Antibody drug conjugates and uses thereof

Abstract

The present application relates to an antibody drug conjugate comprising a bispecific antibody or antigen-binding fragment thereof targeting HER2. The present application further relates to a method for producing and using the antibody drug conjugate, and a pharmaceutical composition comprising the antibody drug conjugate. The antibody drug conjugate described in the present application can effectively kill tumors.

Description

[Technical field]

[0001] The present application relates to the biopharmaceutical field, specifically to antibody drug conjugates and uses thereof. [Background technology]

[0002] The HER receptor tyrosine kinase family is an important mediator of cell proliferation, differentiation and survival. The receptor family contains four distinct members, including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Members of the receptor family are involved in various types of human malignancies.

[0003] Bispecific antibodies (BsAbs) are immunoglobulin molecules that contain two different ligand-binding sites. They substitute the identical sequences of the two arms of a classical antibody Fab with two different Fab sequences, so that the two arms of the Y can bind to different epitopes. The use of bispecific antibodies in the treatment of cancer has been reviewed in many publications (Carter 2001, Chames and Baty 2009, Chames and Baty 2009).

[0004] Antibody-drug conjugates, in which a cytotoxic drug is linked to an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, are expected to be able to selectively deliver the drug to cancer cells, where it will accumulate and kill the cancer cells. Summary of the Invention

[0005] The present application provides an antibody-drug conjugate comprising a bispecific antibody or antigen-binding fragment thereof targeting HER2. The antibody-drug conjugate can specifically bind to at least one (e.g., at least two) epitopes of human HER2. The antibody-drug conjugate described in the present application can have the following characteristics: (1) the antibody portion and the drug portion can exert a synergistic tumor-killing function, and can effectively kill tumor cells; (2) the antibody-drug conjugate can specifically bind to tumor cells; (3) the antibody portion can have good stability in serum; (4) the antibody portion can have a stronger bystander killing effect on tumor cells; (5) the antibody portion can have good ADCC effect; and / or (6) the antibody portion can have good endocytosis efficiency. The present application further provides a method for producing the antibody-drug conjugate, a composition comprising the antibody-drug conjugate, and a use of the antibody-drug conjugate and the composition.

[0006] In one aspect, the present application provides an antibody drug conjugate comprising a bispecific antibody or antigen-binding fragment thereof that targets HER2. In some embodiments, the bispecific antibody or antigen-binding fragment thereof that targets HER2 specifically binds to at least one epitope of human HER2. In some embodiments, the bispecific antibody or antigen-binding fragment thereof targeting HER2 specifically binds to the extracellular domain II of human HER2 and / or the extracellular domain IV of human HER2.

[0007] In some embodiments, the bispecific antibody or antigen-binding fragment thereof that targets HER2 comprises a first light chain and a second light chain. In some embodiments, the first light chain comprises first LCDR1-3, and the first LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the first LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the first LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the first light chain is capable of binding to a heavy chain of pertuzumab.

[0008] In some embodiments, the second light chain comprises second LCDR1-3, and the second LCDR1 comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the second LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the second LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the second light chain is capable of binding to a heavy chain of trastuzumab.

[0009] In some embodiments, the variable regions of the first light chain and the second light chain comprise the amino acid sequence shown in any one of SEQ ID NOs: 7-12. In some embodiments, the variable region of the first light chain and the second light chain comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the first light chain and the second light chain comprise an amino acid sequence set forth in any one of SEQ ID NOs: 13-18.

[0010] In some embodiments, the first light chain is selected from the group consisting of a light chain of pertuzumab or a variant thereof, and a light chain of trastuzumab or a variant thereof, and / or the second light chain is selected from the group consisting of a light chain of pertuzumab or a variant thereof, and a light chain of trastuzumab or a variant thereof.

[0011] In some embodiments, the first light chain and the second light chain have identical amino acid sequences. In some embodiments, the first light chain and the second light chain comprise the amino acid sequence set forth in SEQ ID NO:13.

[0012] In some embodiments, the bispecific antibody or antigen-binding fragment thereof targeting HER2 comprises a first heavy chain and a second heavy chain, and the first heavy chain is capable of correctly binding to the first light chain under physiological conditions or in vitro protein expression conditions. In some embodiments, the second heavy chain is capable of correctly binding to the second light chain under physiological conditions or under in vitro protein expression conditions.

[0013] In some embodiments, the first heavy chain comprises a first heavy chain variable region that is a heavy chain variable region of pertuzumab. In some embodiments, the second heavy chain comprises a second heavy chain variable region that is a heavy chain variable region of trastuzumab.

[0014] In some embodiments, the first heavy chain and the second heavy chain comprise a heavy chain constant region derived from the constant region of human IgG. In some embodiments, the Fc fragment of the first heavy chain and the second heavy chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25-57. In some embodiments, the first heavy chain comprises the amino acid sequence shown in 21 or 23. In some embodiments, the second heavy chain comprises the amino acid sequence shown in 22 or 24.

[0015] In some embodiments, the antibody drug conjugate comprises: M-(L1) a -(L2) b -D (Formula 1), in which M represents a bispecific antibody targeting HER2 described in the present application or an antigen-binding fragment thereof, L1 represents a linker linked to M, L2 represents a linker linked to D, a and b are each independently selected from 0 to 10, and D represents a drug.

[0016] In some embodiments, L1 and / or L2 are selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid based linker. In some embodiments, L1 and M are linked to M via a thiol group, an azide group, or an amide group on M.

[0017] In some embodiments, M comprises a first heavy chain and a second heavy chain, and the first heavy chain and / or the second heavy chain comprises a linking site capable of linking to L1. In some embodiments, the linking site comprises a group capable of linking to the L1 following deglycosylation modification.

[0018] In some embodiments, the group is located flanking the amino acid Q at position 297 of the first heavy chain and / or flanking the amino acid Q at position 298 of the second heavy chain. In some embodiments, the group comprises an amide group.

[0019] In some embodiments, the linking site comprises a group capable of linking to the L1 after glycosylation modification. In some embodiments, the group is located flanking amino acid N at position 299 of the first heavy chain and / or flanking amino acid N at position 300 of the second heavy chain.

[0020] In some embodiments, the group comprises -N3. In some embodiments, the glycosylation modification comprises that M has been contacted with a UDP-GalNAz, β-1,4-galactosyltransferase or a variant thereof.

[0021] In some embodiments, the L1 is capable of participating in a SPAAC reaction. In some embodiments, L1 is selected from the group consisting of maleimide, succinimid-3-yl-N, and DBCO. In some embodiments, L1 is DBCO-(PEG). n1 Among them, n1 is an integer of 0 to 10, or the above L1 is maleimide.

[0022] In some embodiments, L2 is a polypeptide, VC-PAB, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG. NHS, N-4-(maleimidomethyl)cyclohexyl succinimidyl carboxylate (SMCC), N-sulfo(4-maleimidomethyl)cyclohexyl sulfosuccinimidyl carboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidinyl-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecanoic acid ester (CX1-1). In some embodiments, L2 is GGFG.

[0023] In some embodiments, the drug has the ability to kill and / or inhibit the proliferation of tumor cells. In some embodiments, the drug comprises a small molecule drug.

[0024] In some embodiments, the drug is selected from the group consisting of V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphotransfer reactions, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA breakers, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 glucuronic acid, etoposide phosphate, nitrogen mustard, proteasome inhibitors, cytokines, and Toll-like receptor agonists.

[0025] In some embodiments, the drug is selected from the group consisting of DM1, exatecan, DXd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, Duo5 toxin, an SN38 derivative, or a derivative thereof.

[0026] In some embodiments, the antibody drug conjugate comprises: [ka] The compound has a structure selected from the group consisting of:

[0027] In some embodiments, the antibody drug conjugate has a drug / antibody ratio of about 2-6.

[0028] In another aspect, the present application provides a compound for producing the antibody drug conjugate described herein, comprising M-(L1) aThe present invention provides a compound having a structure represented by formula 2, in which M represents a bispecific antibody or antigen-binding fragment thereof targeting HER2 described in the present application, L1 represents a linker connecting to M, and a is selected from 0 to 10.

[0029] In some embodiments, L1 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid based linker. In some embodiments, L1 and M are linked to M via a thiol group, an azide group, or an amide group on M.

[0030] In some embodiments, M comprises a first heavy chain and a second heavy chain, and the first heavy chain and / or the second heavy chain comprises a linking site capable of linking to L1. In some embodiments, the linking site comprises a group capable of linking to the L1 following deglycosylation modification.

[0031] In some embodiments, the group is located flanking the amino acid Q at position 297 of the first heavy chain and / or flanking the amino acid Q at position 298 of the second heavy chain. In some embodiments, the group comprises an amide group.

[0032] In some embodiments, the linking site comprises a group capable of linking to the L1 after glycosylation modification. In some embodiments, the group is located flanking amino acid N at position 299 of the first heavy chain and / or flanking amino acid N at position 300 of the second heavy chain. In some embodiments, the group comprises -N3.

[0033] In some embodiments, the glycosylation modification comprises that M has been contacted with a UDP-GalNAz, β-1,4-galactosyltransferase or a variant thereof. In some embodiments, the L1 is capable of participating in a SPAAC reaction.

[0034] In some embodiments, L1 is selected from the group consisting of maleimide, succinimid-3-yl-N, and DBCO. In some embodiments, L1 is DBCO-(PEG). n1 Among them, n1 is an integer of 0 to 10, or the above L1 is maleimide.

[0035] In some embodiments, the compound is [ka] The structure includes a structure selected from the group consisting of:

[0036] In another aspect, the present application provides a method for producing an antibody drug conjugate described herein, comprising contacting a compound described herein with a drug described herein. In another aspect, the present application provides a pharmaceutical composition comprising an antibody drug conjugate described herein or a pharma- ceutically acceptable carrier.

[0037] In another aspect, the present application provides a method of modulating a tumor microenvironment in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein. In another aspect, the present application provides a method of modulating an immune response in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein.

[0038] In another aspect, the present application provides a use of an antibody drug conjugate described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament, wherein the medicament is capable of preventing and / or treating a tumor. In some embodiments, the tumor comprises a solid tumor and / or a non-solid tumor.

[0039] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the contents of the present application may allow those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention of the present application. Accordingly, the drawings and descriptions in the present application are merely illustrative, not limiting.

[0040] Specific features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is given below. [Brief description of the drawings]

[0041] [Figure 1] 1 shows the glycosylation modification reaction of the bispecific antibody targeting HER2 described in the present application. [Diagram 2] FIG. 1 shows HPLC chromatograms of the glycosylation-modified HER2-targeting bispecifics described in this application. [Diagram 3] 1 shows the reaction steps for obtaining the antibody drug conjugates described in the present application. [Figure 4] 1 shows an HPLC chromatogram of an antibody drug conjugate described in the present application. [Diagram 5] 1 shows the reaction steps for obtaining the antibody drug conjugates described in the present application. [Figure 6] 1 shows the results of Waters Xevo G2-QTOF mass spectrometry of the antibody drug conjugates described herein. [Figure 7] 1 shows the results of Waters Xevo G2-QTOF mass spectrometry of the antibody drug conjugates described herein. [Figure 8] 1 shows the results of Waters Xevo G2-QTOF mass spectrometry of the light chain portion of DS-8201. [Figure 9]1 shows the results of Waters Xevo G2-QTOF mass spectrometry of the heavy chain portion of DS-8201. [Figure 10] 1 shows the binding affinity of the HER2-targeting bispecific antibodies described in the present application to FcγRIIIa. [Figure 11] 1 shows the binding affinity of the antibody drug conjugates described herein for FcγRIIIa. [Figure 12] 1 shows the binding affinity of the HER2-targeting bispecific antibodies described herein to FcγRI. [Figure 13] 1 shows the binding affinity of the antibody drug conjugates described herein for FcγRI. [Figure 14] 1 shows the binding ability of the antibody drug conjugates described herein to tumor cells. [Figure 15] 1 shows the tumor cell killing capacity after 3 days of treatment with antibody drug conjugates described in this application. [Figure 16] 1 shows the tumor cell killing capacity after 5 days of treatment with antibody drug conjugates described in this application. [Figure 17] 1 shows the tumor cell killing capacity after 3 days of treatment with antibody drug conjugates described in this application. [Figure 18] The results of growth inhibition of SK-BR-3 cells for each experimental group are shown. [Figure 19] The growth inhibition results of each experimental group against MDA-MB-468 cells are shown. [Figure 20] 1 shows the results of analyzing the ADCC activity of antibody drug conjugates described in the present application. [Figure 21] 1 shows steps for measuring the ADCC activity of the antibody drug conjugates described in the present application using a PBMC system. [Figure 22] 1 shows the results of measuring the ADCC activity of the antibody drug conjugates described in the present application using a PBMC system. [Diagram 23] 1 shows the results of measuring the ADCC activity of the antibody drug conjugates described in the present application using a PBMC system. [Figure 24]1 shows endocytosis of an antibody drug conjugate described herein in tumor cells. [Diagram 25] 1 shows endocytosis of an antibody drug conjugate described herein in tumor cells. [Figure 26] 1 shows endocytosis of an antibody drug conjugate described herein in tumor cells. [Figure 27] 1 shows endocytosis of an antibody drug conjugate described herein in tumor cells. [Figure 28] 1 shows the results of a pharmacokinetic study of the antibody drug conjugates described in the present application. [Figure 29] 1 shows the percentage results of NCI-N87 positive cells after endocytosis induction by antibody drug conjugates described in the present application. [Diagram 30] The growth curves of mouse tumor volume in each group in a human gastric cancer PDX tumor model are shown. [Diagram 31] Figure 1 shows the growth curve of tumor volume in human gastric cancer HER2 IHC-PDX tumor model mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Hereinafter, the embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0043] Definition of Terms In this application, the term "antibody drug conjugate" generally refers to an ADC, i.e., a binding protein (e.g., an antibody or antigen-binding fragment thereof) linked to one or more chemical agents. The chemical agents may be any therapeutic and / or cytotoxic. The antibody drug conjugate may have any number of drugs conjugated to the antibody, from 1 to 8, and may include, for example, 2, 4, 6, or 8 drug loaded species. In this application, the drugs may include antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemotherapy protective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and / or radiosensitizers.

