Antibody-immune agonist conjugate in combination with anti-pd-1 antibodies and uses thereof

Abstract

This document relates to combinations of antibody-immunologic agonist conjugates (AIACs) with anti-PD-1 antibodies or antigen-binding fragments thereof and uses thereof.

Description

Technical Field

[0001] This article relates to the field of biomedicine, specifically to the combination of antibody-immunoagonist conjugates (AIACs) with anti-PD-1 antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] Immunotherapy, as a novel modality for cancer treatment, has demonstrated potent efficacy. While immune checkpoint inhibitors (essentially T-cell-based therapies), represented by CLTA-4 and PD-1 / L1 monoclonal antibodies, have been approved for various cancer indications, many researchers are exploring other mechanisms of the immune system to combat cancer. Targeting myeloid cells (primarily macrophages or dendritic cells) has emerged as a promising direction. Activating macrophages and dendritic cells through agonists or macrophage checkpoint inhibitors not only enhances their phagocytic capacity to clear tumor cells but also promotes their antigen-presenting function, which will more powerfully stimulate adaptive anti-tumor immunity.

[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 observed in other cancers such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer. (Iqbal N. et al., Mol Biol Int. 2014: 852748) Although HER2-targeted therapies (such as HER2-directed antibodies or antibody-drug conjugates (ADCs)) have significantly improved life expectancy in patients with HER2-positive disease, HER2-positive breast cancer remains inherently a more aggressive form of the disease, with a poorer prognosis and less desirable outcomes compared to patients with HER2-negative (and HR-positive) disease. Furthermore, studies have confirmed that such therapies remain ineffective in other HER2-overexpressing cancers. One of the many reasons for these adverse outcomes is the development of resistance in patients receiving HER2-targeted therapies. Immune escape by tumor cells leads to this process.

[0004] TLR7 and TLR8 are two important pattern recognition receptors located in the endosomal membranes of macrophages, dendritic cells (DCs), and monocytes. They instinctively sense viral ssRNA, thereby mediating the activation of immune cells and the release of pro-inflammatory cytokines. Numerous studies have demonstrated the antitumor activity of TLR7 / 8 agonists. Imiquimod, a TLR7 agonist, has been approved for topical treatment of genital warts, superficial basal cell carcinoma, and actinic keratosis. Resiquimod, a dual TLR7 / 8 agonist, has been approved for the treatment of cutaneous T-cell lymphoma. However, the side effects induced by systemic administration of TLR7 / 8 agonists limit their use in a broader spectrum of cancers.

[0005] Drug resistance is another major obstacle in cancer therapy development. Intrinsic resistance and acquired new resistance mutations can be observed in conventional chemotherapy, targeted therapy, and immunotherapy. Compensatory adaptation processes in oncogenic pathways have also been described as a resistance mechanism. In-depth research is underway to overcome this obstacle. The development of combination therapies often faces challenges such as limited clinical efficacy, more complex dosing regimens, pharmacological compatibility issues, and the resulting need for higher levels of risk management. Cost-effectiveness is also a factor to consider. Summary of the Invention

[0006] This article presents a combination therapy of an antibody-immunoagonist conjugate (AIAC) targeting HER2 with an anti-PD-1 antibody or its antigen-binding fragment, to reduce drug resistance and achieve better anti-tumor therapeutic effects.

[0007] In the first aspect, antibody-immunostimulant conjugates (AIACs) of formula (I-1) and / or formula (I-2) are provided: Formula (I-1) Formula (I-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -; k is an integer from 1 to 5; j is an integer from 1 to 3; d is an integer of 1 or 2; R 1 and R 2 Each is independently selected from hydrogen, -OH, -NH2, and -C. 1-6alkyl; PL is an agonist linked to the B2 portion. Preferably, PL is Resimot. ; z is an integer or non-integer from 1 to 4, preferably 1, 2 or 4; A is a target molecule modified by introducing a ligase donor substrate recognition sequence; preferably, the target molecule is an antibody or its antigen-binding fragment; more preferably, A is an anti-HER2 antibody or its antigen-binding fragment.

[0008] In one embodiment, the anti-HER2 antibody or its antigen-binding fragment comprises a light chain variable region (V... L ) and heavy chain variable region (V H ),in The V L Include: (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 17; (ii) LCDR2 containing the amino acid sequence of SEQ ID NO: 18; and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 19; And / or the V H Include: (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 20; (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 21; and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0009] In one embodiment, the antibody comprises V having the amino acid sequence of SEQ ID NO: 23. L And / or V having the amino acid sequence of SEQ ID NO: 24 H .

[0010] In a second aspect, a pharmaceutical combination is provided, the pharmaceutical combination comprising an antibody-immunostimulant conjugate and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody-immunostimulant conjugate has a structure of formula (I-1) and / or formula (I-2): Formula (I-1) Formula (I-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O)j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -; k is an integer from 1 to 5; j is an integer from 1 to 3; d is an integer of 1 or 2; R 1 and R 2 Each is independently selected from hydrogen, -OH, -NH2, and -C. 1-6 alkyl; PL is an agonist linked to the B2 portion. Preferably, PL is Resimot. ; Z is an integer or non-integer from 1 to 4, preferably 1, 2 or 4; A is a target molecule modified by introducing a ligase donor substrate recognition sequence; preferably, the target molecule is an antibody or its antigen-binding fragment; more preferably, A is an anti-HER2 antibody or its antigen-binding fragment.

[0011] In one embodiment, the anti-HER2 antibody or its antigen-binding fragment comprises a light chain variable region (V... L ) and heavy chain variable region (V H ),in The V L Include: (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 17; (ii) LCDR2 containing the amino acid sequence of SEQ ID NO: 18; and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 19; and / or The V H Include: (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 20; (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 21; and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 22.

[0012] In one embodiment, the antibody comprises V having the amino acid sequence of SEQ ID NO: 23. L And / or V having the amino acid sequence of SEQ ID NO: 24 H .

[0013] In one embodiment, the drug combination further comprises at least one pharmaceutically acceptable carrier.

[0014] In another aspect, a kit is provided that comprises the drug combination.

[0015] In another aspect, the use of the said drug combination or the said kit in the preparation of a medicament for the prevention, relief or treatment of a disease, wherein said disease is a tumor.

[0016] In another aspect, a method is provided for treating a disease suffered by a subject or preventing (or alleviating) the progression of a disease, the method comprising administering the combination of drugs or the kit; wherein the disease is a tumor.

[0017] In one embodiment, the tumor is a HER2-related tumor.

[0018] In one embodiment, the disease includes HER2-positive tumors or HER2-low-expressing tumors. In one embodiment, the disease includes HER2-overexpressing tumors or HER2-mutated tumors. In one embodiment, the HER2-related tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma. In one embodiment, HER2 low expression refers to IHC1. + or IHC2 + (FISH test negative). In one implementation scheme, HER2 positive means IHC2... + (FISH test positive) or IHC3 + .

[0019] In another aspect, a method is provided for treating a disease in a subject or reducing the likelihood of disease progression, the method comprising administering an effective amount of the antibody-immunostimulant conjugate to the subject, and administering an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof to the subject.

[0020] In another aspect, a method is provided for treating a subject with cancer or reducing the likelihood of cancer progression, the method comprising administering an effective amount of the antibody-immunostimulant conjugate to the subject, and administering an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof to the subject.

[0021] In another aspect, the use of an effective amount of an antibody-immunostimulant conjugate for the preparation of a medicament for treating a disease in a subject is provided, said medicament being used in combination with an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0022] In another aspect, the use of an effective amount of an antibody-immunostimulant conjugate for the preparation of a medicament for treating a subject’s cancer is provided, said medicament being used in combination with an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. Attached Figure Description

[0023] Figure 1 The efficacy of AC102-8-1-1 monotherapy or in combination with anti-mPD-1 antibody was demonstrated in the MC38-hHER2 homology model.

[0024] Figure 2 The efficacy of monotherapy with AC102-8-1-1, AC102-6-1-1 or OL-HX20042 alone, or in combination with anti-mPD-1 antibody, was demonstrated in the MC38-hHER2 homology model.

