Antibody drug conjugate and application thereof

Abstract

The antibody drug conjugate comprises an antibody targeting CD73 or an antigen binding fragment of the antibody, the antibody targeting CD73 or the antigen binding fragment of the antibody targeting CD73 comprises HCDR3, and the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3. The invention also provides a preparation method and application of the antibody drug conjugate.

Description

Antibody-drug conjugates and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antibody-drug conjugate and its application. Background Art

[0002] Cluster of differentiation 73 (CD73) is a surface glycoprotein anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) motif. CD73 has been reported to be expressed in many different cancers. Furthermore, CD73 expression in cancer is associated with increased proliferation, migration, angiogenesis, invasiveness, metastasis, and shorter patient survival. In addition to being effective in immunosuppression, CD73 is abnormally activated and expressed in tumor tissues, and compared to other targets, antibodies targeting CD73 are rapidly and extensively internalized, making CD73 an important target for antibody-drug conjugates (ADCs).

[0003] Antibody-drug conjugates (ADCs) typically consist of three components: an antigen-binding moiety, a small molecule drug, and a linker that connects the antigen-binding moiety to the drug. There is an urgent need to develop highly specific and safe ADCs targeting human CD73.

[0004] Summary of the Invention

[0005] On the one hand, the present application provides an antibody-drug conjugate, which comprises an antibody or an antigen-binding fragment thereof targeting CD73, wherein the antibody or the antigen-binding fragment thereof targeting CD73 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.

[0006] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15 or SEQ ID NO:19.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:21.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:20 or SEQ ID NO:22.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 18.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected 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.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 28.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises L-FR4, the N-terminus of the L-FR4 is connected 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.

[0019] In certain 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, and 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.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises any one group of VH and VL selected from the following: the VH comprises the amino acid sequence of SEQ ID NO: 29, and the VL comprises the amino acid sequence of SEQ ID NO: 30; the VH comprises the amino acid sequence of SEQ ID NO: 31, and the VL comprises the amino acid sequence of SEQ ID NO: 32; the VH comprises the amino acid sequence of SEQ ID NO: 33, and the VL comprises the amino acid sequence of SEQ ID NO: 34; the VH comprises the amino acid sequence of SEQ ID NO: 35, and the VL comprises the amino acid sequence of SEQ ID NO: 36; the VH comprises the amino acid sequence of SEQ ID NO: 33, and the VL comprises the amino acid sequence of SEQ ID NO: 36; and the VH comprises the amino acid sequence of SEQ ID NO: 35, and the VL comprises the amino acid sequence of SEQ ID NO: 34.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody heavy chain constant region.

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

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

[0026] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody light chain constant region.

[0028] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody or an antigen-binding fragment thereof.

[0030] In certain 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.

[0031] In certain embodiments, the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0032] In certain embodiments, the CD73 comprises human CD73 and / or monkey CD73.

[0033] In certain embodiments, the antibody drug conjugate is capable of preventing or reducing the activation of CD73.

[0034] In certain embodiments, the antibody drug conjugate is capable of inhibiting the enzymatic activity of CD73.

[0035] In certain embodiments, the antibody-drug conjugate is capable of inducing internalization of cell surface CD73.

[0036] In certain embodiments, the antibody-drug conjugate comprises a structure represented by Formula 1: M-(L1)a-(L2)bD (Formula 1), wherein M represents an antibody or antigen-binding fragment thereof targeting CD73 as described herein; L1 represents a linker connected to M, L2 represents a linker connected to D, b is independently selected from 0 to 10, and D represents a drug.

[0037] In certain 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.

[0038] In certain embodiments, the L1 is linked to the M through a thiol, azide or amide group on the M.

[0039] In certain embodiments, the L1 is capable of reacting with a sulfhydryl group to form a bond.

[0040] In certain embodiments, the L1 is selected from the group consisting of:

[0041] In certain embodiments, the L2 is selected from the group consisting of polypeptides, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), maleimide PEG NHS, succinamidyl N-4-(maleimidomethyl)cyclohexylcarboxylate (SMCC), sulfosuccinamidyl N-sulfo(4-maleimidomethyl)cyclohexylcarboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecane-1-oate (CX1-1).

[0042] In certain embodiments, the L2 is:

[0043] In certain embodiments, L1-L2 are selected from the group consisting of:

[0044] In certain embodiments, the drug has the ability to kill tumor cells and / or inhibit the growth of tumor cells.

[0045] In certain embodiments, the drug comprises a small molecule drug.

[0046] In certain embodiments, the drug is selected from the group consisting of a V-ATPase inhibitor, a Bcl2 inhibitor, an MCL1 inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), a nuclear export inhibitor of protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphotransferase in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalating agent, a DNA minor groove binder, a DHFR inhibitor, a nucleoside analog, an HD AC inhibitor, an anthracycline, a NAMPT inhibitor, SN-38 or a derivative thereof, an etoposide phosphate, a nitrogen mustard, a proteosome inhibitor, a cytokine, a Tubulysin B analog, and a Toll-like receptor agonist.

[0047] In certain embodiments, the drug is selected from the following group: DM1, exatecan, Dxd, MMAE, SN-38, Calichemicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, eribulin, Tub196, Duo5 toxin, SN38 or its derivatives.

[0048] In certain embodiments, the (L1) a -(L2) b -D has a structure selected from the group consisting of:

[0049] in:

[0050] X1 is an alkane chain or a PEG chain, and,

[0051] X2 is H or -C(O)NR 1 R 2 ,and,

[0052] R 1 is selected from hydrogen, halogen, hydroxy, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Hydroxyalkyl, substituted or unsubstituted C 1-6 Aminoalkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 1-6 Alkanoyl and substituted or unsubstituted C 1-6 Alkanal group;

[0053] R 2 selected from hydrogen and substituted or unsubstituted C 1-6 Alkyl; wherein each substituted C 1-6 The alkyl group is optionally substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy, amino, carbonyl, carboxyl, 5-10 membered heteroaryl, and C 1-6 The haloalkyl group is a 5- to 10-membered heteroaryl group having 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur; and n is an integer greater than or equal to 1 and less than or equal to 20.

[0054] In certain embodiments, the (L1) a -(L2) b -D has a structure selected from the group consisting of:

[0055] ;as well as,

[0056] In certain embodiments, the antibody drug conjugate has a structure selected from the group consisting of:

[0057] In certain embodiments, the antibody drug conjugate has a drug / antibody ratio of about 1 to about 8.

[0058] On the other hand, the present application provides a compound that can be used to prepare the antibody drug conjugate described in the present application, which has a structure shown in Formula 2: M-(L1) a (Formula 2), wherein M represents the antibody or antigen-binding fragment thereof targeting CD73 described in the present application; L1 represents a linker connected to M; and a is selected from 0-10.

[0059] In certain embodiments, the L1 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker.

[0060] In certain embodiments, the L1 is linked to the M through a thiol, azide or amide group on the M.

[0061] In certain embodiments, the L1 is capable of reacting with a sulfhydryl group to form a bond.

[0062] In certain embodiments, the L1 is selected from the group consisting of:

[0063] In another aspect, the present application provides a method for preparing the antibody-drug conjugate described herein, comprising the following steps: contacting the compound described herein with the drug described herein.

[0064] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described herein, and optionally, a pharmaceutically acceptable carrier.

[0065] On the other hand, the present application provides a method for regulating the tumor microenvironment of a subject, comprising the following steps: administering the antibody-drug conjugate described herein or the pharmaceutical composition described herein to the subject.

[0066] On the other hand, the present application provides a method for regulating an immune response in a subject, comprising the following steps: administering the antibody-drug conjugate described herein or the pharmaceutical composition described herein to the subject.

[0067] On the other hand, the present application provides a use of the antibody-drug conjugate described herein, or the pharmaceutical composition described herein in the preparation of a drug, wherein the drug can prevent and / or treat tumors.

