Antibody-drug conjugates and uses thereof

Abstract

An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that targets CD73, wherein the antibody or antigen-binding fragment thereof that targets CD73 comprises an HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. Further provided are methods for producing the antibody-drug conjugate and uses thereof.

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to antibody drug conjugates and uses thereof. [Background technology]

[0002] Cluster of differentiation 73 (CD73) is a surface glycoprotein anchored to the outside of the cell membrane by glycosylphosphatidylinositol (GPI). CD73 has been reported to be expressed in many different cancers. CD73 expression in cancer is also associated with increased proliferation, migration, neovascularization, invasiveness, metastasis, and shortened patient survival. In addition to being effective in immunosuppression, CD73 has aberrantly activated expression in tumor tissues, and antibodies targeting CD73 can be rapidly and massively internalized compared to other targets, proving to be an important target for antibody-drug conjugates (ADCs).

[0003] An antibody-drug conjugate generally comprises three parts: an antigen-binding moiety, a small molecule drug, and a linker that couples the antigen-binding moiety to the drug. At present, there is a strong need to obtain a highly specific and safe antibody-drug conjugate against human CD73. Summary of the Invention

[0004] In one aspect, the invention provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that targets CD73, wherein said antibody or antigen-binding fragment thereof that targets CD73 comprises an HCDR3, and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.

[0005] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an LCDR3, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an LCDR2, wherein the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises LCDR1, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises H-FR1, wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:19.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and wherein the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:21.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:22.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 18.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 23.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and wherein the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and wherein the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an L-FR4, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 26.

[0018] In some embodiments, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises one VH and VL selected from the group consisting of: wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:29, and the VL comprises the amino acid sequence set forth in SEQ ID NO:30; the VH comprises the amino acid sequence set forth in SEQ ID NO:31, and the VL comprises the amino acid sequence set forth in SEQ ID NO:32; the VH comprises the amino acid sequence set forth in SEQ ID NO:33, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34; the VH comprises the amino acid sequence set forth in SEQ ID NO:35, and the VL comprises the amino acid sequence set forth in SEQ ID NO:36; the VH comprises the amino acid sequence set forth in SEQ ID NO:33, and the VL comprises the amino acid sequence set forth in SEQ ID NO:36; and the VH comprises the amino acid sequence set forth in SEQ ID NO:35, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody heavy chain constant region.

[0023] In some embodiments, the antibody heavy chain constant region is derived from a human antibody heavy chain constant region.

[0024] In some embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0025] In some embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody light chain constant region.

[0027] In some embodiments, the antibody light chain constant region is derived from a human Igκ constant region.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the antigen-binding fragment comprises a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, and / or a dAb.

[0030] In some embodiments, the antibody is one or more selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0031] In some embodiments, the CD73 comprises human CD73 and / or monkey CD73.

[0032] In some embodiments, the antibody drug conjugate is capable of blocking or reducing activation of CD73.

[0033] In some embodiments, the antibody drug conjugate is capable of inhibiting the enzymatic activity of CD73.

[0034] In some embodiments, the antibody drug conjugate is capable of inducing endocytosis of cell surface CD73.

[0035] In some embodiments, the antibody-drug conjugate comprises the structure shown in Formula 1, M-(L1)a-(L2)bD (Formula 1), where M represents an antibody or antigen-binding fragment thereof targeting CD73 described herein, L1 represents a linker linked to M, L2 represents a linker linked to D, each b is independently selected from 0 to 10, and D represents a drug.

[0036] 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.

[0037] In some embodiments, L1 and M are linked to M via a thiol group, an azide group, or an amide group on M.

[0038] In some embodiments, L1 is capable of reacting with a thiol group to form a linkage.

[0039] In some embodiments, L1 is

[0040] [ka]

[0041] is selected from the group consisting of:

[0042] In some embodiments, L2 is a polypeptide, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)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. It is selected from the group consisting of NHS, N-4-(maleimidomethyl)cyclohexyl succinimidyl carboxylate (SMCC), N-sulfo(4-maleimidomethyl)cyclohexylcarboxylic acid sulfosuccinate ester (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).

[0043] In some embodiments, L2 is

[0044] [ka]

[0045] It is.

[0046] In some embodiments, L1 to L2 are

[0047] [ka]

[0048] is selected from the group consisting of:

[0049] In some embodiments, the drug has the ability to kill and / or inhibit the proliferation of tumor cells.

[0050] In some embodiments, the drug comprises a small molecule drug.

[0051] 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, auristatins, dolasatins, 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 or derivatives thereof, etoposide phosphate, nitrogen mustard, proteasome inhibitors, cytokines, Tubulysin B analogs, and Toll-like receptor agonists.

[0052] In some embodiments, the drug is selected from the group consisting of DM1, exatecan, Dxd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitors SHR153024, PNU-159682, eribulin, Tub196, Duo5 toxin, SN38 or a derivative thereof.

[0053] In some embodiments, the above (L1) a -(L2) b -D is

[0054] [ka]

[0055] having a structure selected from the group consisting of Among them, X 1is an alkane chain or a PEG chain, and X 2 is H or -C(O)NR 1 R 2 and R 1 is hydrogen, halogen, hydroxyl group, substituted or unsubstituted C 1-6 Alkyl groups, substituted or unsubstituted C 1-6 Hydroxyalkyl groups, substituted or unsubstituted C 1-6 Aminoalkyl groups, substituted or unsubstituted C 1-6 Alkoxy groups, substituted or unsubstituted C 1-6 Alkanoyl groups, and substituted or unsubstituted C 1-6 alkyl aldehyde groups, R 2 is hydrogen and substituted or unsubstituted C 1-6 alkyl groups, each of which is selected from the group consisting of substituted C 1-6 The alkyl group is optionally independently selected from halogen, hydroxyl, amino, carbonyl, carboxy, 5- to 10-membered heteroaryl and C 1-6 The 5- to 10-membered heteroaryl group has 1 to 3 heteroatoms selected from oxygen, nitrogen and sulfur, and n is an integer of 1 to 20.

[0056] In some embodiments, the above (L1) a -(L2) b -D is

[0057] [ka]

[0058] The compound has a structure selected from the group consisting of:

[0059] In some embodiments, the antibody drug conjugate comprises:

[0060] [ka]

[0061] The compound has a structure selected from the group consisting of:

[0062] In some embodiments, the antibody-drug conjugate has a drug / antibody ratio of about 1 to about 8.

[0063] In another aspect, the present application provides a compound that can be used to prepare the antibody-drug conjugate described herein, the compound having the structure shown in Formula 2, M-(L1): a (Formula 2): wherein M represents an antibody or antigen-binding fragment thereof targeting CD73 described herein, L1 represents a linker connecting to M, and a is selected from 0 to 10.

[0064] 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.

[0065] In some embodiments, L1 and M are linked to M via a thiol group, an azide group, or an amide group on M.

[0066] In some embodiments, L1 is capable of reacting with a thiol group to form a linkage.

[0067] In some embodiments, L1 is

[0068] [ka]

[0069] is selected from the group consisting of:

[0070] In another aspect, the present application provides a method of making an antibody-drug conjugate described herein, comprising contacting a compound described herein with a drug described herein.

[0071] In another aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate described herein, optionally in a pharma- ceutically acceptable vector.

[0072] In another aspect, the present application provides a method of modulating the tumor microenvironment in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein.

[0073] 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.

[0074] In another aspect, the present application provides the use of an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament, wherein the medicament can prevent and / or treat a tumor.

[0075] In some embodiments, the tumor comprises a solid tumor and / or a non-solid tumor.

[0076] In some embodiments, the tumor comprises a CD73-mediated tumor.

[0077] In some embodiments, the tumor comprises pancreatic cancer and / or breast cancer.

[0078] 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. [Brief description of the drawings]

[0079] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present 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. [Figure 1A-1D] 1 shows a schematic diagram of the structure of the antibody-drug conjugate described in the present application, as well as the HIC detection results and DAR values. [Diagram 2] 1 shows the results of detecting the binding activity of the antibody-drug conjugate described in the present application to the CD73 antigen on the cell membrane surface. [Diagram 3] 1 shows the results of detecting the enzyme activity inhibition status of the antibody-drug conjugate described in the present application and recombinant CD73 protein. [Figure 4] 1 shows the endocytosis efficiency of the antibody-drug conjugate described in the present application in inducing endocytosis of cell membrane surface CD73. [Diagram 5] 1 shows the killing curves of antibody drug conjugates described herein against tumor cells that do not express CD73. [Figure 6] 1 shows the killing curves of antibody drug conjugates described herein against tumor cells highly expressing CD73. [Figure 7] 1 shows the tumor inhibitory effect of the antibody-drug conjugates described herein on an animal model of pancreatic cancer expressing CD73. [Figure 8] 1 shows the tumor inhibitory effect of the antibody-drug conjugates described herein on a breast cancer animal model expressing CD73. [Figure 9] The endocytosis efficiency of different antigen-binding moieties inducing endocytosis of cell membrane surface CD73 is shown. [Figure 10A-10B] 1 shows RP-HPLC chromatograms of the antibody drug conjugates described in the present application. [Figure 11] 1 shows the tumor inhibitory effect of the antibody-drug conjugates described herein on a breast cancer animal model expressing CD73. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] 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.

