Anti-cd73 antibodies and uses thereof

Abstract

The present application belongs to the field of disease treatment and immunology, in particular, the present application discloses an antibody or antigen-binding fragment thereof against CD73, a nucleic acid molecule encoding the same, an immunoconjugate, a bispecific molecule and a pharmaceutical composition comprising the same, and the use thereof for enhancing an immune response and / or treating a tumor.

Description

Technical Field

[0001] This invention relates to the fields of disease treatment and immunology, and more specifically, to antibodies against CD73 or antigen-binding fragments thereof, nucleic acid molecules encoding them, immune conjugates comprising them, bispecific molecules and pharmaceutical compositions, and their use for enhancing immune responses and / or treating tumors. Background Technology

[0002] In recent years, the rapid development of cancer immunotherapy has led to a better understanding of tumor biology and immunology in the scientific community. The tumor microenvironment is a dynamic microenvironment that includes cancer cells, immune cells, fibroblasts, myofibroblasts, cytokines, blood vessels, and the extracellular matrix. Tumors are often in hypoxic conditions, and glucose and other nutrients are also scarce in the environment. In order to survive, cancer cells will reorganize their metabolic mechanisms in such an environment. Among them, adjusting purine metabolism is a very critical step, especially increasing the expression of differentiation cluster 73 (CD73, also known as extracellular 5'-nucleotidase). CD73 is a glycosylphosphatidylinositol-anchored cell surface protein that is usually expressed on a subset of endothelial cells and 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, while the CD73-catalyzed dephosphorylation of adenosine monophosphate to adenosine is the rate-determining step on 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 an immune response. Conversely, the hydrolysis of ATP to adenosine acts as a reverse feedback mechanism, leading to the suppression of the immune response. Adenosine is a well-studied signaling molecule whose biological effects are mediated via 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, constituting an important mechanism for tumor immune escape (Bin Z. Cancer Research 2010; 70:6407-6411). Among other effects, tumor-derived adenosine deeply inhibits invasive effector T cells through A2A receptors activated by adenylate cyclase.

[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 an effective prognostic biomarker in solid tumors, and CD73 overexpression is associated with shorter patient survival or shorter progression-free survival (Rong W et al. Oncotarget 2017; 8(34):57327-57336). CD73 expression in cancer is associated with increased proliferation, migration, angiogenesis, invasiveness, and metastasis. It has been shown that knockdown of siRNA or overexpression of CD73 on tumor cells can regulate tumor growth and metastasis (Paul B et al. PNAS 2013; 110(36):14711-14716); CD73- / - mice are protected from transplanted and spontaneous tumors (John S et al. Cancer Research 2010; 71:2892-2900). In addition to the reported regulation of cell-cell and cell-matrix interactions on tumor cells, CD73 expression and activity are also associated with weakened T-cell responses (Dachuan J et al. Cancer Res 2010; 70:2245-55). CD73 is also involved in resistance to chemotherapy drugs, such as anthracyclines (Loi, S et al. PNAS 2013; 110:11091–11096), and resistance to apoptosis induced by tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL). Therefore, CD73 can regulate cancer progression directly and indirectly, highlighting its potential as a novel therapeutic target.

[0005] In addition, cancer immune checkpoint inhibitors have shown good efficacy in patients with various cancers in recent years. However, a considerable proportion of patients still do not respond to these treatments, and one-third of patients relapse after the initial response (adaptive resistance). This indicates that multiple non-redundant immunosuppressive mechanisms coexist in the tumor microenvironment, and drug targets that can be used in synergy or in combination with the above-mentioned drugs are currently a hot topic in cancer immunology research.

[0006] Therefore, CD73 has shown its potential as a target for anti-tumor therapy, whether as a monotherapy or in combination therapy. Summary of the Invention

[0007] The antibody of this invention can specifically bind to membrane-bound CD73 and non-membrane-bound CD73 on the surface of tumor cells, inhibit their enzymatic activity, enhance the immune response, and exhibit good anti-tumor activity. Furthermore, it possesses superior functional properties compared to known anti-CD73 antibodies. Therefore, the antibody of this invention has the potential for the prevention and / or treatment of tumors, providing an option for clinical tumor immunotherapy.

[0008] The antibody of the present invention

[0009] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, said antibody or antigen-binding fragment comprising:

[0010] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0011] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it;

[0012] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:4 or SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it;

[0013] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it;

[0014] And / or,

[0015] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0016] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it;

[0017] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions); and

[0018] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it.

[0019] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0020] In some embodiments, the antibody or antigen-binding fragment of the present invention is capable of binding to human CD73, such as membrane-bound human CD73 and / or soluble human CD73.

[0021] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD73, said antibody or antigen-binding fragment comprising:

[0022] The following three heavy chain variable regions (CDRs): VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable regions shown in any one of SEQ ID NO: 1, 9, 12, and 13, and

[0023] The following are three light chain variable regions CDRs: VLCDR1, VLCDR2 and VLCDR3 contained in the light chain variable region shown in SEQ ID NO:2 or 10.

[0024] In some implementations, the three CDRs contained in the heavy chain variable region and the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO:1, and VLCDR1, VLCDR2, and VLCDR3 contained in the light chain variable region shown in SEQ ID NO:2. In some embodiments, the antibody or its antigen-binding fragment comprises VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO:9, and VLCDR1, VLCDR2, and VLCDR3 contained in the light chain variable region shown in SEQ ID NO:10.

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO:12, and VLCDR1, VLCDR2, and VLCDR3 contained in the light chain variable region shown in SEQ ID NO:2. In some embodiments, the antibody or its antigen-binding fragment comprises VHCDR1, VHCDR2, and VHCDR3 contained in the heavy chain variable region shown in SEQ ID NO:13, and VLCDR1, VLCDR2, and VLCDR3 contained in the light chain variable region shown in SEQ ID NO:10.

[0027] In some embodiments, the antibody or antigen-binding fragment 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 shown in SEQ ID NO:1 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. In some embodiments, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO:3-5, respectively, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 as shown in SEQ ID NO:6-8, respectively.

[0028] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:12 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. In some embodiments, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO:3, 11, and 5, respectively, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 as shown in SEQ ID NO:6-8, respectively.

[0029] In some embodiments, the antibody or its antigen-binding fragment comprises a framework region sequence derived from human immunoglobulins, wherein the framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human residues to corresponding murine residues. In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence (i.e., the amino acid sequence encoded by the human heavy chain germline gene), and a light chain framework region sequence derived from a human light chain germline sequence (i.e., the amino acid sequence encoded by the human light chain germline gene), wherein the heavy chain framework region and / or the light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human residues to corresponding murine residues.

[0030] In some embodiments, the antibody or its antigen-binding fragment 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, wherein the heavy chain framework region and / or the light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to corresponding murine residues.

[0031] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. In some embodiments, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO:3-5, respectively, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 as shown in SEQ ID NO:6-8, respectively.

[0032] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:13 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10 or a sequence having at least about 85%, 90%, 95%, or 99% sequence identity compared to it. In some embodiments, the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO:3, 11, and 5, respectively, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 as shown in SEQ ID NO:6-8, respectively.

[0033] In some embodiments, the antibody or antigen-binding fragment of the present invention may further comprise a constant region derived from mammalian (e.g., mouse or human) immunoglobulins. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a heavy chain constant region derived from mammalian (e.g., mouse or human) immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment comprises a light chain constant region derived from mammalian (e.g., mouse or human) immunoglobulins (e.g., κ or λ).

[0034] In some embodiments, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0035] The light chain of the antibody or its antigen-binding fragment of the present invention comprises a light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions) compared to the sequence from which it is derived.

[0036] In some embodiments, the constant region may contain amino acid mutations to alter one or more of the following properties of the antibody of the present invention: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, altering the antibody's affinity for effector ligands (such as FcR or complement C1q), thereby changing effector function (e.g., reducing it).