[0068] In certain embodiments, the tumor comprises a solid tumor and / or a non-solid tumor.

[0069] In certain embodiments, the tumor comprises a CD73-mediated tumor.

[0070] In certain embodiments, the tumor comprises pancreatic cancer and / or breast cancer.

[0071] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0073] Figures 1A-1D show the schematic structural diagrams, HIC test results and DAR values ​​of the antibody-drug conjugates described in this application.

[0074] Figure 2 shows the binding activity test results of the antibody-drug conjugate described in this application and the CD73 antigen on the cell membrane surface.

[0075] Figure 3 shows the test results of the antibody-drug conjugate described in this application and the inhibition of recombinant CD73 protease activity.

[0076] Figure 4 shows the endocytosis efficiency of the antibody-drug conjugate described in the present application inducing CD73 endocytosis on the cell membrane surface.

[0077] Figure 5 shows the killing curve of the antibody-drug conjugate described in the present application against tumor cells that do not express CD73.

[0078] Figure 6 shows the killing curve of the antibody-drug conjugate described in the present application on tumor cells that highly express CD73.

[0079] Figure 7 shows the tumor inhibitory effect of the antibody-drug conjugate described in the present application on an animal model of pancreatic cancer expressing CD73.

[0080] Figure 8 shows the tumor inhibitory effect of the antibody-drug conjugate described in the present application on an animal model of breast cancer expressing CD73.

[0081] FIG9 shows the endocytosis efficiency of different antigen-binding moieties in inducing CD73 endocytosis on the cell membrane surface.

[0082] Figures 10A-10B show the RP-HPLC profiles of the antibody-drug conjugates described in the present application.

[0083] Figure 11 shows the tumor inhibitory effect of the antibody-drug conjugate described in the present application on an animal model of breast cancer expressing CD73. DETAILED DESCRIPTION

[0084] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0085] Definition of terms

[0086] In this application, the term "antibody drug conjugate" generally refers to ADC, i.e., a binding protein (e.g., an antibody or an antigen-binding fragment thereof) connected to one or more chemical drugs. The chemical drug can be any therapeutic agent and / or cytotoxic agent. The antibody drug conjugate can have any number of drugs coupled to the antibody from 1 to 8, for example, 2, 4, 6 or 8 drug loaded species can be included. In this application, the drug can include mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic protectants, hormones, anti-hormonal agents, corticosteroids, photosensitizers, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and / or radiosensitizers.

[0087] In this application, the term "drug / antibody ratio" or "DAR" generally refers to the number of drugs attached to the antibody of an ADC. The DAR of an ADC can range from 1-8, or can be higher (e.g., 10), and the range of DAR can depend on the number of attachment sites on the antibody. In this application, the DAR can be the number of drugs loaded onto a single antibody. The DAR can also be the average or mean DAR of a group of ADCs.

[0088] In this application, the term "CD73" is also referred to as "NT5E", "cluster of differentiation 73", "5'-nucleotidase (5'-NT)" or "ectocellular 5'-nucleotidase". The term encompasses "full-length", unprocessed CD73 as well as any form of CD73 produced by cellular processing. In this application, the term "CD73" includes full-length wild-type CD73 and mutants, fragments, variants, isoforms and homologs thereof. In certain embodiments, the CD73 may include human CD73. For example, the sequence for human CD73 can be found under Uniprot accession number P21589. In this application, the CD73 may comprise the amino acid sequence shown in SEQ ID NO:40.

[0089] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. 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 a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia, etc. These numbering systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence of the antigen-binding protein can be numbered according to the IMGT numbering scheme (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, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0090] In this application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as a framework region. Typically, the variable domains of natural heavy and light chains each comprise four FR regions, i.e., 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).

[0091] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0092] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. It is generally concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-pleated sheet configuration and are connected by three CDRs, which form a loop connection and, in some cases, form part of the β-pleated sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0093] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, covering any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specific binding to human CD73). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, consist of two to five basic four-chain units that can combine with J chains to form multivalent combinations. For IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a single covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. The VL corresponds to the VH, and the CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to 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. The L chain from any vertebrate species can be divided into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. According to the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.

[0094] As used herein, 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). As used herein, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0095] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As mentioned above, papain can be used to digest intact antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as described above). The Fab fragment can be composed of a complete L chain with the variable region of a heavy chain and the H chain (V H ) of the first constant region (C H 1) Composition.

[0096] As used herein, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0097] As used herein, the term "F(ab')2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. 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 divalent antigen-binding activity and is capable of cross-linking antigens.

[0098] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of approximately 110 amino acids of the heavy and light chains.

[0099] In this application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in this application, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0100] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, with reference to, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and with reference to, for example, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd.

[0101] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation of single molecule composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristic of the antibody obtained from a substantially homogeneous antibody population, and is not to be construed as needing to produce antibodies by any ad hoc method. For example, the monoclonal antibody used in this application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

[0102] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0103] 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 by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, replacements, or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and / or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0104] The term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, then the fully human antibody may contain mouse sugar chains. 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 or in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0105] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are 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 moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0106] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0107] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0108] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. Cells may be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. In certain embodiments, cells are mammalian cells. In certain embodiments, mammalian cells are HEK293 cells.

[0109] In this application, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology A Synthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0110] In this application, the term "linker" generally refers to a bifunctional or multifunctional chemical moiety used to connect the bispecific antibody or antigen-binding fragment thereof described herein to the drug described herein. The linker may include one coupling component or multiple coupling components.

[0111] As used herein, the term "tumor" generally refers to a physiological condition in mammals that is generally characterized by unregulated cell growth. The tumor comprises one or more cancerous cells. The tumor can include solid tumors and / or non-solid tumors.

[0112] As used herein, the term "tumor microenvironment" generally refers to the environment in which a tumor resides, which is the non-cellular regions within the tumor and the intracellular compartments immediately outside the tumor tissue but not belonging to the cancer cells themselves. Tumors are closely associated with the tumor microenvironment and can constantly interact with it. For example, tumors can alter the tumor microenvironment, which can affect the growth and spread of tumors. Typically, the tumor microenvironment has a low pH ranging from 5.8 to 7.0. The tumor microenvironment can have lower concentrations of glucose and other nutrients, but higher concentrations of lactate. In addition, the temperature of the tumor microenvironment can be 0.3 to 1°C higher than normal physiological temperature. The tumor microenvironment has been described in Gillies et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, Vol. 16, pp. 430-450, 2002.

[0113] In this application, the term "treatment" generally refers to both therapeutic treatment and preventive or preventative measures, wherein the goal is to prevent or slow down (mitigate) unwanted physiological changes or illnesses, such as the growth, formation or spread of hyperproliferative conditions such as cancer. For the present invention, favorable or desired clinical outcomes include, but are not limited to: relieving symptoms, weakening the extent of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing the progression of the disease, improving or mitigating the disease state, and rehabilitation (whether partial or complete), whether detectable or undetectable. "Treatment" or "treatment" can also refer to prolonged survival compared to the expected survival without treatment. Subjects in need of treatment include subjects who have long suffered from a disease or illness and subjects who are prone to developing a disease or illness or subjects who are preventing a condition or illness.

[0114] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target 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 one that binds to that target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding may include, but does not require, exclusive binding.

[0115] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0116] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0117] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to the CD73 protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0118] In the present application, the homolog can 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 homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a CD73 protein).

[0119] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0120] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0121] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 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% above or below the specified value.

[0122] Detailed Description of the Invention

[0123] On the one hand, the present application provides a

[0124] Antigen binding proteins

[0125] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can 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 to the amino acid sequence of the CDR. In certain embodiments, the CDRs are identified by the IMGT numbering scheme.

[0126] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0127] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2.