[0025] Figure 3 The efficacy of AC102-6-1-1 monotherapy or in combination with anti-mPD-1 antibody was demonstrated in the MC38-hHER2 homology model. Detailed Implementation

[0026] The following provides specific implementation schemes to illustrate the technical content of this document. Through the content disclosed in this specification, those skilled in the art can readily understand other advantages and effects of this document. This document can also be implemented or applied through other different specific implementation schemes. Those skilled in the art can make various modifications and changes without departing from the spirit of this document. Definitions

[0027] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein refers to those techniques commonly understood in the art, including variations and equivalents that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be readily understood by one of ordinary skill in the art, the following definitions are provided for the purpose of better illustrating this document. When a trade name appears herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0028] When a quantity, concentration, or other value or parameter is listed as a range, preferred range, or preferred upper or lower limit, it should be understood that this is equivalent to specifically disclosing any range formed by any combination of any upper or preferred value and any lower or preferred value, whether or not the range is explicitly listed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range. For example, the expression "z is an integer from 1 to 4" means that z is any integer from 1 to 4, such that z can be approximately 1, 2, 3, or 4. Other similar expressions (such as j and d) should also be understood in a similar manner.

[0029] Unless the context explicitly indicates otherwise, the singular forms of "a / an" and "the / described" include the plural forms. The expressions "one or more" or "at least one" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0030] When used in conjunction with numerical variables, the terms “about” and “approximately” generally mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0031] The term "stoichiometry" refers to the proportion of various substances in a specific weight ratio. For example, in this document, active ingredients are mixed with fillers, binders, and lubricants in a specified weight ratio.

[0032] The term “optional” or “optionally” means that the event described thereafter may but not necessarily occur, and the description includes the possibility that the event or situation described may or may not occur.

[0033] The expressions "comprising," "including," "containing," and "having" are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of" excludes any unspecified elements, steps, or components. The expression "substantially consisting of" means that the scope is limited to the specified elements, steps, or components, as well as other optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "substantially consisting of" and "consisting of."

[0034] The term "targeting molecule" refers to a molecule that has an affinity for a specific target (such as a receptor, cell surface protein, cytokine, etc.). Targeting molecules can deliver payloads to specific sites in the body through targeted delivery. Targeting molecules can recognize one or more targets. A specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a payload to a site containing a large number of receptors. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins to a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0035] As used herein, the term "antibody" is used broadly and specifically includes complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they possess the desired biological activity. The antibody may have any subtype (such as IgG, IgE, IgM, IgD, and IgA) or subclass and may be derived from any suitable species. In one embodiment, the antibody is human or murine. The antibody may also be a fully human antibody, humanized antibody, or chimeric antibody prepared by recombinant methods.

[0036] In this document, monoclonal antibodies are used to refer to antibodies obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical, except for a small number of possible natural mutations. Monoclonal antibodies exhibit high specificity for a single antigenic site, multiple antigenic sites, or different epitopes of the same antigen. The term "monoclonal" signifies that the antibody is derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring specific methods to produce the antibody.

[0037] A complete or full-length antibody essentially contains an antigen-binding variable region and a light chain constant region (C). L ) and heavy chain constant region (C H The heavy chain constant region may include C H 1. C H 2. C H 3 and / or C H 4. This depends on the antibody subtype. The antigen-binding variable region (also known as the variable region fragment, Fv fragment) typically contains a light chain variable region (V... L ) and heavy chain variable region (V H The constant region can be a constant region with a natural sequence (such as a constant region with a human natural sequence) or a variant of its amino acid sequence. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0038] As used in this article, the term "heavy chain constant region (C)" H")" contains an amino acid sequence derived from the complete antibody or the full-length antibody heavy chain. A polypeptide containing a heavy chain constant region includes at least one of the following: C H 1. Structural domain, hinge (e.g., upper, middle, and / or lower hinge areas), C H 2. Structural Domain, C H 3. Structural domains, C H 4. A domain or a variant or fragment thereof. For example, the antigen-binding polypeptide used in this article may contain: a C... H A polypeptide chain containing a 1-domain structure; containing C H 1. At least a portion of the structural domain, the hinge structural domain, and C H A polypeptide chain with two structural domains; containing C H 1. Structural Domains and C H A polypeptide chain with 3 structural domains; containing C H 1. At least a portion of the structural domain, the hinge structural domain, and C H A polypeptide chain containing a 3-domain structure; or containing C H 1. At least a portion of the structural domain and the hinge structural domain, C H 2. Structural Domains and C H A polypeptide chain with 3 structural domains.

[0039] As used in this article, "C" L "" refers to the constant region of the light chain.

[0040] The subunit structures and three-dimensional configurations of constant regions in various antibody classes are well known. As used herein, the term "V" refers to... H The term "domain" includes the amino-terminal variable domain of the antibody heavy chain, and the term "C" is also used. H The "1 domain" includes the first (farthest amino terminus) constant region domain of the antibody heavy chain. H 1. Structural Domains and V H The domains are adjacent and located at the amino terminus of the hinge region of the antibody heavy chain molecule.

[0041] As used in this article, “V” L "Refers to the variable region of a light chain."

[0042] Antibody fragments may contain a portion of a complete antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and V... H and C H The Fab fragment consists of an Fd fragment, an Fv fragment, a single-domain antibody (dAb) fragment, and a separated complementarity-determining region (CDR). The Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure, generated, for example, through recombinant expression. The Fab fragment contains a light chain (containing V... L and C L) and another chain, wherein the other chain contains a variable structural domain (V) of the heavy chain. H ) and the constant region structural domain of the heavy chain (C H 1) The F(ab')2 fragment is an antibody fragment obtained by digesting immunoglobulins with pepsin at pH 4.0 to 4.5, or by generating a fragment with the same structure, for example, through recombinant expression. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain portion containing a small number of additional amino acids, said small number of additional amino acids including cysteine ​​residues forming a disulfide bond connecting the two fragments. The Fab' fragment (one heavy chain and one light chain) is a fragment containing half of the F(ab')2 fragment. Antibody fragments may comprise multiple chains joined together, for example, via disulfide bonds and / or via peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and generally contain at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework region (e.g., by replacing the corresponding region), is capable of generating an antibody that specifically binds to the antigen.

[0043] Where two or more definitions exist for terms used and / or accepted in this field, the definitions of terms as used herein are intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region (CDR)” to describe discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This specific region has been described in Kabat et al., U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al. J. MoI. Biol References .196:901-917 (1987) are incorporated herein by reference in their entirety. The CDR definition by Kabat and Chothia includes overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition relating to CDRs of antibodies or their variants is intended to fall within the scope of the terminology as defined and used herein. The exact number of residues constituting a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine the residues of a CDR based on the amino acid sequence of the variable region of a given antibody.

[0044] As used in this article, "HCDR" refers to the complementarity-determining region of the heavy chain.

[0045] As used in this article, “LCDR” refers to the complementary determinant region of a light chain.

[0046] The antibodies described herein can be prepared using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, they can be prepared using suitable culture systems (e.g., *Escherichia coli*). E. coli Recombinant antibodies (or antibody mimics) are expressed in mammalian cells (or mammalian cells). Antibody engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its end.

[0047] HER2 refers to human epidermal growth factor receptor 2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this article, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0048] Immune agonists are agonists capable of inducing or enhancing an immune response against a tumor, such as by activating immune cells, including but not limited to dendritic cells (DCs), B cells, macrophages, NK cells, and T cells. Non-limiting examples of immune agonists such as TLR agonists include, but are not limited to, agonists of TLR7 and / or TLR8 and / or TLR9 known in the art (e.g., imiquimod, ralsimod, 852A, and VTX-2337) and STING agonists (e.g., ADU-S100 and MK-1454).

[0049] A linker unit is a functional group that covalently bonds two or more parts of a compound or substance. For example, linkers can be used to covalently connect parts of a target molecule and / or a payload.

