CD73 antigen-binding protein and its use

Abstract

An isolated antigen-binding protein capable of binding to CD73 is provided, the isolated antigen-binding protein comprising an HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. Also provided are methods for preparing and uses of the antigen-binding protein.

Description

[Technical Field]

[0001] This application for approval relates to the field of biopharmaceuticals, specifically to the CD73 antigen-binding protein and its use. [Background technology]

[0002] CD73, also known as NT5E, is an extracellular 5-nuclease (NT5E) with a molecular weight of approximately 70 kD. It is fixed to the cell surface by glycosphatidylinositol (GPI) and exists mainly in dimeric form. Under normal physiological conditions, CD73 can be expressed in various tissues or organs, including the liver, colon, kidneys, spleen, lungs, ovaries, and lymph nodes.

[0003] Numerous preclinical studies have shown that CD73 is abnormally overexpressed on the surface of many tumor cells, including gliomas, breast cancers, melanomas, non-small cell lung cancers, bladder cancers, ovarian cancers, and colorectal cancers. Furthermore, clinical data have shown that high CD73 expression is often associated with poor prognosis in tumor patients, suggesting that CD73 may be a potential target for clinical treatment and prognosis in various tumors.

[0004] However, currently, there are no antibodies or small-molecule drugs that control human CD73, and there is an urgent need to develop more drugs that target tumor CD73 in order to provide new ideas and prospects for treating cancers with abnormal CD73 expression.

[0005] Content of the invention This application provides an isolated antigen-binding protein exhibiting one or more desired functional properties, such as: the ability to specifically bind to CD73 or its functionally activated fragment; the ability to block or reduce the activation of CD73; the ability, after binding to CD73, to bind to at least one amino acid residue in the CD73 sequence at amino acids 143-157, amino acids 178-191, and amino acids 381-386; the ability to inhibit the enzymatic activity of CD73; and the ability to induce endocytosis of CD73 on the cell surface. This application also provides a nucleic acid molecule encoding the isolated antigen-binding protein, an expression vector, host cells, and a method for preparing the isolated antigen-binding protein. The isolated antigen-binding protein described in this application can be used for the prevention and / or treatment of diseases and / or disorders such as tumors.

[0006] On the other hand, this application for approval provides an isolated antigen-binding protein, which contains HCDR3. The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 3.

[0007] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2.

[0008] In some embodiments, the isolated antigen-binding protein comprises HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1.

[0009] In some embodiments, the isolated antigen-binding protein comprises LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6.

[0010] In some embodiments, the isolated antigen-binding protein comprises LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5.

[0011] In some embodiments, the isolated antigen-binding protein comprises LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the isolated antigen-binding protein comprises H-FR1. The C-terminus of H-FR1 is directly or indirectly ligated to the N-terminus of HCDR1, and H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:19.

[0013] In some embodiments, the isolated antigen-binding protein comprises H-FR2, which is located between HCDR1 and HCDR2, and which comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:21.

[0014] In some embodiments, the isolated antigen-binding protein comprises H-FR3, which is located between HCDR2 and HCDR3, and which comprises the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:22.

[0015] In some embodiments, the isolated antigen-binding protein comprises H-FR4. The N-terminus of H-FR4 is ligated to the C-terminus of HCDR3, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18.

[0016] In some embodiments, the isolated antigen-binding protein comprises L-FR1. The C-terminus of L-FR1 is directly or indirectly ligated to the N-terminus of LCDR1, and L-FR1 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:23.

[0017] In some embodiments, the isolated antigen-binding protein comprises L-FR2. The L-FR2 is located between the LCDR1 and LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:24.

[0018] In some embodiments, the isolated antigen-binding protein comprises L-FR3. The L-FR3 is located between the LCDR2 and 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.

[0019] In some embodiments, the isolated antigen-binding protein comprises L-FR4. The N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:26.

[0020] In some embodiments, the isolated antigen-binding protein comprises VH. The VH comprises 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 some embodiments, the isolated antigen-binding protein comprises VL. The VL comprises 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 some embodiments, the isolated antigen-binding protein comprises any one selected from the following VH and VL: 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; 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.

[0023] In some embodiments, the isolated antigen-binding protein includes a constant region of the antibody heavy chain.

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

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

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

[0027] In some embodiments, the isolated antigen-binding protein includes a constant region of the antibody light chain.

[0028] In some embodiments, the antibody light chain constant region is derived from the human IgG constant region.

[0029] In some embodiments, the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0030] In some embodiments, the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

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

[0032] In some embodiments, the isolated antigen-binding protein can specifically bind to CD73 or its functionally activated fragment.

[0033] In some embodiments, the CD73 includes human CD73 and / or monkey CD73.

[0034] In some embodiments, the isolated antigen-binding protein can block or reduce the activation of CD73.

[0035] In some embodiments, when binding to CD73, the isolated antigen-binding protein binds to amino acids 143-157 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, to amino acids 178-191 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, and to at least one amino acid residue in amino acids 381-386 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40.

[0036] In some embodiments, amino acids 143-157 or their corresponding CD73 sequences are located in the N-terminal domain of CD73, amino acids 178-191 or their corresponding CD73 sequences are located in the N-terminal domain of CD73, and amino acids 381-386 or their corresponding CD73 sequences are located in the C-terminal domain of CD73.

[0037] In some embodiments, the isolated antigen-binding protein can inhibit the enzymatic activity of CD73.

[0038] In some embodiments, the isolated antigen-binding protein can induce endocytosis of CD73 on the cell surface.

[0039] On the other hand, this application also provides a polypeptide molecule, which comprises the isolated antigen-binding protein.

[0040] In some embodiments, the polypeptide molecule includes a fusion protein.

[0041] On the other hand, this application for approval provides a single isolated nucleic acid molecule, the nucleic acid molecule encoding the isolated antigen-binding protein or the polypeptide molecule.

[0042] On the other hand, this application for approval provides one type of vector, which includes the nucleic acid molecule.

[0043] On the other hand, this application for approval provides a single type of cell, which comprises the nucleic acid molecule and / or the vector.

[0044] On the other hand, this application for approval provides one type of immune conjugate, which comprises the isolated antigen-binding protein.

[0045] On the other hand, this application for approval provides a pharmaceutical composition comprising the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell and / or immunoconjugate, and optionally a pharmaceutically acceptable carrier.

[0046] On the other hand, this application for approval provides a method for preparing the isolated antigen-binding protein, the method comprising culturing the cells under conditions that express the isolated antigen-binding protein.

[0047] On the other hand, this application for approval provides uses for the isolated antigen-binding proteins, polypeptide molecules, nucleic acid molecules, vectors, cells, immune conjugates and / or pharmaceutical compositions in the preparation of drugs, which are used for the prevention and / or treatment of diseases and / or disorders.

[0048] On the other hand, this application for approval provides a method for preventing and / or treating a disease and / or disorder, the method may include administering an isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immune conjugate and / or pharmaceutical composition described in this application to a subject in need.

[0049] On the other hand, the isolated antigen-binding proteins, polypeptide molecules, nucleic acid molecules, vectors, cells, immune conjugates and / or pharmaceutical compositions described in this application for approval can be used for the prevention and / or treatment of diseases and / or disorders.

[0050] In some embodiments, the disease and / or disorder is a CD73-mediated disease and / or disorder.

[0051] In some embodiments, the disease and / or disorder includes a tumor.

[0052] In some embodiments, the tumor includes solid tumors and / or hematological malignancies.

[0053] In some embodiments, the disease and / or disorder includes breast cancer.

[0054] On the other hand, this application for approval provides a method for detecting CD73 in a sample, the method comprising administering the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition.

[0055] On the other hand, this application also provides a reagent or kit for detecting CD73 in a sample, which comprises the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition.

[0056] On the other hand, this application also provides the use of the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition in the preparation of the kit, which is used to detect the presence and / or content of CD73 in a sample.

[0057] Those skilled in the art will readily recognize other aspects and advantages of this application from the following detailed description. In the following detailed description, only exemplary embodiments of this application are shown and described. As will be apparent to those skilled in the art, the contents of this application can be modified by those skilled in the art, but not to the extent that they deviate from the spirit and scope of the invention relating to this application. Accordingly, the descriptions in the accompanying drawings and description of this application are illustrative and not limiting. [Brief explanation of the drawing]

[0058] Description of the attached drawings The specific features of the invention described in this application are as shown in the attached claims. A better understanding of the features and advantages of the invention described in this application can be gained by referring to the exemplary embodiments and attached drawings described in detail below. A brief description of the attached drawings is as follows: Figure 1 shows the binding activity of antigen-binding proteins 900581 and 900565, described in this approval application, to CD73 on the cell surface.

[0059] Figure 2 shows the binding activity of antigen-binding proteins 900581, 900694-900698, and other proteins described in this approval application to CD73 on the cell surface.

[0060] Figure 3 shows that the antigen-binding proteins 900581, 900694-900698 described in this approval application inhibit the enzyme activity of recombinant CD73.

[0061] Figure 4 shows that the antigen-binding proteins 900581 and 900565 described in this approval application suppress the enzymatic activity of CD73 on the cell surface.

[0062] Figure 5 shows that the antigen-binding proteins 900581, 900697, and 900698 described in this approval application suppress the enzymatic activity of CD73 on the cell surface.

[0063] Figure 6 shows the efficiency of inducing endocytosis of CD73 on the cell surface by antigen-binding protein 900581, as described in this application for approval.

[0064] Figure 7 shows a structural model of the antigen-binding epitope described in this application for approval.

[0065] Figures 8A-8C show the competitive binding activity of the antigen-binding protein described in this approval application with 900609.

[0066] Figure 9 shows the effect of the antigen-binding protein described in this application on treating tumors in vivo.

[0067] Figure 10 shows the efficacy of the antigen-binding protein described in this application for approval in treating tumors in vivo.

[0068] Specific Embodiments The following specific embodiments illustrate embodiments of the invention in this application for approval, and those skilled in the art will be able to easily understand other advantages and effects of the invention in this application from the information disclosed herein.

[0069] Definition of Terms In this application for approval, the term "CD73" is also referred to as "NT5E," "differentiation antigen group 73," "5-nucleotidase" (5'-NT), or "extracellular 5-nucleotidase." The aforementioned terms cover "full-length," untreated CD73, and all forms of CD73 produced by cell processing. In this application for approval, the term "CD73" includes full-length wild-type CD73 and its variants, fragments, variants, isoforms, and homologs. In some embodiments, the CD73 may include human CD73. For example, a sequence targeting human CD73 can be found under Uniprot registry number P21589. In this application for approval, the CD73 may include, for example, the amino acid sequence shown in SEQ ID NO: 40.

