Anti-CD73 antibodies and uses thereof

Abstract

The present invention relates to the fields of disease therapy and immunology. Specifically, anti-CD73 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding same, immunoconjugates, bispecific molecules and pharmaceutical compositions comprising same, and their use in enhancing immune responses and / or treating tumors are disclosed.

Description

Detailed Description of the Invention

[0001] Technical Field The present invention relates to the fields of disease treatment and immunology; in particular, the present invention relates to anti-CD73 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding same, immunoconjugates, bispecific molecules, and pharmaceutical compositions comprising same, and their use for enhancing immune responses and / or treating tumors.

[0002] Background technology In recent years, the rapid development of cancer immunotherapy has provided the scientific community with a better understanding of tumor biology and immunology. The tumor microenvironment is a dynamic microenvironment containing cancer cells, immune cells, fibroblasts, myofibroblasts, cytokines, blood vessels, and extracellular matrix. Tumors are often hypoxic, and the environment is also deficient in glucose and other nutrients. To survive, cancer cells reorganize their metabolic machinery in such an environment. Among these, regulation of purine metabolism is a crucial step, particularly the increased expression of cluster of differentiation 73 (CD73, also known as extracellular 5'-nucleotidase). CD73 is a glycosylphosphatidylinositol-anchored cell surface protein commonly expressed on endothelial cells and a subset of hematopoietic cells (Misumi Y et al., European Journal of Biochemistry 1990;191(3):563-9). Extracellularly, CD73, together with CD39, regulates the conversion of adenosine triphosphate to adenosine, and this step, the CD73-catalyzed dephosphorylation of adenosine monophosphate to adenosine, is the rate-limiting step in the aforementioned conversion axis (Resta R et al., Immunological Reviews 1998;161:95-109).

[0003] In response to cell death and cellular stress, cells release ATP to activate immune responses. In contrast, ATP hydrolysis to adenosine acts as a negative feedback mechanism, resulting in the suppression of immune responses. Adenosine is a widely studied signaling molecule that mediates its biological effects through several receptors, including A1, A2A, A2B, and A3. Adenosine is known to regulate the proliferation and migration of many cancers, and extracellular adenosine accumulates in cancerous tissues, contributing to an important mechanism of tumor immune evasion (Bin Z. Cancer Research 2010;70:6407-6411). Among other effects, tumor-derived adenosine profoundly inhibits infiltrating effector T cells through adenylate cyclase-activating A2A receptors.

[0004] CD73 has been reported to be expressed in many different tumors, including melanoma, colon cancer, lung cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer. CD73 is a powerful prognostic biomarker in solid tumors, and overexpression of CD73 is associated with shorter overall survival or progression-free survival (Rong W et al., Oncotarget 2017;8(34):57327-57336). CD73 expression in cancer is associated with increased tumor cell proliferation, migration, neovascularization, invasiveness, and metastasis; knockdown or overexpression of CD73 in tumor cells using siRNA has been shown to modulate tumor growth and metastasis (Paul B et al., PNAS 2013;110(36):14711-14716), and CD73- / - mice are protected from transplanted and spontaneous cancers (John S et al., Cancer Research 2010;71:2892-2900). In addition to the reported regulation of cell-cell and cell-matrix interactions in tumor cells by CD73, CD73 expression and activity are also associated with attenuated T cell responses (Dachuan J et al., Cancer Res 2010;70:2245-55). CD73 also regulated resistance to chemotherapy drugs such as anthracyclines (Loi, S. et al., PNAS 2013;110:11091-11096) and to the induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, CD73 can regulate cancer progression in both direct and indirect ways, highlighting its potential as a novel therapeutic target.

[0005] In addition, although cancer immune checkpoint inhibitor drugs have shown promising efficacy in patients with various cancers in recent years, a significant proportion of patients remain non-responsive to these treatments, and one-third of patients relapse after an initial response (adaptive drug resistance), indicating that multiple immunosuppressive mechanisms coexist in the tumor microenvironment, and that the targets of these drugs can be used synergistically or in combination, which is a current research hotspot in cancer immunology.

[0006] Thus, CD73 has shown potential as an antitumor therapeutic target, either as a single agent or in combination therapy.

[0007] Contents of the present invention The antibodies of the present invention can specifically bind to membrane-bound CD73 and non-membrane-bound CD73 on the surface of tumor cells, inhibit the enzymatic activity of CD73, and enhance immune responses. Compared with known anti-CD73 antibodies, they have better antitumor activity and functional properties. Therefore, the antibodies of the present invention have potential for preventing and / or treating tumors and are an option for clinical tumor immunotherapy drugs.

[0008] Antibodies of the Invention Accordingly, in one embodiment, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, wherein the antibody or antigen-binding fragment thereof comprises: The three heavy chain variable region CDRs: (i) a VHCDR1 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions compared thereto (e.g., one, two or three amino acid substitutions, deletions or additions); (ii) a VHCDR2 consisting of the following sequence: SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (iii) a VHCDR3 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and the following three light chain variable region CDRs: (iv) VLCDR1 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions compared thereto (e.g., one, two or three amino acid substitutions, deletions or additions); (v) VLCDR2 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); and (vi) VLCDR3 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions, or additions compared thereto (e.g., one, two, or three amino acid substitutions, deletions, or additions); The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0009] In certain embodiments, the substitution described in any of (i) to (vi) is a conservative substitution.

[0010] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can bind to human CD73, eg, membrane-bound human CD73 and / or soluble human CD73.

[0011] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, wherein the antibody or antigen-binding fragment thereof comprises: The following three heavy chain variable region CDRs: VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 1; and The following three light chain variable region CDRs: VLCDR1, VLCDR2, and VLCDR3 contained in the light chain variable region set forth in SEQ ID NO:2 The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0012] In certain embodiments, the three CDRs contained in a heavy chain variable region and the three CDRs contained in a light chain variable region are defined by the Kabat, Chothia, or IMGT numbering systems.

[0013] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can bind to human CD73, eg, membrane-bound human CD73 and / or soluble human CD73.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0015] In certain embodiments, antibodies or antigen-binding fragments thereof of the invention comprise framework region sequences derived from a human immunoglobulin, wherein the framework regions optionally contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to corresponding murine residues. In certain embodiments, antibodies or antigen-binding fragments thereof comprise heavy chain framework region sequences (i.e., amino acid sequences encoded by human heavy chain germline genes) derived from human heavy chain germline sequences, and light chain framework region sequences (i.e., amino acid sequences encoded by human light chain germline genes) derived from human light chain germline sequences, wherein the heavy and / or light chain framework regions optionally contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to corresponding murine residues.

[0016] In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain framework region sequence derived from a heavy chain germline sequence and a light chain framework region sequence derived from a light chain germline sequence, and the heavy chain framework region and / or the light chain framework region optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to the corresponding murine residues.

[0017] In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0018] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention may further comprise a constant region derived from a mammalian (e.g., murine or human) immunoglobulin. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a mammalian (e.g., murine or human) immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (e.g., kappa or lambda) derived from a mammalian (e.g., murine or human) immunoglobulin.

[0019] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof having one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); and / or The light chain of the antibody or antigen-binding fragment thereof of the present invention comprises a human immunoglobulin light chain constant region (CL), or a variant thereof having up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to the sequence from which it is derived.

[0020] In certain embodiments, the constant region may comprise amino acid mutations that alter one or more of the following properties of an antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function. Altered function can be achieved by substituting at least one amino acid residue in the antibody constant region with a different residue; for example, an altered (e.g., decreased) effector function can be achieved by altering the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q).

[0021] Methods for substituting amino acid residues in the Fc region of an antibody to alter its effector function are known in the art. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc. In some cases, these effector functions are necessary for therapeutic antibodies, but in other cases, these effector functions may be unnecessary or even harmful depending on the intended purpose.

[0022] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present invention has reduced or even eliminated effector function (e.g., ADCC and / or CDC activity). In such embodiments, the antibody or antigen-binding fragment thereof of the present invention may comprise a variant of a human IgG heavy chain constant region, wherein, compared to the wild-type sequence from which it is derived, the variant has at least one, at least two, or all three of the following substitutions: L234F, L235E, and P331S (the above-mentioned amino acid positions are positions according to the EU numbering system). See, e.g., Acta Cryst. (2008). D64, pp. 700-704.

[0023] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a human wild-type IgG1 heavy chain constant region. In such embodiments, the antibody or antigen-binding fragment thereof has ADCC and CDC activity.

[0024] In such exemplary embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a variant of a human IgG heavy chain constant region, where, compared to the wild-type sequence from which it is derived, the variant has the following substitutions: L234F, L235E, P331S (positions according to the EU numbering system), e.g., the heavy chain constant region set forth in SEQ ID NO: 15. In such embodiments, the antibody or antigen-binding fragment thereof of the invention has eliminated or reduced ADCC and / or CDC activity.