[0128] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0129] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise HCDR3, HCDR2 and HCDR1. For example, the HCDR3 of the antibody or antigen-binding fragment thereof targeting 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.

[0130] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0131] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may further comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5.

[0132] 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.

[0133] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise LCDR3, LCDR2 and LCDR1. For example, LCDR3 of the antibody or antigen-binding fragment thereof targeting CD73 may comprise the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4.

[0134] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1. For example, the antibody or antigen-binding fragment thereof targeting CD73 described in the present application may comprise an amino acid sequence as shown in SEQ ID NO: 3, HCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 2, HCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 1, LCDR3 may comprise an amino acid sequence as shown in SEQ ID NO: 6, LCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 5, and LCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 4.

[0135] 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 directly or indirectly connected 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.

[0136] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise H-FR2, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 16 or SEQ ID NO: 21.

[0137] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise H-FR3, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 20 or SEQ ID NO: 22.

[0138] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise H-FR4, the N-terminus of the H-FR4 may be connected 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.

[0139] In the present application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, the H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. For example, the H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. For example, the H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 18. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the antibody targeting CD73 or its antigen-binding fragment may respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 18.

[0140] 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 directly or indirectly connected 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.

[0141] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise L-FR2, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24.

[0142] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise L-FR3, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 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.

[0143] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise L-FR4, the N-terminus of the L-FR4 may be connected 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.

[0144] 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 respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27 and SEQ ID NO: 26. For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the antibody or antigen-binding fragment thereof targeting CD73 may respectively comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28 and SEQ ID NO: 26.

[0145] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise 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.

[0146] 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.

[0147] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise the VH and VL. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 29, and the VL may comprise the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 31, and the VL may comprise the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 33, and the VL may comprise the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 35, and the VL may comprise the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 33, and the VL may comprise the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the VH may comprise the amino acid sequence of SEQ ID NO: 35, and the VL may comprise the amino acid sequence of SEQ ID NO: 34.

[0148] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise at least one CDR in the VH described herein. In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise at least one CDR in the VL described herein. The CDRs may be obtained by dividing according to any division method.

[0149] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise HCDR1, HCDR2 and HCDR3 in the 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.

[0150] In the present application, the CD73-targeting antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2, and LCDR3 in the VL described herein. 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.

[0151] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0152] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may include an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0153] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 may comprise an antibody or an antigen-binding fragment thereof.

[0154] In certain embodiments, the antigen-binding fragment may include a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv and / or a dAb.

[0155] In certain embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In addition, it should be noted that the antibodies or antigen-binding fragments thereof targeting CD73 described herein may comprise heavy chain and / or light chain sequences with one or more conservative sequence modifications. The so-called "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the antibody. 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 mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. These amino acid residue groups 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), non-polar 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 certain embodiments, one or more amino acid residues in the CDR region of the CD73-targeting antibodies or antigen-binding fragments thereof described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissrov 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.

[0163] The CD73 antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.

[0164] For example, the antigen binding activity of the antigen-binding protein or fusion protein of the present invention can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0165] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 is capable of specifically binding to CD73 or a functionally active fragment thereof.

[0166] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 can be affinity screened by Biacore. The antibody or antigen-binding fragment thereof targeting CD73 can be affinity screened by 5×10 -9 M or less KD to bind to CD73 protein, for example, the antigen binding protein described herein can bind to CD73 protein with a KD of less than about 4×10 -9 M, less than about 3×10 -9 M, less than about 2×10 -9 M, less than about 1×10 -9 M, less than about 7×10 -10 M, less than about 6×10 -10 M, less than about 5×10 -10 M, less than about 4×10 -10 M, less than about 3×10 -10 M, less than about 2×10 -10 M, less than about 1×10 -10 M, less than about 9×10 - 11 M, less than about 5×10 -11 M, less than about 2×10 -11 M KD binds to CD73. In certain embodiments, the present application also includes using a FACS method to determine the binding activity of the antigen-binding protein to the antigen. For example, the antigen-binding protein described herein can bind to cell surface human CD73 with an EC50 value of less than or equal to about 0.5 μg / mL, less than or equal to about 0.4 μg / mL, less than or equal to about 0.3 μg / mL, less than or equal to about 0.2 μg / mL, or less than or equal to about 0.1 μg / mL. For example, the antigen-binding protein described herein can bind to cell surface human CD73 with an EC50 value of less than or equal to about 0.4 μg / mL, less than or equal to about 0.3 μg / mL, less than or equal to about 0.2 μg / mL, or less than or equal to about 0.1 μg / mL.

[0167] In certain embodiments, the antibodies or antigen-binding fragments thereof targeting CD73 are capable of binding to CD73. In some cases, the antigen-binding proteins described herein may also cross-react with monkey CD73. For example, as detected by FACS. In this application, "cross-reactivity" generally refers to the ability of an antibody to react with homologous proteins from other species.

[0168] In certain embodiments, the CD73-targeting antibodies or antigen-binding fragments thereof described herein do not bind to mouse and / or rat CD73.

[0169] For example, the titer can be tested by ELISA. For example, the binding activity or functional activity of the antibody or antigen-binding fragment thereof targeting CD73 can be tested using FACS. For example, the antibody or antigen-binding fragment thereof targeting CD73 can be subjected to affinity testing.

[0170] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 can prevent or reduce the activation of CD73. For example, the antibody or antigen-binding fragment thereof targeting CD73 can prevent or reduce the conversion of CD73 from a catalytically inactive open conformation to a catalytically active closed conformation. For example, the antibody or antigen-binding fragment thereof targeting CD73 can block the CD73 catalytic center, thereby inhibiting the enzymatic activity of CD73.

[0171] In certain embodiments, the binding epitope of the antibody or antigen-binding fragment thereof targeting CD73 can be determined by hydrogen deuterium exchange mass spectrometry (HDX-MS). In certain embodiments, the antibody or antigen-binding fragment thereof targeting CD73 has competitive binding activity with the CPI-006 antibody (Corvus).

[0172] In the present application, when binding to the CD73, the antibody or antigen-binding fragment thereof targeting CD73 binds to at least one amino acid residue in the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37) of the CD73 N-terminal domain or the CD73 sequence corresponding thereto, the 178-SKETPFLSNPGTNL-191 region (SEQ ID NO: 38) or the CD73 sequence corresponding thereto, and the 381-WNHVSM-386 region (SEQ ID NO: 39) of the CD73 C-terminal domain or the CD73 sequence corresponding thereto.

[0173] In the present application, "the CD73 sequence corresponding thereto" may refer to the amino acid sequence at the corresponding position in its homolog (or CD73 from other species).

[0174] 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 can have an effect of inhibiting the enzymatic activity of recombinant CD73. For example, the antibody or antigen-binding fragment thereof targeting CD73 can have an effect of inhibiting the enzymatic activity of human CD73. The enzyme activity described in the present application can be detected by any method commonly used in the art. The detection of the enzyme activity can include mixing the CD73 antigen binding protein with the recombinant CD73 protein, adding AMP and ATP, and Substrate added to CellTiter- Buffer, mix and equilibrate to room temperature for reaction, and perform full wavelength detection. For example, the antibody or antigen-binding fragment thereof targeting CD73 can inhibit the enzymatic activity of recombinant CD73 protein with an EC50 value of less than or equal to about 0.8 μg / mL, less than or equal to about 0.7 μg / mL, less than or equal to about 0.6 μg / mL, less than or equal to about 0.5 μg / mL, less than or equal to about 0.4 μg / mL, less than or equal to about 0.3 μg / mL, or less than or equal to about 0.2 μg / mL. For example, the antibody or antigen-binding fragment thereof targeting CD73 can inhibit the enzymatic activity of CD73 protein on the cell membrane with an EC50 value of less than or equal to about 6 μg / mL, less than or equal to about 5 μg / mL, less than or equal to about 4 μg / mL, less than or equal to about 3 μg / mL, less than or equal to about 2 μg / mL, less than or equal to about 1 μg / mL, or less than or equal to about 0.5 μg / mL.