[0044] In this application, the term "drug / antibody ratio" or "DAR" generally refers to the number of drugs linked to the antibody of an ADC. The DAR of an ADC may range from 1 to 8, or may be higher loading (e.g., 10), and the range of DAR may be due to the number of linkage sites on the antibody. In this application, the DAR may be the number of drugs loaded onto a single antibody. The DAR may be the average or mean DAR of a set of ADCs.

[0045] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (SwissProt P04626). In this application, the HER2 may also be referred to as rbB-2, NEU, HER-2, or CD340. The HER2 may include any mutant, isoform, and species homolog of HER2 that is naturally expressed by cells, including tumor cells, or that is expressed by cells transfected with the HER2 gene or cDNA. In this application, the term "extracellular domain" generally refers to the extracellular domain of human HER2 (SwissProt P04626), which may include four extracellular domains: I, II, III and IV.

[0046] In this application, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of identical polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and heavy chains further include a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into regions of hypervariability (called complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL), respectively, form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes, among others, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.The antibodies may be of different isotypes, such as, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0047] As used herein, the term "antigen-binding portion" refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., HER2) that the antibody binds and competes with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of whole antibodies. In some cases, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a sufficient portion of an antibody to confer specific antigen-binding ability to the polypeptide. Antigen-binding portions of an antibody (e.g., antibody fragments as described above) can be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and specifically screened for antigen-binding portions of the antibody in the same manner as intact antibodies.

[0048] In this application, the term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544546 (1989)), the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains, and the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.

[0049] In this application, the term "antibody Fc" generally refers to a human immunoglobulin chain constant region defined based on papain cleavage of an antibody. The Fc may be the carboxyl terminus of an immunoglobulin heavy chain constant region or a portion thereof. For example, an immunoglobulin Fc region may include a combination of two or more domains of heavy chain CH2, CH3, CH4 and an immunoglobulin hinge region. Depending on the amino acid sequence of the heavy chain constant region, immunoglobulins can be divided into different classes, and there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. The selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the understanding of one of skill in the art. For example, the Fc may include at least one immunoglobulin hinge region, one CH2 domain, and one CH3 domain, such as human IgG1 Fc.

[0050] In the present application, the term "bispecific antibody" generally refers to an antibody capable of binding to two antigens or epitopes, respectively. The bispecific antibody may comprise a light chain and a heavy chain of an antibody capable of specifically binding to a first antigen or epitope, and a light chain and a heavy chain of an antibody capable of specifically binding to a second antigen or epitope. In one embodiment of the present application, in the bispecific antibody, the light chain of the antibody capable of specifically binding to the first antigen or epitope and the light chain of the antibody capable of specifically binding to the second antigen or epitope have the same sequence. In one embodiment of the present application, in the bispecific antibody, the heavy chain of the antibody capable of specifically binding to the first antigen or epitope and the heavy chain of the antibody capable of specifically binding to the second antigen or epitope have different sequences.

[0051] In this application, "epitope" or "antigenic epitope" generally refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, epitopes usually contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, and may be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all of the interaction sites between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, the sites of interaction span amino acid residues of a protein that are separated from one another.

[0052] In the present application, the term "specific binding" generally refers to binding that is distinguished from non-specific adsorption. Examples of criteria for determining whether a binding is specific include dissociation constants (e.g., "KD"). For example, the KD value of the bispecific antibody or its antigen-binding fragment against the HER2 protein is 1×10 -5 Less than M, 5×10 -6 Less than M, 2 x 10 -6 Less than M or 1×10 -6 Less than M, 5×10 -9 Less than M, 2 x 10 -9 Less than M or 1×10 -9 The binding can be measured by known methods such as surface plasmon resonance, ELISA, and RIA.

[0053] In the present specification, "CDR" means a complementarity determining region (CDR). It is known that an antibody molecule has three CDRs in each of the heavy and light chains. CDRs are also called hypervariable domains, and are sites in which the primary structure is particularly highly variable in the variable regions of the heavy and light chains of an antibody, and are separated into three sites in the primary structure of the heavy and light chain polypeptide chains. In the present specification, the CDRs of an antibody are described as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence for the heavy chain CDRs, and as CDRL1, CDRL2, and CDRL3 from the amino terminal side of the light chain amino acid sequence for the light chain CDRs. These sites are structurally close to each other and determine the specificity for the antigen to which they bind.

[0054] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology A Synthesis (2nd ed., ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0055] In this application, the term "pertuzumab" generally refers to Pertuzumab, also known as 2C4, a trade name of Perjeta. The amino acid sequences of the light and heavy chain variable regions of the pertuzumab can be seen in FIG. 2 of US20090285837A1. The pertuzumab is a recombinant humanized monoclonal antibody that can specifically bind to the extracellular dimerization domain (subdomain II) of epidermal growth factor receptor 2 (HER2). By binding to HER2, the pertuzumab can inhibit the heterodimerization of HER2 with other HER receptors, and can slow tumor growth. The pertuzumab can be used to treat HER2-positive metastatic breast cancer, and can also be used to treat early-stage breast cancer.

[0056] In this application, the term "trastuzumab" generally refers to Trastuzumab, trade name Herceptin®. The amino acid sequences of the light and heavy chains of Trastuzumab can be seen in Figure 16 of US20090285837A1. Trastuzumab is a recombinant DNA-derived humanized monoclonal antibody that can be produced from mammalian cells (Chinese Hamster Ovary Cells CHO) cultured in suspension in a sterile medium. Trastuzumab can specifically bind to the extracellular domain of HER2 and can also stimulate the body's own immune cells to destroy tumor cells. Trastuzumab can be used to treat HER2-positive metastatic breast cancer, early breast cancer, and HER2-positive metastatic gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

[0057] In this application, the term "Exatecan" generally refers to exatecan (DX-8951), a DNA topoisomerase I inhibitor with CAS number 171335-80-1. DXd is an exatecan (DX-8951) derivative with CAS number 1599440-33-1. Exatecan and DXd, as one of the drug camptothecin derivatives, belong to addictive drugs, and such drugs, when applied to ADC, can reduce toxic side effects due to the shedding of toxins.

[0058] In this application, the term "linker" generally refers to a bifunctional or polyfunctional chemical moiety that is used to link a bispecific antibody or antigen-binding fragment thereof described herein to a drug described herein. The linker may comprise one coupling moiety or may comprise multiple coupling moieties.

[0059] As used herein, the term "tumor" generally refers to a physiological condition in a mammal characterized by uncontrolled cell growth. The tumor comprises one or more cancerous cells. The tumor may comprise a solid tumor and / or a non-solid tumor.

[0060] In this application, the term "tumor microenvironment" generally refers to the environment in which the tumor resides, which is the acellular area within the tumor and the area immediately outside the tumor tissue, but not belonging to the intracellular compartment of the cancer cell itself. Tumors can be closely associated with and continuously interact with the tumor microenvironment. For example, tumors can change the tumor microenvironment, which can affect tumor growth and spread. Typically, the tumor microenvironment has a low pH, ranging from 5.8 to 7.0. The tumor microenvironment has lower concentrations of glucose and other nutrients, but may have higher concentrations of lactic acid. The temperature of the tumor microenvironment may be 0.3 to 1°C higher than normal physiological temperature. The tumor microenvironment is described in "MRI of the Tumor Microenvironment" by Gillies et al., Journal of Magnetic Resonance Imaging, Vol. 16, pp. 430-450, 2002.

[0061] In this application, the term "treatment" generally refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or alleviate (reduce) undesirable physiological changes or pathologies, such as hyperproliferative conditions, such as the growth, formation, or spread of cancer. For purposes of the present invention, favorable or desired clinical results include, but are not limited to, amelioration of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or reduction in the progression of disease, improvement or amelioration of the disease state, and rehabilitation (whether partial or complete), whether detectable or undetectable. "Treatment" or "treatment" can also refer to prolonging survival compared to expected survival without treatment. Subjects in need of treatment include those who have previously had a disease or condition, and those who are prone to suffer from a disease or condition, or those in whom a condition or condition is to be prevented.

[0062] In one aspect, the present application provides an antibody drug conjugate comprising a bispecific antibody or antigen-binding fragment thereof that targets HER2. In some embodiments, the antibody drug conjugate is M-(L1) a -(L2)b -D (Formula 1), in which M represents a bispecific antibody targeting HER2 described in the present application or an antigen-binding fragment thereof, L1 represents a linker linked to M, L2 represents a linker linked to D, a and b are each independently selected from 0 to 10, and D represents a drug.

[0063] Bispecific antibodies targeting HER2 or antigen-binding portions thereof In the present application, the bispecific antibody or antigen-binding fragment thereof targeting HER2 can specifically bind to at least one epitope of human HER2. For example, it can specifically bind to two different epitopes of human HER2. In the present application, the at least two epitopes may be located in the same domain in the human HER2 protein, or may be located in at least two different domains in the human HER2 protein.

[0064] For example, the bispecific antibody or antigen-binding fragment thereof that targets HER2 can specifically bind to the extracellular domain II of human HER2 and / or the extracellular domain IV of human HER2. For example, the bispecific antibody or antigen-binding fragment thereof that targets HER2 can specifically bind to the extracellular domain II of human HER2 and the extracellular domain IV of human HER2.

[0065] In the present application, the bispecific antibody or antigen-binding fragment thereof targeting HER2 can comprise a first light chain and a second light chain. In the present application, the first light chain may comprise first LCDR1 to 3, and the first LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4. In the present application, the first LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5. In the present application, the first LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0066] For example, in the first light chain, the first LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the first LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the first LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. In the present application, the first light chain is capable of binding to the heavy chain of Pertuzumab.

[0067] In the present application, the second light chain may comprise a second LCDR1-3, and the second LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1. In the present application, the second LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2. In the present application, the second LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0068] For example, in the second light chain, the second LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the second LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the second LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. In the present application, the second light chain is capable of binding to the heavy chain of trastuzumab.

[0069] In the present application, the first light chain and / or the second light chain may be modified from two original monoclonal antibodies (e.g., may be known monoclonal antibodies). In the present application, the two original monoclonal antibodies may target HER2. For example, the two original monoclonal antibodies may specifically bind to at least one epitope of human HER2. For example, the two original monoclonal antibodies may specifically bind to two different epitopes of human HER2, respectively. For example, the two original monoclonal antibodies may specifically bind to the extracellular domain II of human HER2 and the extracellular domain IV of human HER2, respectively. For example, the two original monoclonal antibodies may be trastuzumab and pertuzumab.

[0070] For example, the first light chain and / or the second light chain may be different from the amino acid sequence of any of the light chains of the two original monoclonal antibodies. Also, for example, the first light chain and / or the second light chain may be identical to the amino acid sequence of any of the light chains of the two original monoclonal antibodies, or the amino acid sequence of the first light chain and / or the second light chain may be obtained by modification based on the amino acid sequence of any of the light chains of the two original monoclonal antibodies. For example, the modification may include amino acid sequence modification. For example, the purpose of the modification may be to maintain the affinity for the antigen or epitope corresponding to the two original monoclonal antibodies as much as possible.

[0071] In this application, such modifications may include mutations, deletions, or additions, such as mutations, deletions, or additions of 3 or fewer amino acids, 2 or fewer amino acids, or 1 or fewer amino acids. In the present application, the amino acid sequence of the variable region of the first light chain may be identical to the amino acid sequence of the variable region of the second light chain.