[0025] In certain preferred embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the present invention comprises a variant of a human immunoglobulin heavy chain constant region (CH), wherein the variant has essentially unchanged effector function compared to the wild-type sequence from which it is derived. In such embodiments, the variant may have up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions).

[0026] In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a human kappa light chain constant region, eg, the light chain constant region set forth in SEQ ID NO:16. In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) set forth in SEQ ID NO:15 and / or a light chain constant region (CL) set forth in SEQ ID NO:16.

[0027] In certain embodiments, the antibody of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In certain embodiments, the antigen-binding fragment of the present invention is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).

[0028] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following characteristics: (a) binds to membrane-bound human CD73 or soluble human CD73, or both; for example, membrane-bound human CD73 is expressed on the surface of tumor cells; (b) inhibiting or reducing the enzymatic activity of CD73 (e.g., membrane-bound human CD73 or soluble human CD73); e.g., inhibiting or reducing human CD73-mediated conversion of adenosine monophosphate (AMP) to adenosine, e.g., as determined by a CellTiter Glo (CTG) assay (e.g., the method described in Example 6); (c) increases the proliferation of anti-CD3 / anti-CD28 stimulated T cells (e.g., CD4+ T cells) in the presence of adenosine monophosphate (AMP), as determined, for example, by the method described in Example 8; (d) induces internalization of CD73 into cells (e.g., tumor cells) that express CD73 on their surface by antibody-mediated receptor internalization; e.g., at an internalization level of at least 10% (e.g., at least 15%, at least 20%, or more) as measured by FACS or flow cytometry (e.g., the methods of Example 7); (e) binds to soluble human CD73 with an EC50 of less than about 0.01 μg / ml or lower; the EC50 is determined by ELISA techniques; (f) binds to soluble human CD73 with a KD of less than about 0.5 nM or lower; KD is measured by Biacore; (g) reducing adenosine levels in CD73-expressing tumor cells; (h) stimulating an immune response; for example, stimulating an immune response against a tumor (e.g., a tumor expressing CD73); (i) preventing and / or treating tumors (e.g., CD73-expressing tumors) have one or more of the following:

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof is 13D12, or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a functional variant thereof, wherein the variant substantially retains the biological function of the antibody or antigen-binding fragment thereof from which it is derived.

[0030] In the present invention, the antibody or antigen-binding fragment thereof of the present invention may include a variant, wherein, compared to the antibody or antigen-binding fragment thereof of the present invention from which it is derived, the variant differs only by conservative substitutions of one or more amino acid residues (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids), or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the antibody or antigen-binding fragment thereof from which it is derived, and substantially retains the above-mentioned biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

[0031] Antibody preparation The antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibody of the present invention.

[0032] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly in recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells, and the Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). In addition, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are known to those skilled in the art.

[0033] Thus, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or the heavy chain variable region and / or light chain variable region thereof. In certain embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the present invention, or the heavy chain variable region and / or light chain variable region thereof.

[0034] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention, and / or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof of the invention.

[0035] In certain embodiments, the first nucleotide sequence comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 11; or (b) a sequence substantially identical to the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity compared to the nucleotide sequence set forth in (a), or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence set forth in (a)); or (c) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (a); The second nucleotide sequence comprises a sequence selected from the group consisting of: (d) the nucleotide sequence set forth in SEQ ID NO: 12; or (e) a sequence substantially identical to the nucleotide sequence set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity compared to the nucleotide sequence set forth in (d), or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence set forth in (d)); or (f) a sequence selected from the group consisting of a sequence that differs by no more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence set forth in (d).

[0036] In certain embodiments, the first nucleotide sequence comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 13; or (b) a sequence substantially identical to the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity compared to the nucleotide sequence set forth in (a), or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence set forth in (a)); or (c) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (a); The second nucleotide sequence comprises a sequence selected from the group consisting of: (d) the nucleotide sequence set forth in SEQ ID NO: 14; or (e) a sequence substantially identical to the nucleotide sequence set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity compared to the nucleotide sequence set forth in (d), or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence set forth in (d)); or (f) a sequence selected from the group consisting of a sequence that differs by no more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence set forth in (d).

[0037] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof of the invention, and / or a second nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof of the invention.

[0038] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain embodiments, a vector of the present invention is, for example, a plasmid, cosmid, phage, etc. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a human).

[0039] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells, such as E. coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell of the present invention is a mammalian cell, such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DG44).

[0040] In another aspect, a method for preparing an antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing a host cell of the present invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0041] derivatized antibodies The antibodies or antigen-binding fragments thereof of the present invention can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to CD73. Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent attachment, or otherwise) to one or more other molecular moieties, e.g., another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or polyhistidine tag) that can mediate binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present invention can also be derivatized with chemical groups, e.g., polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups can be used to improve the biological properties of the antibody, e.g., to increase serum half-life.

[0042] Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32Fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridine esters), magnetic beads (e.g., Dynabeads®), calorimetric labels, such as colloidal gold or colloidal glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin (e.g., streptavidin, etc.) for binding to avidin modified with the aforementioned detectable labels. The detectable labels described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzymatic labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while calorimetric labels are detected by simply visualizing the colored label. In certain embodiments, such labels may be suitable for use in immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels described above can be attached to the antibodies or antigen-binding fragments thereof of the present invention via linkers of various lengths to reduce potential steric hindrance.

[0043] Bispecific or multispecific molecules The antibodies or antigen-binding fragments thereof of the present invention can be used to form bispecific or multispecific molecules. The antibodies or antigen-binding fragments thereof may be part of a bispecific or multispecific molecule, which comprises a second functional module (e.g., a second antibody) with a different binding specificity compared to the antibodies or antigen-binding fragments thereof of the present invention, thereby being capable of binding to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be linked to a second antibody or antigen-binding fragment thereof of the present invention that can specifically bind to any protein that can be used as a potential target for combination therapy. To create such bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be linked (e.g., by chemical conjugation, gene fusion, noncovalent association, or otherwise) to one or more additional binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0044] Thus, in another aspect, the present invention provides bispecific or multispecific molecules comprising an antibody or antigen-binding fragment thereof of the invention.

[0045] In certain embodiments, the bispecific or multispecific molecule specifically binds to CD73 (e.g., membrane-bound human CD73 and / or soluble human CD73) and further specifically binds to one or more additional targets.

[0046] In certain embodiments, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0047] Immunoconjugates The antibodies or antigen-binding fragments thereof of the present invention can be linked to therapeutic agents to form immunoconjugates, which have the ability to selectively deliver one or more therapeutic agents to target tissues (e.g., tumor-associated antigens, e.g., CD73-expressing tumors), and can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention for treating diseases (e.g., cancer).

[0048] Accordingly, in another aspect, the invention provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

[0049] In certain embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0050] In certain embodiments, the therapeutic agent is a cytotoxic agent. In the present invention, a cytotoxic agent includes any agent that is detrimental to cells (e.g., kills cells).

[0051] In certain embodiments, the therapeutic agent is selected from the group consisting of alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof.

[0052] Examples of alkylating agents that can be used in the immunoconjugates of the present invention include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethyleneimines (e.g., thiotepa, etc.), sulfates and polyols (e.g., busulfan, dibromomannitol), nitrosoureas (e.g., carmustine, lomustine, etc.), platinum-based antitumor agents (e.g., cisplatin, oxaliplatin, carboplatin, etc.), and the like.

[0053] Examples of antimitotic agents that can be used in the immunoconjugates of the invention include, but are not limited to, maytansinoids (e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.), taxanes (e.g., docetaxel, paclitaxel, or nanoparticulate paclitaxel, etc.), vinca alkaloids (e.g., vindesine sulfate, vincristine, vinblastine, or vinorelbine, etc.).

[0054] Examples of antitumor antibiotics that can be used in the immunoconjugates of the present invention include, but are not limited to, actinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), calicheamicin, duocarmycin, etc.

[0055] Examples of antimetabolites that can be used in the immunoconjugates of the present invention include, but are not limited to, folate antagonists (e.g., methotrexate, etc.), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, gemcitabine, etc.), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine, etc.), adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, pentostatin, etc.).

[0056] Examples of topoisomerase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, camptothecin and its derivatives (e.g., irinotecan, topotecan, etc.), amsacrine, daunomycin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, rozoxane, teniposide, etc.

[0057] Examples of tyrosine kinase inhibitors that can be used in the immunoconjugates of the invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxinib, sunitinib, vandetanib, and the like.

[0058] Examples of radionuclide agents that can be used in the immunoconjugates of the present invention include, but are not limited to, I 131 , In 111 , Y 90 , Lu 177 Examples include:

[0059] In certain exemplary embodiments, the therapeutic agent is selected from the group consisting of platinum-based anti-neoplastic agents, anthracyclines, taxane compounds, nucleoside analogs, camptothecin compounds, and analogs or homologs thereof, and any combination thereof.