[0070] In this application for approval, the term "isolated" usually refers to a substance obtained by artificial means from its natural state. When an "isolated" substance or component is found in nature, it may be that its natural environment has changed, or that it has been separated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide may naturally exist within the body of an animal, and a highly purified copy of the same polynucleotide or polypeptide isolated from that natural state is said to be "isolated." The term "isolated" does not rule out the presence of artificial or synthetic substances, nor does it rule out the presence of other impurities that do not affect the activity of the substance.

[0071] In this application for approval, the term "isolated antigen-binding protein" usually refers to a protein that has antigen-binding ability but has been removed from its naturally occurring state. Such "isolated antigen-binding protein" may include a portion that binds to the antigen, or optionally a framework or skeletal portion that allows the antigen-binding portion to adopt a conformation that promotes antigen binding by the antigen-binding portion. Antigen-binding proteins may include, for example, antibody-derived protein framework regions (FRs), or candidate protein framework regions or artificial framework regions having transplanted CDRs or CDR derivatives. These frameworks include, but are not limited to, antibody-derived framework regions into which mutations (e.g., mutations that stabilize the three-dimensional structure of the antigen-binding protein) have been introduced, and fully synthetic framework regions including biocompatible polymers. For example, see "Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004)". Examples of antigen-binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and their fragments.

[0072] In this application for approval, the term "CDR" is also called "complementarity-determining region" and generally refers to a region in the variable domain of an antibody whose sequence is highly variable and / or forms a structural loop. Typically, an antibody contains a total of six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of heavy chains function normally and stably even 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 various numbering systems, such as CCG, Kabat, Chothia, and IMGT, but Kabat / Chothia is generally preferred. These numbering systems are known in the art, and for specifics, see, for example, "http: / / www.bioinf.org.uk / abs / index.html#kabatnum". For example, the numbering of the amino acid sequence of the antigen-binding protein can follow 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 CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, "Kabat EA & Wu TT (1971) Ann NY AcadSci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242").

[0073] In this application for approval, the term "FR" generally refers to the framework region, which is a more conservative portion of the antibody's variable domain. Typically, the natural heavy and light chain variable domains each contain four FR regions, namely four in the very high (VH) region (H-FR1, H-FR2, H-FR3, and H-FR4) and four in the very low (VL) region (L-FR1, L-FR2, L-FR3, and L-FR4).

[0074] In this application for approval, the terms “variable domain” and “variable region” may be used interchangeably and generally refer to parts of the heavy and / or light chain of an antibody. The variable domains of the heavy and light chains may also be called “VH” and “VL” (or “VH” and “VL” respectively). These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and also contain the antigen-binding site.

[0075] In this application for approval, the term "variable" generally refers to significant sequence differences in certain segments of the variable domain among antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed across the entire range of the variable domain. It is typically concentrated in three segments called highly variable regions (CDRs or HVRs) in the light and heavy chain variable domains. More conservative portions of the variable domain are called framework regions (FRs). The natural heavy and light chain variable domains each contain four FR regions, most of which are β-fold structures, linked by three CDRs to form a ring-like linkage, and sometimes forming part of a β-fold structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, promote 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)").

[0076] In this application for approval, the term “antibody” generally refers to immunoglobulins or fragments thereof or derivatives thereof, and covers any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, and transplanted antibodies. For the purposes of this invention, unless specifically modified with the term “complete,” such as “complete antibody,” the term “antibody” also includes antibody fragments (e.g., Fab, F(ab')2, Fv, scFv, Fd, dAb) and other antibody fragments that possess antigen-binding function (e.g., specifically bind to human CD73). Typically, such fragments should contain an antigen-binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and a polypeptide called the J chain, and contain 10 antigen-binding sites. IgA antibodies, on the other hand, contain 2 to 5 basic 4-chain units, and these units can bind and polymerize with the J chain to form polyvalent combinations. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain (L chain) is bound to the heavy chain (H chain) by a covalent disulfide bond, but two H chains are bound to each other by one or more disulfide bonds depending on the H chain isotype. In addition, each H chain and L chain has intrachain disulfide bridge bonds at regular intervals. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in the case of α and γ chains, and followed by four CH domains in the case of μ and ε chain isotypes. Each light chain has a variable domain (VL) at the N-terminus and a constant domain at the other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. It is thought that specific amino acid residues form an interface between the variable domains of the light chain and the heavy chain. VH and VL pair up to form a single antigen-binding site.For the structure and properties of different types 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." Light chains from all vertebrate species can be classified into two distinct types based on the amino acid sequence of their constant domain, called κ chains and λ chains. Immunoglobulins can be classified into different types or isotypes based on the amino acid sequence of the constant domain of their heavy chain (CH). Currently, there are five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each possessing a heavy chain named α, δ, ε, γ, and μ, respectively.

[0077] In this application for approval, the term “antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., CD73). In this application for approval, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0078] In this application for approval, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, a complete antibody may be digested using papain. After the antibody is digested by papain, two identical antigen-binding fragments (i.e., "Fab" fragments) and a remaining "Fc" fragment (i.e., the Fc region, same as above) are produced. The "Fab" fragment consists of one complete light chain or one heavy chain variable region, and its heavy chain (V H ) First steady-state region (C H It consists of (1).

[0079] In this application for approval, 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 the entire light chain, the entire heavy chain variable region, and all or part of the primary and secondary constant regions of the heavy chain. For example, a Fab' fragment may also include 220-330 amino acid residues of part or all of the heavy chain.

[0080] In this application for approval, the term "F(ab')2" generally refers to an antibody fragment produced by digesting a complete antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and can bind to an antigen.

[0081] In this application for approval, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which includes all or part of the heavy chain variable region and light chain variable region, and further lacks the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, the CDR. For example, an Fv fragment includes all or part of the amino-terminal variable region of the heavy chain and light chain, which consists of approximately 110 amino acids.

[0082] In this application for approval, the term "scFv" generally refers to a fusion protein comprising an antibody fragment of at least one variable region including a light chain and an antibody fragment of at least one variable region including a heavy chain, wherein the described light chain and heavy chain variable regions are adjacent (e.g., adjacent by a synthetic linker such as a short, flexible polypeptide linker), and can be expressed in the form of a single-chain polypeptide, and the scFv retains its complete antibody-derived specificity. Unless otherwise specified, as used in this application for approval, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and may include VL-linker-VH or VH-linker-VL.

[0083] In this application for approval, the term "dAb" generally refers to having a VH domain, a VL domain, or an antigen-binding fragment of a VH domain or VL domain. For example, see "Ward et al. (Nature, 1989 Oct 12;341(6242):544-6)", "Holt et al., Trends Biotechnol., 2003, 21(11):484-490", and other published patent applications such as WO 06 / 030220, WO 06 / 003388, and Domantis Ltd.

[0084] In this application for approval, the term "monoclonal antibody" generally refers to a preparation of an antibody molecule consisting of a single molecule. Monoclonal antibodies generally have high specificity for a single antigen site. Furthermore, unlike conventional polyclonal antibody preparations (which usually have different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized by hybridoma culture and not being contaminated by other immunoglobulins. The modifier "monoclonal" basically indicates the characteristic of antibodies obtained from a homogeneous antibody population and is not interpreted as requiring the antibody to be produced by a specific method. For example, the monoclonal antibodies used in this application for approval may be prepared in hybridoma cells or by recombinant DNA technology.

[0085] In this approval application, the term "chimeric antibody" generally refers to an antibody in which the variable region originates from one species, but the constant region originates from another species. Typically, the variable region originates from antibodies of experimental animals such as rodents ("parental antibodies"), and the constant region originates from human antibodies. Therefore, the resulting chimeric antibodies are less likely to cause harmful immune responses in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0086] In this application for approval, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced with amino acids derived from human immunoglobulin. Minor additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region are also permitted, as long as the antibody retains its ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulin. Humanized antibodies retain antigen specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may contain, to a minimum, a chimeric antibody derived from the sequence of a non-human immunoglobulin. In some cases, residues in the CDR region of a human immunoglobulin (acceptor antibody) may be replaced with residues in the CDR region of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, residues in the FR region of a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in acceptor or donor antibodies. These modifications are performed to further improve antibody performance, such as binding affinity.

[0087] The term "fully human antibody" typically refers to an antibody containing only the protein sequence of human immunoglobulin. Fully human antibodies may contain mouse glycans when produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, the terms "mouse antibody" or "rat antibody" refer to antibodies containing only the immunoglobulin sequence of mouse or rat, respectively. Fully human antibodies can be produced in humans and in transgenic animals possessing human immunoglobulin germline sequences by phage display or other molecular biological methods. Exemplary techniques that can be used to produce 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 also known in the art.

[0088] In this application for approval, the terms "polypeptide molecule," "polypeptide," and "peptide" may be used interchangeably and generally refer to polymers of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked parts. In this polypeptide, each part may have different properties. These properties may be biological properties such as in vitro or in vivo activity. Alternatively, these properties may be simple chemical or physical properties such as binding to a target molecule or catalysis of a reaction. The two parts may be directly linked by a single peptide bond or by a peptide linker.

[0089] In this application for approval, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides, or ribonucleotides in any isolated form of any length, or analogs thereof isolated from their natural environment or synthesized artificially.

[0090] In this application for approval, the term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted, and which then expresses the protein. Vectors are expressed by transforming, transfecting, or transfecting host cells, thereby causing the genetic material elements they carry to be expressed within the host cells.

[0091] Examples of vectors include plasmids; phage particles; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages; and animal viruses. Animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A single vector may contain various elements that control expression, such as promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, vectors may also contain origins of replication. Vectors may contain, but are not limited to, components that assist in cell entry, such as viral particles, liposomes, or protein shells.

[0092] In this application for approval, the term “cell” generally refers to a single cell, cell line, or cell culture of a possible subject or acceptor of the plasmid or vector of the subject, and includes the nucleic acid molecules or vectors described in the present invention. Cells may include single cell passages. Passages may not necessarily be identical to the original parent cell (in terms of the morphology of the total complementary DNA or the genome) due to spontaneous, accidental, or intentional mutations. Cells include cells transfected in vitro with the vectors described in this application for approval. 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, etc. In some embodiments, cells are mammalian cells. In some embodiments, mammalian cells are HEK293 cells.

[0093] In this application for approval, the term "immune conjugate" generally refers to a conjugate formed by a composite (e.g., covalent bond by molecular linkage) of the aforementioned other drug (e.g., chemotherapeutic drugs, radioactive elements, cell proliferation inhibitors, and cytotoxic drugs) with the aforementioned antibody or its antigen-binding fragment, and the conjugate specifically binds to the antigen on the target cell by the aforementioned antibody or its antigen-binding fragment, thereby delivering the aforementioned other drug to the target cell (e.g., tumor cell).