[0050] A spacer is a structure located between different structural modules and capable of spatially separating these structural modules. The definition of a spacer is not limited to whether it has a function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, where non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" refers to an amino acid sequence used as a spacer, and examples include, but are not limited to, single amino acids such as Leu, Gln, etc.; sequences containing multiple amino acids, such as sequences containing two amino acids such as GA, etc.; or, for example, GGGGS (SEQ ID No. 25), GGGGSGGGGS (SEQ ID No. 26), GGGGSGGGGSGGGGS (SEQ ID No. 27), etc. Other examples of spacers include, for example, self-cleaving spacers, such as PABC (p-benzyloxycarbonyl), and the like.

[0051] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds. Alkyl groups can contain 1 to 6 carbon atoms, involving C1-C6 alkyl groups, such as C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl, C3 alkyl, C4 alkyl, and C3-C6 alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. A divalent radical is a group obtained from a corresponding monovalent radical by removing a hydrogen atom from a carbon atom having free valence electrons. A divalent radical has two connection sites that connect to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a straight-chain or branched divalent hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H4-). 10 -), Hexyl (-C6H) 12 -), 1-methylethylidene (-CH(CH3)CH2-), 2-methylethylidene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, etc.

[0052] As used herein, when one group combines with another, the connection of these groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such combination can be linked to other parts of the molecule via any suitable atom in that structure, preferably via specified chemical bonds. For example, selected from -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 When a heterocyclic group and two or more divalent groups of -(CO)- combine to form a combination, the two or more divalent groups can form linear links with each other, such as -CR. 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-(CO)-, -CR 1R 2 -C 4-10 Cycloalkyl-C 1-10 Alkylene-(CO)-, etc. The resulting divalent structure can be further linked to other parts of the molecule. compound of formula (I')

[0053] In one aspect, an antibody-immune agonist conjugate (AIAC) is provided, wherein the AIAC comprises a compound of formula (I'), the compound of formula (I') comprising formula (I'-1) and / or formula (I'-2): Formula (I'-1) Formula (I'-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -R 3 Or -(CH2) k C(O)-(NH-CR 1 R 2 -(CO)) d -R 3 ; R 1 Selected from hydrogen, -OH, -NH2 and -C 1-6 alkyl; R 2 Selected from hydrogen, -OH, -NH2 and -C 1-6 alkyl; R 3 A group that can be detached when reacting with groups in the payload; k is an integer from 1 to 5, j is an integer from 1 to 3, and d is 1 or 2.

[0054] In one implementation, R 1 and R 2 Each is independently hydrogen or C 1-6 Alkyl group. In a preferred embodiment, R 1 and R 2 Each is either hydrogen or carbon independently. 1-6 Alkyl group. In a more preferred embodiment, R 1 and R 2 Both are hydrogen.

[0055] In one implementation, B2 in equations (I'-1) and (I'-2) is the same.

[0056] In one implementation, k is 2.

[0057] In one implementation, j is 1.

[0058] In one implementation, d is 1.

[0059] In one embodiment, the terminal group R 3 It is hydrogen. In one implementation, R 3 hydroxyl or .

[0060] In one embodiment, the terminal group R 3 This indicates a structural part that does not appear in the product molecule produced by the reaction of B2 with the payload.

[0061] Thiosuccinimide is physiologically unstable and readily undergoes reverse Michael addition, leading to cleavage at the coupling site. Furthermore, thiosuccinimide can undergo thiol exchange with other thiols in the presence of another thiol compound. Both reactions result in payload loss and toxic side effects. In this paper, the open-ring succinimide structure... and The product is more stable because it no longer undergoes reverse Michael addition or thiol exchange. The ring-opening reaction method can be found in WO2015165413A. specific embodiments of compounds of formula (I')

[0062] 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. In one embodiment, the compound of formula (I') has the following structure (LN102-6-1 and / or LN102-6-1'): (LN102-6-1) (LN102-6-1').

[0063] In one embodiment, in the compound of formula (I'), B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -R 3 k is 2, d is 1, R 1 and R 2 The form is hydrogen. In one embodiment, the compound of formula (I') has the following structure (LN102-8-1 and / or LN102-8-1'): (LN102-8-1) (LN102-8-1'), Where R 3 A group that can detach when reacting with groups in the payload.

[0064] In one implementation, R 3 hydroxyl or .

[0065] It should be understood that when there are two or more R... x When x is 1, 2, 3, 4, 5, 6, 7, etc., each R x It is chosen independently. In some implementations, the "x" in the molecule is indicated with or without an additional apostrophe (') or multiple apostrophes (such as '', ''', '''', etc.), for example, R, R 1' R 1'' R 1''' R 2' R 2'' R 2''' Etc. Other R x Such as R 3 It should be understood in a similar way. compounds of formula (I') as linking units

[0066] In one embodiment, the reactive group contained in B2 can be used for covalent coupling with a payload containing another reactive group, such that the compound of formula (I') carries the payload.

[0067] In another embodiment, the ligase recognition sequence GGG (G for glycine) contained in formula (I') can be used for conjugation by ligase with the corresponding ligase recognition sequence LPETGG (SEQ ID No. 28).

[0068] Therefore, compounds of formula (I') can be used as linking units capable of being linked to target molecules (such as antibodies or their antigen-binding fragments) and / or payloads.

[0069] Those skilled in the art can synthesize the connecting units using conventional solid-phase or liquid-phase methods. compound of formula (I') carrying a payload

[0070] The reactive group contained in B2 is covalently coupled with a payload containing another reactive group to obtain a compound of formula (I') carrying the payload.

[0071] In another aspect, an antibody-immune agonist conjugate is provided, wherein the antibody-immune agonist conjugate comprises a compound of formula (II'), the compound of formula (II') comprising formula (II'-1) and / or formula (II'-2): (II'-1) (II'-2) in PL is a payload connected to the B2 portion of the compound of formula (I'), and the payload is an agonist. payload

[0072] In one embodiment, the agonist is selected from TLR agonists, such as TLR agonists (e.g., TLR 7 agonists, TLR 8 agonists, TLR 7 / 8 agonists) and STING agonists. In one embodiment, the immune agonist is selected from TLR agonists.

[0073] In one implementation, the immune agonist is resimod: .

[0074] In one embodiment, any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc., is used to connect the linking unit and the payload via reactive groups as defined above.

[0075] In one embodiment, the payload is an immune agonist, and the antibody-immune agonist conjugate (designated LPx) is one of the compounds shown in the table below: preparation of compounds of formula (I') carrying a payload

[0076] In one embodiment, any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, etc., is used to connect the linking unit and the payload via reactive groups as defined above. compound of formula (III')

[0077] In one respect, a compound of formula (III') is provided: (III'); B2 is defined as in equation (I').

[0078] In one embodiment, the compound of formula (III') can be used to prepare the compound of formula (I') carrying a payload via the following route:

[0079] Compounds of formula (III') carrying a payload can be converted into compounds of formula (I') carrying a payload using methods known in the art or as described herein. For example, one-step or multi-step synthesis can be performed to convert the structural fragments... (LU102) is introduced into the maleimide ring of the loaded compound of formula (III'), and the resulting molecule containing the succinimide moiety can then undergo a ring-opening reaction to open the succinimide ring and obtain the loaded compound of formula (I') (i.e., compound of formula (II')). In one embodiment, LU102 is introduced into the loaded compound of formula (III') by reacting the maleimide group in the compound of formula (III') with the thiol group of LU102. conjugates of formula (I)

[0080] In one aspect, antibody-immunostimulant conjugates of formula (I) are provided, including formula (I-1) and / or formula (I-2): Formula (I-1) Formula (I-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -; k is an integer from 1 to 5; j is an integer from 1 to 3; d is an integer of 1 or 2; R 1 and R 2 Each is independently selected from hydrogen, -OH, -NH2, and -C. 1-6 alkyl; PL is an agonist linked to the B2 portion. Preferably, PL is resimot; z is an integer or non-integer from 1 to 4, preferably 1, 2 or 4; A is a target molecule modified by introducing a ligase donor substrate recognition sequence, such as LPETGG (SEQ ID No. 28) or LPETG (SEQ ID No. 29); preferably, the target molecule is an antibody or its antigen-binding fragment; more preferably, A is an anti-HER2 antibody or its antigen-binding fragment.