[0060] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are conjugated to a therapeutic agent, optionally via a linker.

[0061] In the present invention, cytotoxic agents can be conjugated to the antibody or its antigen-binding fragment of the present invention using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxic agents to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. For example, linkers can be selected from those that are susceptible to cleavage due to low pH in lysosomal compartments or by proteases (e.g., proteases preferentially expressed in tumor tissues, such as cathepsins, e.g., cathepsins B, C, and D).

[0062] Further discussion of types of cytotoxic agents, linkers, and methods of conjugating therapeutic agents to antibodies can be found in Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, PA et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, TM (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug It can also be found in Deliv.Rev. vol. 53:247-264.

[0063] Therapeutic Uses and Pharmaceutical Compositions The antibodies or antigen-binding fragments thereof of the present invention can modulate (e.g., enhance, stimulate, increase, inhibit, decrease, or neutralize) one or more biological activities of CD73. In certain examples, the antibodies or antigen-binding fragments thereof of the present invention result in one or more of the following: inhibition or reduction of the enzymatic activity of CD73; inhibition or reduction of the conversion of adenosine monophosphate (AMP) to adenosine; and increased proliferation of anti-CD3 / anti-CD28-stimulated T cells (e.g., CD4+ T cells) in the presence of adenosine monophosphate (AMP). Thus, the antibodies or antigen-binding fragments thereof of the present invention can be used as single agents to prevent and / or treat tumors by inhibiting or reducing the enzymatic activity of CD73.

[0064] In addition, targeting CD73 has been reported to exhibit synergistic effects with other anticancer drugs. In a prospective, randomized, Phase III clinical trial evaluating the activity of trastuzumab, high levels of CD73 gene expression were significantly associated with poor clinical outcomes. In an immunocompetent mouse model of HER2 / ErbB2-driven breast cancer, CD73 expression by tumor and host cells significantly inhibited immune responses mediated by anti-ErbB2 monoclonal antibodies (Martin T et al., Cancer Research 2017;77(20):5652-63). In addition, in vitro experiments showed that activation of A2A receptors can regulate the upregulation of PD-1 on tumor-infiltrating cytotoxic T cells, while blockade of PD-1 signaling by anti-PD-1 antibodies upregulates A2A receptor expression on tumor-infiltrating cytotoxic T cells (Cekic C et al., Cancer Res 2014;74:7239-49). Anti-CD73 antibodies have been reported to significantly enhance the activity of anti-CTLA-4 and anti-PD-1 antibodies in various mouse tumor models. Both monotherapy and combination therapy depend on host interferon-γ and cytotoxic T cells. The effect of extracellular adenosine on tumor-infiltrating T cells has been shown to enhance PD-1 expression on tumor-specific cytotoxic T cells and helper T cells through receptor activation by adenosine (Bertrand A et al., Clin Cancer Res 2013;19(20):5626-5635). Clinical studies have found that increased CD73 levels positively correlate with disease progression in melanoma patients treated with pembrolizumab (anti-PD-1). The relationship between dynamic upregulation of CD73 and adaptive resistance to anti-PD-1 antibodies is noteworthy (Reinhardt J et al., Cancer Research 2017;77:4697-4709). Another study showed that high levels of soluble CD73 enzyme activity were significantly associated with poor overall survival and progression-free survival in metastatic melanoma patients receiving nivolumab.In multivariate analysis, CD73 enzyme activity emerged as the strongest prognostic factor for overall survival and progression-free survival, and higher basal levels of CD73 enzyme activity before the initiation of nivolumab were associated with lower rates of treatment response (Silvana M et al., J Transl Med 2017;15:244). Accordingly, CD73 and PD-L1 expression levels were also found to complement each other in tumor samples from patients with non-small cell lung cancer. It is understood that the antibody or antigen-binding fragment thereof of the present invention can also be used in combination with immune checkpoint inhibitors or tumor-specific antibodies for tumor prevention and treatment.

[0065] Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and a pharmaceutically acceptable carrier and / or excipient.

[0066] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0067] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer (e.g., gemcitabine, 5-fluorouracil, taxanes, cisplatin, etc.), an anti-angiogenic agent, a cytokine (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), a molecularly targeted drug (e.g., a CD20 antibody such as rituximab, a Her antibody such as trastuzumab, a VEGF antibody such as bevacizumab, an EGFR antibody such as cetuximab, etc.), an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, etc.), an oncolytic virus, etc.

[0068] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of an immune checkpoint inhibitor (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor), an anti-CD39 antibody, an anti-A2AR antibody, or an anti-HER2 / ErbB2 antibody.

[0069] In certain embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and the additional pharmaceutically active agent are provided as isolated components or as a mixture. Thus, the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, or immunoconjugate of the invention and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0070] In certain exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0071] In another aspect, the present invention provides a method for preventing and / or treating a tumor in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention. In another aspect, there is provided a use of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention in the prevention and / or treatment of a tumor in a subject (e.g., a human) or in the manufacture of a medicament for the prevention and / or treatment of a tumor in a subject (e.g., a human).

[0072] In certain embodiments, the tumor expresses CD73. In certain embodiments, the CD73 may be membrane-bound and / or soluble human CD73.

[0073] In certain embodiments, the tumor comprises CD73-expressing tumor cells. In certain embodiments, CD73 is expressed on the surface of tumor cells.

[0074] In certain embodiments, the tumor is selected from the group consisting of melanoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer.

[0075] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is administered in combination with a second therapeutic agent or therapy, which can be administered before, together with, or after administration of the antibody or antigen-binding fragment thereof of the present invention.

[0076] In certain embodiments, the second therapeutic agent is selected from drugs with anti-tumor activity, such as alkylating agents, antimitotics, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

[0077] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is administered in combination with a therapeutic agent selected from the group consisting of an immune checkpoint inhibitor (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor), an anti-CD39 antibody, an anti-A2AR antibody, or an anti-HER2 / ErbB2 antibody.

[0078] In certain exemplary embodiments, the PD-1 inhibitor is selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In certain exemplary embodiments, the PD-L1 inhibitor is selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559.

[0079] In certain exemplary embodiments, the CTLA-4 inhibitor is selected from ipilimumab or tremelimumab.

[0080] In certain exemplary embodiments, the LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767.

[0081] In certain embodiments, the second therapy may be any therapy known to be used against tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0082] In another aspect, the present invention provides a method for stimulating an immune response in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the invention. In another aspect, there is provided use of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the invention in stimulating an immune response in a subject or in the manufacture of a medicament for stimulating an immune response in a subject.

[0083] In certain embodiments, the immune response is a T cell-mediated immune response.

[0084] In certain embodiments, the immune response is an immune response against a tumor (e.g., a CD73-expressing tumor). In certain embodiments, the subject has a tumor (e.g., a CD73-expressing tumor).

[0085] In certain embodiments, the immune response is an immune response to an immunogen. In such embodiments, the method further comprises administering an immunogen to the subject. In some embodiments, the immunogen is selected from a tumor-associated antigen (e.g., a protein, polypeptide, or carbohydrate molecule), tumor cells, dendritic cells primed with the antigen, and any combination thereof. In other embodiments, the immunogen is selected from an antigen (e.g., a protein, polypeptide, or carbohydrate molecule) associated with a pathogen (e.g., a virus), an inactivated or attenuated pathogen, dendritic cells primed with the antigen, and any combination thereof.

[0086] In another aspect, the present invention provides a method for reducing adenosine levels in CD73-expressing tumor cells, comprising contacting the cells with an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention. In certain embodiments, the method is used to reduce adenosine levels in CD73-expressing tumor cells in vitro for non-therapeutic purposes. In another aspect, there is provided use of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the present invention in reducing adenosine levels in CD73-expressing tumor cells or in the manufacture of a medicament for reducing adenosine levels in CD73-expressing tumor cells.

[0087] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical arts, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injectable preparation may be a sterile injectable solution. A sterile injectable solution can be prepared, for example, by incorporating the required amount of the antibody of the present invention in an appropriate solvent, optionally with other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or lyophilization) for ease of storage and use. Such lyophilized powders can be dispersed in a suitable vehicle, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0088] Furthermore, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, pharmaceutical compositions of the invention may be present in pharmaceutical compositions in unit dosage form for ease of administration.

[0089] Antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic plexus, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). One skilled in the art will understand that the route / mode of administration will vary depending on the intended purpose. In a preferred embodiment, antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

[0090] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, immunoconjugate, or pharmaceutical composition of the invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, halt, or delay the onset of disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention may vary according to, among other factors: the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, and any additional therapies administered concomitantly.