[0037] Methods of replacing 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; however, in other cases, these effector functions may be unnecessary or even harmful, depending on the intended purpose.

[0038] Therefore, in some embodiments, the antibody or antigen-binding fragment of the present invention has reduced or even eliminated effector functions (e.g., ADCC and / or CDC activity). In such embodiments, the antibody or antigen-binding fragment of the present invention may comprise a variant of the human IgG heavy chain constant region having at least one, at least two, or all three of the following substitutions compared to its derived wild-type sequence: L234F, L235E, P331S (the amino acid positions mentioned above are according to the EU numbering system), see, for example, Acta Cryst. (2008). D64, 700–704.

[0039] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises a constant region of the human wild-type IgG1 heavy chain. In such embodiments, the antibody or antigen-binding fragment of the present invention has ADCC and CDC activities.

[0040] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises a variant of the human IgG1 heavy chain constant region having the following substitutions compared to its derived wild-type sequence: L234F, L235E, P331S (position according to the EU numbering system), for example, the heavy chain constant region shown in SEQ ID NO:19. In such embodiments, the antibody or antigen-binding fragment of the present invention has eliminated or reduced ADCC and / or CDC activity.

[0041] In some preferred embodiments, the heavy chain of the antibody or its antigen-binding fragment of the present invention comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, the variant having substantially unchanged effector function compared to its derived wild-type sequence. In such embodiments, the variant may have up to 20 conserved amino acid substitutions compared to its derived wild-type sequence (e.g., up to 15, 10, or 5 conserved substitutions; for example, 1, 2, 3, 4, or 5 conserved substitutions).

[0042] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises a human κ light chain constant region, such as the light chain constant region shown in SEQ ID NO:20.

[0043] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises the heavy chain constant region (CH) shown in SEQ ID NO:19; and / or the light chain constant region (CL) shown in SEQ ID NO:20.

[0044] In some embodiments, the antibodies of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies. In some embodiments, the antigen-binding fragments of the present invention are selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0045] In some embodiments, the antibody or antigen-binding fragment of the present invention possesses one or more of the following features:

[0046] (a) Binding to membrane-bound human CD73 or soluble human CD73 or both; for example, said membrane-bound human CD73 is expressed on the surface of tumor cells;

[0047] (b) Inhibit or reduce the enzyme activity of CD73 (e.g., membrane-bound human CD73 or soluble human CD73); for example, inhibit or reduce the conversion of human CD73-mediated adenosine monophosphate (AMP) to adenosine, as determined, for example, by CellTiter Glo (CTG) assay (e.g., the method described in Example 6);

[0048] (c) In the presence of adenosine monophosphate (AMP), increase the proliferation of anti-CD3 / anti-CD28 stimulated T cells (e.g., CD4+ T cells); as determined, for example, by the method described in Example 8;

[0049] (d) CD73 is internalized into cells (e.g., tumor cells) by antibody-mediated receptor internalization, said cells expressing CD73 on their surface; for example, 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 method described in Example 7);

[0050] (e) soluble human CD73 is bound at an EC50 of less than about 0.01 μg / ml or less; said EC50 is determined by ELISA.

[0051] (f) Binding soluble human CD73 with a KD of less than about 0.5 nM or lower; the KD was determined by Biacore;

[0052] (g) Reduce adenosine levels in tumor cells expressing CD73;

[0053] (h) Stimulate an immune response; for example, stimulate an immune response against a tumor (e.g., a tumor expressing CD73);

[0054] (i) Prevention and / or treatment of tumors (e.g., tumors expressing CD73).

[0055] In some embodiments, the antibody or its antigen-binding fragment is 72GB or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or functional variants thereof, which substantially retain the biological function of the antibody or its antigen-binding fragment from which they are derived.

[0056] In this invention, the antibody or antigen-binding fragment thereof may include variants that differ from the antibody or antigen-binding fragment from which they are derived only in the conserved substitution of one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibody or antigen-binding fragment from which they are derived, and substantially retain the aforementioned biological functions of the antibody or antigen-binding fragment from which they are derived.

[0057] Antibody preparation

[0058] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting 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 antibodies of the present invention.

[0059] The antigen-binding fragments of the present invention can be obtained by hydrolyzing 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 also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0060] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0061] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain variable region of the antibody of the present invention or an antigen-binding fragment thereof, and / or a second nucleotide sequence encoding a light chain variable region of the antibody of the present invention or an antigen-binding fragment thereof.

[0062] In some embodiments, the first nucleotide sequence comprises a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO:14, or (b) a sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions), or (c) a sequence differing from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the second nucleotide sequence comprises a sequence selected from: (d) the nucleotide sequence shown in SEQ ID NO:15, or (e) a sequence substantially identical to the nucleotide sequence described in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (d), or a sequence having one or more nucleotide substitutions), or (f) a sequence differing from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0063] In some embodiments, the first nucleotide sequence comprises a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO:16, or (b) a sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions), or (c) a sequence differing from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the second nucleotide sequence comprises a sequence selected from: (d) the nucleotide sequence shown in SEQ ID NO:17, or (e) a sequence substantially identical to the nucleotide sequence described in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (d), or a sequence having one or more nucleotide substitutions), or (f) a sequence differing from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0064] In some embodiments, the first nucleotide sequence comprises a sequence selected from: (a) the nucleotide sequence shown in SEQ ID NO:18, or (b) a sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions), or (c) a sequence differing from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the second nucleotide sequence comprises a sequence selected from: (d) the nucleotide sequence shown in SEQ ID NO:17, or (e) a sequence substantially identical to the nucleotide sequence described in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity compared to the nucleotide sequence described in (d), or a sequence having one or more nucleotide substitutions), or (f) a sequence differing from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0065] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain of the antibody of the present invention or an antigen-binding fragment thereof, and / or a second nucleotide sequence encoding a light chain of the antibody of the present invention or an antigen-binding fragment thereof.

[0066] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecules of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc. In some embodiments, the vector is capable of expressing the antibody or antigen-binding fragment of the present invention in a subject (e.g., a human).

[0067] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecules of the present invention or the vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHOG44).

[0068] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the 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.

[0069] Derived antibodies

[0070] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to CD73. Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0071] Therefore, in some embodiments, the antibody or its antigen-binding fragment of the present invention carries 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 described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such labels are well known in the art, and examples 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 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., The labels include pyrometric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. 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 calculator, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product generated by the enzyme's action on the substrate, and pyrometric markers are detected by simple, visually appealing colored markers. In some embodiments, such labels can be applied to immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0072] Bispecific or multispecific molecules

[0073] 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 of the present invention can be part of a bispecific or multispecific molecule comprising a second functional module (e.g., a second antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, thereby enabling 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 capable of specifically binding to any protein that can be used as a potential target for combination therapy. To generate said bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0074] Therefore, in another aspect, the present invention provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment of the present invention.

[0075] In some embodiments, the bispecific or multispecific molecules specifically bind to CD73 (e.g., membrane-bound human CD73 and / or soluble human CD73) and additionally specifically bind to one or more other targets.

[0076] In some embodiments, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity against a second target.

[0077] Immunoconjugates

[0078] The antibodies or antigen-binding fragments thereof of the present invention can be conjugated with therapeutic agents to form immunoconjugates. Because immunoconjugates have the ability to selectively deliver one or more therapeutic agents to target tissues (e.g., tumor-associated antigens, such as tumors expressing CD73), immunoconjugates can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0079] Therefore, in another aspect, the present invention provides an immunoconjugate comprising an antibody of the present invention or an antigen-binding fragment thereof and a therapeutic agent attached to said antibody or antigen-binding fragment thereof.