[0081] 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 drugs. The chemical drugs may be any therapeutic and / or cytotoxic agent. The antibody drug conjugate may have any number of drugs coupled 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.

[0082] 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.

[0083] As used herein, the term "CD73" may also be referred to as "NT5E", "cluster of differentiation 73", "5'-nucleotidase (5'-NT)" or "extracellular 5'-nucleotidase". The term includes "full-length", unprocessed CD73, and any form of CD73 produced by intracellular processing. As used herein, the term "CD73" includes full-length wild-type CD73 as well as mutants, fragments, variants, isotypes and homologs thereof. In some embodiments, the CD73 may include human CD73. For example, the sequence for human CD73 may be found in Uniprot Accession No. P21589. As used herein, the CD73 may include the amino acid sequence set forth in SEQ ID NO:40.

[0084] In the present application, the term "CDR", also referred to as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or which forms a structurally defined loop. Typically, an antibody comprises six CDRs, three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, a naturally occurring camelid antibody consisting of only heavy chains can function normally and stably even when light chains are absent. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, and Kabat / Chothia when considered together. These numbering systems are known in the art and can be seen, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, amino acid sequence numbering of the antigen binding proteins can follow the IMGT numbering protocol (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr, http: / / imgt.cines.fr; Lefranc et al, 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203).For example, the CDRs of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY AcadSci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242).

[0085] In this application, the term "FR" refers to the more highly conserved parts of antibody variable domains, usually called framework regions. Natural heavy and light chain variable domains each usually contain four FR regions, four in VH (H-FR1, H-FR2, H-FR3 and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3 and L-FR4).

[0086] In this application, the terms "variable domain" and "variable region" may be used interchangeably and generally refer to a portion of an antibody heavy and / or light chain. The variable domains of the heavy and light chains, respectively, are referred to as "V H " and "V L (or "VH" and "VL", respectively). These domains are usually the most variable parts of an antibody (compared to other antibodies of the same type) and contain the antigen-binding sites.

[0087] In this application, the term "variable" usually refers to the fact that certain segments of the variable domains can differ relatively widely in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the scope of the variable domain. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) of the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, most of which adopt a β-fold configuration and are connected by three CDRs that form a cyclic linkage and, in some cases, form part of the β-fold structure. The CDRs in each chain are held in close proximity to each other by the FR regions and, together with the CDRs from another chain, promote the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0088] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, including any polypeptide that contains an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", such as "whole antibody", the term "antibody" also refers, for purposes of the present invention, to Fab, F(ab') and / or F(ab') antibodies. 2Antibody fragments include antibody fragments such as Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically bind to human CD73). Typically, such fragments should contain an antigen-binding domain. The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units with another polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain two to five basic four-chain units that can bind to the J chain and polymerize to form multivalent combinations. For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain further has equally spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain at its other end. VL corresponds to VH and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. VH and VL together form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be assigned to different classes or isotypes.There are currently five immunoglobulins, IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.

[0089] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., CD73). In the present application, the antigen-binding fragment may be any of Fab, Fab', F(ab) and / or Fab' fragments. 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and / or dAb.

[0090] In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As described above, an intact antibody can be digested with papain. An antibody digested with papain produces two identical antigen-binding fragments, a "Fab" fragment and a remaining "Fc" fragment (i.e., Fc region, as described above). The Fab fragment contains the complete L chain and the variable region of the heavy chain, as well as the H chain (V H ) the first constant region (C H It may be composed of 1).

[0091] As used herein, the term "Fab' fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is typically slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the heavy chain variable region, and all or a portion of the first and second constant regions of the heavy chain. For example, a Fab' fragment may further include some or all of the 220-330 amino acid residues of the heavy chain.

[0092] In this application, the term "F(ab')2" refers to an antibody fragment typically generated by digesting an entire antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigen.

[0093] In this application, the term "Fv fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody that typically contains all or part of the heavy and light chain variable regions and lacks the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, the CDRs. For example, an Fv fragment contains all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.

[0094] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, in which the light and heavy chain variable regions are contiguous (e.g., via a linker, such as a short flexible peptide linker) and can be expressed in the form of a single polypeptide chain, in which the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv, as used herein, may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and may comprise a VL-linker-VH or a VH-linker-VL.

[0095] In this application, the term "dAb" generally refers to a VH domain, a VL domain, or an antigen-binding fragment having a VH or VL domain, see, for example, Ward et al. (Nature, 1989 Oct 12;341(6242):544-6), Holt et al. (Trends Biotechnol., 2003,21(11):484-490), and other published patent applications, such as WO 06 / 030220, WO 06 / 003388, and Domantis Ltd.

[0096] As used herein, the term "monoclonal antibody" generally refers to a preparation of an antibody molecule consisting of a single molecule. Monoclonal antibodies are generally highly specific for a single antigenic site. Also, unlike conventional polyclonal antibodies (which generally have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they may be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained, for example, from an essentially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used herein may be produced in hybridoma cells or by recombinant DNA methodologies.

[0097] In this application, the term "chimeric antibody" refers to an antibody whose variable region is usually derived from one species and whose constant region is derived from another species. Usually, the variable region is derived from an antibody of a laboratory animal such as a rodent (the "parent antibody"), and the constant region is derived from a human antibody, thereby reducing the likelihood that the resulting chimeric antibody will provoke a harmful immune response in a human individual compared to the parent (e.g., mouse-derived) antibody.

[0098] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR region can be tolerated as long as the antibody retains the ability to bind to a specific antigen. A humanized antibody may optionally include at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains similar antigen specificity as the original antibody. "Humanized" forms of non-human (e.g., mouse) antibodies may minimally include chimeric antibodies derived from the sequence of a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (acceptor antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (e.g., mouse, rat, rabbit or non-human primate) having the desired properties, affinities and / or capacities. In some cases, FR region residues of a human immunoglobulin may be replaced with the corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in either the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0099] The term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques useful for producing antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as using libraries, are known in the art.

[0100] In this application, the terms "polypeptide molecule" and "polypeptide" may be used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked portions, each of which may have different properties. The properties may be biological properties, such as in vitro or in vivo activity. The properties may be simple chemical or physical properties, such as binding to a target molecule, catalysis of a reaction, etc. The two portions may be directly linked by a single peptide bond or by a peptide linker.

[0101] As used herein, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

[0102] In this application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce, or transfect a host cell so as to express the genetic material elements carried by the vector in the host cell. For example, a vector may include a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC), a phage such as a lambda phage or an M13 phage, and an animal virus. An animal virus as a vector may include a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (e.g., herpes simplex virus), a pox virus, a baculovirus, a papilloma virus, and a papilloma vacuolating virus (e.g., SV40). A vector may include various expression control elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. A vector may also include an origin of replication. A vector may include components that aid in its entry into a cell, such as a viral particle, liposome, or protein coat, but is not limited to these materials.

[0103] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject plasmid or vector, including a nucleic acid molecule or vector described in the present invention. A cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny are not necessarily completely identical (in terms of total DNA complement morphology or genome) to the original parent cell. A cell may include a cell that has been transfected in vitro with a vector described in this application. The cell may be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, or a NS0 cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell.

[0104] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology A Synthesis (2nd ed., E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0105] In the present 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.

[0106] As used herein, the term "tumor" refers to a physiological condition characterized by uncontrolled cell growth, typically in a mammal. The tumor comprises one or more cancerous cells. The tumor may comprise a solid tumor and / or a non-solid tumor.

[0107] In this application, the term "tumor microenvironment" generally refers to the environment in which a tumor resides, which is the acellular area within the tumor and the area immediately outside the tumor tissue, but not within 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 a relatively low concentration of glucose and other nutrients, but may have a relatively high concentration 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.

[0108] 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 symptoms, 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 reduction in the disease state, and recovery (whether partial or complete), whether detectable or undetectable. "Treatment" or "treatment" can also refer to prolonging survival as compared to expected survival without treatment. Subjects in need of treatment include those who have previously had a disease or condition, as well as those who are prone to suffer from a disease or condition, or those in whom a condition or condition is being prevented.

[0109] In this application, the term "specifically binds" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that allows the presence of the target to be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein, the epitope being conserved in proteins of different species. In some embodiments, specific binding may include, but is not required to include, exclusive binding.