[0091] In the present invention, dosage regimens can be adjusted to obtain the optimum response of interest (e.g., a therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dose can be proportionally reduced or increased according to the requirements of the therapeutic situation.

[0092] In the present invention, the subject may be a mammal, for example, a human.

[0093] Detection Methods and Kits The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to CD73 and therefore can be used to detect the presence or level of CD73 in a sample.

[0094] Therefore, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has a detectable label. In other embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0095] In certain embodiments, the detectable label is selected from the group consisting of an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (eg, a chemiluminescent material), or biotin.

[0096] In another aspect, the present invention provides a method for detecting the presence or amount of CD73 in a sample, comprising: (1) contacting a sample with an antibody or antigen-binding fragment thereof of the present invention; (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and CD73, or detecting the amount of the complex. Including, A method is provided.

[0097] The formation of a complex indicates the presence of CD73 or cells expressing CD73.

[0098] In certain embodiments, the sample is a cell sample, i.e., a sample containing cells (e.g., tumor cells). In such embodiments, preferably, a complex is formed between the antibody or antigen-binding fragment thereof and CD73 expressed by cells in the sample.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention may further have a detectable label. In other embodiments, in step (2), the antibody or antigen-binding fragment thereof of the present invention is detected using a reagent having a detectable label.

[0100] The methods can be used for diagnostic or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient). In certain embodiments, the CD73 is human CD73, e.g., membrane-bound and / or soluble human CD73.

[0101] In another aspect, there is provided use of an antibody or antigen-binding fragment thereof of the present invention for determining the presence or amount of CD73 in a sample, or in the manufacture of a kit for determining the presence or amount of CD73 in a sample. In certain embodiments, the CD73 is human CD73, e.g., membrane-bound human CD73 and / or soluble human CD73.

[0102] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the procedures used herein, such as cell culture, biochemistry, nucleic acid chemistry, immunology experiments, etc., are all routine steps widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0103] As used herein, the term "cluster of differentiation 73" or "CD73" also refers to extracellular 5'-nucleotidase, which can convert extracellular 5'-monophosphate nucleosides to nucleosides, i.e., adenosine monophosphate (AMP) is converted to adenosine. The term CD73 includes membrane-bound forms (also known as membrane-bound CD73) or soluble forms (also known as soluble CD73 or non-membrane-bound CD73). CD73 can be isolated from cells or tissues in which it is naturally expressed or can be recombinantly produced using techniques well known in the art. The sequence of CD73 is well known in the art and can be found in the NCBI database accession number NM_002526.

[0104] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in binding of the antibody to the antigen, but exhibits various effector functions, for example, mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into highly diverse regions (also called complementarity-determining regions (CDRs)), which are interspersed with more conserved regions called framework regions (FRs). Each V H and V L Each heavy / light chain pair consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The assignment of amino acids to regions or domains may follow the definitions set forth in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0105] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of ordinary skill in the art will readily identify the CDRs defined by each numbering system. Additionally, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0106] In the present invention, the CDR contained in the antibody or its antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art.In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment of the present invention is preferably determined by Kabat, Chothia or IMGT numbering system.In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment of the present invention is preferably determined by Kabat numbering system.

[0107] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0108] The term "antibody" is not limited to any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0109] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0110] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain consisting of, from N- to C-terminus, a heavy chain variable region (VH), a CH1 domain of the heavy chain constant region, a hinge region (HR), a CH2 domain of the heavy chain constant region, and a CH3 domain of the heavy chain constant region; and optionally, in the case of an IgE isotype, a CH4 domain of the heavy chain constant region. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of, from N- to C-terminus, VH, CH1, HR, CH2, and CH3. A "full-length light chain" is a polypeptide chain consisting of, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL). Two pairs of full-length antibody chains are linked together by a disulfide bond between the CL and CH1, and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human, or may be a chimeric or humanized antibody. Each full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and VL pair, which specifically recognize / bind to the same antigen.

[0111] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment and consisting of an intact light chain and a heavy chain Fd fragment (consisting of the VH and CH1 domains).

[0112] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of one arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming an intact antigen-binding site. In general, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, additional variable regions (e.g., Fd fragments, which contain only three antigen-specific CDRs) can recognize and bind to antigens, although perhaps with lower affinity than the intact binding site.

[0113] As used herein, the term "Fc" refers to an antibody fragment formed by the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding.

[0114] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are linked by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of a repeated GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 and variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) can be used. Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0115] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, causing the domains to pair with complementary domains on another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0116] As used herein, the term "single domain antibody (sdAb)" has the meaning normally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody. Single domain antibodies are also known as nanobodies.

[0117] Each of the foregoing antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or the ability to compete with the full-length antibody for specific binding to the antigen. Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as is used for intact antibodies.

[0118] Herein, unless the context clearly indicates otherwise, when the term "antibody" is referred to, this includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0119] As used herein, the terms "monoclonal antibody," "McAb," and "mAb" have the same meaning and are used interchangeably, referring to a population of highly homologous antibody molecules, i.e., an antibody or a fragment of an antibody, from a population of identical antibody molecules, excluding spontaneously occurring natural mutations. Monoclonal antibodies are highly specific to a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" indicates only that the antibody is characterized as being obtained from a population of highly homologous antibodies, and should not be construed as requiring any particular method for preparing the antibody.

[0120] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application No. 4,816,567), or bacteriophage antibody library technology (see, e.g., Clackson et al., Nature, 352:624-628, 1991, or Marks et al., J. Mol. Biol., 222:581-597, 1991).

[0121] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently, or alternatively, a specific antibody (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography. For the purification of immunoglobulins, this may refer, for example, to D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0122] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light or / and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light or / and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), while still retaining binding activity to a target antigen (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include such antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0123] As used herein, the term "humanized antibody" generally refers to an engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three, receptor CDRs (of the heavy and / or light immunoglobulin chains) of a humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., murine) antibody, and the receptor framework can be a naturally occurring human framework sequence or have about 85%, 90%, 95%, 99%, or more sequence identity thereto. Humanized antibodies generally retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the expected properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0124] In the present application, the expected properties of the antibodies of the present invention include: (1) specifically binding to CD73 (e.g., membrane-bound or soluble human CD73); (2) inhibiting or reducing the enzymatic activity of CD73 (e.g., membrane-bound or soluble human CD73); (3) increasing the proliferation of anti-CD3 / anti-CD28-stimulated T cells (e.g., CD4+ T cells) in the presence of adenosine monophosphate (AMP); (4) mediating CD73 internalization; (5) reducing adenosine levels in CD73-expressing tumor cells; (6) stimulating an immune response (e.g., an immune response against a tumor or an immunogen); and (7) preventing and / or treating tumors (e.g., CD73-expressing tumors). The antibodies of the present invention have one or more of the expected properties described above.

[0125] Chimeric or humanized antibodies of the present invention can be prepared according to the sequences of the mouse monoclonal antibodies prepared above. DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0126] To prepare chimeric antibodies, mouse immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). For example, VH-encoding DNA is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, although IgG1 or IgG4 constant regions are generally preferred. For example, the DNA encoding the VL can be operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, although a kappa constant region is generally preferred.

[0127] To prepare humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, KC, eds., in Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals capable of producing fully human antibody repertoires after immunization that do not produce endogenous immunoglobulins can be utilized. For example, it has been reported that endogenous antibody production can be completely suppressed by homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice, and that the human germ-line immunoglobulin gene array can then be transferred into the germ-line mutant mice, resulting in the mice capable of producing human antibodies upon antigenic stimulation (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include the HuMAb mouse (Medarex, Inc.), which contains a human immunoglobulin gene minilocus encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, and additionally targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM Mouse™," which carries a human heavy chain transgene and a human light chain transchromosome (see patent application WO 02 / 43478).Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0128] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to an immunoglobulin-encoding sequence present in the genome of an organism that has not undergone the maturation process that results in gene rearrangement and mutations that result in the expression of a specific immunoglobulin. In the present invention, the term "heavy chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin heavy chain, comprising a V gene (variable), a D gene (diverse), a J gene (joining), and a C gene (constant). Similarly, the term "light chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin light chain, comprising a V gene (variable), a J gene (joining), and a C gene (constant). In the present invention, the amino acid sequence encoded by a germline antibody gene or germline antibody gene fragment is also referred to as a "germline sequence," the amino acid sequence encoded by a heavy chain germline gene is referred to as a heavy chain germline sequence, and the amino acid sequence encoded by a light chain germline gene is referred to as a light chain germline sequence. Germline antibody genes or germline antibody gene fragments, and their corresponding germline sequences, are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0129] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, for example, between an antibody and its antigen. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K DIn the present invention, "K D The term "dissociation constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) has an affinity of about 10 -9 Less than m, e.g., about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or lower affinity (K D ) refers to an antibody that binds to an antigen. The specific binding properties between two molecules can be determined using methods well known in the art, for example, by surface plasmon resonance (SPR) using a BIACORE device.