[0080] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0081] In some embodiments, the therapeutic agent is a cytotoxic agent. In this invention, the cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells).

[0082] In some embodiments, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

[0083] Examples of alkylating agents that can be used in the immunoconjugates of the present invention include, but are not limited to, nitrogen mustards (such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethyleneimines (such as thiotepa), sulfate esters and polyols (such as busulfan, dibromomannitol), nitrosoureas (such as carmustine, lomustine, etc.), and platinum-based antitumor agents (such as cisplatin, oxaliplatin, carboplatin, etc.).

[0084] Examples of mitotic inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, maytansine derivatives (e.g., maytansine, maytanol, C-3 ester of maytanol, etc.), taxane derivatives (e.g., docetaxel, paclitaxel, or nanoparticle paclitaxel, etc.), and vinblastine alkaloids (e.g., vinorelbine sulfate, vincristine, vinblastine, or vinorelbine, etc.).

[0085] Examples of antitumor antibiotics that can be used in the immunoconjugates of the present invention include, but are not limited to, actinomycin, anthracycline antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), calicimycin, and pyromycin.

[0086] Examples of antimetabolites that can be used in the immunoconjugates of the present invention include, but are not limited to, folic acid antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, fluorouridine, cytarabine, capecitabine, gemcitabine), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nerabine, pentostatin).

[0087] Examples of topoisomerase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, camptothecins and their derivatives (e.g., irinotecan, topotecan, etc.), acridine, doxorubicin, epipodophyllotoxin, rose alkaloids, epirubicin, etoposide, propylamine, teniposide, etc.

[0088] Examples of tyrosine kinase inhibitors that can be used in the immunoconjugates of the present invention include, but are not limited to, axitinib, bosutinib, cidinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, letotinib, nilotinib, semasanib, sunitinib, vandetanib, etc.

[0089] 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 wait.

[0090] In some exemplary embodiments, the therapeutic agent is selected from platinum-based antitumor agents, anthracycline antibiotics, taxanes, nucleoside analogs, camptothecin compounds, and their analogs or homologues, and any combination thereof.

[0091] In some embodiments, the antibody or its antigen-binding fragment of the present invention is optionally conjugated to the therapeutic agent via a linker.

[0092] In this invention, cytotoxic agents can be conjugated to the antibodies or antigen-binding fragments thereof using existing linker techniques in the art. Examples of linker types already used for conjugating cytotoxic agents to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. Linkers that are readily cleaved at low pH within lysosomal compartments or readily cleaved by proteases (e.g., proteases preferentially expressed in tumor tissues, such as cathepsins, such as cathepsins B, C, and D) can be selected.

[0093] Further discussion on the 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 Deliv. Rev. 53: 247-264.

[0094] Therapeutic Uses and Pharmaceutical Compositions

[0095] The antibodies or antigen-binding fragments of the present invention can modulate (e.g., enhance, stimulate, increase, inhibit, reduce, or neutralize) one or more biological activities of CD73. In certain circumstances, the antibodies or antigen-binding fragments 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 AMP. Therefore, the antibodies or antigen-binding fragments of the present invention can be used as monotherapy to prevent and / or treat tumors by inhibiting or reducing the enzymatic activity of CD73.

[0096] Furthermore, there have been reports that targeting CD73 may 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 a HER2 / ErbB2-driven mouse model of breast cancer, CD73 expression in both tumor cells and host cells significantly suppressed immune-mediated responses mediated by anti-ErbB2 monoclonal antibodies (Martin T et al. Cancer Research 2017; 77(20); 5652–63). In addition, in vitro experiments have shown that activation of the A2A receptor regulates the upregulation of PD-1 on tumor-infiltrating cytotoxic T cells, while blocking the PD-1 signaling pathway with anti-PD-1 antibodies upregulates the expression of the A2A receptor on tumor-infiltrating cytotoxic T cells (Cekic C et al. Cancer Res 2014; 74:7239-49). Previous reports have shown that anti-CD73 antibodies significantly enhance the activity of anti-CTLA-4 and anti-PD-1 antibodies in various mouse tumor models, and both monotherapy and combination therapy depend on host interferon-γ and cytotoxic T cells; the effect of extracellular adenosine on tumor-infiltrating T cells shows that adenosine receptor activation enhances PD-1 expression on tumor-specific cytotoxic T cells and helper T cells (Bertrand A et al. Clin Cancer Res 2013; 19(20):5626-5635). Clinical studies have found that increased CD73 levels in melanoma patients treated with pembrolizumab (anti-PD-1) are positively correlated with disease progression, and 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). Other studies have shown that high levels of soluble CD73 enzyme activity in metastatic melanoma patients receiving nivolumab are significantly associated with poorer overall survival and progression-free survival. In multivariate analysis, CD73 enzyme activity was identified as the strongest prognostic factor for both overall survival and progression-free survival, and higher baseline levels of CD73 enzyme activity prior to nivolumab treatment were associated with lower treatment response rates (Silvana M et al. J Transl Med 2017; 15:244). Correspondingly, CD73 expression levels and PD-L1 expression levels have been found to be complementary in tumor samples from non-small cell lung cancer patients. Therefore, the antibody or its antigen-binding fragment of the present invention can also be combined with immune checkpoint inhibitors or tumor-specific antibodies for the prevention and treatment of tumors.

[0097] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, or an immunoconjugate of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0098] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents.

[0099] In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive isotopes, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), antiangiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), molecularly targeted drugs (e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, etc.), oncolytic viruses, etc.

[0100] In some embodiments, the additional pharmaceutically active agent is selected from immune checkpoint inhibitors (e.g., PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors), anti-CD39 antibodies, anti-A2AR antibodies, or anti-HER2 / ErbB2 antibodies.

[0101] In some embodiments, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention, along with the additional pharmaceutically active agent, are provided as separate components or as a mixture in the pharmaceutical composition. Therefore, the antibody or its antigen-binding fragment, bispecific or multispecific molecule, or immunoconjugate of the present invention, along with the additional pharmaceutically active agent, can be administered simultaneously, separately, or sequentially.

[0102] In some exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0103] In another aspect, the present invention provides a method for preventing and / or treating tumors in a subject (e.g., a human), the method comprising administering to a subject in need an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition thereof. In another aspect, the use of the antibody of the present invention or an antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition thereof for preventing and / or treating tumors in a subject (e.g., a human), or for use in the preparation of a medicament for preventing and / or treating tumors in a subject (e.g., a human), is provided.

[0104] In some embodiments, the tumor expresses CD73. In some embodiments, the CD73 may be membrane-bound human CD73 and / or soluble human CD73.

[0105] In some embodiments, the tumor involves tumor cells expressing CD73. In some embodiments, CD73 is expressed on the surface of the tumor cells.

[0106] In some embodiments, the tumor is selected from melanoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer.

[0107] In some embodiments, the antibody of the present invention or its antigen-binding fragment is administered in combination with a second therapeutic agent or treatment. The second therapeutic agent or treatment may be administered before, simultaneously with, or after the administration of the antibody of the present invention or its antigen-binding fragment.

[0108] In some embodiments, the second therapeutic agent is selected from drugs with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

[0109] In some embodiments, the antibody or antigen-binding fragment of the present invention is administered in combination with a therapeutic agent selected from the following: immune checkpoint inhibitors (e.g., PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors), anti-CD39 antibodies, anti-A2AR antibodies, or anti-HER2 / ErbB2 antibodies.

[0110] In some exemplary embodiments, the PD-1 inhibitor is selected from PDR001, nivolumab, pembrolizumab, pildizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224.

[0111] In some exemplary embodiments, the PD-L1 inhibitor is selected from FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559.

[0112] In some exemplary embodiments, the CTLA-4 inhibitor is selected from aspirinumab or trimerumab.