[0072] In the present application, the variable regions of the first light chain and the second light chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 7 to 12. In the present application, the variable region of the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID NO:7.

[0073] In the present application, the first light chain and the second light chain may comprise a light chain constant region. The light chain constant region may be of the κ type or the λ type. For example, the κ type light chain constant region includes various allotypes such as Km1, Km2, Km3, and the λ type light chain constant region includes various allotypes such as CL1, CL2, CL3, CL6, and CL7. In the present application, the first light chain and the second light chain may comprise an amino acid sequence set forth in any one of SEQ ID NOs:13-18.

[0074] In the present application, the first light chain may be selected from the group consisting of a light chain of pertuzumab or a variant thereof, and a light chain of trastuzumab or a variant thereof. In the present application, the second light chain may be selected from the group consisting of a light chain of pertuzumab or a variant thereof, and a light chain of trastuzumab or a variant thereof. In the present application, the first light chain may be the light chain of pertuzumab or a variant thereof. In the present application, the second light chain may be the light chain of trastuzumab or a variant thereof.

[0075] In the present application, the first light chain and the second light chain may have the same amino acid sequence. In the present application, the first light chain and the second light chain may comprise the amino acid sequence set forth in SEQ ID NO:13.

[0076] In the present application, the bispecific antibody or antigen-binding fragment thereof targeting HER2 may comprise a first heavy chain and a second heavy chain, and the first heavy chain can correctly bind to the first light chain under physiological conditions or in vitro protein expression conditions. In the present application, the second heavy chain is capable of correctly binding to the second light chain under physiological conditions or under in vitro protein expression conditions.

[0077] In the present application, the first heavy chain may comprise a first heavy chain variable region, which may be the heavy chain variable region of pertuzumab. In the present application, the second heavy chain may comprise a second heavy chain variable region, which may be the heavy chain variable region of trastuzumab. In the present application, the first heavy chain and the second heavy chain can comprise a heavy chain constant region that can be derived from the constant region of human IgG.

[0078] In some cases, the first heavy chain constant region and / or the second heavy chain constant region may comprise an Fc region, which may be modified, for example, to increase the rate at which the first heavy chain and the second heavy chain form a heterodimer. In this application, the above modification techniques are well known to those skilled in the art, and reference can be made to, for example, Ridgway, Presta et al. 1996, Carter 2001, Patent CN102558355A, and Patent CN103388013A.

[0079] In the present application, the first heavy chain constant region and the second heavy chain constant region may be the same or different in heavy chain type. For example, the first heavy chain constant region and the second heavy chain constant region may be different from the amino acid sequence of the heavy chain constant region of the two original monoclonal antibodies. Also, for example, the amino acid sequence of the first heavy chain constant region may be identical to the amino acid sequence of the heavy chain constant region of one of the original monoclonal antibodies, and / or the amino acid sequence of the second heavy chain constant region may be identical to the amino acid sequence of the heavy chain constant region of the other of the original monoclonal antibodies.

[0080] In the present application, the Fc fragment of the first heavy chain and the Fc fragment of the second heavy chain may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 24 to 57. In the present application, the first heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 21 to 24. For example, the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 21 or 23. In the present application, the second heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 21 to 24. For example, the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 22 or 24.

[0081] In the present application, the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 21 or 23, and the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 22 or 24. For example, in the present application, the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 21, and the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 22. For example, in the present application, the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 23, and the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0082] In the present application, the bispecific antibody or antigen-binding fragment thereof targeting HER2 can be obtained according to conventional technical means in the art. For example, it can be obtained by purification from host cells. The purification method can include chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration.

[0083] Drug Conjugates and Compounds In the present application, the antibody drug conjugate is M-(L1) a -(L2) b -D (Formula 1), in which M represents a bispecific antibody targeting HER2 described in the present application or an antigen-binding fragment thereof, L1 represents a linker linked to M, L2 represents a linker linked to D, a and b are each independently selected from 0 to 10, and D represents a drug. In the present application, the compound for producing the antibody drug conjugate described in the present application is M-(L1) a It has a structure shown in (Formula 2), in which M represents a bispecific antibody targeting HER2 described in the present application or an antigen-binding fragment thereof, L1 represents a linker linked to M, and a is selected from 0 to 10.

[0084] Modification of M and linkage to L1 In the present application, L1 and M may be linked via a thiol group, an azide group, or an amide group on M. In the present application, M may comprise a first heavy chain and a second heavy chain, and the first heavy chain and / or the second heavy chain may comprise a linking site capable of linking to L1.

[0085] In the present application, the linking site may comprise a group capable of linking to the L1 after deglycosylation modification. In the present application, the group may be located at the side group of amino acid Q at position 297 of the first heavy chain and / or at the side group of amino acid Q at position 298 of the second heavy chain. For example, the group may comprise an amide group. For example, transglutaminase (TGs) may be used with the amino acid Q as the linking site to form the antibody drug conjugate with a DAR equal to about 2.

[0086] In the present application, the linking site may comprise a group capable of linking to L1 after glycosylation modification. In the present application, the group may be located side by amino acid N at position 299 of the first heavy chain and / or side by amino acid N at position 300 of the second heavy chain. In the CH2 domain of the heavy chain, N299 or N300 may have one conserved glycosylation site. Site-specific conjugation can be performed by utilizing the glycosylation site at N.

[0087] The numbering of the amino acid positions can be calculated starting from the N-terminal amino acid of the first heavy chain and / or the second heavy chain.

[0088] In the present application, the glycosylation modification site may be glycosylated. In the present application, the glycosylation form may be achieved, for example, by expressing a protein in a cell with an altered glycosylation structure. Cells with altered glycosylation structures have been described in the art and can be used to express a protein with the glycosylation modification (e.g., a bispecific antibody or antigen-binding fragment thereof targeting HER2 described in the present application).

[0089] Glycosylation of a protein can depend on the amino acid sequence of the protein (e.g., a bispecific antibody or antigen-binding fragment thereof targeting HER2 described herein) and the host cell in which the protein is expressed. Different organisms can produce different glycosylases (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to these factors, the glycosylation pattern of a protein and the composition of residues at the glycosylation modification site can vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful in the present application can include glucose, galactose, mannose, fucose, n-acetylglucosamine, and / or sialic acid. For example, the glycosylation modifications can include a pattern of glycosylation modifications adapted for humans.

[0090] In the present application, the above-mentioned glycosylation modifications can change the properties of a protein (eg, a bispecific antibody or antigen-binding fragment thereof targeting HER2 described in the present application). Glycosylation of different proteins can affect and / or result in different protein properties (e.g., changes in expression level, in vivo half-life, protein folding, solubility, susceptibility to proteases, transport, translocation, compartmentalization, secretion, recognition of other proteins or factors, antigenicity or allergenicity, and other properties).

[0091] In the present application, the specific structure and preparation method of glycosylation of the protein can be adjusted according to different glycosylation purposes, and those skilled in the art can adjust it based on the conventional techniques of those skilled in the art. In some cases, the glycosylation modification can be adjusted according to the species specificity of human and / or animal. In some cases, the glycosylation modification can be achieved by a glycosylation enzyme (e.g., a heterologous glycosylation enzyme derived from a host cell). For example, the glycosylation modification can obtain a protein (e.g., a bispecific antibody or an antigen-binding fragment thereof targeting HER2 described in the present application) that exhibits the glycosylation characteristics of human proteins. The glycosylation enzyme can be naturally derived or non-naturally derived.

[0092] In the present application, the glycosylation modification may include that M has been contacted with UDP-GalNAz, β-1,4-galactosyltransferase or a mutant thereof. For example, the β-1,4-galactosyltransferase or a mutant thereof (e.g., β-1,4-Gal-T1-Y289) can be capable of incorporating modified N-acetylgalactosamine (GalNAc) and can be attached to terminal N-acetylglucosamine (GlcNAc) residues on glycans of a protein (e.g., a bispecific antibody or antigen-binding fragment thereof that targets HER2).

[0093] In the present application, after contacting M with UDP-GalNAz, β-1,4-galactosyltransferase or a mutant thereof, M may contain a group as described herein. Position N299 of the first heavy chain and / or the side group of the amino acid N at position N300 of said second heavy chain of said M can be modified with an -N3 group to give a glycosylation-modified M.

[0094] For example, in the antibody drug conjugates described herein and / or the compounds described herein, the Fc region of M can be linked to L1 via the above group. For example, in the antibody drug conjugates described herein and / or the compounds described herein, M can be linked to L1 via the above group. For example, the glycosylated modified M can undergo an addition reaction with L1.

[0095] Linker In the present application, the above a may be an integer selected from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In the present application, the above b may be an integer selected from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0096] In the present application, L1 and / or L2 may be selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker. In the present application, both L1 and L2 can be considered to belong to the linker.

[0097] In the present application, the linker may be a moiety that extends the drug linkage, for example to avoid blocking the active site of an antibody or to increase the solubility of the ADC. The linker may comprise a stretcher and / or an amino acid unit.

[0098] In the present application, the linker can be used to conjugate (e.g., by covalent bonding) an antibody (e.g., a bispecific antibody or antigen-binding fragment thereof targeting HER2 described in the present application) to the drug. For example, the conjugate can form a bond with a functional group of the linker via a cysteine ​​thiol group or an amine (eg, the N-terminus or an amino acid side chain such as lysine) of the antibody.

[0099] For example, the linker can have a functional group that can react with a free cysteine ​​present on the antibody to form a bond (e.g., a covalent bond). For example, the functional group can include active esters such as maleimide, haloacetamide, α-haloacetyl, succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acid chloride, sulfonyl chloride, isocyanate, and / or isothiocyanate.

[0100] In some cases, the linker may comprise a functional group capable of reacting with an electrophilic group present on an antibody. For example, the functional group may comprise an aldehyde, ketocarbonyl, hydrazine, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and / or arylhydrazide.

[0101] In the present application, the linker may include a cleavable linker and a non-cleavable linker. For example, the linker (e.g., L2) may be a cleavable (e.g., self immolative) linker that can facilitate the release of the drug. For example, the linker (e.g., L1) may be a non-cleavable linker that can be linked to the antibody and is non-cleavable. In the present application, the cleavable linker may be cleaved under intracellular conditions. For example, the cleavable linker may include an acid labile linker (e.g., including a hydrazone), a protease-sensitive linker (e.g., peptidase-sensitive), a photolabile linker, and / or a disulfide-containing linker. For example, the cleavable linker may include a peptide linker that can be cleaved by an intracellular protease (e.g., a lysosomal protease) and / or an intracellular protease. In some cases, the linker is cleavable under an intracellular environment. For example, cleavage of the linker can allow the drug in the antibody drug conjugate to exert an effective therapeutic effect.

[0102] In the present application, the structure and / or properties of the linker are stable outside the cell. The antibody-drug conjugate described in the present application may be stable in structure before transport or delivery into the cell. For example, in the antibody-drug conjugate, the bispecific antibody or antigen-binding fragment thereof targeting HER2 remains conjugated to the drug.

[0103] In some cases, the linker in the antibody drug conjugate can retain the specific binding properties of the bispecific antibody or antigen-binding fragment thereof, e.g., targeting HER2, and participate in the delivery of the antibody drug conjugate and / or maintain the therapeutic effect (e.g., cytotoxic effect) of the drug.

[0104] In the present application, the linker may include hydrophilic linkers (e.g., PEG4Mal and sulfo-SPDB) and hydrophobic linkers. For example, the hydrophilic linker may reduce the extent to which the antibody drug conjugate can be pumped out of resistant cancer cells by MDR (multidrug resistance) or functionally similar translocators.

[0105] In the present application, the linker may also function to directly or indirectly inhibit cell growth and / or cell proliferation. For example, the linker may function as an intercalating agent in the cleavage. For example, the linker may play an indirect role to inhibit the biosynthesis of a macromolecule.

[0106] In the present application, the linker can facilitate the entry of the antibody-drug conjugate into cells (e.g., facilitate the "internalization" effect). In the present application, the linker can also be designed to improve the stability of the antibody-drug conjugate.

[0107] In the present application, the L1 can participate in the SPAAC reaction. The SPAAC reaction is an azide-alkyne cycloaddition reaction. The SPAAC reaction can be applied to the manufacturing process of the antibody-drug conjugate as one of the addition reactions. In the present application, L1 may be selected from the group consisting of maleimide, succinimid-3-yl-N, and DBCO.

[0108] In the present application, L1 is DBCO-(PEG) n1 Among them, n1 is an integer of 0 to 10, or the L1 is maleimide. For example, the L1 may be DBCO-(PEG)4, DBCO-(PEG)3. In the present application, the PEG molecule can be used to increase the hydrophilicity of the antibody-drug conjugate. The PEG molecule can be used to meet the spatial distance requirement due to the cleavage (e.g., enzymatic cleavage) of L2 (e.g., GGFG).