[0130] As used herein, the term "cytotoxic agent" includes any agent that is detrimental to (e.g., kills) cells, examples of which include a chemotherapeutic drug, a bacterial toxin, a plant toxin, or a radioactive isotope.

[0131] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain replication origins.

[0132] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell or human cell.

[0133] As used herein, the term "identity" refers to the degree of correspondence between two polypeptides or two nucleic acids. If two sequences for comparison have the same base or amino acid monomer subunit at a particular site (for example, two DNA molecules each have adenine at a particular site, or two polypeptides each have lysine at a particular site), the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences to the total number of sites for comparison x 100. For example, if 6 out of 10 sites in two sequences are identical, these two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 sites are identical). Generally, the comparison of two sequences is performed in a manner that produces maximum identity. Such alignments can be performed using computer programs, such as the Align program (DNAstar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0134] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the intended properties of the protein / polypeptide that contains the amino acid sequence.For example, conservative substitution can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitution includes the replacement of an amino acid residue with an amino acid residue that has a similar side chain, for example, the replacement of a corresponding amino acid residue with a residue that is physically or functionally similar (for example, has a similar size, shape, charge, chemical properties, includes the ability to form covalent or hydrogen bonds, etc.).Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace a corresponding amino acid with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0135] The 20 common amino acids referred to herein are written according to common usage.See, for example, Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably.Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations that are well known in the art.For example, alanine can be represented by A or Ala.

[0136] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. This is well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as p-hydroxybenzoic acid esters, chloretone, phenol, sorbic acid, and the like. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, etc. Agents for delaying absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chloretone, phenol, sorbic acid, etc.The stabilizer has the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, and includes, but is not limited to, sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, glucan or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0137] As used herein, the term "prevention" refers to a method performed to prevent or delay the appearance of a disease or disorder or symptom (e.g., tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of the extent of disease, stabilization (i.e., not worsening) of the disease state, delaying or slowing the progression of the disease, amelioration or remission of the disease state, and alleviation of symptoms (partially or completely), whether detectable or undetectable. In addition, "treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0138] As used herein, the term "subject" refers to a mammal, e.g., a primate, e.g., a human. In certain embodiments, the subject (e.g., a human) has a tumor (e.g., a CD73-expressing tumor) or is at risk for the aforementioned disease.

[0139] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, suppress, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients with the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, the effective amount for therapeutic use depends on the severity of the disease being treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, and other co-administered treatments.

[0140] As used herein, the term "antibody-mediated internalization" refers to the phenomenon in which an antibody crosses the cell membrane after binding to a cell surface antigen. Internalization includes antibody-mediated internalization of a receptor (e.g., CD73).

[0141] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen-presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, complement, etc.) produced by immune cells or the liver, which result in selective damage, destruction, or clearance of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in an autoimmune or pathogenic inflammatory state. In certain embodiments, the immune response refers to a T cell-mediated immune response that occurs upon stimulation of T cells with an antigen specific for the T cell. Non-limiting examples of responses generated by T cells upon antigen-specific stimulation include T cell proliferation and cytokine production.

[0142] Advantageous Effects of the Invention The antibodies of the present invention specifically bind to membrane-bound and soluble CD73 on the surface of tumor cells, significantly inhibit the enzymatic activity of CD73, and enhance immune responses. Therefore, the antibodies of the present invention have potential for use in the prevention and / or treatment of tumors, particularly CD73-expressing tumors. In addition, the humanized antibodies of the present invention not only retain the functions and properties of the mouse parent antibodies but also have a high degree of humanization, so that they can be safely administered to human subjects without eliciting an immunogenic response. It is particularly surprising that the antibodies of the present invention can more significantly restore AMP-mediated CD4+ T cell suppression and enhance the killing effect on CD73-expressing tumor cells compared to known anti-CD73 antibodies. Therefore, the antibodies of the present invention (especially humanized antibodies) have great clinical value.

[0143] Although embodiments of the present invention are described in detail below with reference to figures and examples, those skilled in the art will understand that the following figures and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0144] [Figure 1] FIG. 1 shows the binding curve of murine antibody 13D12 to CD73 on the surface of tumor cells. [Figure 2] FIG. 2 shows a scatter plot of binding of murine antibody 13D12 to monkey CD3+CD8+ T cells. [Figure 3] FIG. 3 shows the binding of humanized antibody 7002-01 to soluble recombinant CD73. [Figure 4A] FIG. 4A shows the inhibition of CD73 enzymatic activity by humanized antibody 7002-01 in the serum of patients with liver cancer (A) and melanoma (B). [Figure 4B]FIG. 4B shows the inhibition of CD73 enzymatic activity by humanized antibody 7002-01 in the serum of patients with liver cancer (A) and melanoma (B). [Figure 5] FIG. 5 shows the alleviating effect of humanized antibody 7002-01 on AMP-mediated CD4+ T cell suppression. [Figure 6] FIG. 6 shows the restorative effect of humanized antibody 7002-01 on tumor cell killing by PBMCs.

[0145] Sequence information The partial sequence information relevant to the present invention is provided below.

[0146] [Table 1]

[0147] [Table 2]

[0148] [Table 3] [Example]

[0149] Example The invention will now be described with reference to the following examples, which are intended to illustrate but not limit the invention.

[0150] Unless otherwise specified, molecular biology experimental methods and immunoassays used in the present invention were essentially performed by referring to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMA Usubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples are intended to describe the present invention by way of example and are not intended to limit the scope of protection sought by the present invention.

[0151] Example 1: Production of mouse anti-human CD37 antibodies To obtain mouse anti-human CD73 antibodies, mice (Balb / c, Shanghai Lingchang Biotechnology) were immunized using different immunization strategies (Table 1). The antigens used included CD73 protein (i.e., recombinantly expressed human CD73 having the sequence set forth in SEQ ID NO: 17) and CHOS-human CD73 (i.e., a CD73-overexpressing CHOS cell line expressing CD73 having the sequence set forth in SEQ ID NO: 17). The adjuvants included Freund's complete adjuvant CFA (InvivoGen company, catalog number: vac-cfa-60), IFA (InvivoGen company, catalog number: vac-ifa-60), and QuickAntibody (Beijing Boaolong Immune Technology Co., Ltd., catalog number: KX0210041). The routes of administration included intraperitoneal (ip) and subcutaneous (sc). Three days after the booster immunization, spleen cells from the immunized mice were fused with mouse myeloma cells SP2 / 0 using polyethylene glycol to obtain B cells capable of antibody expression and unlimited proliferation in vitro, which were then cultured in HAT selection medium. The fused hybridoma cells were plated in 96-well cell culture plates, and positive clones selected by primary screening were subjected to two to three rounds of subcloning.

[0152] [Table 4]

[0153] Primary screening: The binding ability of the culture supernatant of the clones to CD73 on the cell surface was determined in the primary screening by using human CD73-expressing tumor cell lines or overexpressing cell lines. The presence of reactive antibodies in the supernatant was revealed using a secondary antibody, DyLight488 goat anti-mouse IgG (Abeam catalog number ab97015), and the binding ability was evaluated using a whole-field cell scanning analyzer (see Example 2 for detailed experimental procedures). Secondary screening: CD73-expressing cells were used to screen for the ability to block CD73 enzyme activity to evaluate the ability of the antibodies to block CD73 enzyme activity on the cell membrane surface, and human serum soluble CD73 was used to screen for the ability to block CD73 enzyme activity to evaluate the ability of the antibodies to block soluble CD73 enzyme activity (see Example 6 for detailed experimental procedures).

[0154] The mouse monoclonal antibody 13D12 was isolated and purified from the culture supernatant of the final positive hybridoma monoclonal cell line.

[0155] Example 2: Evaluation of antigen-binding activity of mouse anti-CD73 antibodies 2.1 Detection of murine antibody binding to CD73-positive cells by cell scanning analyzer Cells used were: MDA-MB-231 (human breast cancer cell line endogenously expressing human CD73), SK-ME-S (human lung squamous cell carcinoma cell line endogenously expressing human CD73), H2030 (human non-small cell lung carcinoma cell line endogenously expressing human CD73), SKLU1 (human lung adenocarcinoma cell line endogenously expressing human CD73), BT549 (human ductal carcinoma cell line endogenously expressing human CD73), A375 (human melanoma cell line endogenously expressing human CD73), Calu6 (human degenerative carcinoma cell line endogenously expressing human CD73), 4T1 (mouse breast cancer cell line endogenously expressing mouse CD73), CHOS-human CD73 (transfected with human CD73), and CHOS (CD73-negative) cells.