[0081] In one implementation, B2 is -(CH2). k (CO)-NH-(C2H4-O) j -

[0082] In one implementation, B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -

[0083] In one implementation, k is 2.

[0084] In one implementation, j is 1.

[0085] In one implementation, R 1 It is hydrogen or C 1-6 Alkyl group. In a preferred embodiment, R 1 It is hydrogen.

[0086] In one implementation, R 2 It is hydrogen or C 1-6 Alkyl group. In a preferred embodiment, R 2 It is hydrogen.

[0087] In one implementation, R 1 and R 2 Both are hydrogen or both are carbon 1-6 Alkyl group. In a preferred embodiment, R 1 and R 2 Both are hydrogen.

[0088] In one implementation, d is 1.

[0089] In one implementation, B2 is -(CH2). k (CO)-NH-(C2H4-O) j -, k is 2, j is 1, and PL is Resimot. In one embodiment, equation (I) has the following structure (AC102-6), including equation (AC102-6-1) and / or equation (AC102-6-1'): (AC102-6-1), (AC102-6-1') Where z is 2 or 4.

[0090] In one implementation, B2 is -(CH2). kC(O)-(NH-CR 1 R 2 -C(O)) d -, k is 2, d is 1, R 1 and R 2 Both are hydrogen, and PL is resimot. In one embodiment, formula (I) has the following structure (AC102-8), including formula (AC102-8-1) and / or formula (AC102-8-1'): (AC102-8-1), (AC102-8-1'), Where z is 2 or 4. Targeted molecules

[0091] In one embodiment, the target molecule is an antibody or its antigen-binding fragment.

[0092] In one embodiment of this document, the target recognized by the target molecule (such as an antibody or its antigen-binding fragment) is ErbB2 / HER2, Trop2, B7H3, or Claudin18.2, etc.

[0093] In one embodiment, the targeting molecule is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include, but are not limited to, trastuzumab and pertuzumab. Trastuzumab binds to the fourth extracellular domain (ECD4) of HER2, and this antibody is approved for the treatment of HER2-positive breast cancer and gastric cancer, among others.

[0094] In a preferred embodiment, the anti-human HER2 antibody is selected from one or more engineered anti-HER2 antibodies based on trastuzumab.

[0095] In a preferred embodiment, the anti-human HER2 antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimics. In one embodiment, the antibody mimic is selected from scFv, microantibodies, biantibodies, and nanobodies. For conjugation with a compound of formula (II'), the target molecule herein may include a modified moiety for linking with the compound of formula (II'). The location of such modified moiety introduction is not limited; for example, when the target molecule is an antibody, the introduction site may be, but is not limited to, located at the C-terminus and / or N-terminus of the antibody heavy chain and / or light chain.

[0096] In one embodiment, the target molecule described herein is an antibody or its antigen-binding fragment, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus and / or N-terminus of the antibody heavy chain and / or light chain, which may include, for example, a ligase recognition sequence. In another embodiment, the terminal modification may also include a spacer Sp1 containing 2 to 10 amino acids, wherein the antibody, Sp1, and the ligase recognition sequence are sequentially linked. In a specific embodiment, Sp1 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, particularly GA.

[0097] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment comprises three types: wild-type (LC); C-terminal modified light chain (LCCT), which is modified by direct introduction of the ligase donor substrate recognition sequence LPETGG; and C-terminal modified light chain (LCCT). L The heavy chain of an antibody or its antigen-binding fragment is modified by introducing a short peptide spacer and adding the ligase donor substrate recognition sequence LPETGG. There are three types of heavy chains: wild-type (HC); C-terminal modified heavy chains (HCCT), which are modified by directly introducing the ligase donor substrate recognition sequence LPETGG; and C-terminal modified heavy chains (HCCT). L The compound is modified by introducing a short peptide spacer and adding the ligase donor substrate recognition sequence LPETGG. When z in the compound of formula (I) is 1 or 2, the above combination of heavy and light chains can form 8 preferred antibody molecules, see the amino acid sequence listing.

[0098] In one implementation, the target molecule described herein is one containing V L and V H The antibody, wherein the V L LCDR1 comprising the amino acid sequence of SEQ ID NO: 17 (RASQDVNTAVA), LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 (SASFLYS), and LCDR3 comprising the amino acid sequence of SEQ ID NO: 19 (QQHYTTPPT), wherein the V H The antibody comprises HCDR1 having the amino acid sequence SEQ ID NO: 20 (DTYIH), HCDR2 having the amino acid sequence SEQ ID NO: 21 (RIYPTNGYTRYADSVKG), and HCDR3 having the amino acid sequence SEQ ID NO: 22 (WGGDGFYAMDY). In one embodiment, the antibody is modified by introducing a ligase donor substrate recognition sequence. In one embodiment, the antibody comprises V having the amino acid sequence SEQ ID NO: 23.L and V having the amino acid sequence of SEQ ID NO: 24 H .

[0099] In one embodiment, the sequences of the CDR and variable domains are defined according to the Kabat numbering system. In one embodiment, the target molecule described herein 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 described herein 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 described herein 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 described herein 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 described herein 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 herein 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 herein 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 herein 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. preparation of conjugates

[0100] The conjugates described herein can be prepared by any method known in the art. In one embodiment, the conjugates are prepared by conjugating a target molecule to a compound of formula (I') carrying a payload via a ligase-catalyzed site-specific conjugation method, wherein the target molecule is modified with a ligase-recognized sequence (such as a ligase donor-substrate recognition sequence). The preparation procedure is described in WO2022188740A1 or WO2022188743A1. specific conjugate table

[0101] In one embodiment, the payload is an immune agonist. In one embodiment, the antibody-immune agonist conjugate is shown in the table below: pharmaceutical combinations and pharmaceutical preparations

[0102] Another aspect of this article provides a pharmaceutical combination comprising a preventive or therapeutically effective amount of an antibody-immunoagonist conjugate and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody-immunoagonist conjugate has a structure of formula (I), including formula (I-1) and / or formula (I-2): Formula (I-1) Formula (I-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -; k is an integer from 1 to 5; j is an integer from 1 to 3; d is an integer of 1 or 2; R 1 and R 2 Each is independently selected from hydrogen, -OH, -NH2, and -C. 1-6 alkyl; PL is an agonist linked to the B2 portion. Preferably, PL is resimot; z is an integer or non-integer from 1 to 4, preferably 1, 2, 3 or 4; A is the target molecule, which is an antibody or antigen-binding fragment. In one embodiment, the target recognized by the target molecule is selected from: CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvI II. SLC44A4 / AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, cadherin 6, FGFR2, FGFR3, CA6, CanAg, integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2. In one embodiment, the antibody is an anti-HER2 antibody or its antigen-binding fragment, which is modified by introducing a ligase donor substrate recognition sequence.

[0103] In one implementation, R 1 and R 2 Each is independently hydrogen or C 1-6 Alkyl group. In a preferred embodiment, R 1 and R 2 Each is either hydrogen or carbon, and each is independent of the others. 1-6 Alkyl group. In a more preferred embodiment, R 1 and R 2 Both are hydrogen.

[0104] In one implementation, k is 2.

[0105] In one implementation, j is 1.

[0106] In one implementation, d is 1.

[0107] In one embodiment, formula (I) has the following structure (AC102-6), including formula (AC102-6-1) and / or formula (AC102-6-1'): (AC102-6-1), (AC102-6-1') Where z is 2 or 4.

[0108] In one embodiment, formula (I) has the following structure (AC102-8), including formula (AC102-8-1) and / or formula (AC102-8-1'): (AC102-8-1), (AC102-8-1'), Where z is 2 or 4.

[0109] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of integer or non-integer values ​​from 1 to 4.

[0110] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of integer or non-integer value in the range of 1 to 3.5.

[0111] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of integer or non-integer values ​​in the range of 1 to 3.