[0109] In the present application, the above L2 is a polypeptide, VC-PAB, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG. may be selected from the group consisting of NHS, N-4-(maleimidomethyl)cyclohexyl succinimidyl carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidinyl-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecanoic-1-oic acid ester (CX1-1).

[0110] In the present application, the above-mentioned L2 is GGFG, LP, VK, VC, GFG, GGFGG, GGFGS, GGFGGG, GGFGGE, GGFGGGFG, DGGF, DGGFG, D d G.F.G., D.G. Me L2 may be GFG, DGGFS, DDGGFG, KDGGFG, KGGFG, EGGFG or SGGFG. For example, L2 may be GGFG. The cathepsin B cleavable tetrapeptide Gly-Gly-Phe-Gly (GGFG) is a highly hydrophilic linker (e.g., more hydrophilic than Gly-Phe-Leu-Gly).

[0111] Drugs In this application, the drug may have the ability to kill tumor cells and / or inhibit the proliferation of tumor cells. In this application, the drug can include a small molecule drug.

[0112] In the present application, the drug may be selected from the group consisting of V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphotransfer reaction, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA breakers, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 glucuronic acid, etoposide phosphate, nitrogen mustard, proteasome inhibitors, cytokines, and Toll-like receptor agonists.

[0113] In the present application, the drug may be selected from the group consisting of DM1, exatecan, DXd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, Duo5 toxin, SN38 derivatives or derivatives thereof.

[0114] In the present application, the drug may be camptothecin or a derivative thereof. In the present application, the drug may be a DNA topoisomerase I inhibitor. In the present application, the drug may be Exatecan (DX-8951), DXd, duocarmycin, mitomycin C, talimycin, maytansine, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, or a TLR9 agonist.

[0115] For example, the drug may be [ka] The structure may be:

[0116] In the present application, the antibody drug conjugate is [ka] The compound may have a structure selected from the group consisting of:

[0117] In the present application, the antibody drug conjugate can have a drug / antibody ratio of about 2 to 6. For example, the drug / antibody ratio can be about 1, about 2, about 3, about 4, about 5, or about 6. For example, the drug / antibody ratio can be 4. For example, the drug / antibody ratio can be 3.

[0118] Compounds and methods of manufacture In another embodiment, the present application provides M-(L1) aThe present invention provides a compound for an antibody-drug conjugate described herein, having a structure as shown in Formula 2, wherein M represents a bispecific antibody targeting HER2 described herein or an antigen-binding fragment thereof, L1 represents a linker connecting to M, and a is selected from 0 to 10.

[0119] In the present application, the compound is [ka] The compound may comprise a structure selected from the group consisting of:

[0120] In another aspect, the present application provides a method for producing an antibody drug conjugate described herein, comprising contacting a compound described herein with a drug described herein. In some cases, the method for producing the antibody drug conjugate includes the steps of: (1) contacting L1, L2 described herein with the drug; and (2) contacting compound (L1). a -(L2) b (3) reacting M as defined herein with compound (L1) a -(L2) b and contacting M with -D to obtain the antibody-drug conjugate described herein. For example, M in step (3) has been modified by glycosylation as described above.

[0121] Pharmaceutical Compositions and Uses In another aspect, the present application provides a pharmaceutical composition comprising an antibody drug conjugate described herein or a pharma- ceutically acceptable carrier.

[0122] In the present application, the pharmaceutical composition may comprise a "therapeutically effective dose" or a "prophylactically effective dose" of the antibody drug conjugate described herein. The "therapeutically effective dose" may be an amount effective to achieve a desired therapeutic result at the required dose and duration. The therapeutically effective dose can be determined by one skilled in the art and may vary depending on factors such as the disease state, the subject's age, sex, weight, and the ability of the antibody drug conjugate to elicit a desired response in the subject. The "prophylactically effective dose" may be an amount effective to achieve a desired prophylactic result at the required dose and duration. For example, the prophylactically effective dose may be lower than the therapeutically effective dose.

[0123] In the present application, the pharmaceutical composition may be arranged in a form suitable for administration (e.g., suitable for parenteral, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous, intratumoral and / or mucosal administration). In the present application, the pharmaceutical composition may contain other active pharmaceutical ingredients.

[0124] In this application, the pharma- ceutically acceptable carrier can include any or all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The pharma- ceutically acceptable carrier can include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The pharma- ceutically acceptable carrier can further include an isotonic agent, a wetting agent, an emulsifying agent, a preservative, and / or a buffering agent.

[0125] In another aspect, the present application provides a method of modulating a tumor microenvironment in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein. In another aspect, the present application provides a method of modulating an immune response in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein.

[0126] The present application provides the use of an antibody drug conjugate as described herein, or a pharmaceutical composition as described herein, in the manufacture of a medicament capable of modulating the tumor microenvironment of a subject and / or modulating the immune response of a subject. The present application provides an antibody drug conjugate as described above, or a pharmaceutical composition as described herein, for modulating the tumor microenvironment of a subject and / or modulating the immune response of a subject.

[0127] In another aspect, the present application provides a use of an antibody drug conjugate described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament, wherein the medicament is capable of preventing and / or treating a tumor. The present application provides a method for preventing and / or treating a tumor, comprising administering to a subject an antibody drug conjugate described herein, or a pharmaceutical composition manufactured according to the present application. The present application provides the antibody-drug conjugate or the pharmaceutical composition described herein for preventing and / or treating tumors.

[0128] In the present application, the tumor may include a solid tumor and / or a non-solid tumor. For example, the tumor may include gastric cancer, breast cancer (e.g., ductal carcinoma) and / or pancreatic cancer. For example, the tumor of the present application may include HER2 weakly positive tumor and / or HER2 negative tumor, for example, the technical solution of the present application may have a bystander killing effect.

[0129] Without intending to be limited by any theory, the following examples are merely intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0130] A bispecific antibody targeting HER2 (hereinafter abbreviated as Antibody A) comprises a first light chain, a second light chain, a first heavy chain and a second heavy chain, wherein the first light chain and the second light chain have the amino acid sequence shown in SEQ ID NO: 13, the heavy chain variable region of the first heavy chain has the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain variable region of the second heavy chain has the amino acid sequence shown in SEQ ID NO: 20. The first heavy chain has the amino acid sequence shown in SEQ ID NO: 21, and the second heavy chain has the amino acid sequence shown in SEQ ID NO: 22.

[0131] Antibody A was artificially synthesized according to the amino acid sequence of antibody A, and then isolated and purified. DS-8201 (also known as T-DXd, trade name ENHERTU) is manufactured by Daiichi Sankyo and AstraZeneca. DS-8201 is an anti-HER2 IgG1 monoclonal antibody conjugated via a linker to the topoisomerase I inhibitor DXd. U3-1402 was manufactured by Daiichi Sankyo and is a covalent conjugate of the HER3 antibody Patritumab and the topoisomerase inhibitor DXd.

[0132] [Example 1 Production of Antibody A-ADC1 Molecule] (1) Preparation of a compound containing a linker and a toxin Compound 1: DBCO-PEG4-GGFG-DXd containing linker and toxin was obtained, which has the following structural formula: DXd (i.e., exatecan DX-8951 derivative, purchased from Lianying Biology): [ka]

[0133] (2) Preparation of Glycosylated Antibody A Antibody A was contacted with β-1,4-galactosyltransferase (β-1,4-al-T1) (purchased from Shanghai Baosen Biotechnology Co., Ltd.) in the presence of UDP-GalNAz. The structural formula of UDP-GalNAz is [ka] It is.

[0134] After the contact, the N299 site of the first heavy chain and the N300 site of the second heavy chain of antibody A were modified with -N3 groups to give glycosylation-modified antibody A. The glycosylation modification reaction of antibody A is shown in FIG. 1. The obtained glycosylated antibody A was detected by HPLC, and the detection results are shown in FIG. 2. As can be seen from the results in FIG. 2, the glycosylated antibody A has a unique characteristic peak and a molecular weight of 148,796 Da.

[0135] (3) Production of Antibody A-ADC1 Molecule DBCO-PEG4-GGFG-DXd from step (1) was contacted and reacted with glycosylated modified antibody A obtained in step (2) for 10 to 15 hours under conditions of 25 to 35°C, and DBCO reacted with the -N3 group in glycosylated modified antibody A underwent SPAAC reaction to obtain one antibody A-ADC molecule. The reaction for obtaining the antibody A-ADC1 molecule is shown in Figure 3. The antibody A-ADC1 was detected by HPLC, and the detection results are shown in Figure 4. As can be seen from the results in Figure 4, the antibody A-ADC1 molecule has a unique characteristic peak and a molecular weight of 154299.

[0136] (4) Production of Herceptin-ADC1 molecule Herceptin was glycosylated and reacted with DBCO-PEG4-GGFG-DXd according to the method described in Example 1 to obtain a Herceptin-ADC1 molecule with a DAR of 8.

[0137] [Example 2 Production of Antibody A-ADC2 Molecules] (1) Preparation of a compound containing a linker and a toxin The maleimide was reacted with GGFG-DXd to give compound 2, which contains the linker and the toxin and has the following structural formula: [ka]

[0138] (2) Preparation of Modified Antibody A The antibody A was reacted with TECP so that the antibody A had thiol groups (eg, eight -SH groups) to obtain a modified antibody A.

[0139] (3) Production of antibody A-ADC2 molecules The modified antibody A obtained in step (2) was contacted with compound 2 containing a linker and a toxin obtained in step (1) to obtain antibody A-ADC2 molecules. The reaction for obtaining two antibody A-ADC molecules is shown in FIG.

[0140] [Example 3] Stability analysis of ADC molecules containing antibody A The stability in human serum of antibody A-ADC1 produced in Example 1, antibody A-ADC2 produced in Example 2, and DS-8201 was examined. (1) Stability of anti-A-ADC1 in serum When antibody A-ADC1 added to serum was purified with Protein L, no obvious thiol exchange was observed after 3 or 7 days.

[0141] The specific experimental steps are as follows: 100 μL of each treated human serum was added to a 1.5 mL centrifuge tube, and then antibody A-ADC1 was added to make the final concentration of antibody A-ADC1 0.1 mg / mL. The tubes were then placed in a 37°C incubator and incubated for 3 and 7 days, respectively. 20 μL of Protein L magnetic bead suspension was added to a 1.5 mL centrifuge tube and placed on a magnetic stand to separate and remove the storage solution. 200 μL of DPBS was added to mix the magnetic beads evenly and placed on a magnetic stand to separate and remove the supernatant (this step was repeated twice).

[0142] Antibody A-ADC1 incubated for 3 or 7 days was added to a 1.5 mL centrifuge tube with magnetic beads, mixed uniformly, placed in a mixer at room temperature for 1 hour, then placed on a magnetic stand to remove the supernatant. Then, 200 μL of DPBS was added, mixed uniformly, placed on a magnetic stand to separate and remove the supernatant (this step was repeated three times). Finally, 20 μL of elution solution (100 mM Glycine, pH=2.8) was added, placed on a magnetic stand to mix uniformly at room temperature, and the supernatant was collected. Then, 20 μL of neutralization buffer (200 mM tris, pH=8.0) was added to the collected supernatant, its concentration was measured, and the purified antibody A-ADC1 was analyzed by Waters Xevo G2-QTOF mass spectrometry.

[0143] The results are shown in Figure 6. From the results in Figure 6, the molecular weight (about 154299) of antibody A-ADC1 after incubation in human serum for 0, 3 and 7 days did not change substantially, and the DAR value after 3 and 7 days in serum was still maintained at 4, indicating that the conjugated drug molecule did not undergo obvious thiol exchange after incubation in serum for 3 and 7 days, indicating that the stability of antibody A-ADC1 in serum is good, and the toxin is unlikely to be released into the blood during in vivo transport, which is more favorable for the transport of the conjugated small molecule toxin drug to the tumor target, and can reduce the risk of toxicity and side effects on normal biological tissues caused by detargeting of the toxin molecule.

[0144] (2) Stability of Anti-A-ADC2 in Serum The stability of antibody A-ADC2 in serum was detected according to the method of step (1). The results of Waters Xevo G2-QTOF mass spectrometry are shown in FIG. 7. The results in FIG. 7 show the cases where antibody A-ADC2 was incubated in human serum for 0 days, 3 days, and 7 days, respectively. After incubation for 3 days, the maleimide on the light chain can stably exist in human serum due to ring-opening hydrolysis, while the DAR value of the heavy chain decreased from 6 to about 2 after incubation for 3 days, and the DAR value of the heavy chain was almost 0 after incubation for 7 days, indicating that the heavy chain of antibody A-ADC2 was removed in serum after 7 days.