[0156] Construction of CD73-expressing CHOS cells: Human CD73 (SEQ ID NO: 17) was overexpressed in CHOS cells (Invitrogen) by lentiviral infection and antibiotic resistance screening (MOI = 3-10, 5 μg / ml polybrene). Lentivirus was provided by Shanghai Genechem Co., Ltd. 72 hours after infection, the corresponding antibiotic was administered, and the culture was continued for 2-4 weeks, followed by expansion and cryopreservation for subsequent experiments.

[0157] Experimental Procedure: 10,000 cells were plated in a flat-bottom 96-well plate with 100 μL of DMEM + 10% FBS per well and cultured overnight to allow cells to adhere to the wall. The supernatant was discarded the following day. Eight 3-fold serial dilutions of antibodies were performed by diluting 1 / 3 of the total volume (100 μL) into 200 μL of DMEM. 100 μL of diluted antibody was added to each well of the cell plate (supernatants from fused clones or subclones were used for screening), and 100 μL of DMEM was added to the corresponding negative control wells. The incubation was carried out at room temperature for 1 hour. After discarding the supernatant, 100 μL of secondary antibody (DyLight488 goat anti-mouse IgG (Abcam, catalog no. ab97015)) was added to each well at a concentration of 5 μL / mL (diluted in DMEM) and incubated at room temperature for 0.5 hours. After staining, the supernatant was discarded, followed by washing once with PBS + 2% FBS. Then, 100 μL of PBS + 2% FBS was added to each well, and then readings were performed on the analyzer. The experimental plate readings were measured using a full-field cell scanning analyzer (Nexcelom Company, Model Celigo® Image Cytometer). During the measurement, high-speed scanning and imaging of cells in the wells were performed simultaneously in the green fluorescent channel corresponding to the secondary antibody and the bright-field channel. Using the images obtained by the fluorescent channel, antibody-bound cells were counted according to the parameters set for the morphology and fluorescence intensity of fluorescently labeled cells, and using the images obtained by the bright-field channel, adherent cells were counted according to the parameters set for cell morphology. The percentage of fluorescent, antibody-bound cells among the total number of cells was obtained by dividing the two counting results. The binding efficiency of anti-CD73 antibodies to CD73-expressing cell lines was determined according to the percentage. GraphPad was used for data analysis, where the horizontal axis indicates the logarithm of the antibody concentration, the vertical axis indicates the percentage of cells bound to the CD73 antibody with green fluorescence out of the total number of viable cells, and the EC50 value of anti-CD73 antibody binding to each of the cells was obtained by curve fitting.

[0158] The EC50 values ​​for 13D12 binding to each of the tumor cells are shown in Table 2-1 and Table 2-2, where NB stands for not detected within the concentration range for detection, and the binding curves for some cells are shown in Figure 1. The results showed that 13D12 was able to bind to cells naturally expressing CD73 and CHOS cells recombinantly expressing human CD73, but the antibody did not bind to cells not expressing CD73 (CHOS) or cells expressing mouse CD73 (4T1).

[0159] [Table 5]

[0160] [Table 6]

[0161] 2.2 Binding of mouse antibodies to cynomolgus monkey T cells Monkey blood samples from two donors (1132F and 1300M) were obtained from Medicilon. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient centrifugation system. PBMCs were incubated with the antibody to be tested, and then the cell-bound antibodies were stained with fluorochrome-labeled secondary antibodies (DyLight488 goat anti-mouse IgG, Abcam catalog number ab97015; DyLight488 goat anti-human IgG, Abcam catalog number ab97003). Fluorescently labeled antibodies against CD3+ and CD8+ were used to recognize T cells. All samples, along with unstained and fluorescence-compensated control samples, were run on a flow cytometer to detect antibody binding to cynomolgus monkey T cells.

[0162] Figure 2 shows the flow cytometry scatter plot of 13D12 binding to CD3+CD8+ T cells of cynomolgus monkey 1132F. The fold change in mean fluorescence intensity for the binding of mouse antibody 13D12 is shown in the table below. The results showed that 13D12 was able to bind to cynomolgus monkey CD8+ T cells.

[0163] [Table 7]

[0164] Example 3: Determination of variable region sequences of mouse anti-CD73 antibodies and preparation of chimeric antibodies Hybridoma cells were collected by centrifugation and diluted to 5-10 x 10 6The cells were added to 1 ml of TRIzol and 0.2 ml of chloroform, shaken vigorously for 15 seconds, and left at room temperature for 3 minutes. After centrifugation, the aqueous phase was collected, 0.5 ml of isopropanol was added, and the mixture was left at room temperature for 10 minutes. The precipitate was collected, washed with ethanol, and dried to obtain RNA. The template RNA and primers were added to the centrifuge tubes in an ice bath to ensure proper pairing of the primers and template, followed by reverse transcription and PCR amplification. 2.5 μL of dNTP / ddNTP mixture was added to each of the four microcentrifuge tubes, and the mixture was placed at 37°C for 5 minutes for later use. In an empty microcentrifuge tube, 1 pmol of PCR-amplified double-stranded DNA, 10 pmol of sequencing primer, and 2 μl of 5x sequencing buffer were added, followed by double distilled water to a total volume of 10 μl. This was followed by heating at 96°C for 8 minutes, cooling in an ice bath for 1 minute, and centrifugation at 10,000 g for 10 seconds at 4°C. 2 μl of pre-chilled labeling mixture (dCTP, dGTP, and dTTP, 0.75 μmol / L each), 5 μCi of α-32P-dATP, 1 μl of 0.1 mol / L DDT, and 2 U of sequencing enzyme were added, followed by water to a volume of 15 μl. The mixture was mixed thoroughly and then left on ice for 2 minutes to label the newly synthesized DNA strands. 3.5 μl of the labeling reaction mixture was added to four previously prepared microcentrifuge tubes and incubated at 37°C for 5 minutes. 4 μl of stop solution was added to each tube. Samples were heat denatured at 80°C for 5 minutes in a water bath, 2 μl was added to each lane of a sequencing gel, fragments were separated by electrophoresis, and sequence information was collected.

[0165] The VH and VL sequences of mouse antibody 13D12 are shown in the table below, and the CDR sequences of mouse monoclonal antibody 13D12 were further determined by the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669).

[0166] [Table 8]

[0167] The DNA sequences encoding the heavy and light chain variable regions of the above-mentioned mouse antibody (SEQ ID NOs: 11-12) were ligated to sequences encoding the heavy chain constant region (SEQ ID NO: 15) and light chain constant region (SEQ ID NO: 16) of a human antibody, respectively, and recombinantly expressed in HEK293 cells (ATCC). Cell supernatants containing the antibody clones in culture flasks were collected and purified using a Protein A column. The antibody protein was eluted with 100 mM acetic acid at pH 3. The purified antibody protein was then loaded onto a size-exclusion chromatography column for further separation and purification. The antibody protein corresponding to the monomer was formulated in PBS buffer supplemented with 20% glycerol. In this way, the chimeric antibody ch132D12 was obtained.

[0168] Example 4: Humanization of a murine anti-CD37 antibody To improve sequence homology between candidate antibodies and human antibodies and to reduce the immunogenicity of the antibodies to humans, humanized versions of the murine antibodies provided in the above examples were designed and prepared, in which the murine CDR regions were grafted onto human framework sequences by methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, KC (eds.), in Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004).

[0169] Specifically, the heavy and light chain CDR regions of mouse antibody 13D12 were grafted into the FR framework of the corresponding humanized template, and a series of back mutations were performed on the amino acid residues in the FR region of the humanized template, so that the humanized antibody retains the antigen-binding ability of the mouse antibody as much as possible. According to the above method, the present inventors prepared and obtained a humanized antibody of mouse antibody 13D12, named 7002-01 (its heavy chain variable region and light chain variable region are shown in SEQ ID NO: 9 and 10, respectively). The heavy chain constant region of the antibody was shown in SEQ ID NO: 15, and the light chain constant region was shown in SEQ ID NO: 16.