[0110] As used herein, the term "subject" refers generally to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0111] In the present application, such proteins, polypeptides and / or amino acid sequences should also be understood to include variants or homologues having at least the same or similar function as the above proteins or polypeptides.

[0112] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to a CD73 protein). For example, the functional variant may include a protein or polypeptide that already has an amino acid modification by substitution, deletion, and / or insertion of at least one, for example 1 to 30, 1 to 20, or 1 to 10, for example 1, 2, 3, 4, or 5 amino acids. The functional variant can basically retain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0113] In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence identity with the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to a CD73 protein).

[0114] In this application, the above-mentioned homology generally refers to the similarity, similarity or relationship between two or more sequences. The "sequence homology percentage" can be calculated by the following method: comparing two aligned sequences in a comparison window, determining the number of positions in which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exists in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to generate the sequence homology percentage. Alignment for determining sequence homology percentage can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment within the full length sequences being compared or within the region of the target sequence. The homology can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see WR Pearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and DJ Lipman and WR Pearson, "Rapid and Sensitive Protein Similarity Searching," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, EW Myers, and D. Lipman, "Basic local alignment search tools," Journal of Molecular Biology, 215:403-410, 1990.

[0115] In this application, the term "comprise" generally refers to including, collecting, containing or covering. In some cases, it also means "is" or "consists of."

[0116] In this application, the term "about" generally refers to a variation within 0.5% to 10% more or less than a specified value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% more or less than a specified value.

[0117] [Detailed Description of the Invention] In one aspect, the present application provides the following:

[0118] Antigen-binding proteins The CDR of an antibody is also called the complementarity determining region, and is a part of the variable region. The amino acid residues of the region can contact the antigen or antigen epitope. The antibody CDR can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, and Kabat / Chothia when considered together. These numbering systems are known in the art, and can be specifically referred to, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different numbering systems depending on the sequence and structure of the antibody. Using different numbering systems may result in differences in the CDR region. In the present application, the CDR covers the CDR sequence divided according to any CDR division method, and also covers its variants, and the variants include substitutions, deletions, and / or additions of one or more amino acids in the amino acid sequence of the CDR. For example, 1 to 30, 1 to 20, or 1 to 10, further for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions, and also covers homologs thereof, which may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDRs are determined by the IMGT numbering protocol.

[0119] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 comprises an HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0120] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise HCDR2, which may comprise the amino acid sequence shown in SEQ ID NO:2.

[0121] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise HCDR1, which may comprise the amino acid sequence shown in SEQ ID NO:1.

[0122] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise HCDR3, HCDR2 and HCDR1. For example, the HCDR3 of the antibody or antigen-binding fragment thereof that targets CD73 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0123] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise an LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0124] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise an LCDR2, which may comprise the amino acid sequence shown in SEQ ID NO:5.

[0125] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4.

[0126] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise LCDR3, LCDR2 and LCDR1. For example, the LCDR3 of the antibody or antigen-binding fragment thereof that targets CD73 may comprise the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:4.

[0127] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1. For example, HCDR3 of the antibody or antigen-binding fragment thereof targeting CD73 described in the present application may comprise the amino acid sequence set forth in SEQ ID NO:3, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:2, HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:1, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:6, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, and LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:4.

[0128] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise H-FR1, the C-terminus of the H-FR1 may be linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:19.

[0129] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an H-FR2, which may be located between the HCDR1 and the HCDR2, and which may comprise the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:21.

[0130] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an H-FR3, which may be located between the HCDR2 and the HCDR3, and which may comprise the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:22.

[0131] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 18.

[0132] In the present application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively, in that order. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively, in that order. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:18, respectively, in that order. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the above-mentioned CD73-targeting antibody or antigen-binding fragment thereof may comprise the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:18, respectively, in that order.

[0133] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise L-FR1, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 23.

[0134] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an L-FR2, which may be located between the LCDR1 and the LCDR2, and which may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24.

[0135] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an L-FR3, which may be located between the LCDR2 and the LCDR3, and which may comprise the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 28.

[0136] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an L-FR4, the N-terminus of the L-FR4 being linked to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 26.

[0137] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise L-FR1, L-FR2, L-FR3 and L-FR4. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, in that order. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively, in that order. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:26, respectively, in that order. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the above-mentioned CD73-targeting antibody or antigen-binding fragment thereof may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28 and SEQ ID NO:26, respectively, in that order.

[0138] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise a VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35.

[0139] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.

[0140] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise a VH and a VL. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:29, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:31, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:32. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:33, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:35, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:33, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO:35, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:34.

[0141] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise at least one CDR in the VH described in the present application. In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise at least one CDR in the VL described in the present application. The CDRs can be obtained by splitting according to any splitting method.

[0142] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise HCDR1, HCDR2 and HCDR3 in a VH described in the present application. The VH may comprise the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35.

[0143] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise LCDR1, LCDR2 and LCDR3 in a VL described in the present application. The VL may comprise the amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.

[0144] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof that targets CD73 may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0145] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0146] In the present application, the antibody or antigen-binding fragment thereof that targets CD73 may comprise an antibody or antigen-binding fragment thereof.

[0147] In some embodiments, the antigen-binding fragment is a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and / or dAb.

[0148] In some embodiments, the antibody may comprise a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0149] For example, the VH of the chimeric antibody may comprise the amino acid sequence shown in SEQ ID NO:29, and the VL of the chimeric antibody may comprise the amino acid sequence shown in SEQ ID NO:30.

[0150] For example, the VH of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:31, and the VL of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:32.

[0151] For example, the VH of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:33, and the VL of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:34.

[0152] For example, the VH of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:35, and the VL of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:36.

[0153] For example, the VH of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:33, and the VL of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:36.

[0154] For example, the VH of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:35, and the VL of the humanized antibody may comprise the amino acid sequence shown in SEQ ID NO:34.

[0155] It should further be explained that the antibodies or antigen-binding fragments thereof targeting CD73 described herein may comprise heavy and / or light chain sequences in which one or more conservative sequence modifications are present. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antigen-binding fragments thereof targeting CD73 described herein by standard techniques known in the art, such as point mutation and PCR-mediated mutagenesis. A conservative amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR regions of an antibody or antigen-binding fragment thereof that targets CD73 described herein may be substituted with another amino acid residue from the same side chain group.Those skilled in the art are aware that some conservative sequence modifications do not abolish antigen binding, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6; and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0156] The CD73 antigen binding proteins described herein can be identified, screened or characterized by a variety of assays known in the art.

[0157] For example, the antigen-binding activity of an antigen-binding protein or fusion protein of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

[0158] In the present application, the CD73-targeting antibody or antigen-binding fragment thereof is capable of specifically binding to CD73 or a functionally active fragment thereof.

[0159] In some embodiments, the CD73-targeting antibody or antigen-binding fragment thereof may be affinity screened by Biacore. The CD73-targeting antibody or antigen-binding fragment thereof may be affinity screened at a concentration of 5×10 -9 For example, the antigen binding proteins described herein can bind to CD73 protein with a KD of about 4×10 -9 Less than M, about 3 x 10 -9 Less than M, about 2 x 10 -9Less than M, approx. 1×10 -9 Less than M, about 7 x 10 -10 Less than M, approx. 6 x 10 -10 Less than M, about 5 x 10 -10 Less than M, approx. 4 x 10 -10 Less than M, about 3 x 10 -10 Less than M, about 2 x 10 -10 Less than M, approx. 1×10 -10 Less than M, approx. 9 x 10 -11 Less than M, about 5 x 10 -11 Less than M, about 2 x 10 -11 The antigen-binding protein may bind to CD73 with a KD of less than M. In some embodiments, the present application further comprises measuring the binding activity of the antigen-binding protein to the antigen using a FACS method. For example, the antigen-binding protein described herein may bind to cell-surface human CD73 with an EC50 value of about 0.5 μg / mL or less, about 0.4 μg / mL or less, about 0.3 μg / mL or less, about 0.2 μg / mL or less, or about 0.1 μg / mL or less. For example, the antigen-binding protein described herein may bind to cell-surface human CD73 with an EC50 value of about 0.4 μg / mL or less, about 0.3 μg / mL or less, about 0.2 μg / mL or less, or about 0.1 μg / mL or less.

[0160] In some embodiments, the CD73-targeting antibody or antigen-binding fragment thereof can bind to CD73. In some cases, the antigen-binding proteins described herein can also cross-react with monkey CD73, e.g., as detected by FACS. In this application, "cross-reaction" generally refers to the ability of an antibody to react with homologous proteins from other species.

[0161] In some embodiments, the antibodies or antigen-binding fragments thereof that target CD73 described herein do not bind to mouse and / or rat CD73.

[0162] For example, the titer can be detected by ELISA. For example, the binding activity or functional activity of the CD73-targeting antibody or antigen-binding fragment thereof can be detected using FACS detection. For example, affinity detection can be performed on the CD73-targeting antibody or antigen-binding fragment thereof.