[0094] In this application for approval, the term "pharmaceutical composition" generally refers to a composition used for the prevention / treatment of a disease or disorder. The pharmaceutical composition may include the isolated antigen-binding protein, nucleic acid molecule, vector and / or cell described in this application, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the acceptor at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, cryogenic, and lyophilized compositions.

[0095] In this application for approval, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable vectors, excipients, or stabilizers that are non-toxic to cells or mammals to which they are exposed at commonly used doses and concentrations. Examples of physiologically acceptable vectors include buffers, antioxidants, low molecular weight (less than approximately 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium, and / or nonionic surfactants.

[0096] In this application for approval, the terms “specific binding” or “specific” generally refer to measurable and reproducible interactions that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules), such as the binding of a target to an antibody. For example, an antibody that specifically binds to a target (which may be an epitope) may have greater affinity, affinity, and bind to that target more easily and / or for a longer time than an antibody that binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein, and the epitope is conservative across different species of protein. In some embodiments, specific binding may include, but does not require, exclusive binding.

[0097] In this application for approval, the term "subject" generally refers to humans or non-human animals, including but not limited to cats, dogs, horses, pigs, dairy cows, sheep, rabbits, mice, rats, or monkeys.

[0098] The term “tumor” typically refers to any new pathological tissue hyperplasia. Tumor cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. In this application for approval, “tumor” includes both benign and malignant tumors. In this application for approval, “tumor” includes solid tumors and / or hematological malignancies. In this application for approval, “tumor” includes cancer. In this application for approval, examples of “tumor” include, but are not limited to, glioma, breast cancer, melanoma, non-small cell lung cancer, bladder cancer, ovarian cancer, and colorectal cancer.

[0099] In this application for approval, the term "activation" generally refers to the conversion from an inactive state to an active state. In some embodiments, blocking or reducing the activation of CD73 refers to blocking or reducing the conversion of CD73 from an extended conformation without catalytic activity to a bent conformation with catalytic activity.

[0100] In this application for approval, the term “reduction” generally refers to a reduction in quantity and / or degree compared to the original level. For example, the reduction of CD73 activation means reducing the CD73 activation to at least about 0.5 times, at least about 1 time, at least about 1.5 times, at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times compared to the absence of the isolated antigen-binding protein.

[0101] In this application for approval, the proteins, polypeptides, and / or amino acid sequences involved should be understood to include at least variants or homologs having the same or similar function as the aforementioned proteins or polypeptides.

[0102] In this application for approval, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted, or added to the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to the CD73 protein). For example, the functional variant may include a protein or polypeptide having an amino acid change due to at least one, for example, 1 to 30, 1 to 20, or 1 to 10, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant can substantially maintain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion, or addition). For example, the functional variant can maintain 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.

[0103] In this application for approval, the homolog may be a protein or polypeptide having at least about 85% sequence identity (for example, 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 identity) with the amino acid sequence of the protein and / or polypeptide (for example, an antibody or fragment thereof that specifically binds to the CD73 protein).

[0104] In this application for approval, the aforementioned identity generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence identity percentage" is calculated by the following method: comparing two sequences as comparison subjects within a comparison window, determining the number of matching positions by determining the number of nucleic acid bases (e.g., A, T, C, G, I) or amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) that match in both sequences, dividing the total number of positions within the comparison window (i.e., the window range) by the number of matching positions, and multiplying the result by 100 to calculate the sequence identity percentage. The comparison performed to confirm the sequence identity percentage can be achieved by various methods known in the industry. For example, using disclosed computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for comparing sequences, including any algorithm necessary to achieve the maximum possible comparison within the full-length sequence range or target sequence region being compared. The identity can also be measured by, for example, FASTA and BLAST. For a description of the FASTA algorithm, see "Improved Tools for Biological Sequence Comparison" by WR. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988, and "Fast and Sensitive Protein Similarity Search" by D.J. Lipman and WR. Pearson, Science, 227: 1435-1441, 1989. For a description of the BLAST algorithm, see "Basic Local Alignment Search Tools" by S. Altschul, W. Gish, W. Miller, EWMyers, and D. Lipman, Journal of Molecular Biology, 215: 403-410, 1990.

[0105] In this application for approval, the term "includes" generally means "to encompass," "to summarize," "to contain," or "to include." In some cases, it may also mean "to be" or "to consist of."

[0106] In this application for approval, the term "approximately" typically refers to a variation within a range of 0.5% to 10% greater than or less than the specified number. 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% greater than or less than the specified number.

[0107] Detailed description of the invention Isolated antigen-binding protein 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 come into contact with an antigen or antigenic epitope. Antibody CDRs can be identified using various numbering systems, such as CCG, Kabat, Chothia, and IMGT; Kabat / Chothia is generally preferred. These numbering systems are well-known in the art; for specifics, see, for example, "http: / / www.bioinf.org.uk / abs / index.html#kabatnum". Those skilled in the art can identify the CDR region using different numbering systems based on the antibody sequence and structure. When using different numbering systems, differences in the CDR region may exist. In this application for approval, the CDR covers CDR sequences obtained according to any CDR classification scheme; and also covers variants thereof, the variants comprising the amino acid sequence of the CDR after one or more amino acid substitutions, deletions and / or additions, for example, 1 to 30, 1 to 20, or 1 to 10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions and / or insertions; and further covers homologs thereof, the homologs may be amino acid sequences having at least about 85% sequence identity with the amino acid sequence of the CDR (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity). In some embodiments, the CDR is verified by the IMGT numbering scheme.

[0108] On the other hand, this application for approval provides an isolated antigen-binding protein containing HCDR3, wherein HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 3.

[0109] In this application for approval, the isolated antigen-binding protein may also contain HCDR2, and the HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 2.

[0110] In this application for approval, the isolated antigen-binding protein may also contain HCDR1, and the HCDR1 may contain the amino acid sequence shown in SEQ ID NO: 1.

[0111] In this application for approval, the isolated antigen-binding protein may include HCDR3, HCDR2, and HCDR1. For example, the isolated antigen-binding protein HCDR3 may include the amino acid sequence shown in SEQ ID NO: 3, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, and HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1.

[0112] In this application for approval, the isolated antigen-binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6.

[0113] In this application for approval, the isolated antigen-binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5.

[0114] In this application for approval, the isolated antigen-binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4.

[0115] In this application for approval, the isolated antigen-binding protein may include LCDR3, LCDR2, and LCDR1. For example, the isolated antigen-binding protein LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5, and LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4.

[0116] In this application for approval, the isolated antigen-binding protein may include HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1. For example, HCDR3 of the isolated antigen-binding protein described in this application for approval may include the amino acid sequence shown in SEQ ID NO: 3, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, HCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5, and LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4.

[0117] In this application for approval, the isolated antigen-binding protein may include H-FR1. The C-terminus of H-FR1 may be directly or indirectly ligated to the N-terminus of HCDR1. H-FR1 may include the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 15, or SEQ ID NO: 19.

[0118] In this application for approval, the isolated antigen-binding protein may include H-FR2. The H-FR2 may be located between HCDR1 and HCDR2. The H-FR2 may include the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 16, or SEQ ID NO: 21.

[0119] In this application for approval, the isolated antigen-binding protein may contain H-FR3. The H-FR3 may be located between HCDR2 and HCDR3. The H-FR3 may contain the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 22.

[0120] In this application for approval, the isolated antigen-binding protein may include H-FR4. The N-terminus of H-FR4 may be ligated to the C-terminus of HCDR3. The H-FR4 may include the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18.

[0121] In this application for approval, the antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 18, respectively.

[0122] In this application for approval, the isolated antigen-binding protein may include L-FR1. The C-terminus of L-FR1 may be directly or indirectly ligated to the N-terminus of LCDR1. L-FR1 may include the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:23.

[0123] In this application for approval, the isolated antigen-binding protein may include L-FR2. The L-FR2 may be located between LCDR1 and LCDR2. The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24.

[0124] In this application for approval, the isolated antigen-binding protein may include the L-FR3. The L-FR3 may be located between the LCDR2 and LCDR3. The L-FR3 may include the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 28.

[0125] In this application for approval, the isolated antigen-binding protein may include L-FR4. The N-terminus of L-FR4 may be ligated to the C-terminus of LCDR3. The L-FR4 may include the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:26.

[0126] In this application for approval, the isolated antigen-binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4. For example, the isolated antigen-binding protein L-FR1, L-FR2, L-FR3, and L-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. For example, the isolated antigen-binding protein L-FR1, L-FR2, L-FR3, and L-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively. For example, the isolated antigen-binding proteins L-FR1, L-FR2, L-FR3, and L-FR4 may each contain the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 26, respectively.

[0127] In this application for approval, the isolated antigen-binding protein may contain VH. The VH may contain the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 35.

[0128] In this application for approval, the isolated antigen-binding protein may include VL. The VL may include the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

[0129] In this application for approval, the isolated antigen-binding protein may include VH and VL. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 29, and VL may include the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 31, and VL may include the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 33, and VL may include the amino acid sequence shown in SEQ ID NO: 34. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 35, and VL may include the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 33, and VL may include the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, VH may include the amino acid sequence shown in SEQ ID NO: 35, and VL may include the amino acid sequence shown in SEQ ID NO: 34.

[0130] In this application for approval, the isolated antigen-binding protein may contain at least one CDR of VH as described in this application for approval. In this application for approval, the isolated antigen-binding protein may contain at least one CDR of VL as described in this application for approval. The CDR may be obtained according to any classification scheme.

[0131] In this application for approval, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3 of the VH described in this application for approval. The VH may also include the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35.

[0132] In this application for approval, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3 of the VL described in this application for approval. The VL may include the amino acid sequences shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.

[0133] In this application for approval, the isolated antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from the human IgG heavy chain constant region. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the human IgG1 heavy chain constant region.

[0134] In this application for approval, the isolated antigen-binding protein may include the constant region of the antibody light chain. The constant region of the antibody light chain may be derived from the human Igκ constant region.

[0135] In this application for approval, the isolated antigen-binding protein may include an antibody or its antigen-binding fragment.

[0136] In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0137] In some embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0138] For example, the VH portion of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO: 29, and the VL portion of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO: 30.

[0139] For example, the VH of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 31, and the VL of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 32.

[0140] For example, the VH of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 33, and the VL of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 34.

[0141] For example, the VH of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 35, and the VL of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 36.

[0142] For example, the VH of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 33, and the VL of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 36.

[0143] For example, the VH of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 35, and the VL of the humanized antibody may include the amino acid sequence shown in SEQ ID NO: 34.

