that at pH 7.0, 8.0, and 9.0, AIAC has poor molecular stability, the protein is more likely to undergo degradation, the small molecule conjugated to the antibody is more likely to be released, and the DAR value decreases more significantly. Therefore, AIAC is not suitable for long-term storage at pH 7.0 to 9.0.

[0182] II. The optimal pH range of AIAC was further screened in the range of pH 4.0 to pH 6.0.

[0183] II.1 Experimental materials Sample: AC102-8-1-1; Batch number 20200815.

[0184] II.2 Experimental Method Four groups of formulation buffers with different pH (pH values ​​are 4.0, 5.0, 5.5, 6.0, respectively) were prepared, and the four groups of formulation buffers were as follows: [Table 17]

[0185] The AC102-8-1-1 samples were buffer-exchanged using four buffers by dialysis, and the protein concentration was adjusted to approximately 20 mg / ml. The stable pH range of AC102-8-1-1 was determined by testing its stability in buffers of different pH values ​​at 2-8°C, 25°C, and 40°C. The sample preparation procedure was the same as in Example I.2.

[0186] The stability test conditions and times are shown in the table below: [Table 18]

[0187] Test items: Purity (SE-HPLC), Charge Variant (CEX-HPLC), T m / T agg .

[0188] Detection Method Purity (SE-HPLC) Sample purity was confirmed by size-exclusion high-performance liquid chromatography (SE-HPLC). The analytical column was a TSKgel G3000SWXL, 7.8 × 300 mm. The mobile phase was a 0.2 mol / L potassium dihydrogen phosphate-0.25 mol / L potassium chloride solution with a pH of 6.2. The test sample was diluted with ultrapure water to approximately 2 mg per mL, and 50 μL of the test solution was injected into the HPLC. 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 min. The percentages of aggregate, monomer, and fragment components were calculated using area normalization.

[0189] Charge variants (CEX-HPLC) Charge heterogeneity was detected by cation exchange high-performance liquid chromatography (CEX-HPLC). The 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, and 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 with ultrapure water to approximately 2 mg per mL to prepare 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. [Table 19]

[0190] The percentages of major, acidic, and basic components were reported by calculating according to the area normalization method.

[0191] T m / T agg Using the Uncle analysis system (Unchained Labs), m and T aggThe analysis was carried out with the following analytical parameters: SLS settings: Experiment: Example Experiment 1; Start temperature (°C): 25; Incubation time (sec): 180; Ramp rate (°C / min): 0.7; Plate hold (sec): 45; End temperature (°C): 95. DLS settings: Initial acquisition: Yes; Final acquisition: Yes; Number of acquisitions: 4; Acquisition time (sec): 5; Attenuator control: Auto; Laser control: Auto.

[0192] II.3 Results Purity (%) (SEC-HPLC) test results: [Table 20]

[0193] Charge variant (%) (CEX-HPLC) analysis results: [Table 21]

[0194] Protein thermostability analysis results: [Table 22]

[0195] II.4 Conclusion At pH 4.0 and 5.0, more fragments were produced and the purity decreased more rapidly. AIAC molecules have a lower T m and T agg The results of protein thermal stability analysis also showed that the protein had a high pH and easily aggregated. At pH 6.0, the acid component increased more rapidly. Therefore, the optimal pH was finally screened as 5.5.

[0196] 2. Screening of fillers 2.1 Purpose of the experiment Screen fillers (stabilizers) to stabilize AIAC.

[0197] 2.2 Experimental materials Sample: AC102-8-1-1; Batch number 20200815.

[0198] .3 Experimental Method Commonly used protein stabilizers in biological products include sucrose, trehalose, and sorbitol. In this example, 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 formulations of the three formulations were as follows: [Table 23]

[0199] The AC102-8-1-1 sample was buffer exchanged using three buffers by dialysis, and the protein concentration was adjusted to approximately 20 mg / ml. The stability of AC102-8-1-1 in different formulations was examined to screen for suitable stabilizers. The sample preparation procedure was the same as in Example I.2.