[0113] In some exemplary embodiments, the LAG-3 inhibitor is selected from LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767.

[0114] In some implementations, the second treatment may be any therapy known for use on tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

[0115] In another aspect, the present invention provides a method for stimulating an immune response in a subject, the method comprising administering to a subject in need an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition thereof. In another aspect, the present invention is provided for use in stimulating an immune response in a subject, or for use in the preparation of a medicament for stimulating an immune response in a subject.

[0116] In some implementations, the immune response is a T-cell-mediated immune response.

[0117] In some embodiments, the immune response is an immune response against a tumor (e.g., a tumor expressing CDD73). In some embodiments, the subject has a tumor (e.g., a tumor expressing CDD73).

[0118] In some embodiments, the immune response is an immune response against an immunogen. In such embodiments, the method further includes administering the immunogen to the subject. In some embodiments, the immunogen is selected from tumor-associated antigens (e.g., proteins, peptides, or carbohydrate molecules), tumor cells, dendritic cells sensitized by the antigen, and any combination thereof. In other embodiments, the immunogen is selected from pathogen-associated antigens (e.g., proteins, peptides, or carbohydrate molecules), inactivated or attenuated pathogens, dendritic cells sensitized by the antigen, and any combination thereof.

[0119] In another aspect, the present invention provides a method for reducing adenosine levels in CD73-expressing tumor cells, comprising contacting said cells with an antibody or antigen-binding fragment thereof of the present invention, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition thereof. In some embodiments, said method is used to reduce adenosine levels in CD73-expressing tumor cells in vitro for non-therapeutic purposes. In another aspect, the use of the antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition of the present invention for reducing adenosine levels in CD73-expressing tumor cells is provided, or for use in the preparation of a medicament for reducing adenosine levels in CD73-expressing tumor cells.

[0120] 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 field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0121] Furthermore, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention can be present in the pharmaceutical composition in unit dose form for ease of administration.

[0122] The antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intrabladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, bispecific or multispecific molecules, immunoconjugates, and pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0123] The pharmaceutical compositions of the present invention may include, in a “therapeutic effective amount” or “preventative effective amount”, the antibody or antigen-binding fragment thereof, a bispecific or multispecific molecule, an immunoconjugate, or a pharmaceutical composition thereof. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0124] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., therapeutic or preventative response). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0125] In this invention, the subject can be a mammal, such as a human.

[0126] Detection uses and kits

[0127] The antibody or its antigen-binding fragment of the present invention can specifically bind to CD73, thereby enabling it to be used to detect the presence or level of CD73 in a sample.

[0128] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the antibody of the present invention or an antigen-binding fragment thereof is labeled with a detectable marker. In other embodiments, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable marker.

[0129] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin.

[0130] In another aspect, the present invention provides a method for detecting the presence or amount of CD73 in a sample, comprising the following steps:

[0131] (1) Contact the sample with the antibody or its antigen-binding fragment of the present invention;

[0132] (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and CD73 or detect the amount of the complex.

[0133] The formation of the complex indicates the presence of CD73 or cells expressing CD73.

[0134] In some embodiments, the sample is a cell sample, i.e., a sample containing cells (e.g., tumor cells). In such embodiments, preferably, the complex is formed between the antibody or its antigen-binding fragment and CD73 expressed by the cells in the sample.

[0135] In some embodiments, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable marker. In other embodiments, in step (2), a reagent labeled with a detectable marker is used to detect the antibody or antigen-binding fragment of the present invention.

[0136] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In some embodiments, the CD73 is human CD73, such as membrane-bound and / or soluble human CD73.

[0137] In another aspect, the invention provides the use of the antibody or antigen-binding fragment thereof for determining the presence or amount of CD73 in a sample, or in the preparation of a detection reagent for determining the presence or amount of CD73 in a sample. In some embodiments, the CD73 is human CD73, such as membrane-bound and / or soluble human CD73.

[0138] Terminology Definition

[0139] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0140] As used herein, the term “differentiation cluster 73” or “CD73” also refers to an extracellular 5'-nucleotidase that converts extracellular 5' monophosphate nucleosides into nucleosides, specifically adenosine monophosphate (AMP) into adenosine. The term CD73 includes membrane-bound forms (also known as membrane-bound CD73) or soluble forms (also known as soluble or non-membrane-bound CD73). CD73 can be isolated from cells or tissues that naturally express them, or generated recombinantly using techniques well known in the art. The sequences of CD73 are well known in the art and can be found in the NCBI database accession number NM_002526.

[0141] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of 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 are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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. Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V L It 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 carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0142] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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 & 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, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, 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).

[0143] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat numbering system.

[0144] As used herein, the term "framework region" or "FR" residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0145] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0146] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the 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 the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0147] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0148] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0149] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0150] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0151] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, 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, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this 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 VH and VL of scFv.

[0152] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0153] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0154] Each of the above antibody fragments 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 the antigen.

[0155] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0156] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0157] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0158] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0159] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0160] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In some embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0161] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is called the "donor," and the immunoglobulin providing the framework is called the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework or a sequence having approximately 85%, 90%, 95%, 99%, or higher identity with it. Humanized antibodies typically retain the intended properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0162] In this application, the intended properties of the antibodies of the present invention include: (1) specifically binding to CD73 (e.g., membrane-bound human CD73 or soluble human CD73); (2) inhibiting or reducing the enzymatic activity of CD73 (e.g., membrane-bound human CD73 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 immune responses (e.g., immune responses against tumors or immunogens); and (7) preventing and / or treating tumors (e.g., tumors expressing CD73). The antibodies of the present invention have one or more of the above-described intended properties.

[0163] The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0164] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of mouse immunoglobulins to the constant region of human immunoglobulins (see, for example, U.S. Patent No. 4,816,567, Cabilly et al.). For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the 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, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, 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 can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated 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 regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0165] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies, and then transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon encountering antigen stimulation (see, for example, 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 the aforementioned transgenic animals include HuMAb mice (Medarex, Inc.) containing miniloci of human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or "KM mice" carrying human heavy chain transgenes and human light chain transchromosomal transgenes. TM (See patent application WO02 / 43478). Other methods for humanizing antibodies 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).

[0166] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence in the genome of an organism that encodes an immunoglobulin, which has not undergone a maturation process involving genetic rearrangements and mutations that would lead to the expression of a specific immunoglobulin. In this invention, the term "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding the heavy chain of immunoglobulins, including the V gene (variable), D gene (diversity), J gene (joining), and C gene (constant); similarly, the term "light chain germline gene" refers to a germline antibody gene or gene segment encoding the light chain of immunoglobulins, including the V gene (variable), J gene (joining), and C gene (constant). In this invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also referred to as a "germline sequence." The amino acid sequence encoded by the heavy chain germline gene is called the heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene is called the 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 professional databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0167] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody binds at a dissociation equilibrium constant of less than about 10-1. -9 M, for example, less than about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or lower affinity (K) D The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0168] As used herein, the term "cytotoxic agent" includes any agent that is harmful to cells (e.g., kills cells), such as chemotherapy drugs, bacterial toxins, plant toxins, or radioactive isotopes.

[0169] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0170] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0171] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0172] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0173] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0174] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which 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, osmotic pressure maintainers, absorption delayers, 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 parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0175] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0176] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor (e.g., a tumor expressing CD73), or is at risk of having the aforementioned disease.

[0177] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0178] As used herein, the term "antibody-mediated internalization" refers to the phenomenon where an antibody crosses the cell membrane after binding to a cell surface antigen. Internalization includes antibody-mediated receptor (e.g., CD73) internalization.