[0163] In the present application, the CD73-targeting antibody or antigen-binding fragment thereof can prevent or reduce activation of CD73. For example, the CD73-targeting antibody or antigen-binding fragment thereof can prevent or reduce conversion of CD73 from a catalytically inactive open conformation to a catalytically active closed conformation. For example, the CD73-targeting antibody or antigen-binding fragment thereof can block the CD73 catalytic center, thereby inhibiting the enzymatic activity of CD73.

[0164] In some embodiments, the binding epitope of the CD73-targeting antibody or antigen-binding fragment thereof can be determined by hydrogen deuterium exchange mass spectrometry (HDX-MS). In some embodiments, the CD73-targeting antibody or antigen-binding fragment thereof has competitive binding activity with the CPI-006 antibody (Corvus).

[0165] In the present application, the antibody or antigen-binding fragment thereof that targets CD73, when binding to CD73, binds to at least one amino acid residue in the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37) of the N-terminal domain of CD73 or a corresponding CD73 sequence, the 178-SKETPFLSNPGTNL-191 region (SEQ ID NO: 38) of the N-terminal domain of CD73 or a corresponding CD73 sequence, and the 381-WNHVSM-386 region (SEQ ID NO: 39) of the C-terminal domain of CD73 or a corresponding CD73 sequence.

[0166] In this application, "the CD73 sequence corresponding thereto" can refer to the amino acid sequence at the corresponding position of the homologue thereof (or CD73 from another species).

[0167] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 can inhibit the enzymatic activity of CD73. For example, the antibody or antigen-binding fragment thereof targeting CD73 may have an enzymatic activity inhibitory effect on recombinant CD73. For example, the antibody or antigen-binding fragment thereof targeting CD73 may have an enzymatic activity inhibitory effect on human CD73. The enzymatic activity described in the present application can be detected by any method commonly used in the art. The detection of the enzymatic activity may include homogenously mixing the CD73 antigen-binding protein with the recombinant CD73 protein, adding AMP and ATP, adding CellTiter-Glo® substrate to CellTiter-Glo® buffer, homogenously mixing, equilibrating to room temperature, reacting, and performing full wavelength detection. For example, the CD73-targeting antibody or antigen-binding fragment thereof can inhibit the enzymatic activity of recombinant CD73 protein with an EC50 value of about 0.8 μg / mL or less, about 0.7 μg / mL or less, about 0.6 μg / mL or less, about 0.5 μg / mL or less, about 0.4 μg / mL or less, about 0.3 μg / mL or less, or about 0.2 μg / mL or less. For example, the CD73-targeting antibody or antigen-binding fragment thereof can inhibit the enzymatic activity of CD73 protein on a cell membrane with an EC50 value of about 6 μg / mL or less, about 5 μg / mL or less, about 4 μg / mL or less, about 3 μg / mL or less, about 2 μg / mL or less, about 1 μg / mL or less, or about 0.5 μg / mL or less.

[0168] In the present application, the CD73-targeting antibody or antigen-binding fragment thereof can induce endocytosis of cell surface CD73. In some embodiments, the endocytosis status of a protein can be determined by a fluorescent labeling method. The efficiency with which the antigen-binding protein induces cell surface CD73 endocytosis can be obtained, for example, by detecting the fluorescence intensity of cells using flow cytometry. In some cases, the CD73-targeting antibody or antigen-binding fragment thereof can induce endocytosis of CD73 with an efficiency of about 50% or more.

[0169] Antibody-drug conjugates and compounds In another aspect, the present application provides an antibody-drug conjugate.

[0170] In the present application, the antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof that targets CD73 as described in the present application.

[0171] In the present application, the antibody-drug conjugate has the structure shown in formula 1, M-(L1) a -(L2) b -D (Formula 1), in which M represents an antibody or antigen-binding fragment thereof targeting CD73 described herein, 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.

[0172] In the present application, the compound for preparing the antibody-drug conjugate described in the present application has the structure shown in Formula 2, M-(L1): a (Formula 2), in which M represents an antibody or antigen-binding fragment thereof targeting CD73 described in the present application, L1 represents a linker linked to M, and a is selected from 0 to 10.

[0173] 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.

[0174] In the present application, M may be a complete antibody structure (e.g., a complete IgG antibody structure including two identical light chains and two identical heavy chains). In the present application, the light chain constant region, CH1 of the heavy chain constant region and / or hinge region of M may comprise a linking site linkable to L1. For example, the light chain constant region, CH1 of the heavy chain constant region and hinge region of M may comprise a linking site linkable to L1.

[0175] In the present application, the linking site may include a cysteine. In the present application, the linking site may have a linkable thiol group. In the present application, the number of the linking sites may be eight. In the present application, the linking sites may include two linking sites located in the light chain constant region, two links located in the CH1 of the heavy chain constant region, and four links located in the hinge region.

[0176] 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 linking site may comprise a group capable of linking to the L1 after glycosylation modification.

[0177] Glycosylation of a protein can depend on the amino acid sequence of the protein (e.g., an antibody or antigen-binding fragment thereof that targets CD73 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.

[0178] 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, etc.).

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In the present application, both L1 and L2 can be considered to belong to the linker.

[0183] 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 include a stretcher and / or amino acid unit.

[0184] In the present application, the linker can be used to couple (e.g., by covalent bonding) an antibody (e.g., an antibody or antigen-binding fragment thereof that targets CD73 described in the present application) to the drug.

[0185] For example, the coupling can be via a cysteine ​​thiol group or an amine (eg, the N-terminus or an amino acid side chain such as lysine) of the antibody to form a bond with a functional group on the linker.

[0186] 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.

[0187] 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.

[0188] 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 an intracellular protease (e.g., a lysosomal protease) and / or a peptide linker that can be cleaved by an intracellular protease. In some cases, the linker is cleavable in the intracellular environment. For example, cleavage of the linker may enable the drug in the antibody-drug conjugate to exert an effective therapeutic effect.

[0189] In the present application, the structure and / or properties of the linker are stable outside the cell. The antibody-drug conjugates described in the present application may be stable in structure before transport or delivery into the cell. For example, in the antibody-drug conjugates, the CD73-targeting antibody or antigen-binding fragment thereof remains coupled to the drug.

[0190] In some cases, the linker in the antibody-drug conjugate can retain the specific binding properties of, for example, the CD73-targeting antibody or antigen-binding fragment thereof, and participate in the delivery of the antibody-drug conjugate and / or maintain the therapeutic effect (e.g., cytotoxic effect) of the drug.

[0191] 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.

[0192] 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 inhibit the biosynthesis of a macromolecule.

[0193] 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.

[0194] In the present application, the above L1 can react with a thiol group to form a linkage.

[0195] In the present application, the above L1 is

[0196] [ka]

[0197] may be selected from the group consisting of:

[0198] In the present application, the above L2 is a polypeptide, 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), N-sulfo(4-maleimidomethyl)cyclohexylcarboxylic acid sulfosuccinate ester (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).

[0199] 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).

[0200] In the present application, the above L2 is

[0201] [ka]

[0202] may be also possible.

[0203] In the present application, the above L1 to L2 are

[0204] [ka]

[0205] may be selected from the group consisting of:

[0206] Drugs In this application, the drug may have the ability to kill tumor cells and / or inhibit the proliferation of tumor cells.

[0207] In this application, the drug may include a small molecule drug.

[0208] 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 phosphoryl transfer 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, Tubulysin B analogs, and Toll-like receptor agonists.

[0209] In the present application, the drug may be selected from the group consisting of DM1, exatecan, Dxd, MMAE, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitors SHR153024, PNU-159682, eribulin, Tub196, Duo5 toxin, SN38 derivatives or derivatives thereof.

[0210] In the present application, the drug may be a DNA topoisomerase I inhibitor. In the present application, the drug may be a camptothecin derivative.

[0211] In the present application, Tubulysin B may be a tubulysin having cytotoxic activity that inhibits tubulin polymerization to cause cell cycle arrest and apoptosis. The CAS number of Tubulysin B is 205304-87-6. In the present application, MMAE, i.e., Monomethyl auristatin E, is a synthetic derivative of dolastatin 10, which can exert an effective mitosis inhibitory effect by inhibiting tubulin polymerization. The CAS number of MMAE is 474645-27-7. In the present application, SN38 is an active metabolite of the topoisomerase I inhibitor irinotecan. Its CAS number is 119577-28-5. In the present application, the drug may be SN38 or MMAE.