[0175] In the present application, the antibody or antigen-binding fragment thereof targeting CD73 can induce endocytosis of CD73 on the cell surface. In certain embodiments, the endocytosis of the protein can be determined by a fluorescent labeling method. For example, the efficiency of the antigen-binding protein in inducing endocytosis of CD73 on the cell surface can be obtained by detecting the fluorescence intensity of the cells using flow cytometry. In some cases, the antibody or antigen-binding fragment thereof targeting CD73 can induce endocytosis of CD73 with an efficiency of about 50% or higher.

[0176] Antibody-drug conjugates and compounds

[0177] In another aspect, the present application provides an antibody-drug conjugate.

[0178] In the present application, the antibody-drug conjugate comprises the CD73-targeting antibody or its antigen-binding fragment as described in the present application.

[0179] In the present application, the antibody drug conjugate may comprise a structure as shown in Formula 1: M-(L1) a -(L2) b-D (Formula 1), wherein M represents an antibody or antigen-binding fragment thereof targeting CD73 as described in the present application; L1 represents a linker connected to M, L2 represents a linker connected to D, a and b are each independently selected from 0-10, and D represents a drug.

[0180] In the present application, a compound for preparing the antibody drug conjugate described in the present application has a structure shown in Formula 2: M-(L1) a (Formula 2), wherein M represents the antibody or antigen-binding fragment thereof targeting CD73 described in the present application; L1 represents a linker connected to M; and a is selected from 0-10.

[0181] Modification of M and connection with L1

[0182] In the present application, the L1 and the M can be connected via the thiol, azide or amide group on the M.

[0183] In the present application, the M can be a complete antibody structure (e.g., a complete IgG antibody structure, comprising two identical light chains and two identical heavy chains). In the present application, the light chain constant region, the CH1 of the heavy chain constant region, and / or the hinge region of the M can comprise a connection site capable of connecting to the L1. For example, the light chain constant region, the CH1 of the heavy chain constant region, and the hinge region of the M can comprise a connection site capable of connecting to the L1.

[0184] In the present application, the attachment site may comprise a cysteine. In the present application, the attachment site may comprise a sulfhydryl group in a ligatable state. In the present application, the number of the attachment sites may be 8. In the present application, the attachment sites may include: 2 attachment sites located in the light chain constant region, 2 attachment sites located in CH1 of the heavy chain constant region, and 4 attachment sites located in the hinge region.

[0185] In the present application, the linking site may comprise a group that can be linked to the L1 after deglycosylation modification. In the present application, the linking site may comprise a group that can be linked to the L1 after glycosylation modification.

[0186] The glycosylation of a protein may depend on the amino acid sequence of the protein (e.g., an antibody or antigen-binding fragment thereof targeting CD73 as described herein) and the host cell in which the protein is expressed. Different organisms may produce different glycosylases (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to these factors, the protein glycosylation pattern and the composition of the residues at the glycosylation modification site may vary depending on the host system in which the particular protein is expressed. Glycosyl residues that can be used in the present application may include glucose, galactose, mannose, fucose, n-acetylglucosamine and / or sialic acid. For example, the glycosylation modification may include a pattern of glycosylation modification adapted to humans.

[0187] In the present application, different protein glycosylation can affect and / or lead to different protein properties (e.g., changes in expression level, in vivo half-life, protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or allergenicity, etc.).

[0188] Linker

[0189] In the present application, a may be an integer selected from 0-10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0190] In the present application, b may be an integer selected from 0-10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0191] In the present application, the 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.

[0192] In the present application, both the L1 and the L2 can be considered as a linker.

[0193] In the present application, the linker can be a portion that extends the drug connection to avoid, for example, shielding the active site of the antibody or improving the solubility of the ADC. The linker can include a stretcher unit and / or an amino acid unit.

[0194] In the present application, the linker can be used to couple the antibody (e.g., the antibody targeting CD73 or its antigen-binding fragment described in the present application) to the drug (e.g., by covalent linkage).

[0195] For example, the conjugation can be through a bond between a cysteine ​​thiol or an amine (eg, the N-terminus or an amino acid side chain such as lysine) of the antibody and a functional group of the linker.

[0196] For example, the linker can have a functional group capable of reacting with a free cysteine ​​present on the antibody to form a bond (e.g., a covalent bond). For example, the functional group can include: maleimide, haloacetamides, α-haloacetyl, active esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and / or isothiocyanates.

[0197] In some cases, the linker can include a functional group that is capable of reacting with an electrophilic group present on the antibody. For example, the functional group can include: an aldehyde, a ketocarbonyl, a hydrazine, an oxime, an amino, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and / or an arylhydrazide.

[0198] 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, which may facilitate the release of the drug. For example, the linker (e.g., L1) may be a non-cleavable linker, which may be attached 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., containing a hydrazone), a protease-sensitive linker (e.g., sensitive to peptidases), a photolabile linker, and / or a linker containing a disulfide. For example, the cleavable linker may include a peptide linker that can be cleaved by an intracellular protease (e.g., a lysosomal protease) and / or an endosomal protease. In some cases, the linker is cleavable in the intracellular environment. For example, the cleavage of the linker may allow the drug in the antibody-drug conjugate to exert an effective therapeutic effect.

[0199] In the present application, the structure and / or properties of the linker are stable outside the cell. The structure of the antibody-drug conjugate described in the present application can be stable before being transported or delivered into the cell. For example, in the antibody-drug conjugate, the CD73-targeting antibody or antigen-binding fragment thereof and the drug remain coupled.

[0200] In certain cases, if the linker in the antibody drug conjugate is capable of, for example, maintaining the specific binding properties of the antibody or antigen-binding fragment thereof targeting CD73; participating in the delivery of the antibody drug conjugate, and / or maintaining the therapeutic effect of the drug (e.g., cytotoxic effect).

[0201] In the present application, the linker may include a hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB) and a hydrophobic linker. For example, the hydrophilic linker may reduce the extent to which the antibody drug conjugate can be pumped out of resistant cancer cells via MDR (multidrug resistance) or functionally similar transporters.

[0202] In the present application, the linker can also function to directly or indirectly inhibit cell growth and / or cell proliferation. For example, the linker can function as an intercalator in the cleavage. For example, the linker can inhibit macromolecular biosynthesis.

[0203] In the present application, the linker can facilitate the entry of the antibody drug conjugate into cells (eg, can facilitate "internalization"), and can also be designed to improve the stability of the antibody drug conjugate.

[0204] In the present application, the L1 can react with a thiol group to form a bond.

[0205] In the present application, the L1 can be selected from the following groups:

[0206] In the present application, the L2 can be selected from the following group: polypeptide, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), maleimide PEG NHS, succinamidyl N-4-(maleimidomethyl)cyclohexylcarboxylate (SMCC), sulfosuccinamidyl N-sulfo(4-maleimidomethyl)cyclohexylcarboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecane-1-oate (CX1-1).

[0207] In the present application, L2 can be GGFG, LP, VK, VC, GFG, GGFGG, GGFGS, GGFGGG, GGFGGE, GGFGGGFG, DGGF, DGGFG, D d GGFG、DG MeGFG, DGGFS, DDGGFG, KDGGFG, KGGFG, EGGFG or SGGFG. For example, L2 can be GGFG. The tetrapeptide Gly-Gly-Phe-Gly (GGFG) cleavable by cathepsin B is a highly hydrophilic linker (e.g., stronger than Gly-Phe-Leu-Gly).

[0208] In this application, the L2 can be

[0209] In the present application, the L1-L2 can be selected from the following groups:

[0210] drug

[0211] In the present application, the drug may have the ability to kill tumor cells and / or inhibit the growth of tumor cells.