[0170] Example 5: Evaluation of antigen-binding activity of humanized anti-CD73 antibodies 5.1 Determining antibody binding to CD73-expressing cells by flow cytometry 500,000 CD73-expressing cells (see Example 2) were plated in 100 μL of FACS buffer (PBS + 2% FBS) per well in a round-bottom, low-binding 96-well plate for later use. Antibody samples were subjected to 12-point, 3-fold serial dilutions by diluting half of the total volume (100 μL) into 200 μL of FACS buffer. 100 μL of diluted antibody was added to each well of the cell plate, and 100 μL of FACS buffer was added to the corresponding negative control well, followed by incubation at 4° C. for 1 hour. After discarding the supernatant by centrifugation, washing was performed twice with FACS buffer. 100 μL of secondary antibody (DyLight488 goat anti-mouse IgG, Abcam, catalog number ab97015; DyLight488 goat anti-human IgG, Abcam, catalog number ab97003) (5 μg / mL, diluted in FACS buffer) was added to each well, and incubation was performed for another 0.5 hours at 4°C. After staining, the supernatant was removed by centrifugation, washing was performed twice with FACS buffer, 100 μL of FACS buffer was added to each well to resuspend the cells, and then readings were performed in a flow cytometer. The cells in the plate were measured using a flow cytometer (BD, model ACCURI C6 PLUS). During measurement, the cells were first gated according to FCS and SSC, and then analyzed by the green fluorescent channel (FITC) corresponding to the secondary antibody and SSC. GraphPad was used for data analysis, where the horizontal axis represents the logarithm of the antibody concentration and the vertical axis represents the mean fluorescence intensity. The EC50 values ​​of the anti-CD73 antibodies were obtained by curve fitting. The binding of humanized antibody 7002-01 to cells naturally expressing CD73 and cells recombinantly expressing human CD73 is shown in Table 5, where ND represents no detection. The results showed that humanized antibody 7002-01 had good binding activity to membrane-bound CD73.

[0171] [Table 9]

[0172] 5.2 Determining antibody binding to soluble human CD73 protein by ELISA 1 μg / ml recombinant human CD73 protein (Baiying Bio, recombinant human CD73 protein) was coated onto an ELISA plate overnight at 4°C in PBS. The plate was washed three times with wash buffer (PBS, 0.05% Tween-20), and nonspecific sites were blocked by adding 200 μl / well of PBS + 2% BSA. 100 μL of a gradient dilution of anti-CD73 antibody was added to the antigen-coated ELISA plate and incubated at 37°C for 1 hour. The plate was washed three times with wash buffer, and HRP-coupled goat anti-human or goat anti-mouse IgG Fc fragment secondary antibody was added for 1 hour at room temperature to detect captured anti-CD73 antibody. The plate was washed three times with wash buffer, and bound secondary antibody was revealed by adding TMB (HRP substrate) and incubating the plate in the dark at room temperature for 5–10 minutes. The enzyme reaction was stopped by adding 1 M sulfuric acid solution, and light absorption was measured at 450 nm. A graph was plotted with absorbance values ​​on the vertical axis and log antibody concentration values ​​on the horizontal axis, and the EC50 was calculated using GraphPad Prism software. The results are shown in Figure 3 and indicate that humanized antibody 7002-01 had good binding activity to soluble recombinant CD73, with an EC50 of 0.0081 μg / ml.

[0173] 5.3 Determining the affinity of humanized antibodies to recombinant human CD73 protein by Biacore Antibody affinity was measured by SPR on a Biacore T200 (GE) at 25°C. Antibodies were diluted to 1 μg / ml in 1x HBS-EP+ running buffer and captured on the chip surface (Protein A chip, GE, catalog number 29127556) at a flow rate of 10 μl / min for 30 seconds. A series of concentrations of CD73 protein (Baiying Bio, recombinant human CD73 protein) were then injected into the antibody channel at a flow rate of 30 μl / min for 180 seconds of association, followed by 900 seconds of dissociation. 10 mM Gly-HCl, pH 1.5, was used for regeneration. Sensorgram data were fitted using a 1:1 kinetic binding model. The bivalent affinity and kinetic association and dissociation rate constants are shown in the table below.

[0174] [Table 10]

[0175] Example 6: Evaluation of the inhibitory activity of anti-CD73 antibodies against CD73 enzyme activity 6.1 Inhibition assay of CD73 enzyme activity in tumor cells Excess AMP is known to block ATP-dependent luciferase activity. CD73, which cleaves AMP to adenosine + inorganic phosphate, restores luciferase activity and light emission by reducing AMP. Therefore, antibodies that block the enzymatic activity of CD73 will reduce light emission.

[0176] Human CD73-positive cells were harvested and counted, and 20,000 cells were seeded per well in 100 μL of complete medium in a flat-bottom 96-well plate. Antibody samples were subjected to eight three-fold serial dilutions by diluting 1 / 3 of the total volume (100 μL) into 200 μL of DMEM, and 100 μL of the diluted sample was added to the corresponding well of the plate. The negative control was an isotype control antibody (ISO). After 1 hour of incubation at 37°C, the supernatant was removed, and the cells were washed twice with PBS. A solution of AMP at a concentration of 125 μM was prepared in incomplete medium, and 100 μL of AMP was added to each well. The plate was then incubated for an additional 2 hours at 37°C. After centrifuging the reaction plate, 50 μL of the reaction solution was removed and added to another 96-well fluorescence plate (OptiPlate-96, Perkin Elmer, No. 6005290), and the same volume of 50 μM ATP solution and 50 μL of CTG reagent (Promega, G7572) were added per well. The plate was incubated in the dark at room temperature for 15 minutes, and the fluorescence (Lum) was measured using a microplate reader. GraphPad was used for data analysis, with the horizontal axis representing the logarithm of the antibody concentration and the vertical axis representing the inhibition rate. An enzyme activity inhibition curve was drawn and the IC50 was calculated. The inhibition rate was calculated as follows: Inhibition rate = 100-(Lum 陽性対照 -Lum 抗体 ) / (Lum 陽性対照 -Lum 陰性対照 ) x 100

[0177] The IC50 values ​​of the antibodies blocking endogenous CD73 in different human tumor cell lines are shown in the table below. The results showed that the humanized antibody 7002-01 could significantly inhibit the enzymatic activity of CD73 on the surface of tumor cells.

[0178] [Table 11]

[0179] 6.2 Inhibition assay of CD73 enzyme activity in tumor patient sera Tumor patient serum was diluted in phosphate buffer (125 mM Tris, 25 mM MgCl2, 125 mM NaCl), and 12.5 μL of diluted serum was added to each well of a white, flat-bottom 96-well plate for subsequent use. Antibody samples were subjected to 10-point serial 2- to 10-fold dilutions by diluting 1 / 1.5 of the total volume (100 μL) into 50 μL of phosphate buffer and 1 / 10 of the total volume (10 μL) into 90 μL of phosphate buffer. 12.5 μL of diluted antibody was added to each well of the cell plate, and 12.5 μL of phosphate buffer was added as a negative control. After centrifugation, incubation was performed at 37°C for 1.5 hours. AMP was diluted with phosphate buffer to obtain a 20 μM solution, and 25 μL of AMP was added to each well (except the positive control). After centrifugation, incubation was continued for another hour at 37°C. After the reaction, 25 μL of AMP was added to the positive control. 25 μL of AMP-Glo™ Reagent I (Promega, Cat. No. V5012) was immediately added to each well, and the plate was centrifuged and incubated at room temperature for 1 hour. 50 μL of AMP Detection Solution (Promega, Cat. No. V5012) was added to each well, and after centrifugation, the plate was incubated at room temperature for 1 hour. Fluorescence (Lum) was measured using a microplate reader. GraphPad was used for data analysis, with the horizontal axis representing the logarithm of the antibody concentration and the vertical axis representing the inhibition rate. Enzyme activity inhibition curves were plotted, and IC50 values ​​were calculated. The inhibition rate was calculated as follows: Inhibition rate = 100-(Lum 陽性対照 -Lum 抗体 ) / (Lum 陽性対照 -Lum 陰性対照 ) x 100

[0180] The results are shown in Figures 4A-4B, which indicate that the anti-CD73 antibody was able to effectively inhibit the dephosphorylation of AMP by CD73 and inhibit the enzymatic activity of CD73 in serum samples from patients with liver cancer (A) and melanoma (B).

[0181] Example 7: Anti-CD73 antibody-mediated internalization of CD73 Anti-CD73 antibody-mediated internalization of CD73 was examined by flow cytometry. To determine the relationship between antibody-induced internalization and incubation time, cells were incubated with 10 μg / mL of antibody for various periods at 37°C. After washing several times with PBS containing 2% FBS, 10 μg / mL of secondary antibody was added for 30 minutes at 4°C for staining, and then the CD73 expression of the cells was analyzed by flow cytometry. To compare the degree of antibody-induced internalization, cells were incubated with 10 μg / mL of antibody for 20 hours in parallel at both 4°C and 37°C. After washing several times with PBS containing 2% FBS, 10 μg / mL of secondary antibody was added for 30 minutes at 4°C for staining, and then the CD73 expression of the cells was analyzed by flow cytometry. MFI 37 is the MFI value of the sample incubated at 37°C, and MFI4 is the MFI value of the sample incubated at 4°C. Only binding occurs under these conditions without internalization, and MFI バックグラウンド was the MFI of the secondary antibody alone. The percentage of antibody-mediated internalization of cell surface CD73 was calculated by the following formula: Percentage of internalized CD73 = 100 - 100 × (MFI4 - MFI 37 ) / MFI4

[0182] The results are shown in Table 8 and demonstrated that the antibodies mediated the internalization of CD73 on the surface of tumor cells to varying degrees.