[0212] For example, the drug may be

[0213] [ka]

[0214] The structure may be as follows:

[0215] In the present application, the above (L1) a -(L2) b -D is

[0216] [ka]

[0217] and may have a structure selected from the group consisting of: Among them, X 1 is an alkane chain or a PEG chain, and X 2 is H or -C(O)NR 1 R 2 and R 1 is hydrogen, halogen, hydroxyl group, substituted or unsubstituted C 1-6 Alkyl groups, substituted or unsubstituted C 1-6 Hydroxyalkyl groups, substituted or unsubstituted C 1-6 Aminoalkyl groups, substituted or unsubstituted C 1-6 Alkoxy groups, substituted or unsubstituted C 1-6 Alkanoyl groups, and substituted or unsubstituted C 1-6 alkyl aldehyde groups, R 2 is hydrogen and substituted or unsubstituted C 1-6 alkyl groups, each of which is selected from the group consisting of substituted C 1-6 The alkyl group is optionally independently selected from halogen, hydroxyl, amino, carbonyl, carboxy, 5- to 10-membered heteroaryl and C 1-6 The 5- to 10-membered heteroaryl group has 1 to 3 heteroatoms selected from oxygen, nitrogen and sulfur, and n is an integer of 1 to 20.

[0218] In the present application, the above (L1) a -(L2) b -D is

[0219] [ka]

[0220] It may have a structure selected from the group consisting of:

[0221] In the present application, the antibody-drug conjugate is

[0222] [ka]

[0223] It may have a structure selected from the group consisting of:

[0224] In the present application, the antibody-drug conjugate is

[0225] [ka]

[0226] It may have a structure selected from the group consisting of:

[0227] In the present application, the antibody-drug conjugate may have a drug / antibody ratio of about 1 to 8. For example, the drug / antibody ratio may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. For example, the drug / antibody ratio may be 8. For example, the drug / antibody ratio may be 7.8.

[0228] Compounds and methods of manufacture In another embodiment, the present application provides M-(L1) a The present invention provides a compound for an antibody-drug conjugate described herein, having a structure as shown in Formula 2, wherein M represents an antibody or antigen-binding fragment thereof targeting CD73 described herein, L1 represents a linker linked to M, and a is selected from 0 to 10.

[0229] In the present application, the compound is

[0230] [ka]

[0231] The compound may include a structure selected from the group consisting of:

[0232] In another aspect, the present application provides a method for making an antibody-drug conjugate described herein, comprising contacting a compound described herein with a drug described herein.

[0233] In some cases, the method for producing the antibody-drug conjugate includes the steps of: (1) contacting L1 and L2 described herein with the drug; and (2) contacting compound (L1) with the drug. 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 subjected to the above-mentioned glycosylation modification.

[0234] Pharmaceutical Compositions and Uses In another aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate or a pharma- ceutically acceptable vector described herein.

[0235] 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 of skill 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.

[0236] 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.

[0237] In this application, the pharma- ceutically acceptable vector may include any or all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The pharma- ceutically acceptable vector may include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. The pharma- ceutically acceptable vector may further include isotonic agents, wetting agents, emulsifying agents, preservatives, and / or buffers.

[0238] In another aspect, the present application provides a method of modulating the tumor microenvironment in a subject, comprising administering to the subject an antibody drug conjugate described herein, or a pharmaceutical composition described herein.

[0239] 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.

[0240] The present application provides the 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 modulating the tumor microenvironment of a subject and / or the immune response of a subject.

[0241] The present application provides an antibody-drug conjugate as described above, or a pharmaceutical composition as described herein, for modulating a tumor microenvironment in a subject and / or an immune response in a subject.

[0242] In another aspect, the present application provides the use of an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament, wherein the medicament can prevent and / or treat a tumor.

[0243] 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 using the pharmaceutical composition described herein.

[0244] The present application provides the above-mentioned antibody-drug conjugate or the pharmaceutical composition described herein for preventing and / or treating tumors.

[0245] In the present application, the tumor may include a solid tumor and / or a non-solid tumor.

[0246] For example, the tumor may include breast cancer (eg, triple-negative breast cancer) and / or pancreatic cancer.

[0247] 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

[0248] Working Example The chemical formula of the SN-38 small molecule is:

[0249] [ka]

[0250] 900698 antibody: the amino acid sequence of its heavy chain variable region VH is shown in SEQ ID NO: 35, and the amino acid sequence of its light chain variable region VL is shown in SEQ ID NO: 34; 900725 antibody: the amino acid sequence of its heavy chain variable region VH is shown in SEQ ID NO:53, and the amino acid sequence of its light chain variable region VL is shown in SEQ ID NO:54.

[0251] Example 1 Preparation of antibody-drug conjugates The SN-38 small molecule was coupled to the 900698 antibody by adopting the cysteine ​​coupling method, and the drug-to-antibody ratio (DAR value) of the ADC molecule was measured by adopting the hydrophobic chromatography method, and the corresponding structures and DAR values ​​are shown in Figures 1A-1D. FIG. 1A is a schematic diagram of the structure of CD73-ADC, in which the antibody molecule is derived from 900698, and D represents the small molecule drug SN-38; FIG. 1B is the molecular structures of two different linkers-SN38; FIG. 1C is a graph showing the HIC detection results and DAR value of the CD73 antibody-Mc-VC-PAB-DMEDA-PEG-SN38 molecule (i.e., antibody-drug conjugate 1 formed by the binding of 900698 and molecule 1 in FIG. 1B); and FIG. 1D is a graph showing the HIC detection results and DAR value of the CD73 antibody-MP2-VC-PAB-DMEDA-PEG-SN38 molecule (i.e., antibody-drug conjugate 2 formed by the binding of 900698 and molecule 2 in FIG. 1B).

[0252] The results in Figure 1 show that the DAR values ​​of the CD73 antibody-Mc-VC-PAB-DMEDA-PEG-SN38 (abbreviation: antibody-drug conjugate 1) and CD73 antibody-MP2-VC-PAB-DMEDA-PEG-SN38 (abbreviation: antibody-drug conjugate 2) molecules are both approximately 7.8.

[0253] Example 2 Detection of the binding activity of antibody-drug conjugates to cells expressing human CD73 antigen on their membranes The binding activity of antibody-drug conjugate 1, antibody-drug conjugate 2, and naked antibody 900698 prepared in Example 1 to cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Biopharm) was detected.

[0254] All antigen-binding proteins were diluted to 30 μg / mL in PBS solution containing 1% BSA (1% BSA / PBS), and further diluted three-fold in a gradient of 10. 20 μL was added per well of a 96-well U-shaped plate, and a negative control (1% BSA / PBS only) was set up at the same time. Cells expressing human CD73 in logarithmic growth phase (CHOK1-huCD73-3F4, Huabo Biopharm) were taken and centrifuged (1000 rpm x 5 min) to discard the culture medium, and the cells were diluted to a viable cell density of 1 x 10 in 1% BSA / PBS. 6 Resuspend at 10 / mL and add 20 µL (2 x 10) per well of a 96-well U-shaped plate with anti-CD73 antigen-binding protein. 4 After the reaction, the 96-well U-shaped plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3 min) to discard the supernatant, washed once in this manner, and PE-sheep anti-human-Fc (Jackson ImmunoResearch, #109-115-098) diluted 1:200 was added and reacted at room temperature in the dark for 15 min. After the reaction, the 96-well U-shaped plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3 min) to discard the supernatant, washed three times in this manner, and finally resuspended in 100 μL of 1% BSA / PBS per well. The fluorescence intensity of the PE channel was detected using a flow cytometer (BD, #CantoII).

[0255] The binding activity results of antibody drug conjugate 1, antibody drug conjugate 2, and 900698 naked antibody are shown in Figure 2 (represented by 1 to 3 in Figure 2, respectively), and the results show that the three proteins have very good affinity to cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Biopharm), and the affinity of antibody drug conjugate 1, antibody drug conjugate 2, and 900698 naked antibody to cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Biopharm) is comparable, indicating that coupling of SN38 small molecule toxin with 900698 naked antibody does not affect its affinity to cell surface CD73 antigen.

[0256] Example 3 Detection of the inhibitory effect of antibody-drug conjugates on the enzyme activity of soluble recombinant CD73 antigen protein The inhibitory effects of antibody-drug conjugate 1, antibody-drug conjugate 2, and naked antibody 900698 prepared in Example 1 on the enzyme activity of recombinant CD73 protein (ACRO, #CD3-H52H7-100 μg) were detected.

[0257] Specifically, the CD73 antigen-binding protein series was diluted to 120 μg / mL with TM Buffer (ROCKLAND, #MB-059), further diluted 3-fold to 10, and 25 μL / well was added to a 96-well plate. The recombinant CD73 protein was further diluted to 0.3 μg / mL with TM Buffer (ROCKLAND, #MB-059), and 25 μL / well of each was added to a 96-well plate and mixed uniformly. Next, the diluted AMP and ATP were mixed 1:1, and 50 μL / well was added to the above 96-well plate and reacted for 15 min. Finally, CellTiter-Glo® substrate was added to CellTiter-Glo® buffer, mixed uniformly, and equilibrated to room temperature, and then added to the wells waiting for measurement at 100 μL / well, reacted at room temperature for 10 min, and full wavelength detection was performed.