[0179] 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, and complement) produced by immune cells or the liver, resulting in selective damage, destruction, or clearance from the body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. In some embodiments, the immune response refers to a T cell-mediated immune response that arises upon stimulation of the T cell by an antigen specific to that T cell. Non-limiting examples of responses generated by T cells upon antigen-specific stimulation include T cell proliferation and cytokine production.

[0180] Beneficial effects of the invention

[0181] The antibodies of this invention specifically bind to membrane-bound CD73 and soluble CD73 on the surface of tumor cells, significantly inhibiting their enzymatic activity and enhancing the immune response. Therefore, the antibodies of this invention have the potential for the prevention and / or treatment of tumors (especially those expressing CD73). Furthermore, the humanized antibodies of this invention not only retain the function and properties of the murine parental antibodies but also have a high degree of humanization, thus allowing safe administration to human subjects without inducing immunogenic reactions. Particularly surprisingly, the antibodies of this invention more significantly restore AMP-mediated CD4+ T cell suppression and enhance the killing of CD73-expressing tumor cells compared to known anti-CD73 antibodies. Therefore, the antibodies of this invention (especially the humanized antibodies) have significant clinical value.

[0182] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0183] Figure 1 The binding curve of murine antibody 72G8 to CD73 on the surface of tumor cells is shown.

[0184] Figure 2A-2B The binding of humanized antibody 7002-04 to MDA-MB-231 and A375 tumor cells naturally expressing human CD73 is shown. ISO is an isotype control antibody.

[0185] Figure 3 The binding of humanized antibody 7002-04 to soluble recombinant CD73 was shown.

[0186] Figure 4 The study demonstrated the inhibitory effect of humanized antibody 7002-04 on the enzyme activity of CD73 on the surface of tumor cells.

[0187] Figures 5A-5C The study showed the inhibitory effect of humanized antibody 7002-04 on the activity of CD73 enzyme in the serum of patients with colorectal cancer (A), liver cancer (B), and melanoma (C).

[0188] Figures 6A-6B The study demonstrated the alleviating effect of humanized antibody 7002-04 on AMP-mediated CD4+ T cell suppression.

[0189] Figures 7A-7BThis study demonstrates the restorative effect of humanized antibody 7002-04 on the killing of tumor cells by PBMCs.

[0190] Figures 8A-8C The results show the evaluation of the binding ability of the CDR mutant antibody 7002-04-N56Q to CD73 on the cell surface (A), its inhibitory ability on the activity of CD73 enzyme on the cell surface (B), and its inhibitory ability on the activity of CD73 enzyme in the serum of tumor patients (C).

[0191] Sequence information

[0192] Information on a portion of the sequence involved in this invention is provided below.

[0193]

[0194]

[0195] Detailed Implementation

[0196] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0197] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0198] Example 1: Production of mouse-derived anti-human CD73 antibody

[0199] To obtain murine anti-human CD73 antibodies, different immunization strategies (Table 1) were used to immunize mice (Balb / c, Shanghai Lingchang Biotechnology). The antigens used included: CD73 protein (i.e., recombinantly expressed human CD73, its sequence as shown in SEQ ID NO:21) and CHOS-human CD73 (i.e., CHOS cell line overexpressing CD73, the expressed CD73 sequence as shown in SEQ ID NO:21); adjuvants included: complete Freund's adjuvant CFA (InvivoGen, catalog number vac-cfa-60), IFA (InvivoGen, catalog number vac-ifa-60), and QuickAntibody (Beijing Biolong Immunotherapy Co., Ltd., catalog number KX0210041); administration routes included: intraperitoneal (ip) and subcutaneous (sc). Three days after booster immunization, spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 using the polyethylene glycol method to obtain B cell fusions that could both express antibodies and proliferate indefinitely in vitro, and these fusions were cultured in HAT selective medium. The fused hybridoma cells were seeded in 96-well cell culture plates, and positive clones were selected through primary screening for 2-3 rounds of subcloning.

[0200] Table 1: Immune Strategies

[0201]

[0202] Primary screening: In primary screening, the binding ability of the supernatant of the growing clones to cell surface CD73 was tested using human CD73-expressing tumor cells or overexpressing cell lines. The presence of the reactive antibody in the supernatant was revealed by using the DyLight488 goat anti-mouse IgG (Abcam catalog number ab97015) secondary antibody, and the binding ability was evaluated on a full-field cell scanning analyzer (detailed experimental procedures can be found in Example 2). Secondary screening: The ability to block CD73 enzyme activity was screened on CD73-expressing cells to evaluate the cell membrane surface CD73 enzyme activity blocking properties of the antibody; the ability to block CD73 enzyme activity was screened using human serum-soluble CD73 to evaluate the soluble CD73 enzyme activity blocking properties of the antibody (detailed experimental procedures can be found in Example 6).

[0203] The mouse monoclonal antibody 72G8 was isolated and purified from the culture supernatant of the finally obtained positive hybridoma monoclonal cell line.

[0204] Example 2: Evaluation of antigen-binding activity of murine anti-CD73 antibody

[0205] 2.1 The binding of mouse antibodies to CD73-positive cells was detected using a cell scanning analyzer.

[0206] The cells used were: MDA-MB-231 (endogenously expressing human CD73; human breast cancer cell line), SK-ME-S (endogenously expressing human CD73; human lung squamous cell carcinoma cell line), H2030 (endogenously expressing human CD73; human non-small cell lung cancer cell line), SKLU1 (endogenously expressing human CD73; human lung adenocarcinoma cell line), BT549 (endogenously expressing human CD73; human breast duct carcinoma cell line), A375 (endogenously expressing human CD73; human melanoma cell line), Calu6 (endogenously expressing human CD73; human degenerative cancer cell line), 4T1 (endogenously expressing mouse CD73; mouse breast cancer cell line), CHOS-human CD73 (transfected with human CD73) and CHOS (CD73 negative) cells.

[0207] Construction of CHOS cells expressing CD73: Human CD73 (SEQ ID NO:21) was overexpressed in CHOS cells (Invitrogen) using lentiviral infection and antibiotic selection (MOI = 3-10, 5 μg / ml polybrene). Lentiviral cells were provided by Shanghai Jikai Gene Chemical Technology Co., Ltd. After 72 hours of cell infection, cells were cultured with appropriate antibiotics for 2-4 weeks, amplified, and cryopreserved for subsequent experiments.

[0208] Experimental Methods: 10,000 cells were seeded in 100 μL DMEM + 10% FBS per well using a flat-bottomed 96-well plate. Cells were allowed to adhere overnight or settle to the bottom of the wells. The supernatant was removed the next day. Eight consecutive 3-fold dilutions were performed by diluting 1 / 3 volume (100 μL) in 200 μL DMEM. 100 μL of diluted antibody (using fusion clone supernatant or subclone supernatant for selection) was added to each well of the cell plate, and 100 μL of DMEM was added to the corresponding negative control well. The plates were incubated at room temperature for 1 hour. After removing the supernatant, add 100 μL of the secondary antibody (DyLight488 goat anti-mouse IgG (Abcam catalog number ab97015), 5 μg / mL (diluted in DMEM) to each well, and incubate at room temperature for 0.5 hours. After staining, remove the supernatant, wash once with PBS + 2% FBS, and then add 100-100 μL of PBS + 2% FBS to each well before reading the data using a full-field cell scanning analyzer (Nexcelom, model [model number missing]). The experimental plate was measured using an Image Cytometer. During measurement, both the fluorescence channel and bright-field channel corresponding to the second antibody were selected for high-speed scanning imaging of the cells in the wells. The fluorescence channel image was used to count antibody-bound cells based on parameters set according to the morphology and fluorescence intensity of the fluorescently labeled cells. The bright-field channel image was used to count adherent cells based on parameters set according to cell morphology. The two sets of data were then divided to obtain the percentage of fluorescent cells bound to the antibody. This percentage was used to determine the binding effect of the anti-CD73 antibody with the CD73-expressing cell line. Data analysis was performed using GraphPad. The horizontal axis represented the logarithm of antibody concentration, and the vertical axis represented the percentage of cells binding to the CD73 antibody and showing green fluorescence, as well as the total number of viable cells. The EC50 value of anti-CD73 antibody binding on each cell type was obtained by curve fitting.