[0145] (3) Stability of DS-8201 in serum The stability of DS-8201 in serum was detected according to the method of step (1). The results of the heavy and light chains of DS-8201 incubated in serum by Waters Xevo G2-QTOF mass spectrometry are shown in Figures 8 and 9, respectively. The results in Figures 8 and 9 show that DS-8201 was incubated in human serum for 0, 3, and 7 days, respectively. After 3 days of incubation, the maleimide on the light chain can stably exist in human serum due to ring-opening hydrolysis, while after 7 days, the DAR value of the heavy chain decreased from 6 to about 2, the heavy chain of DS-8201 was obviously removed in serum, and the small molecule toxin on the heavy chain was significantly detargeted.

[0146] [Example 3] Measurement of binding affinity between ADC molecules containing antibody A and FcγR The affinity of antibody A and antibody A-ADC1 produced in Example 1 to FcγRI, FcγRIIIa, and the like was measured using biofilm interferometry (BLI) (Fortebio polymer interaction meter manufactured by Danner-Hertz).

[0147] Specific steps for affinity measurement: Antibody A-WS-161018 (parent molecule antibody A) or antibody A-ADC1 was immobilized on a FAB2G biosensor, the curing concentration was 10 μg / mL, and the curing height was 2 nm. FcγRIIIa was diluted to 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM, and the baseline was 60 seconds, the binding was 30 seconds, and the dissociation was 150 seconds. The diluent was a kinetics buffer, the regeneration solution was glycine-HCl (pH 1.7), and the neutralization solution was the diluent. The dissociation time was 10 seconds during fitting, and the experimental results are shown in Figures 10 to 11 and Table 1. Figures 10 and 11 show the binding affinity between antibody A and antibody A-ADC1 and FcγRIIIa, respectively.

[0148] [Table 1]

[0149] Antibody A-WS-161018 (parent molecule antibody A) or antibody A-ADC1 was immobilized on a FAB2G biosensor, the curing concentration was 10 μg / mL, and the curing height was 2 nm. FcγRI was diluted to 200 nM, 100 nM, 250 nM, 50 nM, and 25 nM, and the baseline was 60 seconds, the binding was 30 seconds, and the dissociation was 150 seconds. The diluent was 0.02% PBST20, the regenerating solution was glycine-HCl (pH 1.7), and the neutralizing solution was the diluent. The dissociation time was fitted at 10 seconds during fitting, and the experimental results are shown in Figures 12 to 13 and Table 2. Figures 12 and 13 show the binding affinity of antibody A and antibody A-ADC1 with FcγRI, respectively.

[0150] [Table 2]

[0151] As can be seen from the above results, the affinity of antibody A-ADC1 to FcγRIIIa is consistent with that of the parent antibody A, and antibody A-ADC1 has high binding affinity to FcγRI, with a KD value of 6.05×10 -10 M, which was similar to the affinity of the parent antibody A. FcγR binding experiments showed that site-specific conjugation involving antibody A-ADC1 did not affect the Fc function of the antibody moiety in the ADC, antibody A-ADC1 still retained the Fc function of the parent molecule antibody A, and the antibody moiety in antibody A-ADC1 could act synergistically with toxin to kill tumor cells.

[0152] [Example 4] Binding ability of ADC molecules containing antibody A to tumor cells The binding activity of antibody A-ADC1 prepared in Example 1, antibody A-ADC2 prepared in Example 2, and antibody A against NCI-N87 cells was examined.

[0153] Specific test steps: (1) Prepare the drug in 1% BSA / PBS to a maximum concentration of 120 μg / mL (working concentration 60 μg / mL), then dilute it three-fold from lowest to highest to give a total concentration of 10 μg / mL. (2) NCI-N87 cells (purchased from Shanghai Chinese Academy of Sciences) were harvested, centrifuged, counted, blocked with 5% BSA / PBS for 15 min, and then diluted to 2 × 10 in each 1.5 mL centrifuge tube. 5 cells / 50 μL, 50 μL of antibody drug was added to each tube and left at 4° C. for 1 hour. (3) The plate was centrifuged and washed twice at 2000 rpm for 5 minutes, and 1000 μL of 1% BSA / PBS was added to each wash. The plate was then dried by suction. 90 μL of APC-IgG (1:100 dilution) diluted with 1% BSA / PBS was added to each well and the plate was left in the dark at 4°C for 30 minutes. (4) Centrifuge and wash twice at 2000 rpm for 5 min, add 1000 μL of 1% BSA / PBS to each wash, and finally suction dry. Finally, add 300 μL of 1% BSA / PBS to each tube and perform FACS detection in the dark. The results are shown in FIG.

[0154] [Table 3] As can be seen from the results in Figure 14 and Table 3, antibody A at a high concentration showed the highest binding activity, antibody A-ADC1 was slightly lower than antibody A, and the mean fluorescence intensity (MFI) of the two tended to stabilize after reaching a peak value, and antibody A-ADC2 had low activity. This indicates that the binding of antibody A-ADC1 and its parent molecule antibody A to NCI-N87 cells is more stable. As can also be seen from the EC50 values, antibody A-ADC1 had an EC50 that was consistent with that of parent antibody A, both of which were approximately 1.1 μg / mL, indicating that the binding activity of antibody A-ADC1 and parent antibody A to NCI-N87 cells with high HER2 expression was consistent, and the peak value of antibody A-ADC1 was greater than that of antibody A-ADC2, indicating that the binding activity of antibody A-ADC1 to NCI-N87 cells with high HER2 expression was slightly higher than that of antibody A-ADC2.

[0155] [Example 5] Killing ability of ADC molecules containing antibody A against tumor cells The killing abilities of antibody A-ADC1 produced in Example 1, antibody A-ADC2 produced in Example 2, antibody A, and DS-8201 against NCI-N87 cells and BT474 cells were examined.

[0156] Specific test steps: (a) NCI-N87 cells after 5 days of treatment (1) Preparation of growth medium: RPMI-1640 + 10% FBS + 1% P / S. NCI-N87 cells were digested with pancreatin and inoculated into a 96-well plate at 10,000 cells / well / 150 μL and cultured overnight at 37°C. (2) Preparation of experimental medium: RPMI-1640 + 0.1% FBS + 1% P / S. Antibody A, Antibody A-ADC1, Antibody A-ADC2 and DS-8201 were each prepared to the highest working concentration of 15 μg / mL, and diluted 3-fold, for a total of 9 concentration points. (3) 150 μL of each concentration of drug dilution was added per well, and negative control wells were set up and incubated at 37° C. for 72±3 hours. (4) 70 μL of the supernatant was aspirated and discarded per well, and 70 μL of freshly prepared 15 μg / mL antibody A, antibody A-ADC1, antibody A-ADC2, or DS-8201 was added and incubated at 37° C. for 48±2 hours. (5) 15 μL of CCK-8 was added per well to a 96-well plate, the plate was gently tapped to shake evenly, and the plate was left at 37°C for approximately 2 hours. (6) The reference wavelength was 650 nm, and the absorbance value was read at 450 nm.

[0157] (b) NCI-N87 and BT474 cells after 3 days of treatment (1) Preparation of growth medium: RPMI-1640 + 10% FBS + 1% P / S. NCI-N87 cells or BT474 cells were digested with pancreatin and inoculated into a 96-well plate at 10,000 cells / well / 150 μL and incubated overnight at 37°C. (2) Preparation of experimental medium: RPMI-1640 + 0.1% FBS + 1% P / S. Antibody A, antibody A-ADC1, antibody A-ADC2 and DS-8201 were each prepared to a maximum working concentration of 30 μg / mL, and diluted 8 times, for a total of 5 concentration points. (3) 150 μL of each concentration of drug dilution was added per well, and negative control wells were set up and incubated at 37° C. for 72±3 hours. (4) 15 μL of CCK-8 was added per well to a 96-well plate, the plate was gently tapped to shake evenly, and the plate was left at 37°C for approximately 2 hours. (6) The reference wavelength was 650 nm, and the absorbance value was read at 450 nm.

[0158] Test results: (1) NCI-N87 tumor cell killing test The test results after treating NCI-N87 cells for 3 and 5 days are shown in Figures 15 and 16, respectively. The results of Figures 15-16 show that after treating NCI-N87 cells for 3 days respectively according to the above method steps, the inhibition rate of antibody A-ADC1 against NCI-N87 reaches more than 80%, followed by antibody A, and DS8201 (Herceptin-ADC1) is the lowest, only 60%. After treating NCI-N87 cells for 5 days, the inhibition rate of antibody A-ADC1 still remains the highest, slightly higher than its parent molecule and DS-8201 (Herceptin-ADC1). This shows that the killing effect of antibody A-ADC1 against NCI-N87 cells is significantly higher than that of DS-8201 (Herceptin-ADC1).

[0159] (2) Killing test of BT474 tumor cells The test results after treating BT474 cells for 3 days are shown in FIG. The results in FIG. 17 show that after treating BT474 cells for 3 days respectively according to the above method steps, the inhibition rate of antibody A-ADC1 against BT474 cells reaches 55%, antibody A-ADC2 reaches 36%, and DS-8201 is only 23%, and the inhibition rate of antibody A-ADC1 against BT474 cells is much better than antibodies A-ADC2 and DS-8201, and the killing power is 2.4 times that of DS-8201. Killing tests on NCI-N87 and BT474 tumor cells showed that both antibody A-ADC1 and antibody A-ADC2 had relatively high killing ability against HER2 highly expressing positive tumor cells (NCI-N87, BT474 cells), and that antibody A-ADC1 and antibody A-ADC2 had superior killing ability against HER2 highly expressing positive tumor cells (NCI-N87, BT474 cells) to DS-8201.

[0160] [Example 6] Bystander killing effect of ADC molecules containing antibody A against HER2-negative cells ADC drugs usually bind to antigens on cell membranes first, then the antibody-antigen complex enters cells by endocytosis and forms inclusions, which then undergo further maturation and fuse with lysosomes, where cytotoxic drugs are released (either by degrading the linker with a corresponding specific protease such as cathepsin B, or by degrading the entire ADC drug in the lysosome), and the generated cytotoxic drugs penetrate the lysosomal membrane and bind to DNA or microtubules, resulting in cell apoptosis. These drugs can also be pumped into the tumor microenvironment by protein transport on the cell membrane, enter adjacent tumor cells, and cause a killing effect on them, which can result in a "bystander effect."

[0161] Antibody A-ADC1 is a glycosite-specific conjugate drug of a dual epitope antibody targeting HER2, and uses a cleavable linker. To examine the bystander effect of antibody A-ADC1, SK-BR-3 (highly HER2 expressing) single cell line, MDA-MB-468 (non-HER2 expressing) single cell line, and a mixed cell line of SK-BR-3 and MDA-MB-468 (1:1) were treated with antibody A-ADC1 and DS-8201, respectively, and after 3 days of treatment, all sample well cells were counted, and the mixed cell lines were stained with APC anti-CD340 Antibody fluorescent antibody and detected by flow cytometry.

[0162] Experimental Method For SK-BR-3 and MDA-MB-468 cells in logarithmic growth phase in T75 culture flasks, the medium was discarded, washed twice with PBS, the liquid was aspirated completely, 2 mL of pancreatin was added to digest, and the cells were placed in a 37°C incubator for 4 minutes, the cell digestion liquid was transferred to a 15 mL centrifuge tube, 8 mL of growth medium was added to stop the digestion, and the cells were pipetted into a single cell suspension and centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 3 mL of medium (RPMI-1640 + 1% FBS) was added to resuspend the cells, and 20 μL of the cell suspension was aspirated and mixed with 20 μL of 0.2% trypan blue.

[0163] Counting: Single cell inoculation (mono-culture): Using medium (RPMI-1640 + 1% FBS), both cells were grown to a density of 1 × 10 5 The cells were inoculated into a 96-well plate at 100 μL / well, and 1 × 10 cells / mL were added per well. 4 The cells were cultured overnight in a 37°C, 5% CO2 incubator. Co-culture: both cells were grown at a cell density of 2 × 10 using medium (RPMI-1640 + 1% FBS). 5 The total number of cells per well was adjusted to 2 × 10 cells / mL, and 50 μL of SK-BR-3 cells and 50 μL of MDA-MB-468 cells were added per well. 4 The cells were cultured overnight in an incubator at 37°C and 5% CO2.