[0200] Detection Method Binding activity (ELISA) The binding activity was measured by enzyme-linked immunosorbent assay (ELISA). The antigen (human HER2 protein) was added to a microtiter plate, coated overnight at 2-8°C, and blocked with a blocking solution. After washing the plate with a washing solution, the reference solution and test solution were added, respectively, and incubated at 25°C for 1 hour. After washing the plate with a washing solution, a horseradish peroxidase-labeled mouse anti-human IgG-Fc antibody solution 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 the reaction was terminated with a termination solution, the absorbance was measured at a wavelength of 450 nm. The EC values ​​of the reference and test articles were calculated using a four-parameter logarithmic regression (4PL) model. 50The relative activity of the test article was calculated using the EC 50 / Test Article EC 50 It was calculated by

[0201] 2.4 Results Purity (SEC-HPLC) test results (aggregation %): [Table 24]

[0202] Relative binding activity assay results: [Table 25]

[0203] 2.5 Conclusion Combining the SEC and binding activity results, sucrose was superior to trehalose and sorbitol as a bulking agent (stabilizer).

[0204] 3. Research into the effects of surfactants 3.1 Purpose of the experiment The effect of surfactants on the formulation stability of AIAC was investigated.

[0205] 3.2 Experimental materials Sample: AC102-8-1-1; Batch number 20200815.

[0206] 3.3 Experimental method Preparation groups 1-3 contained no Tween 20, and groups 4-7 contained 0.2 mg / mL Tween 20. The buffer, pH value, and protein concentration of the formulations of all seven groups remained the same. The formulations of the seven groups were as follows: [Table 26]

[0207] The AC102-8-1-1 samples were buffer exchanged using seven buffers by dialysis, and the protein concentration was adjusted to approximately 20 mg / ml. The stability of AC102-8-1-1 in different formulations was examined to determine the protective effect of surfactants (Tween 20, PS20) on AIAC proteins. The sample preparation procedures were the same as in Example I.2.

[0208] Detection parameters: inspection for visible foreign matter (visual method) (F / T is an abbreviation for freeze / thaw).

[0209] 3.4 Results [Table 27]

[0210] 3.5 Conclusion Tween 20 could effectively inhibit the aggregation and precipitation of AIAC molecules during freeze-thaw and freezing.

[0211] Combined with all previous screening results, the formulation of AIAC was determined to be: 20 mM His, pH 5.5 (buffer) 9% (W / V) sucrose (stabilizer) 0.2 mg / mL PS 20 (stabilizer and surfactant).

[0212] 4. AIAC formulation concentration 4.1 Purpose of the experiment After the formulation composition is determined, a suitable protein concentration range is further selected.

[0213] 4.2 Experimental materials Sample: AC102-6-1-1; Batch number 20210114; [ka] z is an integer from 1 to 4, A is the modified antibody trastuzumab.

[0214] 4.3 Experimental Method As shown in the table below, the protein concentrations of Groups 1 to 3 were 20 mg / mL, 40 mg / mL, and 60 mg / mL, respectively. The other formulation components were the same. The formulations of the three groups were as follows: [Table 28]

[0215] The AC102-6-1-1 samples were buffer exchanged using three buffers by dialysis, and the protein concentrations were adjusted to approximately 20 mg / mL, 40 mg / mL, and 60 mg / mL, respectively. The appropriate protein concentration range was determined by monitoring the stability of AC102-6-1-1 at different protein concentrations. The sample preparation procedure was the same as in Example I.2.

[0216] The inspection conditions and sampling time points were as follows (F / T is an abbreviation for freeze / thaw): [Table 29]

[0217] Detection parameters: visible particle inspection (visual method), SEC-HPLC purity detection, non-reduced CE-SDS purity detection, reduced CE-SDS purity detection, DAR value detection, free drug detection. The test methods are similar to those described above.