[0209] The binding curves of 72G8 to various tumor cells are as follows: Figure 1 As shown in Tables 2-1 and 2-2, the EC50 values ​​are indicated by NB, which indicates that no detection was detected within the measured concentration range. The results show that 72G8 can bind to cells naturally expressing CD73 and CHOS cells recombinantly expressing human CD73, but the antibody does not bind to cells that do not express CD73 (CHOS) or cells that express mouse CD73 (4T1).

[0210] Table 2-1: Antibody binding to EC50 of tumor cells endogenously expressing human CD73

[0211]

[0212] Table 2-2: EC50 of antibody binding to recombinant human CD73-expressing CHOS cells and other cells that do not express human CD73

[0213]

[0214] 2.2 Binding of murine antibodies to cynomolgus monkey T cells

[0215] Monkey blood was obtained from two donors (1132F and 1300M) from Medicilon. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient centrifugation system. PBMCs were incubated with the test antibodies, and the bound antibodies were stained with fluorescently labeled secondary antibodies (DyLight488 goat anti-mouse IgG, Abcam catalog number ab97015; DyLight488 goat anti-human IgG, Abcam catalog number ab97003). T cells were identified using fluorescently labeled antibodies against CD3+ and CD8+. Unstained control samples and fluorescence-compensated control samples were collected and flow cytometry was used to detect antibody binding to cynomolgus monkey T cells.

[0216] The results are shown in Table 3. 72G8 was able to bind to CD8+ T cells of cynomolgus monkeys.

[0217] Table 3: Antibody binding to monkey PBMCs

[0218]

[0219] Example 3: Determination of the variable region sequence of murine anti-CD73 antibody and preparation of chimeric antibody

[0220] Hybridoma cells were collected by centrifugation, at a rate of 5-10 × 10⁻⁶ cells per cell. 6 Cells were treated with 1 ml TRIzol and 0.2 ml chloroform, vortexed vigorously for 15 seconds, incubated at room temperature for 3 minutes, centrifuged, and the aqueous phase was collected. 0.5 ml isopropanol was added, and the mixture was incubated at room temperature for 10 minutes. The precipitate was collected, washed with ethanol, and dried to obtain RNA. Template RNA and primers were added to ice-cold centrifuge tubes, and reverse transcription was performed after ensuring proper primer-template pairing. PCR amplification was then performed. 2.5 μl of a dNTP / ddNTP mixture was added to each of four microcentrifuge tubes, and the mixture was incubated at 37°C for 5 minutes. In an empty microcentrifuge tube, 1 pmol of PCR amplification double-stranded DNA, 10 pmol of sequencing primers, 2 μl of 5× sequencing buffer, and double-distilled water were added to a total volume of 10 μl. The tube was heated at 96°C for 8 minutes, cooled on ice for 1 minute, and centrifuged at 10000g for 10 seconds at 4°C. Add 2 μl of pre-chilled labeling mixture (0.75 μmol / L each of dCTP, dGTP, and dTTP), 5 μCi of α-32P-dATP, 1 μl of 0.1 mol / L DDT, and 2 U of sequencing enzyme. Add water to a final volume of 15 μl, mix well, and incubate on ice for 2 min to label the newly synthesized DNA strand. Add 3.5 μl of the labeling reaction mixture to four prepared microcentrifuge tubes and incubate at 37°C for 5 min. Add 4 μl of stop solution to each tube. Denature the samples in an 80°C water bath for 5 min. Add 2 μl of the mixture to each lane of the sequencing gel, separate the fragments by electrophoresis, and collect the sequence information.

[0221] The VH and VL sequences of the murine antibody 72G8 are shown in the table below. Furthermore, the CDR sequence of this murine monoclonal antibody was determined using 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, Maryland (1991), pp. 647-669).

[0222] Table 4: Sequence information of murine antibodies

[0223]

[0224]

[0225] The DNA sequences (SEQ ID NOs: 14-15) encoding the variable regions of the heavy and light chains of the aforementioned murine antibody were linked to the sequences encoding the constant regions of the heavy and light chains of the human antibody (SEQ ID NO: 19 and SEQ ID NO: 20), respectively. Recombinant expression was then performed in HEK293 cells (ATCC). Cell supernatants containing antibody clones were harvested from culture flasks, purified using a Protein A column, and the antibody protein was eluted with 100 mM acetic acid at pH 3.0. The purified antibody protein was then loaded onto a size exclusion chromatography column for further purification. The antibody protein corresponding to the monomer was prepared in PBS buffer, supplemented with 20% glycerol. This yielded the corresponding chimeric antibody ch72G8.

[0226] Example 4: Humanization of mouse anti-CD73 antibody

[0227] To improve the sequence homology between candidate antibodies and human antibodies and reduce the immunogenicity of antibodies to humans, the mouse antibodies provided in the above embodiments can be humanized by designing and preparing them, and inserting the mouse CDR region into the human frame sequence using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0228] Specifically, the heavy and light chain CDR regions of the murine antibody 72G8 were transplanted into the FR framework of the corresponding humanized template, and a series of reversion mutations were performed on the amino acid residues of the FR region of the humanized template to ensure that the humanized antibody retained as much of the antigen-binding ability of the murine antibody as possible. Based on the above method, the inventors prepared a humanized antibody of the murine antibody 72G8, named 7002-04 (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 is SEQ ID NO:19, and the light chain constant region is SEQ ID NO:20.

[0229] Example 5: Evaluation of antigen-binding activity of humanized anti-CD73 antibody

[0230] 5.1 The binding of the antibody to CD73-expressing cells was determined by flow cytometry.

[0231] 500,000 CD73-expressing cells (see Example 2) were placed in 100 μL FACS buffer (PBS + 2% FBS) per well using a round-bottom low-absorption 96-well plate. Antibody samples were serially diluted 3-fold at 12 spots by diluting 1 / 2 volume (100 μL) in 200 μL 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. The plates were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed twice with FACS buffer. 100 μL of the 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 the plates were incubated at 4°C for another 0.5 hours. After staining, the cells were centrifuged to remove the supernatant, washed twice with FACS buffer, and then resuspended in 100 μL of FACS buffer per well before being read. A flow cytometer (BD, ACCURI C6 PLUS) was used to analyze the cells in the experimental plate. During the assay, cells were first located using the FCS and SSC, and then the corresponding green fluorescence channel (FITC) and SSC of the secondary antibody were selected for cell analysis. Data analysis was performed using GraphPad, with the logarithm of antibody concentration on the x-axis and the average fluorescence intensity on the y-axis. The EC50 of the anti-CD73 antibody was calculated by curve fitting. The binding of the humanized antibody 7002-04 and the corresponding chimeric antibody ch72G8 to naturally expressing human CD73 MDA-MB-231 and A375 cells are shown below. Figure 2A-2B As shown in Table 5, the binding of humanized antibody 7002-04 to cells naturally expressing CD73 and recombinant cells expressing human CD73 is illustrated. ND indicates no detection was performed. The results indicate that humanized antibody 7002-04 possesses good binding activity to membrane-bound CD73, even comparable to chimeric antibodies.

[0232] Table 5: Antibody binding to EC50 of tumor cells expressing human CD73

[0233]

[0234] 5.2 ELISA was used to determine the binding of the antibody to soluble human CD73 protein.