[0112] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of integer or non-integer value in the range of 1 to 2.5.

[0113] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of integer or non-integer value of 1-2.

[0114] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of 1.5-2, which can be either an integer or a non-integer value.

[0115] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of 1.6-2, which can be either an integer or a non-integer value.

[0116] In one embodiment, the conjugate of formula (I) has a drug-antibody ratio (DAR) of 1.7-2, which can be either an integer or a non-integer value.

[0117] In one embodiment, the anti-HER2 antibody of formula (I) or its antigen-binding fragment comprises a light chain variable region (V). L ) and heavy chain variable region (V H In one embodiment, the antibody or its antigen-binding fragment has a light chain variable region (V... LThe antibody comprises (i) an LCDR1 containing the amino acid sequence of SEQ ID NO: 17; (ii) an LCDR2 containing the amino acid sequence of SEQ ID NO: 18; and (iii) an LCDR3 containing the amino acid sequence of SEQ ID NO: 19. In one embodiment, the heavy chain variable region (V) of the antibody or its antigen-binding fragment... H The antibody comprises (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 20; (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 21; and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 22. In one embodiment, the light chain variable region (V) of the antibody or its antigen-binding fragment... L The antibody comprises (i) an LCDR1 containing the amino acid sequence of SEQ ID NO: 17; (ii) an LCDR2 containing the amino acid sequence of SEQ ID NO: 18; and (iii) an LCDR3 containing the amino acid sequence of SEQ ID NO: 19; and the heavy chain variable region (V) of the antibody or its antigen-binding fragment. H The HCDR1 contains (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and (iii) the amino acid sequence of SEQ ID NO: 22.

[0118] In one embodiment, the light chain variable region (V) of the antibody or its antigen-binding fragment L The antibody contains an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 23. In one embodiment, the heavy chain variable region (V) of the antibody or its antigen-binding fragment... H The antibody contains an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 24. In one embodiment, the antibody or its antigen-binding fragment contains a light chain variable region (V... L The antibody contains an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 23; and the heavy chain variable region (V) of the antibody or its antigen-binding fragment contains... H It contains an amino acid sequence that has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 24.

[0119] In one embodiment, the anti-HER2 antibody or its antigen-binding fragment comprises V having the amino acid sequence of SEQ ID NO: 23. LIn one embodiment, the antibody or its antigen-binding fragment comprises V having the amino acid sequence of SEQ ID NO:24. H In one embodiment, the antibody or its antigen-binding fragment comprises V having the amino acid sequence of SEQ ID NO: 23. L and V having the amino acid sequence of SEQ ID NO: 24 H .

[0120] In one embodiment, the anti-HER2 antibody or antigen-binding fragment comprises a light chain having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In one embodiment, the antibody or antigen-binding fragment comprises a light chain having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7; and a heavy chain having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0121] In one embodiment, the modified antibody or its antigen-binding fragment comprises a light chain having any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7. In one embodiment, the modified antibody or its antigen-binding fragment comprises a heavy chain having any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In one embodiment, the modified antibody or its antigen-binding fragment comprises a light chain having any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7; and a heavy chain having any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0122] In one embodiment, the anti-PD-1 antibody is a murine antibody, a humanized antibody, or a fully human antibody. In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and / or monkey PD-1 and / or mouse PD-1. In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment binds to both human PD-1 and monkey PD-1, but does not bind to mouse PD-1.

[0123] In one embodiment, the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, cimiprimab, palolizumab, dostalimab, penaprilimab, cepalimumab, slulilimumab, putelizumab, riverimab, and camrelizumab.

[0124] In one embodiment, a pharmaceutical combination comprising an antibody-immunoagonist conjugate and an anti-PD-1 antibody or an antigen-binding fragment thereof, optionally further comprising a pharmaceutically acceptable carrier.

[0125] The drug combinations described in this article can be administered in any manner, as long as they achieve the effect of relieving, preventing, or curing symptoms in humans or animals. For example, various suitable dosage forms can be prepared depending on the route of administration.

[0126] The term "pharmaceutical acceptable" means that when in contact with patient tissues within the bounds of normal medical judgment, it will not produce inappropriate toxicity, irritation, or allergic reactions, has a reasonable risk-benefit ratio, and is effective for its intended use.

[0127] The term pharmaceutically acceptable carrier refers to those carrier materials that are pharmaceutically acceptable and do not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers also include carrier materials that bring the composition close to physiological conditions, such as pH adjusters, buffers, toxicity modifiers, and sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In one embodiment, the term "carrier" refers to a diluent, adjuvant, excipient, or solvent administered with the active ingredient for treatment. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug composition is administered intravenously. Solutions of saline and glucose in water or glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers, such as acetates, citrates, or phosphates.

[0128] In one embodiment, a kit is provided that comprises the drug combination.

[0129] In one embodiment, the kit comprises: The first packaging unit contains an antibody-immunostimulant conjugate of formula (I). The second packaging unit contains an anti-PD-1 antibody or its antigen-binding fragment; and Optionally, administer the antibody-immunoagonist conjugate and the anti-PD-1 antibody or its antigen-binding fragment to the subject according to the instructions. methods of treatment and uses

[0130] A pharmaceutical combination comprising an antibody-immunoagonal conjugate of formula (I) and an anti-PD-1 antibody or an antigen-binding fragment thereof, or a kit comprising an antibody-immunoagonal conjugate of formula (I) and an anti-PD-1 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising an antibody-immunoagonal conjugate of formula (I) and an anti-PD-1 antibody or an antigen-binding fragment thereof, is intended for the prevention, relief, or treatment of tumors and / or autoimmune diseases. Tumors sensitive to conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors, and these tumors will be recognized by the targeting molecule in the conjugate and can be killed by the agonist in the conjugate.

[0131] Therefore, in another aspect, the use of the pharmaceutical combinations, kits or pharmaceutical compositions described herein in the preparation of medicaments for the prevention, relief or treatment of diseases, conditions or symptoms selected from tumors or autoimmune diseases is also provided.

[0132] In another aspect, the drug combinations, kits, or drug compositions described herein are provided for the prevention, relief, or treatment of tumors or autoimmune diseases.

[0133] In another aspect, a method for preventing, alleviating, or treating tumors or autoimmune diseases is provided, the method comprising administering an effective amount of the present invention’s combination of drugs, kits, or pharmaceutical compositions to a subject in need.

[0134] In another aspect, a method is provided for treating a disease in a subject or reducing the likelihood of disease progression, the method comprising administering to the subject an effective amount of an antibody-immunostimulator conjugate having the structure of formula (I), and administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0135] In some implementations, the disease is a tumor or an autoimmune disease.

[0136] In another aspect, a method is provided for treating a subject with cancer or reducing the likelihood of cancer progression, the method comprising administering to the subject an effective amount of an antibody-immunostimulant conjugate having the structure of formula (I), and administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0137] In one embodiment, prevention, mitigation, or treatment of HER2-related tumor diseases is provided. In one embodiment, the HER2-related tumors include HER2-overexpressing tumors or HER2-mutated tumors. In one embodiment, the disease is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic carcinoma, etc. Adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; preferably, the diseases are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma. In one embodiment, the diseases are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma. In one embodiment, the HER2-related tumors are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0138] In one embodiment, the anti-PD-1 antibody is a murine antibody, a humanized antibody, or a fully human antibody; and / or The anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and / or monkey PD-1 and / or mouse PD-1; or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and monkey PD-1, but not to mouse PD-1.

[0139] In one embodiment, the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, cimiprimab, palolizumab, dostalimab, penaprilimab, cepalimumab, slulilimumab, putelizumab, riverimab, and camrelizumab.

[0140] In one embodiment, the antibody-immunoagonist conjugate is: and / or , or The antibody-immunoagonist conjugate is and / or .

[0141] In one embodiment, the antibody-immunoagonist conjugate is: or .

[0142] In one embodiment, the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of the same pharmaceutical formulation.

[0143] In one embodiment, the antibody-immunoagonist conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of different pharmaceutical formulations.

[0144] In one embodiment, the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered at different times.