[0212] In the present application, the drug may include a small molecule drug.

[0213] In the present application, the drug can be selected from the following group: V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansinoids, MetAP (methionine aminopeptidase), nuclear export inhibitors of protein CRM1, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphorylation reaction inhibitors, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HD AC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 glucuronide, etoposide phosphate, nitrogen mustard, proteosome inhibitors, cytokines, Tubulysin B analogs and Toll-like receptor agonists.

[0214] In the present application, the drug can be selected from the following group: DM1, exatecan, Dxd, MMAE, Calichemicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, eribulin, Tub196, Duo5 toxin, SN38 derivatives or their derivatives.

[0215] In the present application, the drug may be an inhibitor of DNA topoisomerase I. In the present application, the drug may be a camptothecin derivative.

[0216] In the present application, Tubulysin B can be a microtubule lysin with cytotoxic activity, which can inhibit tubulin polymerization and cause cell cycle arrest and apoptosis. The CAS number of Tubulysin B is 205304-87-6. In the present application, MMAE, namely Monomethyl auristatin E, is a synthetic derivative of dolastatin 10, which can effectively inhibit mitosis 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 can be SN38 or MMAE.

[0217] For example, the drug may have the following structure:

[0218] or

[0219] In this application, the (L1) a -(L2) b -D may have a structure selected from the group consisting of:

[0220] in:

[0221] X1 is an alkane chain or a PEG chain, and,

[0222] X2 is H or -C(O)NR 1 R 2 ,and,

[0223] R 1 is selected from hydrogen, halogen, hydroxy, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Hydroxyalkyl, substituted or unsubstituted C 1-6 Aminoalkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 1-6 Alkanoyl and substituted or unsubstituted C 1-6 Alkanal group;

[0224] R 2 selected from hydrogen and substituted or unsubstituted C 1-6 Alkyl; wherein each substituted C 1-6The alkyl group is optionally substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy, amino, carbonyl, carboxyl, 5-10 membered heteroaryl, and C 1-6 The haloalkyl group is a 5- to 10-membered heteroaryl group having 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur; and n is an integer greater than or equal to 1 and less than or equal to 20.

[0225] In this application, the (L1) a -(L2) b -D may have a structure selected from the group consisting of:

[0226] as well as

[0227] In the present application, the antibody drug conjugate may have a structure selected from the following group:

[0228] In the present application, the antibody drug conjugate may have a structure selected from the following group:

[0229] In the present application, the drug / antibody ratio of the antibody drug conjugate can be about 1-8. For example, the drug / antibody ratio can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. For example, the drug / antibody ratio can be about 8. For example, the drug / antibody ratio can be about 7.8.

[0230] Compounds and preparation methods

[0231] On the other hand, the present application provides a compound for use in the antibody drug conjugate described in the present application, which has a structure shown in Formula 2: M-(L1) a (Formula 2), wherein M is the antibody or antigen-binding fragment thereof targeting CD73 described in the present application; L1 represents a linker connected to M; and a is selected from 0-10.

[0232] In the present application, the compound may include a structure selected from the group consisting of:

[0233] In another aspect, the present application provides a method for preparing the antibody-drug conjugate described herein, comprising the following steps: contacting the compound described herein with the drug described herein.

[0234] In some cases, the method for preparing the antibody-drug conjugate may include the following steps: (1) contacting L1, L2 and the drug described in the present application; (2) obtaining compound (L1) a -(L2) b-D; (3) M described in this application and the compound (L1) a -(L2) b -D to obtain the antibody drug conjugate described in the present application. For example, M in step (3) is subjected to the glycosylation modification.

[0235] Pharmaceutical compositions and applications

[0236] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described herein, or a pharmaceutically acceptable carrier.

[0237] In the present application, the pharmaceutical composition may include a "therapeutically effective dose" or a "prophylactically effective dose" of the antibody drug conjugate described herein. The "therapeutically effective dose" may be an amount that is effective at the necessary dose and duration to achieve the desired therapeutic outcome. The therapeutically effective dose can be determined by a person skilled in the art and may vary, for example, based on factors such as the disease state, the subject's age, sex, weight, and the ability of the antibody drug conjugate to elicit the desired response in the subject. The "prophylactically effective dose" may be an amount that is effective at the necessary dose and duration to achieve the desired prophylactic outcome. For example, the prophylactically effective dose may be lower than the therapeutically effective dose.

[0238] In the present application, the pharmaceutical composition can be configured to be 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 can include other pharmaceutically active ingredients.

[0239] In the present application, the pharmaceutically acceptable carrier may include any or all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents. The pharmaceutically acceptable carrier may include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The pharmaceutically acceptable carrier may also include isotonic agents, wetting agents, emulsifiers, preservatives, and / or buffers.

[0240] On the other hand, the present application provides a method for regulating the tumor microenvironment of a subject, comprising the following steps: administering the antibody-drug conjugate described herein or the pharmaceutical composition described herein to the subject.

[0241] In another aspect, the present application provides a method for regulating an immune response in a subject, comprising the following steps: administering the antibody-drug conjugate described herein or the pharmaceutical composition described herein to the subject.

[0242] The present application provides the use of the antibody-drug conjugate described herein or the pharmaceutical composition described herein in the preparation of a drug, wherein the drug can regulate the tumor microenvironment of a subject and / or regulate the immune response of the subject.

[0243] The present application provides the antibody-drug conjugate or the pharmaceutical composition described herein, which is used to regulate the tumor microenvironment of a subject and / or regulate the immune response of the subject.

[0244] On the other hand, the present application provides the use of the antibody-drug conjugate described herein or the pharmaceutical composition described herein in the preparation of a drug, wherein the drug can prevent and / or treat tumors.

[0245] The present application provides a method for preventing and / or treating tumors, comprising the following steps: administering the antibody-drug conjugate described herein or the pharmaceutical composition described herein to a subject to prepare a drug.

[0246] The present application provides the antibody-drug conjugate or the pharmaceutical composition described herein, which is used to prevent and / or treat tumors.

[0247] In the present application, the tumor may include solid tumors and / or non-solid tumors.

[0248] For example, the tumor may include breast cancer (eg, triple-negative breast cancer) and / or pancreatic cancer.

[0249] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0250] Example

[0251] The chemical formula of the SN-38 small molecule is as follows:

[0252] 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;

[0253] 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;

[0254] Example 1 Preparation of Antibody Drug Conjugates

[0255] The SN-38 small molecule was conjugated to the 900698 antibody using cysteine ​​coupling, and the drug-to-antibody ratio (DAR) of the ADC molecule was determined by hydrophobic chromatography. The corresponding structure and DAR value are shown in Figures 1A-1D below. Figure 1A is a schematic structural diagram of CD73-ADC, in which the antibody molecule is derived from 900698 and D represents the small molecule drug SN-38; Figure 1B is the molecular structure of two different linker-SN38s; Figure 1C is the HIC test result graph and DAR value of the CD73 antibody-Mc-VC-PAB-DMEDA-PEG-SN38 molecule (i.e., antibody-drug conjugate 1 formed by connecting 900698-molecule 1 in Figure 1B); Figure 1D is the HIC test result graph and DAR value of the CD73 antibody-MP2-VC-PAB-DMEDA-PEG-SN38 molecule (i.e., antibody-drug conjugate 2 formed by connecting 900698-molecule 2 in Figure 1B).

[0256] The results in Figure 1 show that the DAR values ​​of CD73 antibody-Mc-VC-PAB-DMEDA-PEG-SN38 (abbreviated as: antibody-drug conjugate 1) and CD73 antibody-MP2-VC-PAB-DMEDA-PEG-SN38 (abbreviated as antibody-drug conjugate 2) molecules are both around 7.8.