[0144] It should be noted that the isolated antigen-binding proteins described in this application may contain one or more conservative sequence modifications in their heavy and / or light chain sequences. “Conservative sequence modifications” refer to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described in this application by standard techniques known in the art (e.g., point mutations or PCR-mediated mutations). Conservative amino acid substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in this application may be substituted with other amino acid residues from the same side chain group.Those skilled in the art know that modifications to certain conserved sequences do not result in the loss of antigen-binding properties, specifically, for example, "Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. See 6:2835-43.

[0145] The CD73 antigen-binding proteins described in this application can be identified, screened, or represented by various assay methods known in the art.

[0146] For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this approval application can be measured by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, and immunofluorescence.

[0147] In this application for approval, the isolated antigen-binding protein can specifically bind to CD73 or its functionally activated fragment.

[0148] In some embodiments, the isolated antigen-binding protein can be subjected to affinity screening using Biacore. The isolated antigen-binding protein is 5 × 10 -9 The CD73 protein can be bound to KD values ​​of M or less. For example, the antigen-binding protein described in this application has a binding capacity of approximately 4 × 10⁶. -9 Less than M, approximately 3 x 10 -9Less than M, approximately 2×10 -9 Less than M, approximately 1×10 -9 Less than M, approximately 7×10 -10 Less than M, approximately 6×10 -10 Less than M, approximately 5×10 -10 Less than M, approximately 4×10 -10 Less than M, approximately 3×10 -10 Less than M, approximately 2×10 -10 Less than M, approximately 1×10 -10 Less than M, approximately 9×10 -11 Less than M, approximately 5×1×10 -11 Less than M, approximately 2×10 -11 It can bind to CD73 with KD less than M. In some embodiments, this approval application also includes measuring the binding activity between the antigen-binding protein and the antigen by the FACS method. For example, the antigen-binding protein described in this approval application can bind to human CD73 on the cell surface with an EC50 value of about 0.5 μg / mL or less, about 0.4 μg / mL or less, about 0.3 μg / mL or less, about 0.2 μg / mL or less, about 0.1 μg / mL or less. For example, the antigen-binding protein described in this approval application can bind to human CD73 on the cell surface with an EC50 value of about 0.4 μg / mL or less, about 0.3 μg / mL or less, about 0.2 μg / mL or less, about 0.1 μg / mL or less.

[0149] In some embodiments, the isolated antigen-binding protein can bind to CD73. In some embodiments, the antigen-binding protein described in this approval application can also cross-react with cynomolgus CD73. For example, it is measured by FACS. In this approval application, "cross-reaction" generally refers to the ability of an antibody to react with an identical protein from another species.

[0150] In some embodiments, the isolated antigen-binding protein described in this approval application does not bind to mouse and / or rat CD73.

[0151] For example, efficacy can be measured by ELISA. For example, the binding activity or functional activity of the isolated antigen-binding protein can be measured by FACS. For example, affinity can be measured for the isolated antigen-binding protein.

[0152] In this application for approval, the isolated antigen-binding protein can block or reduce the activation of CD73. For example, the isolated antigen-binding protein can block or reduce the conversion of CD73 from an extended conformation without catalytic activity to a bent conformation with catalytic activity. For example, the isolated antigen-binding protein can suppress the enzymatic activity of CD73 by closing the catalytic center of CD73.

[0153] In some embodiments, the binding epitope of the isolated antigen-binding protein can be measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). In some embodiments, the isolated antigen-binding protein has competitive binding activity with the CPI-006 antibody (Corvus).

[0154] In this approval application, when the isolated antigen-binding protein binds to CD73, it binds to at least one amino acid residue in the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37) or the corresponding CD73 sequence of the N-terminal domain of CD73, the 178-SKETPFLSNPGTNL-191 region (SEQ ID NO: 38) or the corresponding CD73 sequence, and the 381-WNHVSM-386 region (SEQ ID NO: 39) or the corresponding CD73 sequence of the C-terminal domain of CD73.

[0155] In this approval application, "corresponding CD73 sequence" refers to the amino acid sequence at the corresponding position in its homolog (or CD73 from another species).

[0156] In this application for approval, the isolated antigen-binding protein can inhibit the enzymatic activity of CD73. For example, the isolated antigen-binding protein has an enzymatic activity inhibitory effect on recombinant CD73. For example, the isolated antigen-binding protein has an enzymatic activity inhibitory effect on human CD73. The enzymatic activity described in this application for approval can be measured by any method commonly used in the art. The measurement of the enzymatic activity may include mixing the CD73 antigen-binding protein and recombinant CD73 protein, adding AMP and ATP, adding the CellTiter-Glo® substrate to the CellTiter-Glo® buffer, mixing well, equilibrating to room temperature and allowing the reaction to proceed, and measuring the activity at all wavelengths. For example, the isolated antigen-binding protein can inhibit the enzymatic activity of recombinant CD73 protein at EC50 values ​​of approximately 0.8 μg / mL or less, approximately 0.7 μg / mL or less, approximately 0.6 μg / mL or less, approximately 0.5 μg / mL or less, approximately 0.4 μg / mL or less, approximately 0.3 μg / mL or less, and approximately 0.2 μg / mL or less. For example, the isolated antigen-binding protein can inhibit the enzymatic activity of CD73 protein on the cell membrane at EC50 values ​​of approximately 6 μg / mL or less, approximately 5 μg / mL or less, approximately 4 μg / mL or less, approximately 3 μg / mL or less, approximately 2 μg / mL or less, approximately 1 μg / mL or less, and approximately 0.5 μg / mL or less.

[0157] In this application for approval, the isolated antigen-binding protein can induce endocytosis of CD73 on the cell surface. In some embodiments, protein endocytosis can be determined by a fluorescent labeling method. For example, by using flow cytometry and measuring the fluorescence intensity of cells, the efficiency of the antigen-binding protein inducing endocytosis of CD73 on the cell surface can be determined. In some embodiments, the isolated antigen-binding protein can induce endocytosis of CD73 with an efficiency of about 50% or more.

[0158] Polypeptide molecules, nucleic acid molecules, vectors, cells, immune conjugates, and pharmaceutical compositions On the other hand, this application for approval provides a polypeptide molecule, which may contain the isolated antigen-binding protein described in this application for approval.

[0159] In some embodiments, the polypeptide molecule may include a fusion protein.

[0160] On the other hand, this application for approval provides an isolated nucleic acid molecule that can encode an isolated antigen-binding protein described in this application. For example, it can be produced or synthesized by the following methods: (i) amplification in vitro, e.g., by polymerase chain reaction (PCR); (ii) production by clonal recombination; (iii) purification, e.g., by classification and separation by enzymatic treatment and gel electrophoresis; or (iv) synthesis, e.g., by chemical synthesis.

[0161] On the other hand, this application for approval provides a single type of vector, which may contain the nucleic acid molecule described in this application. The vector may also contain other genes. For example, a marker gene of the vector can be selected in a suitable host cell under suitable conditions. The vector may also contain expression regulatory elements that enable the correct expression of the coding region in a suitable host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that control gene transcription or mRNA translation. The vector can be expressed by transforming, transfecting, or transfecting host cells so that the genetic material elements supported therein are expressed within the host cells. The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in genetic engineering. For example, the vector is an expression vector. The vector may contain, but is not limited to, components that assist in cell entry, such as viral particles, liposomes, or protein shells.

[0162] On the other hand, this application provides a single type of cell, which may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each type or individual host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each type or individual host cell may contain more (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vector described in this application may be introduced into the host cell (e.g., a eukaryotic cell such as a plant-derived cell, fungal or yeast cell). In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or another eukaryotic cell, such as a COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell, or myeloma cell. The vector described in this application can be introduced into the host cell by methods known in the art. For example, electroporation, lipofectin transfection, lipofectamine transfection, etc.

[0163] On the other hand, this application also provides an immune conjugate, which may contain the isolated antigen-binding protein described in this application.

[0164] In some embodiments, the isolated antigen-binding proteins or fragments thereof described in this application can be ligated with other agents such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes, and beam splitter probes. The ligation may involve interaction by one or more covalent or non-covalent bonds, and may also involve chelation. Various linkers (such as those known in the art) may be used to form an immunoconjugate. The immunoconjugate may also be provided in the form of a fusion protein and expressed by a polynucleotide encoding the immunoconjugate. The immunoconjugate may also include, for example, an antibody-drug conjugate (ADC). Suitable agents include cytotoxic agents, alkylating agents, DNA groove binding molecules, DNA intercalator agents, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, protease inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, antibodies and therapeutic agents can be crosslinked by cleavable linkers, such as peptide linkers, disulfide linkers, or hydrazine linkers.

[0165] On the other hand, this application also provides a pharmaceutical composition which may include the isolated antigen-binding protein, peptide molecule, immunoconjugate, nucleic acid molecule, vector and / or cell described in this application, and optionally a pharmaceutically acceptable carrier.

[0166] In some embodiments, the pharmaceutical composition may contain one or more suitable formulations such as (pharmaceutically effective) auxiliaries, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably nontoxic to the acceptor at the dose and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, cryogenic, and freeze-dried compositions.

[0167] In some embodiments, the pharmaceutical composition may also contain one or more active compounds, typically compounds having complementary activities that do not adversely affect each other. The type and effective amount of such drugs are determined, for example, by the amount and type of antagonists present in the formulation and the target clinical parameters.

[0168] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersions, coatings, isotonic agents, and absorption retarders suitable for drug administration, and is generally safe and non-toxic.

[0169] In some embodiments, the method of administering the pharmaceutical composition may include parenteral, transdermal, intracavitary, intra-arterial, intrathecal and / or nasal administration, or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the administration of the pharmaceutical composition may be carried out by different means such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the pharmaceutical composition may be administered without interruption. Such uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the amount of therapeutic agent entering the patient's body, as described in WO2015 / 036583.

[0170] Preparation method On the other hand, this application for approval provides a method for preparing the antigen-binding protein. The method may include culturing the host cells described in this application under conditions that express the antigen-binding protein. This may be done, for example, by using methods known to those skilled in the art, such as appropriate culture medium, appropriate temperature, and culture time.

[0171] Any method suitable for the production of monoclonal antibodies can be used to produce the antigen-binding protein of this application. For example, animals can be immunized with the linked or naturally occurring CD73 or a fragment thereof. Appropriate immunization methods such as adjuvants, immunostimulants, or repeated booster inoculations may be used, and one or more approaches may be used. For example, spleen cells from already immunized mice can be obtained using a hybridoma preparation method, fused with SP2 / 0 myeloma cells, and the hybridoma cell line can be screened by HAT.