[0218] 4.4 Results Visible particle detection results: [Table 30]

[0219] SEC-HPLC results for % aggregation detection: [Table 31]

[0220] Non-reduced CE-SDS Purity (%) Test Results: [Table 32]

[0221] Reduced CE-SDS Purity (%) Test Results: [Table 33]

[0222] DAR value test results: [Table 34]

[0223] Free drug detection results (linker-payload, ppm): [Table 35]

[0224] Free drug detection results (agonist, ppm): [Table 36]

[0225] 4.5 Conclusion 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 had very good stability in the concentration range of 20 to 60 mg / mL under this formulation.

[0226] 5.High protein concentration of AIAC 5.1 Purpose of the experiment The high protein concentration formulation will be further developed and its stability verified.

[0227] 5.2 Experimental materials Sample: AC102-6-1-1.

[0228] 5.3 Experimental Method The formulation of the high protein concentration formulation was as follows: [Table 37]

[0229] The AC102-6-1-1 samples were buffer-exchanged using six buffers by dialysis, 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 procedures were the same as in Example I.2.

[0230] The inspection conditions and sampling time points were as follows (F / T is an abbreviation for freeze / thaw): [Table 38]

[0231] Detection parameters: inspection for visible particles (visual method), purity detection by SEC-HPLC, purity detection by non-reduced CE-SDS.

[0232] 5.4 Results Visible particle detection results: [Table 39]

[0233] [Table 40]

[0234] SEC-HPLC Purity (%) Test Results: [Table 41]

[0235] Non-reduced CE-SDS Purity (%) Test Results: [Table 42]

[0236] 5.5 Conclusion The results of visible particle inspection, SEC-HPLC purity detection, and non-reduced CE-SDS purity detection showed that AC102-6-1-1 has very good stability in high protein concentration formulations, such as 62.2 mg / ml, 82.2 mg / ml, and 100.4 mg / ml. Therefore, AIAC can be used at high concentrations under the formulation of the present disclosure.

[0237] While specific embodiments of the present disclosure have been described above, it should be understood that these are merely for illustrative purposes, and that the scope of the present disclosure is defined by the appended claims. Those skilled in the art may make various changes or modifications to the embodiments without departing from the spirit and scope of the present disclosure, and such changes and modifications are included within the scope of the present disclosure.

[0238] Sequence Listing SEQ ID NO: 1: Ab0001-LCCT L -HC light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETG SEQ ID NO: 2: Ab0001-LCCT L -HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 3: Ab0001-LC-HCCT Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 4: Ab0001-LC-HCCT Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID NO:5:Ab0001-LC-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:6:Ab0001-LC-HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG SEQ ID NO: 7: Ab0001-LCCT-HC Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID NO: 8: Ab0001-LCCT-HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK <0'001439>Sequence number 9: Ab0001-LCCT-HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG Sequence number 10: Ab0001-LCCT-HCCT heavy chain: It should be noted that in the original text, " " seems to have an incorrect format. It is written as "0'001439" in the translation for the sake of showing the difference. You may want to double-check the original text for accuracy.EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID NO:11:Ab0001-LCCT-HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID NO:12:Ab0001-LCCT-HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG Sequence number 13: Ab0001-LCCT L -HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 14: Ab0001-LCCT L -HCCT heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG sequence number 15:Ab0001-LCCT L -HCCT L Light weight: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG sequence number 16:Ab0001-LCCT L -HCCT L heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG SEQ ID NO: 17: LCDR1: RASQDVNTAVA SEQ ID NO: 18: LCDR2: SASFLYS SEQ ID NO: 19: LCDR3: QQHYTTPPT SEQ ID NO: 20: HCDR1:DTYIH SEQ ID NO: 21: HCDR2: RIYPTNGYTRYADSVKG SEQ ID NO: 22: HCDR3: WGGDGFYAMDY SEQ ID NO:23:V L : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV SEQ ID NO:24:V H : EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