[0235] 1 μg / ml of recombinant human CD73 protein (BioBio, recombinant human CD73 protein) was coated onto an ELISA plate in PBS and incubated overnight at 4°C. The plate was washed three times in wash buffer (PBS, 0.05% Tween 20) and saturated with nonspecific sites by adding 200 μl / w PBS + 2% BSA. 100 μL of serially diluted anti-CD73 antibody was added to each antigen-coated ELISA plate and incubated at 37°C for 1 h. The plate was washed three times in wash buffer and HRP-conjugated goat anti-human or goat anti-mouse IgG Fc fragment secondary antibody was added at room temperature for 1 h to detect the bound anti-CD73 antibody. The plate was washed three times in wash buffer and the bound secondary antibody was revealed by adding TMB (HRP substrate) and incubating in the dark at room temperature for 5 to 10 minutes. The enzyme reaction was terminated by adding 1 M sulfuric acid solution, and the absorbance was measured at 450 nm. A curve was plotted with absorbance as the ordinate and antibody concentration log value as the abscissa, and the EC50 was calculated using GraphPad Prism software. The results are as follows: Figure 3 As shown, the humanized antibody 7002-04 exhibits good binding activity against soluble recombinant CD73, with an EC50 of 0.0047 μg / ml.

[0236] 5.3 The affinity between the humanized antibody and recombinant human CD73 protein was determined using Biacore.

[0237] SPR measurements of antibody affinity were performed at 25°C on a Biacore T200 (GE) sensor. Antibodies were diluted to 1 μg / ml with running buffer 1*HBS-EP+ and captured onto the chip surface (Protein A chip, GE, Cat#29127556) at a flow rate of 10 μl / min for 30 s. Subsequently, a series of concentrations of CD73 protein (Biacore Biotech, recombinant human CD73 protein) were injected into the corresponding antibody channels at a flow rate of 30 μl / min, with an association phase of 180 s followed by dissociation of 900 s. Regeneration was performed using 10 mM pH 1.5 Gly-HCl. A 1:1 dynamic binding model was used to fit the entire sensor dataset. Bivalent affinity and dynamic binding and dissociation rate constants are shown in the table below.

[0238] Table 6: Affinity constants of antibodies binding to recombinant CD73

[0239] Antibody ka(1 / Ms) kd(1 / s) KD(M) 7002-04 1.32E+06 2.75E-04 2.073E-10

[0240] Example 6: Evaluation of the inhibitory activity of anti-CD73 antibody against CD73 enzyme activity

[0241] 6.1 Inhibition assay of CD73 enzyme activity in tumor cells

[0242] It is known that excess AMP blocks ATP-dependent luciferase activity. Cleavage of AMP into adenosine + inorganic phosphate at CD73 restores luciferase activity and light emission by reducing AMP. Therefore, antibodies that block the enzymatic activity of CD73 will reduce light emission.

[0243] Harvest and count human CD73-positive cells. Seed 20,000 cells per well in 100 μL of complete medium in a flat-bottomed 96 plate. Antibody samples were serially diluted 8 times by diluting 1 / 3 volume (100 μL) in 200 μL of DMEM. 100 μL of the diluted sample was added to the corresponding well. The negative control was an isotype control antibody (ISO). Incubate at 37°C for 1 hour, remove the supernatant, and wash the cells twice with PBS. Prepare a 125 μM AMP solution in incomplete medium, add 100 μL of AMP to each well, and incubate the plate at 37°C for another two hours. Centrifuge the reaction plate, remove 50 μL and add it to another 96 fluorescence plate (OptiPlate-96, Perkin Elmer, #6005290), along with the same volume of 50 μM ATP solution, and add 50 μL of CTG reagent (Promega, G7572) to each well. Incubate the plate in the dark at room temperature for 15 minutes, and measure the fluorescence (Lum) using a microplate reader. Data analysis was performed using GraphPad, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the inhibition rate. The inhibition rate was calculated as follows:

[0244] Inhibition rate = (Lum) 阳性对照 -Lum 抗体 ) / (Lum 阳性对照 -Lum 阴性对照 )*100

[0245] The IC50 values ​​of antibodies blocking endogenous CD73 in different human tumor cell lines are shown in the table below. Figure 4 The inhibition curves of humanized antibody 7002-04 and its corresponding chimeric antibody ch72GB on CD73 enzyme activity on the surface of A375 cells are shown. The results indicate that humanized antibody 7002-04 can significantly inhibit the enzyme activity of CD73 on the surface of tumor cells.

[0246] Table 7: IC50 of antibody inhibition of CD73 enzyme activity on the surface of different human tumor cells

[0247]

[0248] 6.2 Inhibition test of serum CD73 enzyme activity in cancer patients

[0249] Serum from tumor patients was diluted in a phosphate buffer (Tris 125mM, MgCl2 25mM, NaCl 125mM), 12.5 μL per well, using a white flat-bottomed 96-well plate. Antibody samples were serially diluted 2-10 times at 10 points by diluting 1 / 1.5 volume (100 μL) in 50 μL of phosphate buffer and 1 / 10 volume (10 μL) in 90 μL of phosphate buffer. 12.5 μL of the diluted antibody was added to each well of the cell plate, and 12.5 μL of phosphate buffer was added to the negative control. After centrifugation, the plates were incubated at 37°C for 1.5 hours. AMP was diluted to a 20 μM solution using phosphate buffer, and 25 μL of AMP was added to each well (except for the positive control). After centrifugation, the plates were incubated at 37°C for an additional 1 hour. After the reaction, 25 μL of AMP was added to the positive control. Immediately afterward, 25 μL of AMP-Glo ​​was added to each well. TM Reagent I (Promega, catalog number V5012) was used. The reaction plate was centrifuged and incubated at room temperature for 1 hour. 50 μL of AMP Detection Solution (Promega, catalog number V5012) was added to each well, centrifuged, and incubated at room temperature for 1 hour. Fluorescence (Lum) was measured using a microplate reader. Data analysis was performed using GraphPad. The x-axis represents the logarithm of antibody concentration, and the y-axis represents the inhibition rate. The inhibition rate was calculated as follows:

[0250] Inhibition rate = 100 - (Lum) 阳性对照 -Lum 抗体 ) / (Lum 阳性对照 -Lum 阴性对照 )*100

[0251] The IC50 of the anti-CD73 antibody was determined by curve fitting. The results are as follows: Figures 5A-5C As shown, anti-CD73 antibody can effectively inhibit the dephosphorylation of AMP by CD73 in the serum of patients with colorectal cancer (A), liver cancer (B), and melanoma (C), and inhibit CD73 enzyme activity.

[0252] Example 7: Anti-CD73 antibody-mediated CD73 internalization

[0253] Anti-CD73 antibody-mediated CD73 internalization was tested by flow cytometry. To determine the relationship between antibody-induced internalization and time, indicator cells were incubated with 10 μg / mL antibody at 37°C for different times. After washing several times with PBS containing 2% FBS, 10 μg / mL secondary antibody was added and stained at 4°C for 30 minutes. CD73 expression in the cells was then analyzed by flow cytometry. To compare the different degrees of antibody-induced internalization, indicator cells were incubated with 10 μg / mL 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 secondary antibody was added and stained at 4°C for 30 minutes. CD73 expression in the cells was then analyzed by flow cytometry.

[0254] MFI 37 MFI 1 represents the MFI of samples incubated at 37°C; MFI 4 represents the MFI of samples incubated at 4°C, under which only binding and not endocytosis occur. 背景 For MFI with only a secondary antibody, the percentage of antibody-mediated cell surface CD73 internalization is calculated using the following formula:

[0255] Percentage of CD73 internalized = 100 – 100 × (MFI4 - MFI) 37 ) / MFI4

[0256] The results are shown in Table 8. These antibodies mediated the internalization of CD73 on the surface of tumor cells to varying degrees.