[0172] Any suitable form of CD73 can be used as an immunogen (antigen) to generate specific non-human antibodies against CD73 and to screen the biological activity of said antibodies. For example, the immunogen for immune stimulation may be full-length mature human CD73, such as a natural homodimer, or a peptide containing one or more epitopes. The immunogen may be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0173] Chimeric human antibodies may be selected from any type of immunoglobulin, such as IgM, IgD, IgG, IgA, and IgE. In this application, the antibody may be an IgG antibody, and the IgG1 isotype may be used. By screening the antibody using the biological assay described in the embodiments below, the desired constant domain sequence optimization is achieved to generate the desired biological activity. Similarly, any type of light chain can be used in the compound and method of this application. For example, a κ chain or a variant thereof can be used in the compound and method of this application.

[0174] Methods and Uses On the other hand, this application for approval provides uses for the isolated antigen-binding proteins, polypeptide molecules, nucleic acid molecules, vectors, cells, immune conjugates and / or pharmaceutical compositions in the preparation of drugs, which are used for the prevention and / or treatment of diseases and / or disorders.

[0175] On the other hand, this application for approval provides a method for preventing and / or treating a disease and / or disorder, the method may include administering an isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immune conjugate and / or pharmaceutical composition described in this application to a subject in need.

[0176] In this application for approval, the administration may be carried out by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration.

[0177] On the other hand, the isolated antigen-binding proteins, polypeptide molecules, nucleic acid molecules, vectors, cells, immune conjugates and / or pharmaceutical compositions described in this application for approval can be used for the prevention and / or treatment of diseases and / or disorders.

[0178] In this application for approval, the aforementioned diseases and / or disorders may be CD73-mediated diseases and / or disorders.

[0179] In this application for approval, the aforementioned diseases and / or disorders include tumors.

[0180] In this application for approval, the term "tumor" includes solid tumors and / or hematological malignancies.

[0181] In this application for approval, the aforementioned diseases and / or disorders include breast cancer.

[0182] On the other hand, this application for approval provides a method for detecting CD73 in a sample, the method comprising administering the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition.

[0183] In some embodiments, the method for detecting CD73 in a sample is an in vitro method. In some embodiments, the method for detecting CD73 in a sample is a method for non-therapeutic purposes. In some embodiments, the method for detecting CD73 in a sample is not a diagnostic method.

[0184] On the other hand, this application for approval provides a reagent or kit for detecting CD73 in a sample, which comprises the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition.

[0185] On the other hand, this application for approval provides the use of the isolated antigen-binding protein, polypeptide molecule, nucleic acid molecule, vector, cell, immunoconjugate and / or pharmaceutical composition in the preparation of a kit, the kit being used to detect the presence and / or content of CD73 in a sample.

[0186] We do not wish to be limited by any theory, and the following examples are solely for the purpose of illustrating the fusion protein, preparation method, and uses of this application, and are not intended to limit the scope of the invention.

[0187] Examples Example 1: Method for preparing mouse-derived antigen-binding protein for anti-human CD73 1.1 Preparation of hybridoma cells that produce mouse-derived antigen-binding proteins The preparation of mouse-derived monoclonal antigen-binding proteins was carried out using the hybridoma preparation technique invented by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). First, human CD73 with the His-tagged protein (Met 1-Lys 547) was used as the immunoantigen. Some were treated with Freund's Adjuvant, Complete (Sigma cat no. F5881, also known as FCA) and Freund's Adjuvant, Incomplete (Sigma cat no. F5506, also known as FICA), while some were treated with Quick Antibody - Mouse 5w (Biodragon cat no. KX0210041) adjuvant. Several BALB / c, CD1 mice were immunized with each via multiple subcutaneous injections. After three immunizations, serum was collected and efficacy was measured by ELISA, and binding activity and functional activity were measured by FACS. Finally, the optimal mouse was selected, spleen cells were collected and fused with SP2 / 0 myeloma cells. After screening hybridoma cell lines by HAT, the cell culture supernatant was collected and monoclonal hybridoma cell lines that specifically bind to human CD73 and monkey CD73 were screened using FACS assay. Monoclonal cell lines that inhibit CD73 enzyme activity were screened again, and affinity (Biacore) screening was performed on the screened monoclonal cell lines. Finally, monoclonal hybridoma cell lines expressing human CD73 antigen-binding protein were obtained, and sequence analysis was performed. The screening data is shown in Table 1.

[0188] [Table 1]

[0189] High-throughput screening ultimately yielded monoclonal hybridoma cell lines that exhibited high affinity and binding activity to both humans and monkeys.

[0190] Example 2 Gene sequencing of the variable region of the anti-CD73 antigen-binding protein and preparation of each antigen-binding protein 2.1 Gene sequencing and cloning of the variable region of antigen-binding proteins in hybridoma cells Based on TAKARA's 5'RACE technology principle, the cDNA sequence of the variable region of a mouse antigen-binding protein expressed by hybridoma cell line 3A9 was cloned. Simply put, using the SMARTer 5'RACE synthesis kit (TAKARA, #634859), gene-specific cDNAs of the heavy and light chain variable regions were synthesized according to the instructions. The 5' and 3' ends of the cDNA sequence were modified with PCR primers. Since these primers are designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, the resulting PCR products could be seamlessly cloned into the heavy chain vector pHB-Fc and light chain vector pHB-Cκ, which are expressed by existing recombinant antigen-binding proteins. pHB-Fc expresses that the vector contains the gene sequence of the human IgG1 heavy chain constant region, and within this, CH2 has L234A and L235A (Eu numbering) mutations due to the ADCC knockout (KO) effect of the antigen-binding protein. The pHB-Cκ vector contains the gene sequence of the human κ light chain constant region. PCR amplification products of the heavy chain and light chain variable regions were cloned into an expression vector using an in-fusion cloning reagent (TAKARA, #639650) and transformed into competent cells of E. coli DH5α (Yi Sheng Biotechnology, #FYE607-80VL). By selecting monoclonal colonies, Sanger and NGS sequencing were performed and analyzed to obtain the sequence of the antigen-binding protein variable region. Among these, the sequence of the variable region of the anti-CD73 antigen-binding protein expressed by 3A9 is shown below.

[0191] 3A9 VH SEQ ID NO:29 EVQLVESGGGLVKPGGSLKLSCAAS GFTFSKYA MSWVRQSPEKRLEWVAE ISSGGGYI NYPDTVTGRFTISRDNAKNTLYLEMSSLRSGDTAMYYC ARAIYYYGSSYNYYAMDY WGQGTSVTVSS 3A9 VL SEQ ID NO:30 DIVMTQSHKFMSTSVGDRVSITCKAS QDVGTA EAWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFC QQYSSYPLT FGGGTKLEIK

[0192] Within that, the underlined parts are CDRs (IMGT definition, as shown below for each CDR list).

[0193] [Table 2]

[0194] 2.2 Expression of antigen-binding proteins The expression vector obtained in Example 2.1 was amplified using Escherichia coli, and a sufficient amount of plasmid was prepared using an endotoxin removal plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117). This plasmid was then used for transient transfection to express the antigen-binding protein. The host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). The two prepared heavy chain vectors were separately mixed with a light chain vector and polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes. Subsequently, CHO-S cells were transfected and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a protein A affinity column to obtain human mouse antigen-binding protein (antigen-binding protein expressing 3A9, No. 900581).

[0195] 2.3 Preparation of antigen-binding proteins 900694-900698 Humanization of antigen-binding proteins is performed using 3D modeling. First, mouse-derived antigen-binding proteins are 3D modeled to select the optimal structural model. Specifically, Discovery Studio and Schrodinger Antibody Modeling are used, respectively, to select 5-10 optimal structural solutions using homology modeling. Loop regions are generally modeled using homology modeling. If the result of CDR amino acid sequence comparison is less than 50% identity, the ab initio modeling method is used to construct a CDR3 structural model. Ten antibody crystal structure models (structural resolution of 2.5 Å or higher) with the closest sequence are searched using PDB BLAST, compared with automated modeling models, and the optimal structural model is selected. Finally, the sequence of the variable region of the antigen-binding protein is compared with available sequences in the NCBI IgBlast database, identified, and analyzed to determine the human-derived framework region (FR region) suitable for constructing the heavy and light chains of CDR transplantation on top of it.

[0196] During modification, modification sites were designed based on conservative amino acid residues in the human antibody FR region and important amino acid residues in the antibody FR region. Humanization mutations were designed for the variable regions of the heavy and light chains of the antigen-binding protein 900581 for which approval is being sought. The designed humanization sequences must meet requirements such as not affecting antibody structural stability, not affecting the binding of the antigen-binding protein to the antigen, not introducing protein modification sites such as glycosylation or phosphorylation, not introducing sites that are sensitive to oxidation or amination, and enhancing structural stability. As a result of the analysis, three humanized heavy chain sequences and three humanized light chain sequences were designed for the mouse-derived antigen-binding protein sequence of 900581. Humanization point mutation antigen-binding protein expression plasmids were expressed in CHO-S cells, and after purification, humanized antigen-binding proteins were obtained. Using measurement methods such as ELISA, Biacore, and flow cytometry, we screened humanized antigen-binding proteins for receptor binding ability, functional inhibitory activity, nonspecific binding characteristics, and thermal stability, and obtained five humanized anti-CD73 antigen-binding proteins with superior performance. The protein numbers and corresponding VH and VL sequences of the obtained humanized anti-CD73 antigen-binding proteins are shown below.

[0197] 900694 VH SEQ ID NO: 31 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSKYA MSWVRQAPGKGLEWVAE ISSGGGYI NYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARAIYYYGSSYNYYAMDY WGQGTTVTVSS 900694 VL SEQ ID NO: 32 DIQMTQSPSSLSASVGDRVTITCRAS QDVGTA EAWYQQKPGKAPKLLIY WAS TRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSSYPLT FGQGTKLEIK 900695 and 900697 VH SEQ ID NO: 33 QVQLVESGGGLVKPGGSLRLSCAAS GFTFSKYA MSWVRQAPGKGLEWVAE ISSGGGYI NYADSVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARAIYYYGSSYNYYAMDY WGQGTTVTVSS 900695 and 900698 VL SEQ ID NO: 34 DIQMTQSPSSLSASVGDRVTITCRAS QDVGTA EAWYQQKPGKAPKLLIY WAS TRHTGVPSRFSGSGSGTFTLTISSLQPEDFATYYC QQYSSYPLT FGQGTKLEIK 900696 and 900698 VH SEQ ID NO: 35 QVQLVESGGGLVKPGGSLRLSCAAS GFTFSKYA MSWIRQAPGKGLEWVAE ISSGGGYI NYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARAIYYYGSSYNYYAMDY WGQGTTVTVSS 900696 and 900697 VL SEQ ID NO: 36 DIQMTQSPSSLSASVGDRVTITCRAS QDVGTA EAWYQQKPGKAPKLLIY WAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSSYPLT FGQGTKLEIK

[0198] Within that, the underlined parts are CDRs (IMGT definition), and 900694, 900695, 900696, 900697, and 900698 are the numbers of five different anti-CD73 humanized antigen-binding proteins, respectively.