[0164] Antibody drug configuration: Dilute the test sample with RPMI-1640 medium containing 1% FBS, and dilute 5-fold from the highest working concentration of 50 nM to a total of 4 concentrations. At the same time, set up a control group, i.e., a drug-free group (Negative Control), add 100 μL of the diluted test sample per well, and directly add 100 μL of RPMI-1640 + 1% FBS medium to the control group (Negative Control), and culture at 37 ° C and 5% CO2 for 72 hours. Aspirate the supernatant of the 96-well plate, wash once with 200 μL of 1XPBS, then add 20 μL of pancreatin per well to digest, place in a 37 ° C incubator for 4 minutes, add 100 μL of medium (RPMI-1640 + 10% FBS) to stop digestion, and pipette the cells into a single cell suspension, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 40 μL of pancreatin per well. Add 1 μL of 1XPBS to resuspend the cells, aspirate 20 μL of the cell suspension and mix with 20 μL of 0.2% trypan blue to count, add 100 μL (APC anti-human CD340 Antibody diluted 1:100 times in 1XPBS) fluorescent antibody to each well of the mixed cell inoculation group after counting, mix evenly, incubate at 2-8 °C in the dark for 15 minutes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 200 μL of 1XPBS per well, wash once by centrifugation at 1000 rpm for 5 minutes, aspirate the supernatant completely, add 100 μL of 1XPBS to resuspend, and detect with a flow cytometer.

[0165] Figure 18 shows the results of the growth inhibition of each experimental group against SK-BR-3 cells, and Figure 19 shows the results of the growth inhibition of each experimental group against MDA-MB-468 cells. As can be seen from the results, (a) antibodies A-ADC1 and ENHERTU (i.e., DS-8201) have a bystander killing effect on SK-BR-3 cells in both SK-BR-3 single cell system and SK-BR-3 and MDA-MB-468 mixed system, and (b) antibodies A-ADC1 and ENHERTU have no obvious killing effect on HER2-negative MDA-MB-468 single cell system, but have obvious killing effect on MDA-MB-468 cells in the mixed system. When the sample concentrations were 50 nM, 10 nM, 2 nM, and 0.4 nM, the proliferation inhibition rates of antibody A-ADC1 against MDA-MB-468 cells in the mixed cell system were 68.65%, 46.87%, 31.61%, and -4.95%, respectively, and the proliferation inhibition rates of ENHERTU against MDA-MB-468 cells in the mixed cell system were 45.02%, 25.4%, -20.02%, and -22.08%, respectively. At a concentration of 50 nM, the bystander killing ability of antibody A-ADC1 against HER2-negative cells is approximately 1.5 times that of ENHERTU, at a concentration of 10 nM, the bystander killing ability of antibody A-ADC1 against HER2-negative cells is approximately 1.8 times that of ENHERTU, and at a concentration of 2 nM, antibody A-ADC1 still has a bystander killing ability of 31.61%, while ENHERTU has no bystander killing ability; therefore, antibody A-ADC1 has a higher bystander killing ability than ENHERTU at concentrations of 2 to 50 nM.

[0166] The table below shows the counting results of CountStar cells. TIFF2024525624000012.tif81170

[0167] The table below shows the ratios of HER2-positive and HER2-negative cells in the mixed cell groups. TIFF2024525624000013.tif73170

[0168] The table below shows the results of the proliferation inhibition rate of SK-BR-3 cells. TIFF2024525624000014.tif63170

[0169] The table below shows the results of the proliferation inhibition rate of MDA-MB-468 cells. TIFF2024525624000015.tif63170

[0170] Results and Conclusions At a concentration of 50 nM, the bystander killing ability of antibody A-ADC1 against HER2-negative cells is approximately 1.5 times that of ENHERTU (i.e., DS-8201); at a concentration of 10 nM, the bystander killing ability of antibody A-ADC1 against HER2-negative cells is approximately 1.8 times that of ENHERTU; at a concentration of 2 nM, antibody A-ADC1 still has a bystander killing ability of 31.61%, whereas ENHERTU has no bystander killing ability; therefore, antibody A-ADC1 has a higher bystander killing ability than ENHERTU at concentrations of 2 to 50 nM.

[0171] [Example 7] Analysis of ADCC activity of ADC molecules containing antibody A (a) Measurement of ADCC activity of antibody A-ADC1 using a reporter gene system The target cells NCI-N87 and the effector cells Jurkat-FcγRIIIa-V158-NFAT were co-cultured with the antibody A-ADC1 produced in Example 1 for 6 hours, and the ADCC activity was measured by detecting the fluorescence RUL value.

[0172] Specific experimental steps: (1) Preparation of reaction medium: 2% FBS+1640. (2) Using the reaction medium prepared in step (1), different concentrations of antibody A-ADC1, antibody A-ADC2 and antibody A were prepared, with the maximum concentration being 15,000 ng / mL (final concentration 10,000 ng / mL), and diluted 4-fold to nine concentrations. (3) Collect NCI-N87 cells and dilute them to a cell number of 1 × 10 using the reaction medium prepared in step (1). 6 The Jurkat-FcγRIIIa-V158-NFAT cells were harvested and the cell number was adjusted to 4 × 10 cells / mL using the reaction medium prepared in step (1). 6 Adjusted to cells / mL. (4) 25 μL of antibody A or ADC containing antibody A produced in step (2), 25 μL of N8 cells, and 25 μL of Jurkat-FcγRIIIa-V158-NFAT cells were added to a 96-well plate and placed in an incubator for 6 hours. (5) Add 10 μL of Bio-Glo to each well. TM Luciferase substrate was added, and the RLU value of each well was immediately detected using a fluorescence microplate reader.

[0173] The results are shown in Figure 20, with 1 to 3 showing the results for antibody A, antibody A-ADC1, and antibody A-ADC2, respectively. The antibody A-ADC1 molecule produced in Example 1 still retained good ADCC activity even in HER2-positive / highly expressing cells (NCI-N87 cells), with its ADCC activity being 87.7% (antibody A EC50 / ADC1 EC50) of that of the parent molecule antibody A, while the antibody A-ADC2 produced in Example 2 almost lost its ADCC activity, with its activity being only 37.2% (antibody A EC50 / ADC2 EC50) of that of the parent molecule antibody A. It was shown that the conjugation method has a large effect on the ADCC activity of ADC.

[0174] (b) The ADCC activity of ADCs containing antibody A was measured using a PBMC system. Specific test steps (BT474 cells): (1) PBMCs activated with IL-2 for 24 hours (activation medium: 10% FBS + 1640 + 300 IU / mL) were collected at 300 IU / mL, and the cell number was increased to 4.5 × 10 6 Adjusted to cells / mL. (2) Antibody A-ADC1, antibody A-ADC2, DS8201 and human IgG1 were prepared at different concentrations in activation medium, with the maximum concentration being 3 μg / mL (final concentration 1 μg / mL) and diluted 10-fold to three concentrations. 3. Harvest BT474 cells and increase the cell number to 3 x 10 in activation medium. 5 Adjusted to cells / mL. 4. 50 μL of the ADC containing antibody A prepared in step (2), 50 μL of BT474, and 50 μL of PBMC were added to a 96-well plate and placed in an incubator for 4 hours. 5. 30 min before detection, 10 μL of cell lysate was added to TMR and VCC wells. 6. 1500 rpm / 5 min, 50 μL of the supernatant was transferred to a new 96-well plate, 50 μL of LDH detection substrate was added per well, and the plate was left at room temperature for 30 min. 7, OD values ​​were detected at 490 nm.

[0175] Specific test steps (NCI-N87 cells): (1) PBMCs activated with IL-2 for 24 hours (activation medium: 10% FBS + 1640 + 300 IU / mL) were collected at 300 IU / mL, and the cell number was increased to 4.5 × 10 6 Adjusted to cells / mL. (2) Antibody A-ADC1, antibody A-ADC2, DS8201 and human IgG1 were prepared at different concentrations in activation medium, with the maximum concentration being 3000 ng / mL (final concentration 1000 ng / mL) and diluted 6-fold to five concentrations. 3. Collect NCI-N87 cells and dilute them to 3 × 10 in activation medium. 5 Adjusted to cells / mL. 4. 50 μL of the ADC containing antibody A prepared in step (2), 50 μL of BT474, and 50 μL of PBMC were added to a 96-well plate and placed in an incubator for 4 hours. 5. 30 min before detection, 10 μL of cell lysate was added to TMR and VCC wells. 6. 1500 rpm / 5 min, 50 μL of the supernatant was transferred to a new 96-well plate, 50 μL of LDH detection substrate was added per well, and the plate was left at room temperature for 30 min. 7, OD values ​​were detected at 490 nm.

[0176] According to the above method steps, the ADCC activities of antibody A-ADC1, antibody A-ADC2, DS8201, antibody A, etc. in HER2 highly expressing cell lines were measured, and HER2 highly expressing cells BT747 and NCI-N87 were selected as target cells, and effector cells were PBMCs (PBMC-qc and PBMC-z) in peripheral blood of two donors. The detection flow is shown in Figure 21, and the detection results are shown in Figures 22 to 23. The results showed that antibody A-ADC1 had higher activity than both antibodies A-ADC2 and DS-8201 in the PBMC-qc:BT474 (effective target ratio 15:1) and PBMC-z:NCI-N87 (effective target ratio 15:1) cell lines, and that antibody A-ADC1 exhibited higher ADCC activity than DS-8201 in the HER2-high expressing cell lines, demonstrating that the ADCs described in this application are able to retain the ADCC activity of the conjugated parent molecule, antibody A.

[0177] [Example 8] Detection of endocytosis efficiency of ADC molecules containing antibody A in tumor cells Specific experimental steps (NCI-N87 cells, purchased from Shanghai Chinese Academy of Sciences): (1) Antibody A-ADC1, Antibody A-ADC2, DS8201, and Antibody A (to be mixed with the endocytosis reagent) were prepared at various concentrations using 1640 medium containing 10% FBS, and 50 μL of antibody A-ADC1, antibody A-ADC2, DS8201, or antibody A at final concentrations of 50 nM, 10 nM, or 2 nM, respectively, were added to a 96-well plate. (2) NCI-N87 cells were digested, counted, and added to each drug-containing well at 4 × 10 4 Cells were added at 50 μL / well and placed in the incubator for 2, 5, and 23 hours. (3) The supernatant of the cells in the 96-well plate was discarded, and 50 μL of pancreatin was added per well and digested for 5 minutes. (4) 200 μL of 1640 medium containing 10% FBS was added to each well, the cells were resuspended, and pipetted into a single-cell suspension. (5) The mixture was transferred to a 1.5 mL EP tube and subjected to FACS detection.

[0178] Specific experimental steps (BT474 cells): (1) Antibody A-ADC1, Antibody A-ADC2, DS8201, and Antibody A (to be mixed with the endocytosis reagent) were prepared at various concentrations using 1640 medium containing 10% FBS, and 50 μL of antibody A-ADC1, antibody A-ADC2, DS8201, or antibody A at final concentrations of 40 nM, 8 nM, or 1.6 nM, respectively, were added to a 96-well plate. (2) BT474 cells were digested, counted, and added to each drug-containing well at 4 × 10 4 Cells were added at 50 μL / well and placed in an incubator for 24 hours. (3) The supernatant of the cells in the 96-well plate was discarded, and 50 μL of pancreatin was added per well and digested for 5 minutes. (4) 200 μL of 1640 medium containing 10% FBS was added to each well, the cells were resuspended, and pipetted into a single-cell suspension. (5) The mixture was transferred to a 1.5 mL EP tube and subjected to FACS detection.

[0179] According to the above method steps, the antibody-endocytosis reagent mixtures such as antibody A-ADC1, antibody A-ADC2, DS-8201, and antibody A were incubated in tumor cell lines, respectively, and then the endocytosis efficiency of cells for each molecule was detected by flow cytometer.

[0180] (1) Endocytosis in NCI-N87 tumor cells After incubating each molecule of Antibody A-ADC1, Antibody A-ADC2, DS-8201, and Antibody A in the NCI-N87 cell line for 2, 5, and 23 hours, respectively, the endocytosis of each molecule by NCI-N87 cells was detected by flow cytometer. The results of incubation for 2, 5, and 23 hours are shown in Figures 24, 25, and 26, respectively.

[0181] (a) When the incubation reached 2 hours, the endocytosis efficiency of cells for each molecule was antibody A> antibody A-ADC1> antibody A-ADC2> DS-8201, and when incubated for 5 hours, the endocytosis efficiency was antibody A=antibody A-ADC1>> antibody A-ADC2>> DS-8201, in which case antibody A-ADC1 was equivalent to the endocytosis efficiency of the parent molecule antibody A, and was much higher than DS-8201 and antibody A-ADC2. It was shown that antibody A-ADC1 retained the tumor cell endocytosis effect equivalent to the parent molecule over a short incubation period (5 hours) and was much superior to DS-8201, indicating that the ADC described in this application can rapidly bind to HER2 on the tumor cell surface to achieve endocytosis, block the HER2 signaling pathway, and exert tumor killing effects.