[0257] Example 2 Detection of the Binding Activity of Antibody Drug Conjugates to Cells Expressing Human CD73 Antigen on the Membrane

[0258] The binding activity of antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 naked antibody prepared in Example 1 to cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio) was detected.

[0259] All antigen binding proteins were diluted to 30 μg / ml with PBS solution containing 1% BSA (1% BSA / PBS) and then diluted 3-fold in 10 gradients. 20 μL was added to each well of a 96-well U-shaped plate, and a negative control (1% BSA / PBS alone) was set up simultaneously. A suspension of cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio) in the logarithmic growth phase was taken, centrifuged (1000 rpm × 5 min), the culture medium was discarded, and the cells were resuspended in 1% BSA / PBS to a viable cell density of 1×10 6 / mL, 20 μL per well (2×10 4Cells were added to a 96-well U-shaped plate containing anti-CD73 antigen binding protein and reacted at room temperature for 30 minutes. The 96-well U-shaped plate was resuspended in 1% BSA / PBS and centrifuged (300g × 3 min). The supernatant was discarded and washed once. A 1:200 dilution of PE-goat anti-human-Fc (Jackson ImmunoResearch, #109-115-098) was added and reacted at room temperature in the dark for 15 minutes. The 96-well U-shaped plate was resuspended in 1% BSA / PBS and centrifuged (300g × 3 min). The supernatant was discarded and washed three times. Finally, each well was resuspended in 100 μL of 1% BSA / PBS. The fluorescence intensity of the PE channel was measured using a flow cytometer (BD, #CantoⅡ).

[0260] The binding activity results of antibody drug conjugate 1, antibody drug conjugate 2 and 900698 naked antibody are shown in Figure 2 (represented by 1-3 in Figure 2 respectively). The results show that the three proteins have good affinity to cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio). Antibody drug conjugate 1, antibody drug conjugate 2 and 900698 naked antibody have similar affinities for cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio), indicating that the conjugation of 900698 naked antibody to SN38 small molecule poison did not affect its affinity to cell surface CD73 antigen.

[0261] Example 3 Detection of the Effect of Antibody Drug Conjugates on the Inhibition of Soluble Recombinant CD73 Antigen Protease Activity

[0262] The inhibitory effects of antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 naked antibody prepared in Example 1 on the protease activity of recombinant CD73 protein (ACRO, #CD3-H52H7-100 μg) were detected.

[0263] The specific operation is as follows: Dilute the series of CD73 antigen binding proteins to 120μg / mL with TM Buffer (ROCKLAND, #MB-059), dilute them 3-fold in 10 gradients, and add 25μL / well to a 96-well plate. Then dilute the recombinant CD73 protein to 0.3μg / ml with TM Buffer (ROCKLAND, #MB-059), add 25μL / well to a 96-well plate, and mix well. Then mix the diluted AMP and ATP in a 1:1 ratio, add 50μL / well to the above 96-well plate, and react for 15 minutes. Finally, add CellTiter- Substrate addition Buffer, mix well and equilibrate to room temperature, then add to the wells to be tested, 100 μL / well, react at room temperature for 10 minutes, and detect at full wavelength.

[0264] The results of the inhibition of the enzyme activity of soluble recombinant CD73 antigen protein (ACRO, #CD3-H52H7-100 μg) by antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 naked antibody are shown in Figure 3 (represented by 1-3 in Figure 3 respectively). The results show that antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 have similar effects on the inhibition of soluble recombinant CD73 protease activity, indicating that the coupling of 900698 naked antibody to SN38 small molecule poison has no effect on its ability to inhibit the protease activity of recombinant CD73 antigen.

[0265] Example 4 Binding affinity detection of antibody drug conjugates to human and monkey CD73 antigen proteins

[0266] The binding kinetics of antibody-drug conjugate 1, antibody-drug conjugate 2, and naked antibody 900698 prepared in Example 1 to human CD73 protein (manufacturer: ACRO Biosystems, catalog number: CD3-H52H7) and monkey CD73 protein (manufacturer: Novoprotein, catalog number: CI21) were detected using Biacore (GE, model T200) with HBS-EP+ (pH 7.4) as the experimental buffer and protein A chip (manufacturer: GE, catalog number: 29-1275-56).

[0267] The sample chamber and flow path temperature were set at 25°C. Protein A chips were used to capture antigen-binding proteins as ligands, with approximately 100 RU immobilized. Human CD73 protein was diluted in assay buffer to a series of concentrations, including 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.57 nM, as the analyte. Assay buffer was used as a zero concentration control. Binding was performed for 120 s and dissociation for 600 s at a flow rate of 30 μL / min. Multiple cycles of kinetic analysis were performed using 10 mM Glycine pH 1.5 at a flow rate of 50 μL / min, followed by a regeneration period of 60 s. A 1:1 binding model was used, and the Fit local mode was selected to fit the association rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium rate constant (KD). The fitting results are shown in Table 1, showing that all antigen-binding proteins bound to human and monkey CD73 antigen proteins, and the binding affinities of the molecules antibody-drug conjugate 1 and antibody-drug conjugate 2 to human and CD73 proteins were not significantly different from those of the 900698 naked antibody itself.

[0268] Table 1 Binding affinity test results with human and monkey CD73

[0269] Example 5 Detection of the ability of antibody-drug conjugates to induce internalization of cell surface CD73 antigen

[0270] The antibody drug conjugate 1, antibody drug conjugate 2 and naked antibody 900698 prepared in Example 1 were diluted to 1 μg / ml with PBS solution, and each concentration was repeated in 2 wells. Calu6 cells in the logarithmic growth phase were taken and the cells were prepared into 2×10 5 / ml cell suspension, 100 μL per well (approximately 20,000 cells) was added to a 96-well U-shaped plate, centrifuged at 300g × 3 min, and the supernatant was removed. The cells were then resuspended with diluted antigen-binding protein, 100 μL / well, mixed, and incubated at 4°C for 30 min. After the incubation, the cells were centrifuged at 300g × 3 min, the supernatant was removed, and the cells were resuspended in 1% BSA. Half of the cells were incubated at 37°C and the other half at 4°C for 5 hours. After the incubation, the wells corresponding to the time were removed, centrifuged at 300g × 3 min, the supernatant was removed, and PE anti-huIgG Fc (1:200) was added, 50 μL / well, mixed, and reacted at 4°C for 30 minutes. After the incubation, the cells were washed twice, centrifuged at 300g × 3 min, and the fluorescence intensity was detected by flow cytometry. The results of the anti-CD73 antigen binding protein-induced cell surface CD73 endocytosis test are shown in Table 2 and Figure 4 below (antibody drug conjugate 1, antibody drug conjugate 2, and 900698 are represented by 1-3 in Figure 4, respectively, and NC represents the addition of only 1% BSA solution without the addition of any antibody or antibody drug conjugate). The results show that the ADC molecules antibody drug conjugate 1 and antibody drug conjugate 2 have similar abilities to induce cell surface CD73 endocytosis as the naked antibody 900698, with excellent performance and an endocytosis efficiency of about 95%.

[0271] Table 2 Calculation of the efficiency of inducing cell surface CD73 antigen internalization

[0272] Example 6 Detection of the killing ability of antibody drug conjugates against tumor cells with different CD73 antigen expression levels

[0273] The killing effects of antibody-drug conjugate 1, antibody-drug conjugate 2 and 900698 naked antibody prepared in Example 1 on different breast cancer cells (MDA-MB-231 and MCF-7) were detected, among which MDA-MB-231 highly expressed CD73 while MCF-7 did not express CD73.