[0199] Example 3: Measurement of the binding activity of anti-CD73 antigen-binding protein to human CD73 on the cell surface. The binding activity of anti-CD73 antigen-binding proteins 900581, 900565, and humanized antigen-binding proteins 900694, 900695, 900696, 900697, and 900698 of the 900581 antigen-binding protein to cells (CHOK1-huCD73-3F4, Huabo Bio) possessing human CD73 on the cell surface was measured. Among these, the chimeric antibody 900565 was derived from the hybridoma cell line 115B10. The amino acid sequence of the heavy chain variable region (VH) of 900565 is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region (VL) of 900565 is shown in SEQ ID NO: 42.

[0200] All antigen-binding proteins are diluted to 30 μg / ml in PBS solution containing 1% BSA (1% BSA / PBS), and then 3-fold diluted with an 11-fold gradient. 20 μL is added per well to a 96-well U plate, and a negative control (adding only 1% BSA / PBS) is simultaneously established. A suspension of cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio) during the logarithmic growth phase is collected, centrifuged (1000 rpm × 5 minutes), the culture medium is discarded, and the viable cell density is increased to 1 × 10⁶ with 1% BSA / PBS. 6 Resuspend to 20 μL / mL and place in a 96-well U-plate already containing anti-CD73 antigen-binding protein, 20 μL per well (2 × 10⁶). 4 Add individual cells and allow to react at room temperature for 30 minutes. After the reaction, resuspend the 96-well U plate in 1% BSA / PBS, centrifuge (300 g x 3 minutes), discard the upper layer, wash once in the same manner, add 1:200 dilution of PE-sheep anti-human-Fc (Jackson ImmunoResearch, #109-115-098), and allow to react at room temperature for 15 minutes. After the reaction, resuspend the 96-well U plate in 1% BSA / PBS, centrifuge (300 g x 3 minutes), discard the upper layer, wash three times in the same manner, and finally resuspend in 100 μL of 1% BSA / PBS per well, and measure the fluorescence intensity of the PE channel by flow cytometry (BD, #Canto II).

[0201] The binding activity results for antigen-binding protein 900581 are shown in Figure 1 and Table 3, and the binding activity results for antigen-binding proteins 900694-900698 are shown in Figure 2 and Table 4. These results demonstrate good affinity for antigen-binding protein 900581 with cells expressing human CD73 on the cell surface (CHOK1-huCD73-3F4, Huabo Bio). 900694-900698 exhibited equivalent binding activity to 900581.

[0202] [Table 3]

[0203] [Table 4]

[0204] Example 4: Measurement of the enzyme activity inhibitory effect of recombinant CD73 by anti-CD73 antigen-binding protein. The protease activity inhibitory effect of recombinant CD73 protein (ACRO,#CD3-H52H7-100 μg) was measured using 900581, 900694, 900695, 900696, 900697, and 900698. The specific procedure was as follows: The CD73 antigen-binding protein series was diluted to 120 μg / mL with TM Buffer (ROCKLAND, #MB-059), then diluted 5-fold with a 10-fold gradient, and 25 μL per well was added to a 96-well plate. Next, the recombinant CD73 protein was diluted to 0.3 μg / mL with TM Buffer (ROCKLAND, #MB-059), and 25 μL / well of each was added to the 96-well plate and mixed well. Then, diluted AMP and ATP were mixed in a 1:1 ratio, and 50 μL / well was added to the above 96-well plate and allowed to react for 15 minutes. Finally, add the CellTiter-Glo® substrate to the CellTiter-Glo® buffer, mix well, and equilibrate to room temperature. Then, add 100 μL / well to the test well, allow to react at room temperature for 10 minutes, and measure at all wavelengths.

[0205] Figure 3 and Table 5 show the results of enzyme activity inhibition of recombinant CD73 protein (ACRO, #CD3-H52H7-100 μg) by 900581, 900694, 900695, 900696, 900697, and 900698. These results demonstrate that 900694, 900695, 900696, 900697, and 900698 are equivalent to the antigen-binding protein 900581 in their inhibitory effect on recombinant CD73 protease activity.

[0206] [Table 5]

[0207] Example 5: Measurement of the inhibitory effect of anti-CD73 antigen-binding protein on the enzymatic activity of CD73 on the cell membrane. All antigen-binding proteins were diluted to 30 μg / ml in PBS solution, then diluted 5-fold with a 6-gradient. A suspension of cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Bio) was collected during the logarithmic growth phase and incubated in medium for 4 × 10⁻⁶ days. 5Prepare a cell suspension at 1 / ml, add 100 ul (approximately 40,000 cells) per well to a 96-well U plate, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Then, using sequentially diluted resuspended cells with antigen-binding protein, add 100 ul to each well, mix well, and incubate at 37°C for 20 minutes. After incubation, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash once with 200 ul / well PBS, resuspend in 100 μL of 180 μM AMP per well, and incubate at 37°C for 60 minutes. After incubation, centrifuge at 1500 rpm for 5 minutes, collect 50 μL / well of supernatant, transfer to a 96-well black plate, add the previously prepared ATP at 50 ul / well, mix well, and react at 37°C for 15 minutes. Finally, the CellTiter-Glo® substrate was added to the CellTiter-Glo® buffer, mixed well, and equilibrated to room temperature. Then, 100 μL / well was added to the test wells, reacted at room temperature for 10 minutes, and measured at all wavelengths. The percentage of enzyme activity was evaluated as follows. The activity of the residual CD73 was assumed to be (CHOK1-huCD73-3F4 cells + Ab + ATP + AMP) - (ATP + AMP) / (CHOK1-huCD73-3F4 cells + ATP + AMP) - (ATP + AMP) * 100.

[0208] Figure 4 and Table 6 show the enzyme activity inhibitory effects of anti-CD73 chimeric antibodies 900581 and 900565 on CD73 cells (CHOK1-huCD73-3F4, Huabo Bio). Figure 5 and Table 7 show the enzyme activity inhibitory effects of 900581, 900697, and 900698 on CD73 cells (CHOK1-huCD73-3F4, Huabo Bio). The results indicate that 900697 and 900698 are essentially equivalent to 900581 in inhibiting the enzyme activity of CD73 on the cell membrane.

[0209] [Table 6]

[0210] [Table 7]

[0211] Example 6: Measurement of the binding affinity between anti-CD73 antigen-binding protein and human CD73. Using Biacore (GE, model number: T200), the binding kinetics of 900581, 900694, 900695, 900696, 900697, and 900698 with human CD73 protein (manufacturer: ACRO Biosystems, catalog number: CD3-H52H7) were measured. The experimental buffer was HBS-EP+ (pH 7.4), and a Protein A tip (manufacturer: GE, catalog number: 29-1275-56) was used.

[0212] The sample compartment and flow path temperatures were set to 25°C, and antigen-binding proteins captured by the Protein A tip were used as ligands and immobilized at approximately 100 RU. Human CD73 protein was diluted with experimental buffer at concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.57 nM to be used as the analyte. Experimental buffer was used as the 0 concentration control, and binding was performed at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 600 seconds. A multicyclic kinetic assay was performed by regenerating 10 mM Glycine pH 1.5 at a flow rate of 50 μL / min for 60 seconds. Using a 1:1 binding model, the Fit local mode was selected, and the binding rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium rate constant (KD) were fitted. The fitting results are shown in Table 8. All antigen-binding proteins bound to human CD73 protein, and most antigen-binding proteins did not show a significant difference in binding affinity to human CD73 protein.

[0213] [Table 8]

[0214] Example 7: Measurement of binding affinity between anti-CD73 antigen-binding protein and CD73 from different species (human, mouse, rat, monkey). Using Biacore (GE, model number: T200), the binding kinetics of human CD73 protein (manufacturer: ACRO Biosystems, catalog number: CD3-H52H7), mouse CD73 / NT5E (manufacturer: ACRO Biosystems, catalog number: CD3-M52H9), rat CD73 (manufacturer: Novoprotein, catalog number: CB16), and monkey CD73 (manufacturer: Novoprotein, catalog number: CI21) were measured with 900581, 900694, 900695, 900696, 900697, and 900698 respectively. Protein A chips (manufacturer: GE, catalog number: 29-1275-56) were used, and the experimental buffer was HBS-EP+ (pH 7.4).

[0215] The sample compartment and flow path temperatures were set to 25°C, and antigen-binding proteins captured by the Protein A tip were used as ligands and immobilized at approximately 100 RU. Human CD73 protein / monkey CD73 / rat CD73 / mouse CD73 were diluted in experimental buffer at concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.57 nM, respectively, and used as analytes. Experimental buffer was used as the 0 concentration control, and binding was performed at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 600 seconds. A multicyclic kinetic assay was performed by regenerating 10 mM Glycine pH 1.5 at a flow rate of 50 μL / min for 60 seconds. Using a 1:1 binding model, the Fit local mode was selected, and the binding rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium rate constant (KD) were fitted. The fitting results are shown in Table 9. The antigen-binding proteins described above were able to bind to human CD73 protein and monkey CD73 (manufacturer: Novoprotein, catalog number: CI21). Most antigen-binding proteins showed no significant difference in binding affinity to human CD73 and monkey CD73 proteins. Furthermore, it was confirmed that none of the antigen-binding proteins bound to mouse (manufacturer: ACRO Biosystems, catalog number: CD3-M52H9) or rat (manufacturer: Novoprotein, catalog number: CB16) CD73 protein.

[0216] [Table 9]

[0217] Example 8: Measurement of the endocytotic ability of CD73 on the cell surface induced by anti-CD73 antigen-binding protein. Control protein 900222, chimeric protein 900581, and humanized proteins 900697 and 900698 were diluted to 1 μg / ml in PBS solution, and two wells were repeated for each concentration. The sequence of control protein 900222 was derived from the mAb-CD73.4-IgG2 antibody sequence in Bristol-Myers Squibb's CD73 antibody patent US20190284293A1. Calu6 cells were collected during the logarithmic growth phase, and cells were divided into 2 × 10⁶ cells. 5 Prepare a cell suspension at 1 / ml / ml, add 100 μL (approximately 20,000 cells) per well to a 96-well U plate, centrifuge at 300 g for 3 minutes, and remove the supernatant. Alternatively, use the resuspended cells with diluted antigen-binding protein, mix well at 100 μL / well, and incubate at 4°C for 30 minutes. After incubation, centrifuge at 300 g for 3 minutes, remove the supernatant, resuspend in 1% BSA, and incubate half at 37°C and the other half at 4°C for 5 hours. After incubation, remove the wells with the appropriate incubation time, centrifuge at 300 g for 3 minutes, remove the supernatant, add PE anti-huIgG Fc (1:200), mix well at 50 μL / well, and react at 4°C for 30 minutes. After incubation, wash twice and measure the fluorescence intensity by flow cytometry at 300 g for 3 minutes. The results of endocytosis of cell surface CD73 induced by anti-CD73 antigen-binding protein are shown in Table 10 and Figure 6 below. The results showed that the endocytosis effect of CD73 induced by chimeric protein 900581 and humanized proteins 900697 and 900698 was significantly superior to that of the control protein 900222, and that the endocytosis effect induced by humanized proteins 900697 and 900698 was superior to that of the chimeric protein 900581.