[0145] In a preferred embodiment, the antibody-immunoagonist conjugate of this invention, formed by conjugating an anti-human HER2 antibody and an agonist, specifically binds to HER2 on the surface of tumor cells and selectively kills HER2-expressing tumor cells. In another preferred embodiment, the use of the conjugate of this invention or the pharmaceutical composition or combination thereof of this invention in the preparation of a medicament for treating a disease, condition, or symptom selected from HER2-related tumors is provided. In a more preferred embodiment, the disease, condition, or symptom is a HER2-positive tumor or a HER2-low-expressing tumor. In one embodiment, the HER2-positive tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma, etc.

[0146] The dosage of the antibody-immunoagonist conjugate and anti-PD-1 antibody administered to the subject can be adjusted over a wide range. The dosage can be varied depending on the specific route of administration and the subject's needs, and can be determined by a healthcare professional. beneficial effects

[0147] The antibody-immunostimulant conjugate of the present invention uses a specially designed linker-loader, which has higher stability and can achieve excellent efficacy through appropriate DAR, thereby reducing side effects and improving the therapeutic index.

[0148] The antibody-immunoagonist conjugates presented in this paper exhibit good homogeneity, high activity, and high selectivity. Furthermore, the toxicity of the linker-support intermediate is significantly lower than that of the free support, thus reducing the adverse effects on drug preparation and facilitating industrial production.

[0149] The combination of antibody-immunoagonist conjugate and anti-PD-1 antibody in this paper achieved the following technical effects: (1) Good anticancer effects, especially in improving overall response rate (ORR) and / or complete response (CR) and / or partial response (PR) and / or tumor growth inhibition rate (TGI). (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action). (4) Good safety profile (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider therapeutic window), etc. (5) It has good therapeutic effects on subjects who are resistant to antibody therapy, especially trastuzumab or pertuzumab therapy. (6) It has good therapeutic effects on subjects who are resistant to checkpoint inhibitor therapy, especially PD-1 inhibitor therapy. examples

[0150] Preparation Examples

[0151] To more clearly illustrate the objectives and technical solutions, the following description further illustrates specific embodiments. It should be understood that these embodiments are not intended to limit the scope of this document. Specific experimental methods not mentioned in the following embodiments were performed according to conventional experimental methods.

[0152] Unless otherwise stated, the instruments and reagents used in the examples are commercially available. The reagents can be used directly without further purification.

[0153] The preparation process of antibody-immunostimulant conjugates AC102-6-1-1 and AC102-8-1-1 is described in accordance with WO2022188740A and / or WO2022188743A, the entire contents of which are incorporated herein by reference.

[0154] AC102-6-1-1 has the following formula (DAR=1.74): Where A is Ab0001-LCCT L -HC.

[0155] AC102-8-1-1 has the following formula (DAR=1.79): Where A is Ab0001-LCCT L -HC. Preparation Example 1: Preparation of Antibody-Immune Agonist Conjugate OL-HX20042

[0156] 1.1 Preparation of linker-TLR7 / 8 agonist HX20042

[0157] Agonist HX20040 (1.0 equivalent) was weighed and dissolved in DMF. N-hydroxysuccinimide ester of 3-maleimide propionate (1.1 equivalent) was added to the reaction system and stirred until completely dissolved. Then, diisopropylethylamine (1.5 equivalent) was added dropwise to the reaction mixture, and the reaction was stirred and monitored by HPLC until HX20040 was completely consumed. The reaction mixture was purified by preparative HPLC and lyophilized to obtain product HX20042. LC-MS analysis showed [M+H]... + =589.6, consistent with its theoretical molecular weight.

[0158] 1.2 Preparation of antibody-immunoagonist conjugate OL-HX20042 (OL-HX20042)

[0159] Antibody (Ab0001-LCCT) L -HC) and TCEP (Tris(2-carboxyethyl)phosphine) were added to a phosphate buffer liquid system (pH 4-10) at a molar ratio of 1:1 to 1:20 to obtain a reaction mixture. The reaction mixture was reacted at 37°C for 2 hours. HX20042 dissolved in an organic solvent (DMA, DMSO, or DMF) (molar ratio of the above antibody to HX20042 of 1:1 to 1:100) was added to the above reaction system, and the reaction was continued at 4°C-40°C for 0.5 to 20 hours. Then, cysteine ​​(molar ratio of the above antibody to cysteine ​​of 1:1 to 1:20) was added, followed by DHAA (dehydroascorbic acid) (molar ratio of the above antibody to DHAA of 1:1 to 1:20) to react with excess thiol groups. The resulting mixture was purified, ultrafiltered, or dialyzed to remove unreacted intermediates to obtain the antibody-immunoagonist conjugate OL-HX20042. The purified OL-HX20042 was stored in 1×PBS pH 7.4 at 4°C or -80°C.

[0160] 1.3 HPLC detection of antibody-immunoagonist conjugate OL-HX20042

[0161] A Proteomix Hic Butyl-NP5 4.6 * 35 mm 5 μm non-porous column (manufacturer: Sepax Technologies, Inc., PN: 431NP5-4603) was used; 2.5 M ammonium sulfate + 25 mM phosphate buffer (pH 7.0) was used as mobile phase A; 25 mM phosphate buffer (pH 7.0) was used as mobile phase B; the flow rate was 1 mL / min; the column temperature was 40 °C; a gradient method was used: phase B was increased from 0% to 100% over 25 minutes; the DAR distribution of the antibody-immunoagonist conjugate OL-HX20042 was detected at 280 nm. The results showed that the mean DAR of OL-HX20042 was approximately 4.0. Pharmacological Example 1: In vivo efficacy evaluation of the combination of AC102-8-1-1 and anti-mPD-1 antibody in the MC38-hHER2 homology model.

[0162] Materials: Tumor tissue from MC38-hHER2 homologous models was extracted from tumor-bearing mice. The MC38-hHER2 homologous model was established by subcutaneous injection of MC38-hHER2 cells (Biocytokines) into the right flank of 7- to 9-week-old C57BL / 6 mice. MC38-hHER2 cells are genetically modified MC38 cells (mouse colon cancer cells) that overexpress human HER2 while simultaneously knocking out mouse HER2.

[0163] Method: The tumor diameter was measured using calipers, and calculated according to the formula V = 0.5a×b 2 Calculate the tumor volume (where a is the long axis of the tumor and b is the short axis). The average tumor volume is approximately 100 mm². 3 -300mm 3Mice were randomly divided into the following groups: solvent group (G1), AC102-8-1-1 0.25 mg / kg group (G2), AC102-8-1-1 0.5 mg / kg group (G3), anti-mPD-1 antibody (mouse antibody, Biocytogen, 780120J2) 3 mg / kg group (G4), combination group of anti-mPD-1 antibody 3 mg / kg and AC102-8-1-1 0.25 mg / kg (G5), and combination group of anti-mPD-1 antibody 3 mg / kg and AC102-8-1-1 0.5 mg / kg (G6). AC102-8-1-1 was administered intravenously to tumor-bearing mice twice, on days 0 and 7. Anti-mPD-1 antibody was administered intraperitoneally to tumor-bearing mice four times, on days 0, 3, 7, and 10. Mice were administered PBS intravenously as a solvent twice, on days 0 and 7. Tumor volume was measured twice weekly in each group. The experiment ended on day 33 after the last administration, and the tumor growth inhibition rate (TGI) on day 31 after grouping was calculated as follows: TGI (%) = [1 - (mean tumor volume of the treatment group on the end day - mean tumor volume of the treatment group on day 1) / (mean tumor volume of the solvent group on the end day - mean tumor volume of the solvent group on day 1)] × 100%. The results are shown in Table 1.

[0164] in conclusion

[0165] The results are shown in Table 1 and Figure 1 As shown, both AC102-8-1-1 (0.25 mg / kg or 0.5 mg / kg) and anti-mPD-1 antibody (3 mg / kg) monotherapy can inhibit tumor growth, while the combination of AC102-8-1-1 (0.25 mg / kg or 0.5 mg / kg) and anti-mPD-1 antibody shows superior antitumor efficacy compared to either of the above monotherapy.