[0274] The specific procedures were as follows: MDA-MB-231 cells and MCF-7 cells were treated to cell suspensions at densities of 8E4 / mL and 7.5E5 / mL, respectively, and plated into 96-well cell culture plates at a volume of 100 μL / well. The 96-well cell culture plates were sealed and incubated in a 37°C, 5% CO2 incubator for 6 hours. Antibody-drug conjugate 1, antibody-drug conjugate 2, and 900698 naked antibody were diluted to 400 μg / mL using assay medium. A two-fold serial dilution was performed across eight concentrations, for a total of nine concentrations. The buffer solution was diluted in the same manner as for antibody-drug conjugate 1. The serial dilutions were added to the 96-well cell culture plates at a volume of 50 μL / well. 50 μL of the corresponding assay medium was then added to each well. The 96-well cell culture plates were incubated in a 37°C, 5% CO2 incubator for an additional 5 days. After incubation, remove the 96-well plate from the incubator, aspirate 100 μL of culture medium, and add 100 μL / well of the equilibrated Celltiter-Glo Luminescent Cell Viability Assay (Promega, #G7573). Incubate at room temperature in the dark for 10 minutes with shaking. Then, transfer 100 μL from each well to the corresponding well on the 96-well plate and analyze using a microplate reader.

[0275] The test results in Figure 5 (corresponding to MCF-7 cells) and Figure 6 (corresponding to MDA-MB-231 cells) show that ADC 1 and ADC 2 have excellent killing effects on MDA-MB-231 cells that highly express CD73, but have no killing effect on MCF-7 cells that do not express CD73. Naked antibody 900698 has no killing effect on either cell line. This indicates that ADC 1 and ADC 2 are highly specific in killing tumor cells.

[0276] Example 7 Antitumor Efficacy Detection of Antibody Drug Conjugates in the BxPC3CDX Pancreatic Cancer Mouse Model

[0277] The BxPC-3 pancreatic cancer animal model was established using NPG mice and the efficacy of the test antibodies was tested. The human pancreatic cancer cells BxPC-3 used in this example were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the tenth generation of cell culture, approximately 1×10 7 BxPC-3 cells were mixed with 100 μL of PBS and an equal volume of Matrigel and inoculated subcutaneously into 18 NPG mice on the right side of the back near the armpit. The inoculation volume was approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7PBMC (100 μL), when the tumor grows to an average size of about 50-80 mm 3 At approximately 4 dpi, 18 tumor-bearing mice were randomly divided into 3 groups based on tumor volume and body weight, with 6 mice in each group. Drugs were administered on the day of grouping. The specific dosing schedule is shown in Table 3 below.

[0278] Among them, 900543 is a negative control antibody that does not bind to the CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca and is currently in Phase 2 clinical trials. Uliledlimab is an anti-CD73 monoclonal antibody developed by Tianjin Biopharma and is currently in Phase 1 clinical trials. 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.

[0279] The results are shown in Table 3 and Figure 7 below. Antibody-drug conjugates 1 and 2 were able to significantly inhibit the growth of BxPC-3 pancreatic cancer at a dose of 1 mg / kg. Moreover, the tumor growth inhibition effect was superior to that of the naked antibody 900698 and the positive control antibodies Oleclumab and Uliledlimab at a dose of 5 mg / kg. However, the negative control antibody 900543 was unable to inhibit tumor growth.

[0280] Table 3 Dosing regimen for pancreatic cancer BxPC3 CDX model

[0281] Example 8 Antitumor Efficacy Detection of Antibody Drug Conjugates in the MDA-MB-231 Triple Negative Mammary Mouse CDX Efficacy Model

[0282] The MDA-MB-231 triple-negative breast cancer model was established using NPG mice and the efficacy of the test antibodies was tested. The human breast cancer cells MDA-MB-231 used in this example were cultured in L-15 medium supplemented with 10% FBS in a 37°C incubator without CO2. Before the tenth generation of cell culture, a medium containing approximately 5.0×10 6MDA-MB-231 cells were suspended in 100 μL PBS and mixed with an equal volume of Matrigel. The cells were then inoculated subcutaneously into the right side of the back of NPG humanized mice near the armpit. The inoculation volume was approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7 PBMC (100 μL), when the tumor grows to an average size of about 50-80 mm 3 Around 6:00 p.m., 18 tumor-bearing mice were randomly divided into 7 groups of 6 mice each based on tumor volume and body weight. Dosing was performed on the day of grouping. The specific dosing schedule is shown in Table 4 below. 900201 is a negative control antibody that does not bind to the CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca and is currently in Phase 2 clinical trials. The amino acid sequence of the heavy chain of 900201 is shown in SEQ ID NO. 43; the amino acid sequence of the light chain of 900201 is shown in SEQ ID NO. 44.

[0283] The results, as shown in Table 4 and Figure 8 below, show that ADCs 1 and 2 significantly inhibited the growth of MDA-MB-231 breast cancer cells, with the tumor inhibition effect positively correlated with the dosage. Furthermore, the tumor growth inhibition effect was significantly superior to that of the 900698 naked antibody and the oleclumab positive control antibody at a dose of 5 mg / kg, while the negative control antibody 900201 failed to inhibit tumor growth.

[0284] Table 4 Dosing regimen for triple-negative breast cancer MDA-MB-231 CDX model

[0285] Example 9: Selection of Antibodies for Preparation of Antibody Drug Conjugates

[0286] The endocytosis efficiency of 900698 and 900725 monoclonal antibodies was compared by endocytosis experiment. The operation was as follows: CD73 positive control antibody BMS-986179, 900698 and 900725 anti-CD73 antigen binding proteins were diluted to 1 μg / ml with PBS solution, and each concentration was repeated in 2 wells. Calu6 cells in the logarithmic growth phase were taken and the cells were prepared into 2×10 5100 μL of the 1% / ml cell suspension (approximately 20,000 cells) was added to each well of a 96-well U-shaped plate and centrifuged at 300g for 3 minutes. The supernatant was removed. The cells were then resuspended in the diluted antigen-binding protein at 100 μL / well, mixed, and incubated at 4°C for 30 minutes. After incubation, the cells were centrifuged at 300g for 3 minutes, the supernatant was removed, and the cells were resuspended in 1% BSA. Half of the cells were incubated at 37°C and the other half at 4°C for 5 hours. After incubation, the wells corresponding to the incubation time were removed and centrifuged at 300g for 3 minutes to remove the supernatant. PE anti-huIgG Fc (1:200) was added at 50 μL / well, mixed, and reacted at 4°C for 30 minutes. After incubation, the cells were washed twice at 300g for 3 minutes, and the fluorescence intensity was measured by flow cytometry. The results of the anti-CD73 antigen binding protein-induced cell surface CD73 endocytosis test are shown in Table 5 and Figure 9 below. The results show that the anti-CD73 antibody 900725 induces cell surface CD73 endocytosis efficiency (37.49%) similar to the endocytosis efficiency of the positive control antibody BMS-986179 (44.18%), while the endocytosis efficiency of 900698 is significantly higher than that of 900725 and BMS-986179, reaching approximately 96%. 34%. Figures 1-3 of Figure 9 respectively show the results of BMS-986179, 900725, and 900698, while NC represents the result of adding only 1% BSA solution without adding any antibody or antibody drug conjugate. These results show that 900698 is more suitable for ADC drug development than 900725. The amino acid sequence of the heavy chain of BMS-986179 is shown in SEQ ID NO. 51; the amino acid sequence of the light chain of BMS-986179 is shown in SEQ ID NO. 52.

[0287] Table 5 Calculated values ​​of endocytosis efficiency of 900698 and 900725

[0288] Example 10 Preparation of Antibody Drug Conjugates and Antitumor Effects

[0289] To verify the anti-tumor effect of ADCs prepared by coupling the anti-CD73 antibody 900698 with different linkers and payloads, the tubulin polymerization inhibitor MMAE small molecule was coupled to the 900698 antibody through two different linkers and cysteine ​​coupling, resulting in 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).