[0218] [Table 10]

[0219] Example 9: Identification of the binding epitope of the anti-CD73 antigen-binding protein. 9.1 Measurement of the binding epitope of the 900581 antigen-binding protein by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Allow 5–10 μM antigen, antigen-binding protein, or antigen-binding protein complex (1:1 molar ratio) (50 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM TCEP) to stabilize at 4°C for 1 hour. Dilute 5 μL of the sample in 20 μL of D2O (deuterium) at 4°C and allow to stand at various HDX time points (e.g., 0, 10, 60, 300, 900 seconds) before performing mass spectrometry. After a certain period of hydrogen-deuterium exchange, stop the reaction by mixing 25 μL of ice-cold 4M guanidine with 1% trifluoroacetic acid. Immediately after stopping the reaction, place the sample tube on dry ice until injecting the sample into the HDX LEAP PAL3.0 platform. After injection into a fully automated hydrogen-deuterium exchange platform, the sample is passed through a fixed pepsin column at a flow rate of 120 μL / min, and the enzymatically degraded peptides are captured on a C18 capture column and desalted. Within 8 minutes, the desalted peptides are separated using a 2.1 mm × 5 cm C18 column (1.9 μm Hypersil Gold, Thermo Fisher) in a linear gradient of 4-40% acetonitrile and 0.3% formic acid. During sample processing, both enzymatic protein degradation and peptide separation are performed at 4°C. Orbitrap mass spectrometer (Orbitrap Fusion) is used. TM Pibrid TM Mass spectral data for hydrogen-deuterium exchange were acquired using a Mass Spectrometer (Thermo Fisher), with a measured resolution of 65,000 (m / z 400). Three HDX measurements (triplicates) were performed for each sample at each time point.

[0220] Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements revealed that the binding epitope of 900581 mainly includes the 143-NIKAKGPLASQISGL-157 region (SEQ ID NO: 37), the 178-SKETPFLSNPGTNL-191 region (SEQ ID NO: 38) of the N-terminal domain of CD73, and the 381-WNHVSM-386 region (SEQ ID NO: 39) of the C-terminal domain of CD73. When the discontinuous regions of these three segments were placed on a three-dimensional structural model of CD73, it was found that the epitope is located on the periphery of the CD73 catalytic active site, as detailed in Figure 7. When 900581 binds to this epitope, it inhibits the enzymatic activity of CD73 with maximum efficiency by very efficiently closing the CD73 catalytic active site. By reviewing patents and literature, we have shown that our 900581 is currently the first reported therapeutic antigen-binding protein that binds to the catalytic center of CD73 and also provides specific epitope information.

[0221] 9.2 Competitive binding assay of APCP, a non-hydrolyzable AMP analog, to validate the binding epitope of the 900581 antigen-binding protein. When CD73 binds to its substrate, AMP, it undergoes a structural change from an extended conformation to a bent conformation. When APCP, a non-hydrolyzable AMP analog, is used instead of AMP, CD73 can be immobilized in the catalytic intermediate state of the bent conformation because APCP cannot be hydrolyzed to produce adenosine. As the concentration of APCP increases, the proportion of CD73 in the catalytic intermediate state of the bent conformation increases, and the proportion of antigen-binding proteins that bind to the catalytic region decreases, while the proportion of antigen-binding proteins that bind to the non-catalytic region remains unchanged. The method is as follows: Prepare cells in a 1E6 / ml cell suspension with 1% BSA and add 20 μL per well to a 96-well U plate. Dilute APCP to 30 nM / 30 μM with 1% BSA and add 20 μL / well to the 96-well U plate, mix well, and leave at room temperature for 30 minutes. The antigen-binding protein to be measured is diluted to 90 μg / ml, and each solution is then 3-fold diluted with a 10-degree gradient. The serially diluted antigen-binding proteins are added to the above 96-well U plate, mixed well with 20 μL / well, and left at room temperature for 30 minutes. After incubation, the solution is centrifuged at 300 g for 3 minutes, the supernatant is removed, and the secondary antibody PE sheep anti-human IgG Fc (1:200) is added again at 20 μL / well, mixed well, and left at room temperature for 15 minutes. After incubation, the solution is washed twice, and PE-MFI is measured by flow cytometry at 300 g for 3 minutes.

[0222] The measurement results are shown in Figures 8A-8C. Among them, 900609 is Corvus's CPI-006 antibody, which has been proven to bind to the area around the catalytic center, and TNP is the negative control antibody. From the results, the antigen-binding protein 900581 showed a similar effect to the CPI-006 antibody, and as the APCP concentration increased, the proportion of antigen-binding protein bound to CD73 decreased, further verifying that 900581 binds to the area around the catalytic active site of CD73.

[0223] Example 10: Efficacy study of in vivo treatment of tumors with anti-CD73 antigen-binding protein 10.1 In this study, an animal model of MDA-MB-231 triple-negative breast cancer was constructed using CD73-humanized mice, and the efficacy of the test antibody was verified. First, 100 μl of MDA-MB-231 (ATCC bank) cells (2E6 cells) were inoculated into CD73-humanized mice to construct an animal model of CD73-humanized mouse MDA-MB-231 triple-negative breast cancer. The tumor-bearing mice were divided equally into four test groups according to tumor volume and body weight, with 6 mice per group, administered intraperitoneally twice a week for a total of 6 times. The specific administration method is shown in the table below. Among them, 900201 is a negative control antibody and does not bind to the CD73 antigen. The heavy chain (HC) amino acid sequence of 900201 is shown in SEQ ID NO: 43, and the light chain (LC) amino acid sequence of 900201 is shown in SEQ ID NO: 44.

[0224] The results showed that 900581, 900697, and 900698 could significantly suppress the progression of MDA-MB-231 triple-negative breast cancer without affecting the weight gain index of mice. On the other hand, the negative control antibody 900201 was unable to suppress tumor progression.

[0225] [Table 11]

[0226] 10.2 An animal model of MDA-MB-231 triple-negative breast cancer will be constructed using NPG mice, and the efficacy of the test antibody will be verified. Human breast cancer cells MDA-MB-231 used in this study will be cultured in L-15 medium supplemented with 10% FBS in a CO2-free incubator at 37°C. Before serial culturing the cells for 10 passages, approximately 5.0 × 10⁶ cells will be cultured. 6 Individual MDA-MB-231 cells were suspended in 100 μL of PBS, mixed with an equal volume of Matrigel, and then inoculated subcutaneously into the right dorsal side near the axilla of NPG humanized mice, with an inoculation volume of approximately 200 μL. Mice were anesthetized with 2-5% isoflurane prior to inoculation. On the day of inoculation, 1.0 × 10¹⁴ cells were extracted from the tail vein. 7PBMC (100 μL) was injected, and the tumor was approximately 50-80 mm in size. 3 Once the mice have grown to a certain size, 18 tumor-bearing mice are randomly divided into three groups of six based on tumor volume and body weight. The drugs are administered on the day of group assignment, and the specific administration regimens are shown in Table 12. Among these, 900201 is a negative control antibody and does not bind to the CD73 antigen. The heavy chain (HC) amino acid sequence of 900201 is shown in SEQ ID NO: 43, and the light chain (LC) amino acid sequence of 900201 is shown in SEQ ID NO: 44. Oleculumab is an anti-CD73 monoclonal antibody developed by AstraZeneca.

[0227] The results are shown in Table 12 and Figure 9 below. Oleculumab and 900698 significantly suppressed the progression of MDA-MB-231 triple-negative breast cancer, but the negative control antibody 900201 was unable to suppress tumor progression.

[0228] [Table 12]

[0229] Example 11: Efficacy study of in vivo treatment of pancreatic cancer with anti-CD73 antigen-binding protein In this study, an animal model of BxPC-3 pancreatic cancer will be constructed using NPG mice, and the efficacy of the test antibody will be verified. The human pancreatic cancer cells BxPC-3 used in this study will be cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before serial culture of 10 passages, approximately 1 × 10⁶ cells will be cultured. 7 100 μL of PBS containing BxPC-3 cells was mixed with an equal volume of Matrigel and administered by subcutaneous injection to the right dorsal side near the axilla of 18 NPG mice, with an inoculation volume of approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0 × 10¹⁴ cells were extracted from the tail vein. 7 PBMC (100 μL) was injected, and the tumor was approximately 50-80 mm in size. 3Once the mice have grown to a certain size, 18 tumor-bearing mice are randomly divided into three groups of six based on tumor volume and body weight. The drugs are administered on the day of group assignment, and the specific administration regimens are shown in Table 13. Among these, 900543 is a negative control antibody (900543 is an ADCC knockout type of 900201) and does not bind to the CD73 antigen. The heavy chain (HC) amino acid sequence of 900543 is shown in SEQ ID NO: 47, and the light chain (LC) amino acid sequence of 900543 is shown in SEQ ID NO: 44. Oleculumab is an anti-CD73 monoclonal antibody developed by AstraZeneca.

[0230] The results are shown in Table 13 and Figure 10 below. Oleculumab and 900698 significantly suppressed the progression of BxPC-3 pancreatic cancer, but the negative control antibody 900543 was unable to suppress tumor progression.