[0166] Throughout the study, the mice maintained good activity and appetite during the administration periods. After each administration, the mice experienced a slight decrease in body weight, which returned to normal within 3-7 days, and a slight increase in body weight was observed during the observation period. Based on these observations, the combined medication regimen demonstrated good safety. Pharmacological Example 2: In vivo efficacy evaluation of AC102-8-1-1, AC102-6-1-1, or OL-HX20042 monotherapy or in combination with anti-mPD-1 antibody in the MC38-hHER2 homology model.

[0167] Materials: Tumor tissue from MC38-hHER2 homologous models was extracted from tumor-bearing mice. The MC38-hHER2 homologous model was established by subcutaneous injection of MC38-hHER2 cells (from Biocytogen) into the right flank of 7- to 9-week-old C57BL / 6 mice. MC38-hHER2 cells are genetically modified MC38 cells (mouse colon cancer cells) that overexpress human HER2 while simultaneously knocking out mouse HER2.

[0168] Method: The tumor diameter was measured using calipers, and calculated according to the formula V = 0.5a×b 2 Calculate the tumor volume (where a is the long axis of the tumor and b is the short axis of the tumor). The average tumor volume is approximately 339 mm². 3 Mice were randomly divided into the following groups: solvent group (G1), anti-mPD-1 antibody 3 mg / kg group (G2), AC102-8-1-1 1 mg / kg group (G3), combination group of anti-mPD-1 antibody 3 mg / kg and AC102-8-1-1 1 mg / kg group (G4), AC102-6-1-1 1 mg / kg group (G5), combination group of anti-mPD-1 antibody 3 mg / kg and AC102-6-1-1 1 mg / kg group (G6), OL-HX20042 1 mg / kg group (G7), and combination group of anti-mPD-1 antibody 3 mg / kg and OL-HX20042 1 mg / kg group (G8).

[0169] On day 0, tumor-bearing mice were intravenously administered single doses of AC102-8-1-1, AC102-6-1-1, and OL-HX20042, respectively. On days 0 and 3, tumor-bearing mice were intraperitoneally administered anti-mPD-1 antibody twice. On day 0, a single dose of PBS was intravenously injected as the solvent. Tumor volume was measured twice weekly in each group of mice. The experiment ended on day 7, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1 - (mean tumor volume of the treatment group on the end day - mean tumor volume of the treatment group on day 1) / (mean tumor volume of the solvent group on the end day - mean tumor volume of the solvent group on day 1)] × 100%. The results are shown in Table 2.

[0170] in conclusion

[0171] The results are shown in Table 2 and Figure 2As shown, monotherapy with AC102-8-1-1, AC102-6-1-1, and OL-HX20042 all inhibited tumor growth, while the combination of anti-mPD-1 antibody, AC102-8-1-1, AC102-6-1-1, and OL-HX20042 showed superior anti-tumor efficacy compared to monotherapy. Among these, AC102-6-1-1 demonstrated better anti-tumor efficacy in both monotherapy and combination therapy.

[0172] During the experiment, no unplanned animal deaths or obvious clinical symptoms occurred, and the combined drug therapy showed good safety. Pharmacological Example 3: In vivo efficacy evaluation of AC102-6-1-1 monotherapy or combination with anti-mPD-1 antibody in the MC38-hHER2 homology model.

[0173] Materials: Tumor tissue from MC38-hHER2 homologous models was extracted from tumor-bearing mice. The MC38-hHER2 homologous model was established by subcutaneous injection of MC38-hHER2 cells (from Biocytogen) into the right flank of 7- to 9-week-old C57BL / 6N mice. MC38-hHER2 cells are genetically modified MC38 cells (mouse colon cancer cells) that overexpress human HER2 while simultaneously knocking out mouse HER2.

[0174] Method: The tumor diameter was measured using calipers, and calculated according to the formula V = 0.5a×b 2 Calculate the tumor volume (where a is the long axis of the tumor and b is the short axis). When the average tumor volume reaches 116 mm... 3Mice were randomly divided into eight groups. These eight groups were: G1: solvent; G2: anti-mPD-1 antibody (3 mg / kg); G3: AC102-6-1-1 (0.2 mg / kg); G4: AC102-6-1-1 (1 mg / kg); G5: AC102-6-1-1 (5 mg / kg); G6: a combination of anti-mPD-1 antibody (3 mg / kg) and AC102-6-1-1 (0.2 mg / kg); G7: a combination of anti-mPD-1 antibody (3 mg / kg) and AC102-6-1-1 (1 mg / kg); and G8: a combination of anti-mPD-1 antibody (3 mg / kg) and AC102-6-1-1 (5 mg / kg). On day 0 after grouping, the tumor-bearing mice were subcutaneously administered AC102-6-1-1 once. Anti-mPD-1 antibody was administered intraperitoneally to tumor-bearing mice four times on days 0, 3, 7, and 10. PBS was injected as the solvent using either of the methods described above. Tumor volume was measured twice weekly for each group of mice. The experiment was terminated on day 29 after grouping, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1 - (mean tumor volume of the treatment group on the end day - mean tumor volume of the treatment group on day 1) / (mean tumor volume of the solvent group on the end day - mean tumor volume of the solvent group on day 1)] × 100%. The results are shown in Table 3. Table 3. Effects of the tested drugs on tumor growth inhibition in the MC38-hHER2 homology model

[0175] in conclusion

[0176] The results are shown in Table 3 and Figure 3 As shown, both 3 mg / kg anti-mPD-1 antibody monotherapy and various tested doses of AC102-6-1-1 monotherapy demonstrated significant anti-tumor activity in terms of tumor volume, and AC102-6-1-1 exhibited a clear dose-dependent anti-tumor effect. Combinations of different doses of AC102-6-1-1 with anti-mPD-1 antibodies produced improved therapeutic efficacy.

[0177] During the study, there were no unplanned animal deaths or obvious clinical symptoms, and all combined drug administrations were deemed safe. sequence listing SEQ ID No. 1: Ab0001-LCCT L -HC Light Chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No. 2: Ab0001-LCCT L -HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 3: Ab0001-LC-HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 4: Heavy chain of Ab0001-LC-HCCT EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID No. 5: Ab0001-LC-HCCT L Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 6: Ab0001-LC-HCCT L Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG SEQ ID No. 7: Ab0001-LCCT-HC Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID No. 8: Ab0001-LCCT-HC heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 9: Ab0001-LCCT-HCCT light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID No. 10: Heavy chain of Ab0001-LCCT-HCCT EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID No. 11: Ab0001-LCCT-HCCT L Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGG SEQ ID No. 12: Ab0001-LCCT-HCCT L Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG SEQ ID No. 13: Ab0001-LCCT L -HCCT Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No. 14: Ab0001-LCCT L -HCCT heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGG SEQ ID No. 15: Ab0001-LCCT L -HCCT L Light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No. 16: Ab0001-LCCT L -HCCT L Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGALPETGG 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 SEQ ID No. 25:GGGGS SEQ ID No. 26:GGGGSGGGGS SEQ ID No. 27:GGGGSGGGGSGGGGS SEQ ID No. 28:LPETGG SEQ ID No. 29:LPETG

Claims

1. A pharmaceutical combination comprising an antibody-immunostimulant conjugate and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody-immunostimulant conjugate has a structure of formula (I-1) and / or formula (I-2): Equation (I-1) Equation (I-2) in, B2 is -(CH2) k (CO)-NH-(C2H4-O) j -or-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -; k is an integer from 1 to 5; j is an integer from 1 to 3; d is an integer of 1 or 2; R 1 and R 2 Each is independently selected from hydrogen, -OH, -NH2, and -C. 1-6 alkyl; PL is an agonist linked to the B2 portion. Preferably, PL is Resimot. ; z is an integer or non-integer from 1 to 4, preferably 1, 2 or 4; A is a target molecule modified by introducing a ligase donor substrate recognition sequence; preferably, the target molecule is an antibody or its antigen-binding fragment; more preferably, A is an anti-HER2 antibody or its antigen-binding fragment.