Claims

1. an antibody-immune agonist complex (AIAC); A buffering agent; A stabilizer and A pharmaceutical composition comprising: The AIAC has the structure of formula (II-1), (II-2), or a mixture thereof. 【Chemical 1】 [Wherein B2 is -(CH ２ ) ｋ (CO)-NH-(C ２ H ４ -O) ｊ - or - (CH ２ ) 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 of 1 or 2, and R １ and R ２ are hydrogen, -OH, and -NH ２ , -C １－６ independently selected from alkyl; PL is a payload linked to the B2 portion, Preferably, the PL is resiquimod, 【Chemistry 2】 and z is an integer of 1 to 4, preferably 1 or 2; A is V Ｌ and V Ｈ an antibody comprising Here, the V Ｌ comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 17, an LCDR2 having the amino acid sequence of SEQ ID NO: 18, and an LCDR3 having the amino acid sequence of SEQ ID NO: 19; The V Ｈ comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 20, an HCDR2 having the amino acid sequence of SEQ ID NO: 21, and an HCDR3 having the amino acid sequence of SEQ ID NO: 22; The antibody is modified by the introduction of a ligase donor substrate recognition sequence.

2. The antibody has the amino acid sequence of SEQ ID NO: 23 Ｌ and V having the amino acid sequence of SEQ ID NO: 24 Ｈ The pharmaceutical composition of claim 1 , comprising:

3. an antibody-immune agonist complex (AIAC); an acidic buffer; A stabilizer and A pharmaceutical composition comprising: The AIAC has the structure of formula (II-1), (II-2), or a mixture thereof. 【Chemistry 3】 [Wherein B2 is -(CH ２ ) ｋ (CO)-NH-(C ２ H ４ -O) ｊ - or - (CH ２ ) 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 of 1 or 2, and R １ and R ２ are hydrogen, -OH, and -NH ２ , -C １－６ independently selected from alkyl; PL is the payload linked to the B2 portion, Preferably, the PL is resiquimod, 【Chemistry 4】 and z is an integer from 1 to 4, preferably 1 to 2; A is, 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; an antibody comprising:

4. R １ and R ２ are each independently hydrogen or C １－６ alkyl, preferably R １ and R ２ are each independently hydrogen or C １－６ alkyl, and more preferably, R １ and R ２ The pharmaceutical composition according to any one of claims 1 to 3, wherein both are hydrogen.

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

6. The AIAC is selected from the following structures, or mixtures thereof: 【Chemistry 5】 z is an integer of 1 to 4, preferably 1 or 2; A is V Ｌ and V Ｈ wherein said V Ｌ comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 17, an LCDR2 having the amino acid sequence of SEQ ID NO: 18, and an LCDR3 having the amino acid sequence of SEQ ID NO: 19, Ｈ comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 20, an HCDR2 having the amino acid sequence of SEQ ID NO: 21, and an HCDR3 having the amino acid sequence of SEQ ID NO: 22; the antibody is modified by the introduction of the 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; The pharmaceutical composition according to claim 4.

7. 7. The pharmaceutical composition of claim 1, wherein the conjugate of the AIAC has an antibody-drug ratio (DAR) that is an integer or non-integer between 1 and 4, between 1 and 4, between 1 and 3.5, between 1 and 3, between 1 and 2.5, between 1 and 2, between 1.5 and 2, between 1.6 and 2, or between 1.7 and 2.

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

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the concentration of the buffering agent is 10 to 40 mM, or the concentration of the buffering agent is 15 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. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the stabiliser comprises one or more of sucrose, trehalose dihydrate, trehalose and sorbitol, preferably sucrose.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the stabiliser comprises 5-15% (W / V) of one or more of sucrose, trehalose dihydrate, trehalose and sorbitol, preferably 9% (W / V) of sucrose, trehalose dihydrate, trehalose and sorbitol, more preferably 9% (W / V) of sucrose.

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

14. 14. The pharmaceutical composition of claim 13, wherein the surfactant comprises 0.05-0.5 mg / ml tween 20 or tween 80, preferably 0.2 mg / ml tween 20.

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

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

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

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