[0258] The inhibition results of antibody drug conjugate 1, antibody drug conjugate 2, and 900698 naked antibody on the enzyme activity of soluble recombinant CD73 antigen protein (ACRO, #CD3-H52H7-100μg) are shown in Figure 3 (shown as 1 to 3 in Figure 3, respectively), and the results show that the inhibitory effects of antibody drug conjugate 1, antibody drug conjugate 2, and 900698 on the enzyme activity of soluble recombinant CD73 protein are comparable, indicating that the coupling of SN38 small molecule toxin with 900698 naked antibody does not affect its inhibitory ability on the enzyme activity of recombinant CD73 antigen protein.

[0259] Example 4 Detection of the binding affinity of antibody-drug conjugates to human and monkey CD73 antigen proteins Binding kinetics experiments of antibody-drug conjugate 1, antibody-drug conjugate 2, and 900698 naked antibody produced in Example 1 to human CD73 protein (manufacturer: ACRO Biosystems, product number: CD3-H52H7) and monkey CD73 protein (manufacturer: Novoprotein, product number: CI21) were performed using Biacore (GE, model number: T200), and the experimental buffer was HBS-EP+ (pH 7.4), protein A chip (manufacturer: GE, product number: 29-1275-56).

[0260] The temperature of the sample bottle and the flow channel was set to 25°C, and the antigen-binding protein was captured on the protein A chip, and approximately 100 RU was immobilized as a ligand. Human CD73 protein was diluted to a series of concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.57 nM using the experimental buffer to prepare the analyte. The experimental buffer was used as a zero concentration control, and the protein was bound at a flow rate of 30 μL / min for 120 s and dissociated for 600 s. 10 mM Glycine pH 1.5 was regenerated at a flow rate of 50 μL / min for 60 s to perform multi-cycle kinetic detection. The results of fitting the association rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium rate constant (KD) using a 1:1 binding model and selecting the Fit local mode are shown in Table 1, which shows that all antigen-binding proteins bind to human and monkey CD73 antigen proteins, and the binding affinities of antibody-drug conjugate 1 and antibody-drug conjugate 2 to human and monkey CD73 proteins are not significantly different from those of the 900698 naked antibody itself.

[0261] [Table 1]

[0262] Example 5. Detection of the Ability of Antibody-Drug Conjugates to Induce Endocytosis of Cell Surface CD73 Antigen Antibody-drug conjugate 1, antibody-drug conjugate 2, and 900698 naked antibody prepared in Example 1 were diluted to 1 μg / mL with PBS solution, and duplicate wells were used for each concentration. Calu6 cells were harvested in the logarithmic growth phase, and 2 × 10 cells were cultured.5 The cells were prepared into a cell suspension of 1:1 / mL, and 100 μL (approximately 20,000 cells) was added per well to a 96-well U-shaped plate, centrifuged at 300g×3min, and the supernatant was removed. The cells were further resuspended with the diluted antigen-binding protein, mixed uniformly at 100 μL / well, and incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 300g×3min, the supernatant was removed, and the cells were resuspended in 1% BSA, and half were incubated at 37°C and the other half was incubated at 4°C for 5 h. After incubation, the wells of the corresponding time were taken out, centrifuged at 300g×3min, the supernatant was removed, PE anti-huIgG Fc (1:200) was added, mixed uniformly at 50 μL / well, and reacted at 4°C for 30 min. After incubation, the cells were washed twice, and the fluorescence intensity was detected by flow cytometry at 300g×3min. The detection results of cell surface CD73 endocytosis induced by anti-CD73 antigen-binding protein are shown in Table 2 below and Figure 4 (antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 are shown as 1 to 3 in Figure 4, respectively, and NC indicates that only 1% BSA solution was added and no antibody or antibody-drug conjugate was added). The results show that the ability of ADC molecule antibody-drug conjugate 1 and antibody-drug conjugate 2 to induce cell surface CD73 endocytosis is similar to that of naked antibody 900698, with excellent expression and endocytosis efficiency of about 95%.

[0263] [Table 2]

[0264] Example 6 Detection of the killing ability of antibody-drug conjugates against tumor cells with different CD73 antigen expression levels The killing effects of antibody-drug conjugate 1, antibody-drug conjugate 2, and naked 900698 antibody prepared in Example 1 on different breast cancer cells (MDA-MB-231 and MCF-7) were detected, among which, MDA-MB-231 highly expresses CD73, and MCF-7 does not express CD73.

[0265] Specifically, MDA-MB-231 cells and MCF-7 cells were processed into cell suspensions with densities of 8E4 / mL and 7.5E5 / mL, respectively, and seeded in a 96-well cell culture plate at 100 μL / well. After sealing the 96-well cell culture plate, the plates were incubated at 37° C., 5% CO 2 The plates were placed in an incubator and cultured for 6 hours. Antibody-drug conjugate 1, antibody-drug conjugate 2, and 900698 naked antibody were each diluted to 400 μg / mL in detection medium, and 8 concentrations were diluted in a 2-fold gradient dilution, for a total of 9 concentrations. The same dilution procedure was carried out for the buffer solution as for antibody-drug conjugate 1. The gradient dilution solution was added at 50 μL / well to the 96-well cell culture plate after the above-mentioned culture. Then, 50 μL of the corresponding detection medium was added to each of all sample wells. The 96-well cell culture plate was placed at 37°C and incubated with 5% CO 2 The plates were placed in an incubator and cultured for 5 days. After the culture was completed, the 96-well plate was removed from the incubator, 100 μL of the culture medium was aspirated, and 100 μL of equilibrated Celltiter-Glo Luminescent Cell Viability Assay detection solution (Promega, #G7573) was added per well. After shaking in the dark at room temperature for 10 minutes, 100 μL was removed from each well and transferred to the corresponding wells of a 96-well black plate, and full wavelength detection was performed using a microplate reader.

[0266] From the detection results in Figure 5 (corresponding to MCF-7 cells) and Figure 6 (corresponding to MDA-MB-231 cells), it can be seen that antibody-drug conjugate 1 and antibody-drug conjugate 2 have excellent killing effect on MDA-MB-231 cells with high CD73 expression, but have no killing effect on MCF-7 cells that do not express CD73, and naked antibody 900698 has no killing effect on either of the two cells. It was shown that antibody-drug conjugate 1 and antibody-drug conjugate 2 have high specificity for killing tumor cells.

[0267] Example 7 Detection of antitumor efficacy of antibody-drug conjugates in a BxPC3 CDX pancreatic cancer mouse model A BxPC-3 pancreatic cancer animal model was established using NPG mice, and the efficacy of the test antibody was tested. The human pancreatic cancer cells BxPC-3 used in this example were cultured in RPMI-1640 medium supplemented with 10% FBS under 5% CO 2 The cells were cultured in an incubator containing 0.1% CO and 10% CO at 37°C. Before the cells were serially cultured for 10 generations, approximately 1 × 10 7 After homogeneously mixing 100 μL of PBS containing BxPC-3 cells and an equal volume of Matrigel, the cells were seeded near the armpit on the right side of the back of 18 NPG mice in a volume of approximately 200 μL by subcutaneous injection. Before inoculation, the mice were anesthetized with 2-5% isoflurane. On the day of inoculation, 1.0 × 10 7 PBMCs (100 μL) were injected via the tail vein, and tumors were grown to an average size of approximately 50–80 mm 3 When tumors had grown to a certain extent, 18 tumor-bearing mice were randomly divided into three groups of six mice each according to tumor volume and body weight. Administration was performed on the day of grouping, and the specific administration schedule is shown in Table 3 below.

[0268] Among them, 900543 is a negative control antibody and does not bind to CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca, which is currently in clinical phase II. Uliledlimab is an anti-CD73 monoclonal antibody developed by Tianjing Biology, which is currently in clinical phase I. The amino acid sequence of the heavy chain of 900543 is shown in SEQ ID NO: 45, the amino acid sequence of the light chain of 900543 is shown in SEQ ID NO: 46, the amino acid sequence of the heavy chain of Oleclumab is shown in SEQ ID NO: 47, the amino acid sequence of the light chain of Oleclumab is shown in SEQ ID NO: 48, the amino acid sequence of the heavy chain of Uliledlimab is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain of Uliledlimab is shown in SEQ ID NO: 50.

[0269] The results are shown in Table 3 and Figure 7 below. Antibody-drug conjugate 1 and antibody-drug conjugate 2 were able to significantly inhibit the proliferation of BxPC-3 pancreatic cancer at a dose of 1 mg / kg, and their tumor growth inhibitory effects were superior to those of naked antibody 900698 and positive control antibodies Oleclumab and Uliledlimab at a dose of 5 mg / kg, while the negative control antibody 900543 was unable to inhibit tumor growth.