[0231] [Table 13]

[0232] All documents referenced in this application are cited as references in this application, just as each document is cited individually. It should be understood that, after reading the aforementioned lecture content of this application, various changes or modifications can be made to this application by those skilled in the art. Equivalent forms of such changes or modifications are also included within the scope of the claims attached to this application. This disclosure includes, for example, the embodiments described in the following sections. [Section 1] The isolated antigen-binding protein contains HCDR3, which has the amino acid sequence shown in SEQ ID NO: 3. [Section 2] The isolated antigen-binding protein described in item 1 comprises HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2. [Section 3] For an isolated antigen-binding protein described in any one of items 1 or 2, it comprises HCDR1, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1. [Section 4] For any isolated antigen-binding protein described in any one of items 1 to 3, it comprises LCDR3, wherein the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6. [Section 5] For any isolated antigen-binding protein described in any one of items 1 to 4, it comprises LCDR2, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5. [Section 6] For any isolated antigen-binding protein described in any one of items 1 to 5, it comprises LCDR1, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4. [Section 7] For any isolated antigen-binding protein described in any one of sections 3 to 6, it comprises H-FR1. The C-terminus of H-FR1 is directly or indirectly ligated to the N-terminus of HCDR1, and H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:19. [Section 8] For an isolated antigen-binding protein described in any one of items 2 to 7, it contains H-FR2. The H-FR2 is located between HCDR1 and HCDR2, and the H-FR2 contains the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:21. [Section 9] For an isolated antigen-binding protein described in any one of items 2 to 8, it contains H-FR3. The H-FR3 is located between the HCDR2 and HCDR3, and the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 22. [Section 10] For any isolated antigen-binding protein described in any one of items 1 to 9, it contains H-FR4. The N-terminus of H-FR4 is ligated to the C-terminus of HCDR3, and H-FR4 contains the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18. [Section 11] For any isolated antigen-binding protein described in any one of items 6 to 10, it contains L-FR1. The C-terminus of L-FR1 is directly or indirectly ligated to the N-terminus of LCDR1, and L-FR1 contains the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:23. [Section 12] For an isolated antigen-binding protein described in any one of items 6 to 11, it comprises L-FR2. The L-FR2 is located between LCDR1 and LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 24. [Section 13] For an isolated antigen-binding protein described in any one of items 5 to 12, it comprises L-FR3. The L-FR3 is located between LCDR2 and 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. [Section 14] For any isolated antigen-binding protein described in any one of sections 4 to 13, it contains L-FR4. The N-terminus of L-FR4 is ligated to the C-terminus of HCDR3, and L-FR4 contains the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:26. [Section 15] For an isolated antigen-binding protein described in any one of items 6 to 14, the HCDR3 therein contains the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 2, and the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 1. Furthermore, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 5, and LCDR1 contains the amino acid sequence shown in SEQ ID NO: 4. [Section 16] For any isolated antigen-binding protein described in any one of items 1 to 15, it contains VH. The VH contains the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 35. [Section 17] For any isolated antigen-binding protein described in any one of items 1 to 16, it contains a VL. The VL contains the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36. [Section 18] The isolated antigen-binding protein described in item 17 comprises one of the following VH and VL: 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; 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. [Section 19] For any isolated antigen-binding protein described in any one of items 1 to 18, it includes an antibody heavy chain constant region. [Section 20] Regarding the isolated antigen-binding proteins described in item 19, the antibody heavy chain constant region described therein is derived from the human antibody heavy chain constant region. [Section 21] For isolated antigen-binding proteins described in any one of items 19-20, the antibody heavy chain constant region described therein is derived from the human IgG heavy chain constant region. [Section 22] For isolated antigen-binding proteins described in any one of sections 19 to 21, the antibody heavy chain constant region described therein is derived from the human IgG1 heavy chain constant region. [Section 23] For an isolated antigen-binding protein described in any one of items 1 to 22, it includes an antibody light chain constant region. [Section 24] Regarding the isolated antigen-binding proteins described in item 23, the antibody light chain constant region described therein is derived from the human Igκ constant region. [Section 25] For an isolated antigen-binding protein described in any one of items 1 to 24, it comprises an antibody or an antigen-binding fragment thereof. [Section 26] With respect to the isolated antigen-binding proteins described in item 25, the antigen-binding fragments described therein include Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb. [Section 27] For any isolated antigen-binding protein described in any one of sections 25-26, the antibody described therein is one of the following monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. One or more of these options will be selected. [Section 28] For any isolated antigen-binding protein described in any one of sections 1 to 27, it can specifically bind to CD73 or its functionally activated fragment. [Section 29] With respect to the isolated antigen-binding protein described in item 28, the CD73 includes human CD73 and / or monkey CD73. [Section 30] For isolated antigen-binding proteins described in any one of sections 1 to 29, it can block or reduce CD73 activation. [Section 31] With respect to the isolated antigen-binding protein described in any one of items 28 to 30, when it binds to CD73, the isolated antigen-binding protein binds to amino acids 143 to 157 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, to amino acids 178 to 191 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, and to at least one amino acid residue in amino acids 381 to 386 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40. [Section 32] With respect to the isolated antigen-binding protein described in item 31, the amino acids 143-157 or the corresponding CD73 sequences are located in the N-terminal domain of CD73, the amino acids 178-191 or the corresponding CD73 sequences are located in the N-terminal domain of CD73, and the amino acids 381-386 or the corresponding CD73 sequences are located in the C-terminal domain of CD73. [Section 33] For any isolated antigen-binding protein described in any one of sections 1 to 32, it can inhibit the enzymatic activity of CD73. [Section 34] For any isolated antigen-binding protein described in any one of sections 1 to 33, it can induce endocytosis of CD73 on the cell surface. [Section 35] Regarding polypeptide molecules, they include isolated antigen-binding proteins as described in any one of sections 1 to 34. [Section 36] The polypeptide molecules described in item 35 include fusion proteins. [Section 37] The isolated nucleic acid molecule encodes an isolated antigen-binding protein as described in any one of sections 1 to 34 or a polypeptide molecule as described in any one of sections 35 to 36. [Section 38] Regarding the vector, it includes the nucleic acid molecule described in Section 37. [Section 39] As for the cell, it includes the nucleic acid molecule described in section 37 and / or the vector described in section 38. [Section 40] Regarding the immunoconjugate, it comprises an isolated antigen-binding protein as described in any one of sections 1 to 34. [Section 41] The pharmaceutical composition comprises an isolated antigen-binding protein as described in any one of claims 1 to 34, a polypeptide molecule as described in any one of claims 35 to 36, a nucleic acid molecule as described in claim 37, a vector as described in claim 38, a cell as described in claim 39 and / or an immunoconjugate as described in claim 40, and optionally a pharmaceutically acceptable carrier. [Section 42] A method for preparing an isolated antigen-binding protein according to any one of items 1 to 34, wherein the method comprises culturing the cells according to item 39 under conditions for expressing the isolated antigen-binding protein. [Section 43] For use in the preparation of a drug, the isolated antigen-binding protein described in any one of items 1 to 34, the polypeptide molecule described in any one of items 35 to 36, the nucleic acid molecule described in item 37, the vector described in item 38, the cell described in item 39, the immune conjugate described in item 40, and / or the pharmaceutical composition described in item 41 is used for the prevention and / or treatment of diseases and / or disorders. [Section 44] With respect to the uses described in paragraph 43, the diseases and / or disorders described therein are CD73-mediated diseases and / or disorders. [Section 45] For the uses described in any one of paragraphs 43 to 44, the diseases and / or disorders described therein include tumors. [Section 46] The uses described in paragraph 45 include solid tumors and / or hematological malignancies. [Section 47] For the uses described in any one of paragraphs 43 to 46, the diseases and / or disorders described therein include breast cancer. [Section 48] A method for measuring CD73 in a sample comprises administering an isolated antigen-binding protein as described in any one of items 1 to 34, a polypeptide molecule as described in any one of items 35 to 36, a nucleic acid molecule as described in item 37, a vector as described in item 38, cells as described in item 39, an immunoconjugate as described in item 40, and / or a pharmaceutical composition as described in item 41. [Section 49] A reagent or kit for detecting CD73 in a sample comprises an isolated antigen-binding protein as described in any one of items 1 to 34, a polypeptide molecule as described in any one of items 35 to 36, a nucleic acid molecule as described in item 37, a vector as described in item 38, a cell as described in item 39, an immunoconjugate as described in item 40, and / or a pharmaceutical composition as described in item 41. [Section 50] For use in the preparation of the kit, the isolated antigen-binding protein described in any one of items 1 to 34, the polypeptide molecule described in any one of items 35 to 36, the nucleic acid molecule described in item 37, the vector described in item 38, the cell described in item 39, the immunoconjugate described in item 40, and / or the pharmaceutical composition described in item 41, the kit is used to detect the presence and / or content of CD73 in a sample.

Claims

1. An antibody or its antigen-binding fragment that can specifically bind to CD73, comprising HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

4.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising VH, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO:

35.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO:

36.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising one of the following selected from VH and VL: 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; 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.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising an antibody light chain constant region, wherein the antibody light chain constant region is derived from a human Igκ constant region.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD73 is human CD73 and / or monkey CD73.

8. The antibody or antigen-binding fragment thereof according to claim 1, which can block or reduce the activation of CD73, suppress the enzymatic activity of CD73, and / or induce endocytosis of CD73 on the cell surface.

9. The antibody or antigen-binding fragment according to claim 1, wherein, when bound to the CD73, the antibody or antigen-binding fragment binds to amino acids 143-157 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, to amino acids 178-191 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO: 40, and to at least one amino acid residue in amino acids 381-386 or the corresponding CD73 sequence of the amino acid sequence shown in SEQ ID NO:

40.

10. The antibody or antigen-binding fragment according to claim 9, wherein amino acids 143-157 of the amino acid sequence shown in SEQ ID NO: 40 or the corresponding CD73 sequence are located in the N-terminal domain of CD73, amino acids 178-191 of the amino acid sequence shown in SEQ ID NO: 40 or the corresponding CD73 sequence are located in the N-terminal domain of CD73, and amino acids 381-386 of the amino acid sequence shown in SEQ ID NO: 40 or the corresponding CD73 sequence are located in the C-terminal domain of CD73.

11. A polypeptide molecule comprising the antibody or antigen-binding fragment thereof as described in claim 1.

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in claim 1.

13. A vector comprising the nucleic acid molecule described in claim 12.

14. A cell comprising the nucleic acid molecule described in claim 12.

15. An immunoconjugate comprising the antibody or antigen-binding fragment thereof as described in claim 1.

16. The antibody or antigen-binding fragment thereof as described in claim 1, and a pharmaceutically acceptable carrier, Pharmaceutical composition.

17. A pharmaceutical composition according to claim 16, used for the prevention, alleviation, and / or treatment of diseases and / or disorders.

18. The pharmaceutical composition according to claim 17, wherein the disease and / or disorder is a CD73-mediated disease and / or disorder.

19. The pharmaceutical composition according to claim 17, wherein the disease and / or disorder includes a tumor.

20. The pharmaceutical composition according to claim 19, wherein the tumor includes a solid tumor and / or a hematological tumor.

21. The pharmaceutical composition according to claim 17, wherein the disease and / or disorder includes breast cancer.

22. A method for measuring CD73 in a sample, wherein the method comprises detecting CD73 with an antibody or antigen-binding fragment thereof as described in claim 1, a polypeptide molecule as described in claim 11, a nucleic acid molecule as described in claim 12, a vector as described in claim 13, a cell as described in claim 14, and / or an immunoconjugate as described in claim 15, and the method is an in vitro method.

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