[0183] [Table 12]

[0184] Example 8: Anti-CD73 antibodies alleviate AMP-mediated CD4+ T cell suppression PBMC cells (obtained from fresh apheresis samples by Ficoll separation) stimulated with anti-CD3 / anti-CD28 for 24 hours before the experiment were collected and sorted using a CD4+ T Cell Isolation Kit for Human (Miltenyi, catalog number 130-096-533) to obtain CD4+ T cells, followed by centrifugation and removal of the supernatant. CD4+ T cells were resuspended in AIMV medium containing 40 μM EHNA and 120 IU / ml IL2 (the final concentration of EHNA was 20 μM, and the final concentration of IL2 was 60 IU / ml). 200,000 CD4+ cells were plated at 100 μL / well in a low-attachment round-bottom 96-well plate. Ten-point, 3-fold serial dilutions were performed by diluting 1 / 3 of the total volume (100 μL) into 200 μL AIMV medium. 50 μL of diluted antibody was added to each well, and 50 μL of AIMV medium was added to the corresponding negative control wells, and incubation was carried out at 37°C for 0.5 hours. 400 μM AMP was prepared with AIMV to reach a final concentration of 100 μM, and 50 μL of the prepared AMP solution was added to each well (control wells received AMP-free medium). After centrifugation, readings were taken on the analyzer. The plates were then incubated at 37°C for 72 hours and then read again on the analyzer. Readings were taken on the cells in the plate using a full-field cell scanning analyzer (Nexcelom, model Celigo® Image Cytometer). During the measurement, fast-scanning imaging of the cells in the wells was performed in the bright-field channel. The effect of anti-CD73 antibodies in alleviating AMP-mediated CD4+ T cell suppression was determined based on the size of clonal clusters. MEDI9447 (MedImmune) and BMS986179 (BMS) were used as reference antibodies and were expressed and purified by Genechem.

[0185] Cell growth on day 4 of T cell expansion is shown in Figure 5, where #18 refers to the number of PBMC donors for internal use. The initial antibody concentration in the figure was 100 μg / mL, with a total of nine 4-fold dilutions. Humanized antibody 7002-01 effectively alleviated AMP-mediated CD4+ T cell suppression, significantly restoring T cell proliferation, with efficacy superior to that of the reference antibodies MEDI9447 and BMS986179.

[0186] Example 9: Anti-CD73 antibody-mediated tumor cell killing Five thousand A375 cells were plated in 100 μL of DMEM + 10% FBS per well in a flat-bottom 96-well plate. Cells were allowed to adhere overnight, and the supernatant was removed the next day. Ten-point, three-fold serial dilutions were performed by diluting 1 / 3 of the total volume (100 μL) into 200 μL of AIMV. 50 μL of diluted antibody was added to each well of the cell plate, and 50 μL of AIMV was added to the corresponding negative control well. Incubation was carried out at 37°C for 0.5 hours. PBMC cells (from fresh apheresis samples by Ficoll separation) stimulated with anti-CD3 / CD28 for 24 hours were collected and resuspended in AIMV containing 40 μM EHNA and 120 IU / ml IL2 (final EHNA concentration was 20 μM, and final IL2 concentration was 60 IU / ml) and added to each well at 5,000 cells / 100 μL. A 400 μM AMP solution was prepared using AIMV, and 50 μL of the solution was added to each well to achieve a final AMP concentration of 100 μM. After centrifugation, incubation was carried out at 37°C for 72 hours. 10 μL of CCK8 kit (Japan Dojindo Co., Ltd., catalog number CK04) was added to each well and incubated at 37°C for 4 hours. OD450 was then measured using a microplate reader. According to the values ​​of the control wells, the OD values ​​were converted into inhibition percentages to appropriately determine the anti-CD73 antibody-mediated tumor cell killing effect. A higher percentage indicates a better anti-CD73 antibody-mediated tumor cell killing effect, and a lower percentage indicates a worse anti-CD73 antibody-mediated tumor cell killing effect. GraphPad was used for data analysis, where the horizontal axis is the logarithm of the antibody concentration and the vertical axis is the percentage of inhibition, and the IC50 values ​​of anti-CD73 antibodies in A375 cells were obtained by curve fitting.

[0187] The results are shown in Figure 6, where #22 refers to the donor number of PBMCs for internal use. The results showed that humanized antibody 7002-01 was able to effectively restore the killing effect of PBMCs on tumor cells.

[0188] While specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings published, and that all such modifications are within the scope of the present invention. The full division of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CD73, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); An antibody or antigen-binding fragment thereof, wherein the VH comprises a VHCDR1 comprising the sequence set forth in SEQ ID NO: 3, a VHCDR2 comprising the sequence set forth in SEQ ID NO: 4, and a VHCDR3 comprising the sequence set forth in SEQ ID NO: 5, and the VL comprises a VLCDR1 comprising the sequence set forth in SEQ ID NO: 6, a VLCDR2 comprising the sequence set forth in SEQ ID NO: 7, and a VLCDR3 comprising the sequence set forth in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 1 or a sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 2 or a sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

3. the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin; or the antibody or antigen-binding fragment thereof comprises a heavy chain framework region sequence derived from a human heavy chain germline sequence and a light chain framework region sequence derived from a human light chain germline sequence. The antibody or antigen-binding fragment thereof according to claim 1.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 9 or a sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 10 or a sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

5. The antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin. The antibody or antigen-binding fragment thereof according to claim 1.

6. 2. The antibody or antigen-binding fragment thereof of claim 1, characterized by one or more of the following: (i) the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin, IgG1, IgG2, IgG3, or IgG4; (ii) the light chain of the antibody or antigen-binding fragment thereof comprises a kappa or gamma light chain constant region derived from a human immunoglobulin; (iii) The antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from (1) a human IgG1 heavy chain constant region, or (2) a variant of a human IgG1 heavy chain constant region having the following substitutions compared to the wild-type sequence from which it is derived: L234F, L235E, P331S, and the above-mentioned amino acid positions are positions according to the EU numbering system. (iv) The antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO:

15. (v) The antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO:

16.

7. The antigen-binding fragment may be Fab, Fab', (Fab') ２ 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is selected from the group consisting of Fv, disulfide-linked Fv, scFv, and diabody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

9. The antibody or antigen-binding fragment thereof has the following characteristics: (a) binds to membrane-bound human CD73 or soluble human CD73, or both; (b) inhibits or reduces the enzymatic activity of membrane-bound human CD73 or soluble human CD73; (c) increasing the proliferation of CD4+ T cells stimulated by anti-CD3 and anti-CD28 antibodies in the presence of adenosine monophosphate (AMP); (d) reducing adenosine levels in CD73-expressing tumor cells; (e) Induce internalization of CD73 into cells by antibody-mediated receptor internalization The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, having one or more of the following:

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. 12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing the host cell according to claim 12 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cell.

14. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

15. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

16. 16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the bispecific or multispecific molecule of claim 14, or the immunoconjugate of claim 15, and a pharmaceutically acceptable carrier and / or excipient.

17. 17. The pharmaceutical composition of claim 16, characterized by one or more of the following: (i) The pharmaceutical composition further comprises an additional pharmaceutically active agent having anti-tumor activity. (ii) said pharmaceutical composition further comprises an additional pharmaceutically active agent selected from an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus. (iii) the pharmaceutical composition further comprises an additional pharmaceutically active agent selected from a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an anti-CD39 antibody, an anti-A2AR antibody, or an anti-HER2 / ErbB2 antibody.

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, (i) the antibody or antigen-binding fragment thereof bears a detectable label; (ii) The kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the second antibody further comprises a detectable label.

19. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the bispecific or multispecific molecule of claim 14, the immunoconjugate of claim 15, or the pharmaceutical composition of claim 16 or claim 17 in the manufacture of a medicament for preventing and / or treating a tumor in a subject expressing CD73.

20. 20. The use of claim 19, wherein the antibody or antigen-binding fragment thereof, the bispecific or multispecific molecule, the immunoconjugate, or the pharmaceutical composition is administered in combination with an additional pharmaceutically active agent.

21. 21. Use according to claim 20, characterized by one or more of the following: (i) The additional pharmaceutically active agent is a drug having anti-tumor activity. (ii) said additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus. (iii) the additional pharmaceutically active agent is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, an anti-CD39 antibody, an anti-A2AR antibody, or an anti-HER2 / ErbB2 antibody.

22. 20. The use according to claim 19, characterized by one or more of the following: (i) the tumor is selected from the group consisting of melanoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer and breast cancer. (ii) the subject is a human;

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 16, in the manufacture of a medicament for stimulating an immune response in a subject.

24. 1. A method for detecting the presence or amount of CD73 in a sample, comprising: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and CD73, or detecting the amount of the complex. Including, method.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the manufacture of a detection reagent for detecting the presence or amount of CD73 in a sample.

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