[0290] The chemical structures of MC-VC-PAB-MMAE and PY-VC-PAB-MMAE are shown below, respectively. The mAb is antibody 900698. In antibody-drug conjugates 3 and 4, the connection between antibody 900698 and MC-VC-PAB-MMAE or PY-VC-PAB-MMAE is shown in Figure 1A.

[0291] The drug-to-antibody ratio (DAR) of antibody drug conjugates 3 and 4 was determined by hydrophobic chromatography. The DAR values ​​and HPLC spectra (wavelength 280 nm) are shown in Table 6 and Figures 10A-10B.

[0292] Table 6 DAR value and purity test

[0293] To compare the in vivo anti-tumor effects of antibody-drug conjugate 3 and antibody-drug conjugate 4, we used the NPG mouse model inoculated with the human triple-negative breast cancer cell line MDA-MB-231 that highly expresses the CD73 antigen.

[0294] 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 in a 37°C incubator without CO2. Before the cells were cultured for ten consecutive generations, a solution containing approximately 5.0×10 6 MDA-MB-231 cells were suspended in 100 μL PBS and mixed with an equal volume of Matrigel. The cells were then inoculated subcutaneously into the right side of the back of NPG humanized mice near the armpit. The inoculation volume was approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7 PBMC (100 μL), when the tumor grows to an average size of about 50-80 mm 3 Around 60 tumor-bearing mice were randomly divided into 7 groups of 6 mice each based on tumor volume and body weight. Dosing was performed on the day of grouping; the specific dosing schedule is shown in Table 7 below. Twenty-eight days after dosing, the mice were dissected and tumor volume and size were measured to assess antitumor efficacy. The results are shown in Figure 11. As can be seen from the results, both ADC 3 and ADC 4 exhibited superior antitumor efficacy compared to the monoclonal antibody, with comparable antitumor efficacy.

[0295] Table 7 Dosage regimen for triple-negative breast cancer model

[0296] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof targeting CD73, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The antibody-drug conjugate according to any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

1.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

5.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

4.

7. The antibody-drug conjugate according to any one of claims 3 to 6, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 15 or SEQ ID NO:

19.

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

21.

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

22.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

18.

11. The antibody-drug conjugate according to any one of claims 6 to 10, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises 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.

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

24.

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

28.

14. The antibody-drug conjugate according to any one of claims 4 to 13, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises L-FR4, the N-terminus of the L-FR4 is connected 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.

15. The antibody-drug conjugate of any one of claims 1 to 14, wherein 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 of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

6.

16. The antibody-drug conjugate of any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO:

35.

17. The antibody-drug conjugate of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO:

36.

18. The antibody drug conjugate according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises any one group of VH and VL selected from the following: 1) the VH comprises the amino acid sequence shown in SEQ ID NO: 29, and the VL comprises the amino acid sequence shown 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 shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 36 The amino acid sequence shown; 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.

19. The antibody drug conjugate of any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody heavy chain constant region. The antibody drug conjugate according to claim 19 , wherein the antibody heavy chain constant region is derived from a human antibody heavy chain constant region.

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

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

23. The antibody drug conjugate of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody light chain constant region. The antibody drug conjugate of claim 23 , wherein the antibody light chain constant region is derived from a human Igκ constant region.

25. The antibody drug conjugate of any one of claims 1-24, wherein the antibody or antigen-binding fragment thereof targeting CD73 comprises an antibody or antigen-binding fragment thereof.

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

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

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

29. The antibody drug conjugate according to any one of claims 1 to 28, which is capable of preventing or reducing the activation of CD73.

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

31. The antibody drug conjugate according to any one of claims 1-30, which is capable of inducing endocytosis of cell surface CD73.

32. The antibody drug conjugate according to any one of claims 1 to 31, comprising 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 connected to M, L2 represents a linker connected to D, a, b are each independently selected from 0-10, D stands for drug.

33. The antibody drug conjugate of claim 32, wherein 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. 34 . The antibody drug conjugate according to claim 32 , wherein the L1 is linked to the M via a sulfhydryl, azide or amide group on the M. The antibody drug conjugate according to any one of claims 32 to 34 , wherein the L1 is capable of reacting with a sulfhydryl group to form a bond.

36. The antibody drug conjugate according to any one of claims 32 to 35, wherein the L1 is selected from the group consisting of:

37. The antibody drug conjugate of any one of claims 32 to 36, wherein L2 is selected from the group consisting of a polypeptide, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), maleimide PEG NHS, succinamidyl N-4-(maleimidomethyl)cyclohexylcarboxylate (SMCC), sulfosuccinamidyl N-sulfo(4-maleimidomethyl)cyclohexylcarboxylate (sulfo-SMCC), and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecane-1-oate (CX1-1).

38. The antibody drug conjugate according to any one of claims 32 to 37, wherein L2 is:

39. The antibody drug conjugate according to any one of claims 32 to 38, wherein said L1-L2 is selected from the group consisting of:

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

41. The antibody drug conjugate of any one of claims 32-40, wherein the drug comprises a small molecule drug.

42. The antibody drug conjugate of any one of claims 32-41, wherein the drug is selected from the group consisting of a V-ATPase inhibitor, a Bcl2 inhibitor, an MCL1 inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), a nuclear export inhibitor of protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphotransferase in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, 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 proteosome inhibitor, a cytokine, a Tubulysin B analog, and a Toll-like receptor agonist.

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

44. The antibody drug conjugate according to any one of claims 32 to 43, wherein (L1) a -(L2) b -D has a structure selected from the group consisting of: in: X1 is an alkane chain or a PEG chain, and, X2 is H or -C(O)NR 1 R 2 ,and, R 1 is selected from hydrogen, halogen, hydroxy, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Hydroxyalkyl, substituted or unsubstituted C 1-6 Aminoalkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 1-6 Alkanoyl and substituted or unsubstituted C 1-6 Alkanal group; R 2 selected from hydrogen and substituted or unsubstituted C 1-6 Alkyl; wherein each substituted C 1-6 The alkyl group is optionally substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy, amino, carbonyl, carboxyl, 5-10 membered heteroaryl, and C 1-6 The haloalkyl group is a 5-10 membered heteroaryl group having 1-3 heteroatoms selected from oxygen, nitrogen and sulfur; and n is an integer greater than or equal to 1 and less than or equal to 20.

45. The antibody drug conjugate according to any one of claims 32 to 44, wherein (L1) a -(L2) b -D has a structure selected from the group consisting of: as well as, 46. ​​The antibody drug conjugate according to any one of claims 32 to 45, having a structure selected from the group consisting of:

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

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

49. The compound of claim 48, wherein the L1 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker.

50. The compound according to any one of claims 48-49, wherein said L1 is linked to said M via a sulfhydryl, azide or amide group on said M.

51. The compound according to any one of claims 48 to 50, wherein said L1 is capable of reacting with a sulfhydryl group to form a bond.

52. A compound according to any one of claims 48-51, wherein said L1 is selected from the group consisting of:

53. A method for preparing the antibody drug conjugate of any one of claims 1-47, comprising the following steps: The compound of any one of claims 48-52 is contacted with the drug of any one of claims 40-43.

54. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-47, optionally comprising a pharmaceutically acceptable carrier.

55. A method of modulating a tumor microenvironment in a subject, comprising the steps of: The antibody drug conjugate of any one of claims 1 to 47 or the pharmaceutical composition of claim 54 is administered to a subject.

56. A method of modulating an immune response in a subject, comprising the steps of: The antibody drug conjugate of any one of claims 1 to 47 or the pharmaceutical composition of claim 54 is administered to a subject.

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 preparation of a medicament, wherein the medicament can prevent and / or treat tumors.

58. The use according to claim 57, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

59. The use according to any one of claims 57-58, wherein the tumor comprises a CD73-mediated tumor.

60. The use according to any one of claims 57 to 59, wherein the tumor comprises pancreatic cancer and / or breast cancer.

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