[0270] [Table 3]

[0271] Example 8: Detection of antitumor efficacy of antibody-drug conjugates in a drug efficacy model of MDA-MB-231 triple-negative breast cancer mice CDX An MDA-MB-231 triple-negative breast cancer animal model was established using NPG mice, and the efficacy of the test antibody was tested. The human breast cancer cells MDA-MB-231 used in this example were cultured in L-15 medium supplemented with 10% FBS under CO 2 The cells were cultured in a 37°C incubator without any added sodium chloride. Before the cells were serially cultured for 10 generations, approximately 5.0 × 10 6 MDA-MB-231 cells were suspended in 100 μL of PBS and uniformly mixed with an equal volume of Matrigel, and then seeded in a volume of approximately 200 μL near the armpit on the right side of the back of NPG-humanized mice by subcutaneous injection. Mice were anesthetized with 2–5% isoflurane before inoculation. On the day of inoculation, 1.0 × 10 7 PBMCs (100 μL) were injected via the tail vein, and tumors were grown to an average size of approximately 50–80 mm 3 When the tumors grew to a certain extent, 18 tumor-bearing mice were randomly divided into 7 groups of 6 mice each according to tumor volume and body weight. Administration was performed on the day of grouping, and the specific administration schedule is shown in Table 4 below. Among them, 900201 is a negative control antibody that does not bind to CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca that is currently in clinical phase II. The amino acid sequence of the heavy chain of 900201 is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain of 900201 is shown in SEQ ID NO: 44.

[0272] The results are shown in Table 4 and Figure 8 below. Antibody-drug conjugate 1 and antibody-drug conjugate 2 can significantly inhibit the proliferation of MDA-MB-231 breast cancer, and the tumor inhibition effect is positively correlated with the dose. At the same time, the tumor growth inhibition effect is significantly superior to that of naked antibody 900698 and positive control antibody Oleclumab at a dose of 5mg / kg, while the negative control antibody 900201 cannot inhibit tumor growth.

[0273] [Table 4]

[0274] Example 9 Selection of antibodies for producing antibody-drug conjugates The endocytosis experiment was conducted to compare the endocytosis efficiency of 900698 and 900725 monoclonal antibodies, in which anti-CD73 antigen-binding proteins, such as CD73 positive control antibody BMS-986179, 900698, and 900725, were diluted to 1 μg / mL in PBS solution, with duplicate wells for each concentration. Calu6 cells were harvested in logarithmic growth phase, and 2 × 10 cells were cultured. 5The cells were prepared into a cell suspension of 1:1 / mL, and 100 μL (approximately 20,000 cells) was added per well to a 96-well U-shaped plate, centrifuged at 300g×3min, and the supernatant was removed. The cells were further resuspended with the diluted antigen-binding protein, mixed uniformly at 100 μL / well, and incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 300g×3min, the supernatant was removed, and the cells were resuspended in 1% BSA, and half were incubated at 37°C and the other half was incubated at 4°C for 5 h. After incubation, the wells of the corresponding time were taken out, centrifuged at 300g×3min, the supernatant was removed, PE anti-huIgG Fc (1:200) was added, mixed uniformly at 50 μL / well, and reacted at 4°C for 30 min. After incubation, the cells were washed twice, and the fluorescence intensity was detected by flow cytometry at 300g×3min. The detection results of cell surface CD73 endocytosis induced by anti-CD73 antigen binding protein are shown in Table 5 and Figure 9 below. The results show that the cell surface CD73 endocytosis efficiency induced by anti-CD73 antibody 900725 (37.49%) is similar to that of the positive control antibody BMS-986179 (44.18%), and the endocytosis efficiency of 900698 is significantly higher than that of 900725 and BMS-986179, up to about 96%. 34%. Figure 9 1-3 show the results of BMS-986179, 900725, and 900698, respectively, and NC shows the results when only 1% BSA solution was added, without any antibody or antibody drug conjugate. These results indicate that 900698 is more suitable for the development of ADC drugs than 900725. The amino acid sequence of the heavy chain of BMS-986179 is set forth in SEQ ID NO:51, and the amino acid sequence of the light chain of BMS-986179 is set forth in SEQ ID NO:52.

[0275] [Table 5]

[0276] Example 10: Preparation of antibody-drug conjugate and its antitumor effect To verify the antitumor effects of ADCs prepared by coupling different linkers and payloads to the anti-CD73 antibody 900698, the tubulin polymerization inhibitor MMAE small molecule was coupled to the 900698 antibody using two different linkers and cysteine ​​coupling methods to obtain two ADC molecules, 900698-MC-VC-PAB-MMAE (hereinafter referred to as antibody-drug conjugate 3) and 900698-PY-VC-PAB-MMAE (hereinafter referred to as antibody-drug conjugate 4).

[0277] Among them, the chemical structural formulas of MC-VC-PAB-MMAE and PY-VC-PAB-MMAE are as follows, and mAb is antibody 900698. The linkage between antibody 900698 and MC-VC-PAB-MMAE or PY-VC-PAB-MMAE in antibody-drug conjugate 3 and antibody-drug conjugate 4 is shown in Figure 1A.

[0278] [ka]

[0279] Then, the drug-to-antibody ratio (DAR value) of antibody-drug conjugate 3 and antibody-drug conjugate 4 was measured using hydrophobic chromatography, and the DAR values ​​and HPLC profiles (wavelength 280 nm) are shown in Table 6 below and Figures 10A to 10B.

[0280] [Table 6]

[0281] To compare the in vivo antitumor effects of antibody-drug conjugate 3 and antibody-drug conjugate 4, we employed an NPG mouse model vaccinated with the human triple-negative breast cancer cell line MDA-MB-231, which highly expresses the CD73 antigen, to detect them.

[0282] The specific method is as follows: The human breast cancer cells MDA-MB-231 used in this experiment were cultured in L-15 medium supplemented with 10% FBS under CO 2 The cells were cultured in a 37°C incubator without any added sodium chloride. Before the cells were serially cultured for 10 generations, approximately 5.0 × 10 6 MDA-MB-231 cells were suspended in 100 μL of PBS and uniformly mixed with an equal volume of Matrigel, and then seeded in a volume of approximately 200 μL near the armpit on the right side of the back of NPG-humanized mice by subcutaneous injection. Mice were anesthetized with 2–5% isoflurane before inoculation. On the day of inoculation, 1.0 × 10 7 PBMCs (100 μL) were injected via the tail vein, and tumors were grown to an average size of approximately 50–80 mm 3 When tumors had grown to about 100% in the 100% control group, 60 tumor-bearing mice were randomly divided into 7 groups of 6 mice each according to tumor volume and body weight. Administration was performed on the day of grouping, and the specific administration schedule is shown in Table 7 below. 28 days after administration, the mice were dissected and the tumor volume and size were measured to evaluate the antitumor effect. The results are shown in Figure 11. The results show that antibody-drug conjugate 3 and antibody-drug conjugate 4 both have superior antitumor effects compared to monoclonal antibodies, and the antitumor effects are comparable.

[0283] [Table 7]

[0284] 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 an antibody or antigen-binding fragment thereof that targets CD73, wherein the antibody or antigen-binding fragment thereof that targets CD73 comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

3. Antibody-drug conjugates.

2. The antibody or antigen-binding fragment thereof that targets CD73 comprises HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:

2. The antibody-drug conjugate of claim 1.

3. The antibody or antigen-binding fragment thereof that targets CD73 comprises HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:

1. The antibody-drug conjugate according to any one of claims 1 to 2.

4. the antibody or antigen-binding fragment thereof that targets CD73 comprises an LCDR3, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; The antibody-drug conjugate according to any one of claims 1 to 3.

5. the antibody or antigen-binding fragment thereof that targets CD73 comprises an LCDR2, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; The antibody-drug conjugate according to any one of claims 1 to 4.

6. the antibody or antigen-binding fragment thereof targeting CD73 comprises an LCDR1, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4; The antibody-drug conjugate according to any one of claims 1 to 5.

7. The antibody or antigen-binding fragment thereof targeting CD73 comprises an H-FR1, wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:

19. The antibody-drug conjugate according to any one of claims 3 to 6.

8. the antibody or antigen-binding fragment thereof targeting CD73 comprises an H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:21; The antibody-drug conjugate according to any one of claims 2 to 7.

9. the antibody or antigen-binding fragment thereof targeting CD73 comprises an H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:22; The antibody-drug conjugate according to any one of claims 2 to 8.

10. the antibody or antigen-binding fragment thereof that targets CD73 comprises an H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 18; The antibody-drug conjugate according to any one of claims 1 to 9.

11. The antibody or antigen-binding fragment thereof targeting CD73 comprises an L-FR1, wherein the C-terminus of the L-FR1 is linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO:

23. The antibody-drug conjugate according to any one of claims 6 to 10.

12. the antibody or antigen-binding fragment thereof targeting CD73 comprises an L-FR2, the L-FR2 being located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 24; The antibody-drug conjugate according to any one of claims 6 to 11.