[0182] (b) When the incubation time reached 23 hours, the endocytosis efficiency of Antibody A-ADC1, Antibody A-ADC2, Antibody A, etc. was comparable, and all reached 55% or more at 50 nM, which was superior to DS-8201; after 23 hours of administration of the ADCs described herein, 55% of the drug (50 nM) was delivered into NCI-N87 tumor target cells, indicating that the tumor targeting was slightly higher than that of DS-8201.

[0183] (2) Endocytosis in BT474 tumor cells Antibody A-ADC1, antibody A-ADC2, and DS-8201 were incubated in BT474 cell line for 24 hours and then detected by flow cytometer. The results of 24-hour incubation are shown in FIG.

[0184] The detection results showed that antibody A-ADC1 had an endocytosis efficiency equivalent to that of antibody A-ADC2, and reached 70% or more at 40 nM, and the endocytosis efficiency of the ADC containing antibody A described in the present application was 2.33 times that of DS-8201 (30%) under comparable conditions, while the former's targeting ability to BT474 cells was much higher than that of DS-8201. It was shown that antibody A-ADC1 has a higher inhibitory ability on target-mediated signal transduction pathways, a much higher tumor-killing ability than DS-8201, and a better tumor-inhibiting ability.

[0185] Example 9: Inhibition of tumor growth by ADC molecules containing antibody A in animal models Group administration test protocol: BxPC-3 cells were subcutaneously injected into the armpits of nude mice to establish the model, with an inoculation concentration of 1 × 10 6 The average tumor size was approximately 190 mm 3 When the mice reached 100 mm, they were randomly assigned to groups according to tumor size and body weight, with 4 mice in each group, all female. The day of random assignment was recorded as day 0. On day 0, the test drug was administered intraperitoneally, and then the test drug was administered intraperitoneally twice, once every 3 days. Unselected animals were euthanized with CO2.

[0186] The test animals were divided into three groups, A, B, and C. Group A was administered PBS, group B was administered antibody A-ADC1 (DAR=4), and group C was administered DS-8201 (DAR=8). The specific group administration protocol is shown in Table 4. [Table 4]

[0187] According to the above test protocol, three test compounds were administered to mice in three groups of test animals, A, B, and C, for 24 days (two animals in group A were euthanized after 20 days of administration of the compounds due to excessive tumor growth in PBS). The tumor inhibition curves of the three compounds against the BxPC-3 tumor model were obtained. As can be seen from the results of the tumor inhibition curves, administration of antibodies A-ADC1 (DAR=4) and DS-8201 (DAR=8) to BxPC-3 tumor model animals significantly inhibited tumor growth, and the tumor inhibition effect was obvious over time, with antibody A-ADC1 being similar to that of DS-8201.

[0188] Example 10: Pharmacokinetic study of ADC molecules containing antibody A in Balb / c-Nu nude mouse NCI-N87 tumor model Balb / c-Nu nude mice were subcutaneously inoculated with human gastric cancer cells NCI-N87. NCI-N87 tumor model was adopted. 1 mg / kg antibody A-ADC1 and 1 mg / kg ENHERTU (i.e. DS-8201) were administered intravenously (iv) in a single dose. Blood samples were taken at different time points, and the total antibody and ADC content in the animal serum samples was detected by ELISA (Method 1: ELISA was used to detect the total antibody content, including ADC and naked antibody; Method 2: ELISA was used to detect the ADC content (including ADC containing 1-4 drugs) without naked antibody after toxin release). Pharmacokinetic parameters were calculated using DAS (3.2.8) software non-compartment model.

[0189] [Table 5] After a single intravenous injection of antibody A-ADC1 into NCI-N87 tumor model mice, the C max were 45422.727 μg / L and 70056.81675 μg / L, respectively, and AUC (0-t) are 1598292.175 μg / L*h and 2098916.987 μg / L*h, respectively, and T max are both 0.033 hours and 0.033 hours, and T1 / 2 are 100.976 hours and 95.32 hours, respectively.

[0190] After a single intravenous injection of ENHERTU (i.e., DS-8201) into NCI-N87 tumor model mice, the C max were 12343.966 μg / L and 19604.51225 μg / L, respectively, and the AUC (0-T) were 421103.345 μg / L*h and 586509.313 μg / L*h, respectively, and T max are both 0.033 hours and 0.033 hours, and T 1 / 2 are 86.744 hours and 65.384 hours, respectively.

[0191] In summary, antibodies A-ADC1 and ENHERTU were administered intravenously once to NCI-N87 tumor model mice, and the results of Elisa method 1 (total antibody concentration) showed that T 1 / 2 were 100.976 hours and 86.744 hours, respectively, and antibody A-ADC1 T 1 / 2 was 1.164-fold higher than ENHERTU, Cmax was 3.680-fold higher than ENHERTU, and AUC (0-t) The results of Method 2 (antibody drug conjugate content) showed that antibody A-ADC1 and ENHERTU T 1 / 2 were 95.32 and 65.384 hours, respectively, and antibody A-ADC1 T 1 / 2 was 1.460-fold higher than ENHERTU, Cmax was 3.574-fold higher than ENHERTU, and AUC (0-T) was 3.580 times that of ENHERTU. The results can be seen in FIG.

[0192] [Example 11] Effect of ADC containing antibody A on NCI-N87 cell surface HER2 endocytosis Monoclonal antibodies targeting Her2 could promote the internalization and degradation of Her2, further inhibit downstream signaling of Her2, and inhibit cell proliferation. The ADC (Antibody A-ADC1) of the present application is an ADC drug in which a small molecule DXd is conjugated to a dual epitope antibody targeting HER2, such as Antibody A, and the endocytosis effects of Antibody A-ADC1, Antibody A, and the commercially available syngeneic targeting drug DS8201 were compared by selecting human gastric cancer cell line NCI-N87 cells overexpressing Her2.

[0193] Experimental steps: 1) Experimental buffer arrangement: 1640 + 10% FBS medium. 2) N87 cells were centrifuged at 350 g for 5 minutes, the supernatant was removed, and the concentration was adjusted to 1*106 cells / mL with experimental buffer. 3) The antibodies required for the experiment were diluted with the experimental buffer to an initial concentration of 200 nM, and pHrodo was diluted with the experimental buffer to a concentration of 600 nM. 4) 200 nM diluted antibody and 600 nM pHrodo were mixed in a 1:1 volume ratio and incubated for 15 minutes, after which the antibody and pHrodo mixture was diluted 5-fold with experimental buffer for a total of three concentration points, and 50 μL of cells were added after 5 minutes of incubation. 5) 50 μL of the antibody and pHrodo mixture and 50 μL of cells were added to the sample wells.

[0194] 6) NC well: 25 μL Buffer + 25 μL PHrodo + 50 μL cells (1E5). 7) BLANK well: 50 μL of Buffer + 50 μL of cell (1E5), the volume of each well was 100 μL, and the mixture was sprayed to mix evenly. 8) Incubate in a 37°C cell incubator for 5 hours. 9) After the incubation was completed, the cells were centrifuged at 350 x g for 5 minutes, the supernatant was removed, trypsin was added to each well to digest the cells, and the medium was resuspended into a single cell suspension, centrifuged at 350 x g for 5 minutes, and 1xPBS was added at 200 μL / well to resuspend the cells. 10) After centrifugation at 350 x g for 5 minutes, 150 μL / well of 1xPBS was added to resuspend the cells, and the cells were transferred to a new 96-well plate in the flow cytometer.

[0195] Test Results: Antibody A, antibody A-ADC1 and DS8201 can all induce HER2 endocytosis on the surface of NCI-N87 cells, and antibody A-ADC1 has the same endocytosis effect as antibody A. The endocytosis effect of antibody A-ADC1 is obviously higher than that of DS8201 at all three concentration points, and the endocytosis rates of antibody A were 92.25%, 91.82% and 65.71% at 50 nM, 10 nM and 2 nM, respectively, the endocytosis rates of antibody A-ADC1 were 85.48%, 84.65% and 58.95%, respectively, and the endocytosis rates of DS8201 were 34.79%, 28.36% and 21.26%, respectively. The endocytosis effect of antibody A-ADC1 on NCI-N87 was equivalent to that of antibody A and obviously higher than that of DS8201. [Table 6]

[0196] Table 6 shows the percentage of positive NCI-N87 cells after endocytosis of Antibody A, Antibody A-ADC1 and DS8201. FIG. 29 shows the ratio of NCI-N87 positive cells after induction of endocytosis by Antibody A, Antibody A-ADC1, and DS8201.

[0197] [Example 12] Inhibitory effect of ADC molecules containing antibody A on PDX subcutaneous xenograft NOD / SCID mouse model of HER2-weakly positive gastric cancer. The antitumor activity of the present ADC (Antibody A-ADC1) was evaluated in comparison with the commercially available positive control drug ENHERTU (i.e., DS-8201) in a PDX subcutaneous xenograft NOD / SCID mouse animal model of human HER2 weakly positive (2+) gastric cancer.

[0198] Model information The table below shows sample information for human gastric cancer PDX xenograft models. TIFF2024525624000019.tif21170

[0199] Experimental procedure Thirty 4- to 5-week-old NOD / SCID female mice were selected from each PDX model and subcutaneously inoculated with gastric cancer PDX tumor masses measuring 2 to 3 mm in diameter into the right anterior scapula, with the average tumor volume reaching 213.40 mm. 3 If the tumor size was 1, the mice were randomly assigned to groups according to tumor size and mouse weight (see the table below), the day of group assignment was counted as day 1, administration began immediately after group assignment, and the day of administration was counted as day 1 after administration.

[0200] The following table shows the experimental design of the antitumor effect of the test drugs in the PDX tumor model of human gastric cancer. TIFF2024525624000020.tif72170

[0201] Test Results: All animals in the control and test drug groups were alive 27 days after the first administration (at this time, the maximum long diameter of the tumor in the control group reached 1.5 cm, the ethical upper limit for mouse experiments), so the tumor volume data at this time was selected to analyze and evaluate the antitumor effect of the test drug, the ADC of the present application (antibody A-ADC1). The PBS control group (CTL) mice had an average tumor volume of 725.39 ± 190.25 mm on the 27th day after being divided into groups and first administration. 3 The relative tumor volume was 3.58 ± 1.11, and the test drug, antibody A-ADC1 (10 mg / kg), had a mean tumor volume of 112.89 ± 61.65 mm on day 27 after the first administration in the groups.3 The relative tumor volume was 0.50±0.16, and the relative tumor inhibition rate (TGI) (%) was 86.05%, which was statistically significant compared to the PBS control group (p<0.0001). The positive control drug ENHERTU (i.e., DS-8201) (10 mg / kg) had a mean tumor volume of 106.41±17.77 mm on the 27th day after the first administration in the groups. 3 The relative tumor volume was 0.50±0.11, and the relative tumor inhibition rate (TGI) (%) was 86.08%, which was statistically significant compared to the PBS control group (p<0.0001). In addition, at the dose level of 10 mg / kg, on the 27th day after the first dose in the group, the mean tumor volume in the test drug antibody A-ADC1 treatment group was similar to that of the ENHERTU treatment group, which was not statistically significant (Antibody A-ADC1 VS ENHERTU, P=0.8362>0.05, ns.). Overall, antibody A-ADC1 had a significant tumor inhibitory effect on human HER2 IHC 2+ gastric cancer PDX tumor models at a dose of 10 mg / kg, and antibody A-ADC1 with a DAR of 4 was equivalent to the antitumor efficacy of the positive control drug ENHERTU with a DAR of 8. FIG. 30 shows the growth curve of mouse tumor volume in each group in a PDX tumor model of human gastric cancer.

[0202] The table below shows the efficacy analysis table for each group in the Case168 human gastric cancer PDX tumor model. TIFF2024525624000021.tif135170

[0203] [Example 13] Inhibitory effect of ADC molecules containing antibody A on PDX subcutaneous xenograft NOD / SCID mouse model of HER2-negative gastric cancer. The antitumor activity of the present ADC (Antibody A-ADC1) was evaluated in a HER2-negative human gastric cancer subcutaneous PDX tumor xenograft NOD / SCID mouse animal model, in comparison with the commercially available positive control drug ENHERTU (i.e., DS-8201).

[0204] Testing Procedure: Model information The table below shows sample information for human gastric cancer HER2 IHC2+ PDX xenograft models. TIFF2024525624000022.tif22170

[0205] Experimental procedure Thirty 4- to 5-week-old NOD / SCID female mice were selected from each PDX model, and gastric cancer PDX tumor masses with a diameter of 2 to 3 mm were subcutaneously inoculated into the right anterior scapula of the mice. The average tumor volume was 166.53 mm. 3 In the case of , the mice were randomly divided into groups according to the size of the tumor and the body weight of the mice, and the day of grouping was counted as day 1. Administration was started immediately after grouping, and the day of administration was counted as day 1 after administration.