2. The drug combination according to claim 1, wherein R 1 and R 2 Each is independently hydrogen or C 1-6 Alkyl, preferably, R 1 and R 2 Both are hydrogen.

3. The pharmaceutical combination according to any one of claims 1 to 2, wherein k is 2, and / or j is 1, and / or d is 1.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein The antibody-immunoagonist conjugate has the structure of formula (AC102-6-1) and / or formula (AC102-6-1'): Equation (AC102-6-1) Formula (AC102-6-1'); or The antibody-immunoagonist conjugate has the structure of formula (AC102-8-1) and / or formula (AC102-8-1'): Equation (AC102-8-1) Equation (AC102-8-1'), z is an integer or non-integer from 1 to 4, preferably 1, 2 or 4; A is a target molecule modified by introducing a ligase donor substrate recognition sequence; preferably, the target molecule is an antibody or its antigen-binding fragment; more preferably, A is an anti-HER2 antibody or its antigen-binding fragment.

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein The antibody-immunostimulant conjugate has a drug-antibody ratio (DAR) of integer or non-integer values ​​of 1-4, 1-3.5, 1-3, 1-2.5, 1-2, 1.5-2, 1.6-2, or 1.7-2.

6. The pharmaceutical combination according to claim 1, wherein... The anti-HER2 antibody or its antigen-binding fragment includes a light chain variable region (V). L ) and heavy chain variable region (V H ),in The V L Include: (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 17; (ii) LCDR2 containing the amino acid sequence of SEQ ID NO: 18; and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 19; And / or the V H Include: (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 20; (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 21; and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO:

22.

7. The pharmaceutical combination according to claim 6, wherein... The V L Contains an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 23; and / or, The V H An amino acid sequence containing at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:

24.

8. The pharmaceutical combination of claim 1, wherein the anti-HER2 antibody or its antigen-binding fragment comprises V with the amino acid sequence of SEQ ID NO: 23 L ; and / or V with the amino acid sequence of SEQ ID NO: 24 H .

9. The pharmaceutical combination of claim 6, wherein the anti-HER2 antibody or antigen-binding fragment comprises... A light chain comprising an amino acid sequence having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7; and / or, The heavy chain comprises an amino acid sequence having at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:

6.

10. The pharmaceutical combination of claim 9, wherein the anti-HER2 antibody or its antigen-binding fragment comprises... A light chain having any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 7; and / or, A heavy chain having any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:

6.

11. The pharmaceutical combination according to any one of claims 1 to 10, wherein The anti-PD-1 antibody is a murine antibody, a humanized antibody, or a fully human antibody; and / or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and / or monkey PD-1 and / or mouse PD-1; or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and monkey PD-1, but does not bind to mouse PD-1.

12. The pharmaceutical combination according to claim 11, wherein... The anti-PD-1 antibody is selected from pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, cimiprimab, palolizumab, dostalimab, penaprilimab, cepalimumab, slulilimumab, putelizumab, riverimab, and camrelizumab.

13. The pharmaceutical combination according to any one of claims 1 to 12, wherein the pharmaceutical combination optionally further comprises a pharmaceutically acceptable carrier.

14. A kit comprising the drug combination of any one of claims 1 to 13.

15. The kit according to claim 14, wherein the kit comprises A first packaging unit comprising an antibody-immunostimulant conjugate as defined in any one of claims 1 to 13. The second packaging unit comprises an anti-PD-1 antibody or its antigen-binding fragment as defined in any one of claims 1 to 13, and Optionally, administer the antibody-immunoagonist conjugate and the anti-PD-1 antibody or its antigen-binding fragment to the subject according to the instructions.

16. A pharmaceutical composition comprising an antibody-immunostimulator conjugate as defined in any one of claims 1 to 13 and an anti-PD-1 antibody or an antigen-binding fragment thereof.

17. Use of the pharmaceutical combination of any one of claims 1 to 13, or the kit of claim 14 or 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament for the prevention, relief or treatment of a disease; wherein the disease is a tumor or an autoimmune disease.

18. The use according to claim 17, wherein the disease is a HER2-related tumor; preferably, the HER2-related tumor is a HER2-positive tumor or a HER2-low-expressing tumor; preferably, the HER2-related tumor is a HER2-overexpressing tumor or a HER2-mutated tumor.

19. The use according to claim 18, wherein the disease is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, nipple carcinoma. Adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; preferably, the diseases are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

20. A method for preventing, alleviating, or treating a disease in a subject or reducing the likelihood of disease progression, the method comprising administering a pharmaceutical combination of any one of claims 1 to 13, or a kit of claim 14 or 15, or a pharmaceutical composition of claim 16, wherein the disease is a tumor.

21. A method for preventing, alleviating, or treating cancer in a subject or reducing the likelihood of cancer progression, the method comprising administering to the subject an effective amount of an antibody-immunostimulant conjugate as defined in any one of claims 1 to 13, and administering to the subject an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof.

22. The method of claim 21, wherein the cancer is a HER2-related cancer; preferably, the cancer overexpresses HER2 or the cancer has a HER2 gene mutation.

23. The method according to claim 21 or 22, wherein the cancer is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma. Papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; preferably, the cancer is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

24. The method according to any one of claims 21 to 23, wherein the anti-PD-1 antibody is a murine antibody, a humanized antibody, or a fully human antibody; and / or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and / or monkey PD-1 and / or mouse PD-1; or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and monkey PD-1, but not to mouse PD-1.

25. The method according to any one of claims 21 to 23, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, cimiprimab, palolizumab, dostalimab, penaprilimab, slulilimab, putelilimab, riverilimab, and camrelizumab.

26. The method according to any one of claims 21 to 25, wherein the antibody-immunostimulant conjugate has a structure of formula (AC102-6-1) and / or formula (AC102-6-1'): Equation (AC102-6-1) Formula (AC102-6-1'); or The antibody-immunoagonist conjugate has the structure of formula (AC102-8-1) and / or formula (AC102-8-1'): Equation (AC102-8-1) Formula (AC102-8-1').

27. The method according to any one of claims 21 to 26, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of the same pharmaceutical formulation.

28. The method according to any one of claims 21 to 26, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of different pharmaceutical formulations.

29. The method according to any one of claims 21 to 26, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered at different times.

30. Use of an effective amount of the antibody-immunostimulant conjugate as defined in any one of claims 1 to 13 for the preparation of a medicament for the prevention, relief or treatment of cancer in a subject, wherein the medicament is used in combination with an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof.

31. The use according to claim 30, wherein the cancer overexpresses HER2 or the cancer has a HER2 gene mutation.

32. The use according to claim 30 or 31, wherein the cancer is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma. Papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; preferably, the diseases are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

33. The use according to any one of claims 30 to 32, wherein the anti-PD-1 antibody is a murine antibody, a humanized antibody, or a fully human antibody; and / or The anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and / or monkey PD-1 and / or mouse PD-1; or the anti-PD-1 antibody or its antigen-binding fragment binds to human PD-1 and monkey PD-1, but not to mouse PD-1.

34. The use according to any one of claims 30 to 33, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, cimiprimab, palolizumab, dostalimab, penaprilimab, slulilimab, putelilimab, leverilimab, riverlilimab, and camrelizumab.

35. The use according to any one of claims 30 to 34, wherein the antibody-immunostimulant conjugate has a structure of formula (AC102-6-1) and / or formula (AC102-6-1'): Equation (AC102-6-1) Formula (AC102-6-1'); or The antibody-immunoagonist conjugate has the structure of formula (AC102-8-1) and / or formula (AC102-8-1'): Equation (AC102-8-1) Formula (AC102-8-1').

36. The use according to any one of claims 30 to 35, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of the same pharmaceutical formulation.

37. The use according to any one of claims 30 to 35, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered simultaneously as part of different pharmaceutical formulations.

38. The use according to any one of claims 30 to 35, wherein the antibody-immunostimulant conjugate and the anti-PD-1 antibody or its antigen-binding fragment are administered at different times.

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