13. the antibody or antigen-binding fragment thereof targeting CD73 comprises an L-FR3, the L-FR3 being located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28; The antibody-drug conjugate according to any one of claims 5 to 12.

14. the antibody or antigen-binding fragment thereof targeting CD73 comprises an L-FR4, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 26; The antibody-drug conjugate according to any one of claims 4 to 13.

15. the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6; The antibody-drug conjugate according to any one of claims 1 to 14.

16. The antibody or antigen-binding fragment thereof that targets CD73 comprises a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:

35. The antibody-drug conjugate according to any one of claims 1 to 15.

17. The antibody or antigen-binding fragment thereof that targets CD73 comprises a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:

36. The antibody-drug conjugate according to any one of claims 1 to 16.

18. The antibody or antigen-binding fragment thereof targeting CD73 comprises: 1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 29, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 30; 2) the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 32; 3) the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 34; 4) the VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 36; 5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 33, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 36; and 6) the VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 34; VH and VL selected from the group consisting of: The antibody-drug conjugate according to any one of claims 1 to 17.

19. The antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody heavy chain constant region, The antibody-drug conjugate according to any one of claims 1 to 18.

20. The antibody heavy chain constant region is derived from a human antibody heavy chain constant region.

20. The antibody-drug conjugate of claim 19.

21. The antibody heavy chain constant region is derived from a human IgG heavy chain constant region. The antibody-drug conjugate according to any one of claims 19 to 20.

22. The antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region. The antibody-drug conjugate according to any one of claims 19 to 21.

23. The antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody light chain constant region, The antibody-drug conjugate according to any one of claims 1 to 22.

24. The antibody light chain constant region is derived from a human Igκ constant region. The antibody-drug conjugate of claim 23.

25. The antibody or antigen-binding fragment thereof that targets CD73 comprises an antibody or antigen-binding fragment thereof. The antibody-drug conjugate according to any one of claims 1 to 24.

26. The antigen-binding fragment may be a Fab, Fab', Fv fragment, F(ab') ２ , F(ab) ２ , scFv, di-scFv and / or dAb; The antibody-drug conjugate of claim 25.

27. The antibody is one or more selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. The antibody-drug conjugate according to any one of claims 25 to 26.

28. The CD73 comprises human CD73 and / or monkey CD73. The antibody-drug conjugate according to any one of claims 1 to 27.

29. Can block or reduce activation of CD73; The antibody-drug conjugate according to any one of claims 1 to 28.

30. It is capable of inhibiting the enzymatic activity of CD73. The antibody-drug conjugate according to any one of claims 1 to 29.

31. It can induce endocytosis of cell surface CD73. The antibody-drug conjugate according to any one of claims 1 to 30.

32. The structure shown in formula 1: M-(L1) ａ - (L2) ｂ -D (Formula 1), wherein M represents an antibody or antigen-binding fragment thereof targeting CD73 according to any one of claims 1 to 31; L1 represents a linker connecting to M, L2 represents a linker connecting to D, a and b are each independently selected from 0 to 10; D represents a drug. The antibody-drug conjugate according to any one of claims 1 to 31.

33. L1 and / or L2 are selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers and dicarboxylic acid-based linkers; The antibody-drug conjugate of claim 32.

34. The L1 and the M are linked via a thiol group, an azide group, or an amide group on the M; The antibody-drug conjugate according to any one of claims 32 to 33.

35. The L1 can react with a thiol group to link the thiol group. The antibody-drug conjugate according to any one of claims 32 to 34.

36. The L1 is 【Chemistry 1】 Selected from the group consisting of: The antibody-drug conjugate according to any one of claims 32 to 35.

37. The L2 may be a polypeptide, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)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 selected from the group consisting of NHS, N-4-(maleimidomethyl)cyclohexyl succinimidyl carboxylate (SMCC), N-sulfo(4-maleimidomethyl)cyclohexyl carboxylic acid sulfosuccinate (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); The antibody-drug conjugate according to any one of claims 32 to 36.

38. The L2 is 【Chemistry 2】 That is, The antibody-drug conjugate according to any one of claims 32 to 37.

39. The L1 to L2 are 【Chemistry 3】 Selected from the group consisting of: The antibody-drug conjugate according to any one of claims 32 to 38.

40. The drug has the ability to kill tumor cells and / or inhibit the proliferation of tumor cells. The antibody-drug conjugate according to any one of claims 32 to 39.

41. The drug comprises a small molecule drug. The antibody-drug conjugate according to any one of claims 32 to 40.

42. The drug may be 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), an inhibitor of nuclear export of protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of mitochondrial phosphoryl transfer reaction, 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 or a derivative thereof, an etoposide phosphate, a nitrogen mustard, a proteasome inhibitor, a cytokine, a tubulysin B analogs and Toll-like receptor agonists; The antibody-drug conjugate according to any one of claims 32 to 41.

43. The drug is selected from the group consisting of DM1, exatecan, Dxd, MMAE, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitors SHR153024, PNU-159682, eribulin, Tub196, Duo5 toxin, SN38 or a derivative thereof; The antibody-drug conjugate according to any one of claims 32 to 42.

44. () ａ -(L2) ｂ -Dは、 【Chemistry 4】 having a structure selected from the group consisting of Among them, X １ is an alkane chain or a PEG chain, and X ２ is H or -C(O)NR １ R ２ and R １ is a hydrogen atom, a halogen atom, a hydroxyl group, a substituted or unsubstituted C １－６ Alkyl group, substituted or unsubstituted C １－６ Hydroxyalkyl group, substituted or unsubstituted C １－６ Aminoalkyl groups, substituted or unsubstituted C １－６ Alkoxy group, substituted or unsubstituted C １－６ Alkanoyl groups, and substituted or unsubstituted C １－６ alkyl aldehyde groups, R ２ represents hydrogen and substituted or unsubstituted C １－６ alkyl groups, each of which is selected from the group consisting of substituted C １－６ The alkyl group is optionally independently selected from halogen, hydroxy, amino, carbonyl, carboxy, 5- to 10-membered heteroaryl, and C １－６ haloalkyl groups, wherein the 5-10 membered heteroaryl group has 1-3 heteroatoms selected from oxygen, nitrogen and sulfur, and n is an integer of 1 to 20; The antibody-drug conjugate according to any one of claims 32 to 43.

45. () ａ -(L2) ｂ -Dは、 【Chemistry 5】 having a structure selected from the group consisting of: The antibody-drug conjugate according to any one of claims 32 to 44.

46. 【Chemical 6】 having a structure selected from the group consisting of: The antibody-drug conjugate according to any one of claims 32 to 45.

47. having a drug / antibody ratio of about 1 to about 8; The antibody-drug conjugate according to any one of claims 1 to 46.

48. A compound for producing the antibody-drug conjugate according to any one of claims 1 to 47, comprising the structure shown in formula 2: M-(L1) ａ (Formula 2), in which M represents an antibody or antigen-binding fragment thereof targeting CD73 according to any one of claims 1 to 31, L1 represents a linker connecting to M; a is selected from 0 to 10; compound.

49. 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; 49. The compound of claim 48.

50. The L1 and the M are linked via a thiol group, an azide group, or an amide group on the M; 50. The compound according to any one of claims 48 to 49.

51. The L1 can react with a thiol group to link the thiol group. A compound according to any one of claims 48 to 50.

52. The L1 is 【Chemistry 7】 Selected from the group consisting of:

52. The compound according to any one of claims 48 to 51.

53. A method for producing an antibody-drug conjugate according to any one of claims 1 to 47, comprising: The method comprises contacting a compound according to any one of claims 48 to 52 with a drug according to any one of claims 40 to 43, method.

54. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 47, and optionally a pharma- ceutically acceptable vector. Pharmaceutical compositions.

55. A method for modulating a tumor microenvironment in a subject, comprising administering to the subject an antibody-drug conjugate according to any one of claims 1 to 47 or a pharmaceutical composition according to claim 54. method.

56. A method for modulating an immune response in a subject, comprising administering to the subject an antibody-drug conjugate according to any one of claims 1 to 47 or a pharmaceutical composition according to claim 54. method.

57. Use of the antibody-drug conjugate according to any one of claims 1 to 47 or the pharmaceutical composition according to claim 54 in the manufacture of a medicament, wherein the medicament is capable of preventing and / or treating a tumor. use.

58. The tumor comprises a solid tumor and / or a non-solid tumor.

58. The use according to claim 57.

59. The tumor comprises a CD73-mediated tumor.

59. Use according to any one of claims 57 to 58.

60. The tumor comprises pancreatic cancer and / or breast cancer.

60. The use according to any one of claims 57 to 59.

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