[0206] The following table shows the experimental design of the antitumor effect of the test drugs in the PDX tumor model of human gastric cancer. TIFF2024525624000023.tif55170

[0207] Test Results: All animals in the control and test drug groups were alive on the 17th day after the first administration (at this time, the maximum long diameter of the tumor in the control group reached the ethical upper limit for mouse experiments of 1.5 cm), so the tumor volume data at this time was selected to evaluate the antitumor effect of the test drug, the ADC of the present application (antibody A-ADC1). The PBS control group mice had an average tumor volume of 629.24 ± 146.59 mm on the 17th day after being grouped and first administered the drug. 3 The relative tumor volume was 4.13 ± 0.97, and the test drug, antibody A-ADC1 (10 mg / kg), had a mean tumor volume of 238.32 ± 84.57 mm on day 17 after the first administration in the groups. 3 The relative tumor volume was 1.68±1.01, and the relative tumor inhibition rate (TGI) (%) was 59.41%, which was statistically significant compared to the PBS control group (p<0.0001). The positive control drug ENHERTU (i.e., DS-8201) (10 mg / kg) had a mean tumor volume of 174.06±71.09 mm on the 17th day after the first administration in the groups. 3The relative tumor volume was 1.02±0.34, and the relative tumor inhibition rate (TGI) (%) was 75.19%, which was statistically significant compared to the PBS control group (p<0.0001). In addition, at the dose level of 10 mg / kg, on the 17th day after the first administration in the group, the mean tumor volume in the test drug antibody A-ADC1 treatment group was slightly larger than that in the positive control drug ENHERTU treatment group, but the difference was not statistically significant (Antibody A-ADC1 VS ENHERTU, P=0.526>0.05, ns.). Overall, antibody A-ADC1 had a significant tumor inhibitory effect on human HER2-gastric cancer PDX tumor models at a dose of 10 mg / kg, and the antitumor efficacy of antibody A-ADC1 with a DAR of 4 was equivalent to that of the positive control drug ENHERTU with a DAR of 8. FIG. 31 shows the growth curve of mouse tumor volume in human gastric cancer HER2 IHC-PDX tumor model (single drug treatment on day 1 only, tail vein injection, 10 mg / kg).

[0208] The table below shows the efficacy analysis table for each group in the Case 111 human gastric cancer PDX tumor model. TIFF2024525624000024.tif137170

[0209] The above detailed description is provided for purposes of explanation and illustration, and is not intended to limit the scope of the appended claims. Various modifications to the present exemplary embodiments will be apparent to those skilled in the art and are intended to remain within the scope of the appended claims and their equivalents.

Claims

1. An antibody-drug conjugate comprising a bispecific antibody targeting HER2 or an antigen-binding fragment thereof.

2. The antibody-drug conjugate according to claim 1, wherein the bispecific antibody targeting HER2 or an antigen-binding fragment thereof specifically binds to at least one epitope of human HER2.

3. The antibody-drug conjugate according to claim 1, wherein the bispecific antibody targeting HER2 or an antigen-binding fragment thereof specifically binds to extracellular domain II of human HER2 and / or extracellular domain IV of human HER2.

4. The antibody-drug conjugate according to claim 1, wherein the bispecific antibody targeting HER2 or an antigen-binding fragment thereof comprises a first light chain and a second light chain.

5. The antibody-drug conjugate according to claim 4, wherein the first light chain comprises first LCDR1-3, and the first LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

4.

6. The antibody-drug conjugate according to claim 5, wherein the first LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

5.

7. The antibody-drug conjugate according to claim 5, wherein the first LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

8. The antibody-drug conjugate according to claim 4, wherein the first light chain can bind to the heavy chain of pertuzumab.

9. The antibody-drug conjugate according to claim 4, wherein the second light chain comprises second LCDR1-3, and the second LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

1.

10. The antibody-drug conjugate according to claim 9, wherein the second LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

11. The second LCDR3 is the antibody-drug conjugate according to claim 9, comprising the amino acid sequence shown in SEQ ID NO:

3.

12. The second light chain is the antibody-drug conjugate according to claim 4, which can bind to the heavy chain of trastuzumab.

13. The variable regions of the first light chain and the second light chain are the antibody-drug conjugate according to claim 4, comprising the amino acid sequence shown in any one of SEQ ID NOs: 7 to 12.

14. The variable regions of the first light chain and the second light chain are the antibody-drug conjugate according to claim 4, comprising the amino acid sequence shown in SEQ ID NO:

7.

15. The first light chain and the second light chain are the antibody-drug conjugate according to claim 4, comprising the amino acid sequence described in any one of SEQ ID NOs: 13 to 18.

16. The first light chain is selected from the group consisting of the light chain of pertuzumab or a variant thereof, and the light chain of trastuzumab or a variant thereof, and / or the second light chain is selected from the group consisting of the light chain of pertuzumab or a variant thereof, and the light chain of trastuzumab or a variant thereof, which is the antibody-drug conjugate according to claim 4.

17. The first light chain and the second light chain are the antibody-drug conjugate according to claim 4, having the same amino acid sequence.

18. The first light chain and the second light chain are the antibody-drug conjugate according to claim 4, comprising the amino acid sequence described in SEQ ID NO:

13.

19. The bispecific antibody targeting HER2 or an antigen-binding fragment thereof comprises a first heavy chain and a second heavy chain, and the first heavy chain can accurately bind to the first light chain under physiological conditions or in the protein expression state in vitro, which is the antibody-drug conjugate according to claim 1.

20. The antibody-drug conjugate according to claim 19, wherein the second heavy chain can accurately bind to the second light chain under physiological conditions or under the protein expression state in vitro.

21. The antibody-drug conjugate according to claim 19, wherein the first heavy chain comprises a first heavy chain variable region which is the heavy chain variable region of pertuzumab.

22. The antibody-drug conjugate according to claim 19, wherein the second heavy chain comprises a second heavy chain variable region which is the heavy chain variable region of trastuzumab.

23. The antibody-drug conjugate according to claim 19, wherein the first heavy chain and the second heavy chain comprise heavy chain constant regions derived from the constant region of human IgG.

24. The antibody-drug conjugate according to claim 19, wherein the Fc fragments of the first heavy chain and the second heavy chain comprise the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 57.

25. The antibody-drug conjugate according to claim 19, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 23.

26. The antibody-drug conjugate according to claim 19, wherein the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22 or 24.

27. M-(L1) a -(L2) b -comprises the structure shown in formula (1), wherein M represents the HER2-targeting bispecific antibody according to claim 1 or an antigen-binding fragment thereof, L1 represents a linker that links to M, and L2 represents a linker that links to D, a and b are independently selected from 0 to 10 respectively, D represents a drug, The antibody-drug conjugate according to claim 1.

28. The antibody-drug conjugate according to claim 27, wherein the L1 and / or L2 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, a non-charged linker, and a dicarboxylic acid-based linker.

29. The antibody-drug conjugate according to claim 27, wherein the L1 and the M are linked via a thiol group, an azide group, or an amide group on the M.

30. The antibody-drug conjugate according to claim 27, wherein the M comprises a first heavy chain and a second heavy chain, and the first heavy chain and / or the second heavy chain comprises a linking site connectable to the L1.

31. The antibody-drug conjugate according to claim 30, wherein the linking site comprises a group connectable to the L1 after deglycosylation modification.

32. The antibody-drug conjugate according to claim 31, wherein the group is located at the side chain of the amino acid Q at position 297 of the first heavy chain and / or at the side chain of the amino acid Q at position 298 of the second heavy chain.

33. The antibody-drug conjugate according to claim 30, wherein the linking site comprises a group connectable to the L1 after glycosylation modification.

34. The antibody-drug conjugate according to claim 33, wherein the group is located at the side chain of the amino acid N at position 299 of the first heavy chain and / or at the side chain of the amino acid N at position 300 of the second heavy chain.

35. The group is -N 3 and is included in the antibody-drug conjugate according to claim 33.

36. The antibody-drug conjugate according to claim 33, wherein the glycosylation modification includes the M contacting UDP-GalNAz, β-1,4-galactosyltransferase, or a variant thereof.

37. The L1 is the antibody-drug conjugate according to claim 27 that can participate in the SPAAC reaction.

38. The L1 is the antibody-drug conjugate according to claim 27 selected from the group consisting of maleimide, succinimid-3-yl-N, and DBCO.

39. The L1 is DBCO-(PEG) n1 wherein n1 is an integer from 0 to 10, or the L1 is maleimide, the antibody-drug conjugate according to claim 27.

40. The L2 is a polypeptide, VC-PAB, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-4-(maleimidomethyl)cyclohexyl succinimidyl carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidinyl-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecan-1-oic acid ester (CX1-1), the antibody-drug conjugate according to claim 27 selected from the group consisting of.

41. The L2 is GGFG, the antibody-drug conjugate according to claim 27.

42. The drug has the ability to kill tumor cells and / or inhibit the growth of tumor cells, the antibody-drug conjugate according to claim 27.

43. The antibody-drug conjugate according to claim 27, wherein the drug comprises a small molecule drug.

44. The drug is selected from the group consisting of a V-ATPase inhibitor, a Bcl2 inhibitor, an MCL1 inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), a nuclear export inhibitor of protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of the phosphate transfer reaction in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA breaker, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a DHFR inhibitor, a nucleoside analog, an HDAC inhibitor, an anthracycline, a NAMPT inhibitor, SN-38 glucuronic acid, etoposide phosphate, nitrogen mustard, a proteasome inhibitor, a cytokine, and a Toll-like receptor agonist. The antibody-drug conjugate according to claim 27.

45. The drug is selected from the group consisting of DM1, exatecan, DXd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, the microtubule inhibitor SHR153024, PNU-159682, Duo5 toxin, an SN38 derivative, or a derivative thereof. The antibody-drug conjugate according to claim 27.

46. 【Chemical Formula 1】 The antibody-drug conjugate according to claim 27, having a structure selected from the group consisting of.

47. The antibody-drug conjugate according to claim 1, having a drug / antibody ratio of about 1 to 6.

48. A compound for producing the antibody-drug conjugate according to claim 1, M-(L1) a having the structure shown in (Formula 2), wherein M represents the HER2-targeting bispecific antibody according to claim 1 or an antigen-binding fragment thereof. L1 represents a linker that connects to M, a is selected from 0 to 10, compound. **Claim 49** The compound according to claim 48, wherein the L1 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker. **Claim 50** The compound according to claim 48, wherein the L1 and the M are linked via a thiol group, an azide group, or an amide group on the M. **Claim 51** The compound according to claim 48, wherein the M includes a first heavy chain and a second heavy chain, and the first heavy chain and / or the second heavy chain includes a linking site connectable to the L1. **Claim 52** The compound according to claim 51, wherein the linking site includes a group connectable to the L1 after deglycosylation modification. **Claim 53** The compound according to claim 51, wherein the group is located at the side chain of amino acid Q at position 297 of the first heavy chain and / or at the side chain of amino acid Q at position 298 of the second heavy chain. **Claim 54** The compound according to claim 51, wherein the linking site includes a group connectable to the L1 after glycosylation modification. **Claim 55** The composition according to claim 54, wherein the group is located at the side chain of amino acid N at position 299 of the first heavy chain and / or at the side chain of amino acid N at position 300 of the second heavy chain. **Claim 56** The group is -N 3 including the compound according to claim 54. **Claim 57** The compound according to claim 53, wherein the glycosylation modification includes the M contacting UDP-GalNAz, β-1,4-galactosyltransferase, or a variant thereof. **Claim 58** L1 is the compound according to claim 48 that can participate in the SPAAC reaction.

59. L1 is the compound according to claim 48 selected from the group consisting of maleimide, succinimid-3-yl-N, and DBCO.

60. L1 is DBCO-(PEG) n1 where n1 is an integer from 0 to 10, or L1 is maleimide, the compound according to claim 48.

61. 【Chemical formula 2】 The compound according to claim 48 comprising a structure selected from the group consisting of.

62. A method for producing an antibody-drug conjugate according to claim 1, comprising the step of contacting the compound according to claim 48 with the drug according to claim 1.

63. A pharmaceutical composition comprising the antibody-drug conjugate according to claim 1 and optionally a pharmaceutically acceptable carrier.

64. A method for regulating the tumor microenvironment in a subject, comprising the step of administering to the subject the antibody-drug conjugate according to claim 1 or the pharmaceutical composition according to claim 63.

65. A method for regulating the immune response of a subject, comprising the step of administering to the subject the antibody-drug conjugate according to claim 1 or the pharmaceutical composition according to claim 63.

66. Use of the antibody-drug conjugate according to claim 1 or the pharmaceutical composition according to claim 63 in the manufacture of a drug, wherein the drug can prevent and / or treat tumors.

67. The use according to claim 66, wherein the tumor comprises solid tumors and / or non-solid tumors.

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