[0257] Table 8: Proportion of CD73 internalization on the surface of tumor cells mediated by antibodies

[0258] cell Antibody 6 hours 4 hours 2 hours 1 hour 0.5 hours 0 hours A375 7002-04 19％ 13％ 11％ 8％ 4％ 0％ MDA-MB-231 7002-04 14％ 10％ 13％ 7％ 9％ 0％ H2030 7002-04 15％ 9％ 10％ 6％ 4％ 0％ HCC44 7002-04 29％ 20％ 16％ 10％ 12％ 0％ Calu6 7002-04 24％ 23％ 15％ 9％ 12％ 0％

[0259] Example 8: Anti-CD73 antibody alleviates AMP-mediated CD4+ T cell suppression

[0260] One day prior to the experiment, PBMCs were stimulated with anti-CD3 / anti-CD28 for 24 hours. PBMCs (obtained from fresh apheresis blood via Ficoll separation) were collected, and CD4+ T cells were sorted using the CD4+ T Cell Isolation Kit human (Medrin, catalog number 130-096-533). Cells were centrifuged to remove the supernatant, and the CD4+ T cells were resuspended in AIMV medium containing 40 μM EHNA and 120 IU / ml IL2 (final EHNA concentration 20 μM, final IL2 concentration 60 IU / ml). 200,000 CD4+ T cells were placed in 100 μL / well using 96-well low-absorption round-bottom plates. Ten consecutive 2- to 10-fold dilutions were performed by diluting 1 / 3 volume (100 μL) in 200 μL of AIMV medium. 50 μL of the diluted antibody was added to each well, and 50 μL of AIMV medium was added to the corresponding negative control wells. The plates were incubated at 37°C for 0.5 hours. 400 μM AMP (final concentration 100 μM) was prepared using AIMV, and 50 μL of the prepared AMP solution was added to each well (control wells were treated with AMP-free medium). After centrifugation, the plates were read. The plates were then incubated at 37°C for 72 hours and read again. Readings were performed using a full-field cell scanning analyzer (Nexcelom, model [model number missing]). The cells within the experimental plate were measured using an ImageCytometer. During measurement, a bright-field channel was selected for high-speed scanning imaging of the cells within the wells. The size of the clonal clusters was used to determine the effect of the anti-CD73 antibody in alleviating AMP-mediated CD4+ T cell suppression. MEDI9447 (MedImmune) and BMS986179 (BMS) were used as reference antibodies, both purified and expressed by GIC.

[0261] The cell growth of T cells on day 5 and day 4 is as follows: Figures 6A-6B As shown in the figure; #20 and #18 are the donor numbers for internal use PBMCs; the antibody concentration used in the figure started at 100 μg / mL, with nine points representing four-fold dilutions. Humanized antibody 7002-04 effectively alleviated AMP-mediated CD4+ T cell suppression, significantly restored T cell proliferation, and showed better results than the reference antibodies MEDI9447 and BMS986179.

[0262] Example 9: Anti-CD73-mediated tumor cell killing

[0263] 5000 A375 cells were seeded in 100 μL DMEM + 10% FBS per well in a flat-bottomed 96-well plate and incubated overnight to allow cell adhesion. The supernatant was removed the next day. Ten consecutive 2- to 10-fold dilutions were performed by diluting 1 / 3 volume (100 μL) in 200 μL 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. The plates were incubated at 37°C for 0.5 hours. PBMCs (fresh apheresis blood separated by Ficoll) were collected 24 hours after stimulation with CD3 / CD28 the day before. The cells were resuspended in AIMV containing 40 μM EHNA and 120 IU / ml IL2 (final EHNA concentration 20 μM, final IL2 concentration 60 IU / ml), and added at a ratio of 5,000 / 100 μL to each well. A 400 μM AMP solution was prepared using AIMV, and 50 μL was added to each well (final AMP concentration 100 μM). After centrifugation, the solution was incubated at 37°C for 72 hours. 10 μL of a CK8 assay kit (Dojin Co., Ltd., Japan, catalog number CK04) was added to each well, and the solution was incubated at 37°C for 4 hours. The OD450 was measured using a microplate reader. The OD values ​​were converted to inhibition percentages based on the control well values ​​to determine the extent of anti-CD73 antibody-mediated tumor cell killing. A higher percentage indicates a better anti-CD73-mediated tumor cell killing effect, while a lower percentage indicates a worse effect. Data analysis was performed using GraphPad, with the logarithm of antibody concentration on the x-axis and the inhibition percentage on the y-axis. The IC50 value of the anti-CD73 antibody on A375 cells was obtained by curve fitting.

[0264] The results are as follows Figures 7A-7B As shown in the figure, #13 and #22 are the donor numbers of PBMCs used internally. The results show that the humanized antibody 7002-04 can effectively restore the killing effect of PBMCs on tumor cells and is significantly better than the reference antibodies MEDI9447 and BMS986179.

[0265] Example 10: Antibodies containing CDR mutations and their activity evaluation

[0266] The N56 variant in the HCDR2 variable region of the 7002-04 heavy chain was mutated to Q, and the resulting variant was named 7002-04-N56Q (its VH and VL are shown in SEQ ID NO:13 and 10, respectively). The binding of the antibody to CD73-expressing human tumor cells MDA-MB-231 was determined by flow cytometry using the method described in 5.1, and the results are as follows: Figure 8A As shown, the binding affinity of 7002-04-N56Q to CD73 on the cell surface is consistent with that of 7002-04. The inhibition of CD73 enzyme activity in tumor cells by the antibody was measured using the method described in section 6.1, and the results are as follows. Figure 8B As shown, the inhibitory effect of 7002-04-N56Q on the CD73 enzyme activity on the surface of MDA-MB-231 is basically the same as that of 7002-04. The inhibitory effect of the antibody on the serum CD73 enzyme activity of tumor patients was measured using the method in section 6.2, and the results are as follows. Figure 8C As shown, the inhibitory effect of 7002-04-N56Q on CD73 enzyme activity in the serum of colorectal cancer patients is consistent with that of 7002-04. This indicates that 7002-04-N56Q, containing the CDR mutation, essentially maintains the excellent biological activity of its parent antibody 7002-04.

[0267] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Promis Biotechnology (Zhuhai) Co., Ltd. <120> Anti-CD73 antibodies and their uses <130> IDC220287 <150> PCT / CN2020 / 089869 <151> 2020-05-12 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 72G8 Heavy Chain Variable Region <400> 1 Glu Val Arg Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Thr Tyr 20 25 30 Pro Ile Glu Trp Met Lys Gln Asn His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Asn Asp Thr Lys Phe Asn Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Met Thr Val Glu Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Tyr Tyr Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 72G8 Light Chain Variable Region <400> 2 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Gln Gln 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 3 Gly Tyr Ser Leu Thr Thr Tyr 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR2-1 <400> 4 His Pro Tyr Asn Asn Asp 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 5 Gly Asp Tyr Tyr Gly Asn Ser Asp 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR1 <400> 6 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 7 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 <400> 8 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 9 <211> 118 <212> PRT <213> Artificial sequence <220> <223> 7002-04 Heavy Chain Variable Region <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Thr Tyr 20 25 30 Pro Ile Glu Trp Met Lys Gln Ala His Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Asn Asp Thr Lys Phe Asn Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ile Asn Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Tyr Tyr Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Leu Thr Val Ser Ser 115 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 7002-04 Light Chain Variable Region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 6 <212> PRT <213> Artificial sequence <220> <223> HCDR2-2 <400> 11 His Pro Tyr Asn Gln Asp 1 5 <210> 12 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 72G8-N56Q heavy chain variable region <400> 12 Glu Val Arg Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Thr Tyr 20 25 30 Pro Ile Glu Trp Met Lys Gln Asn His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Gln Asp Thr Lys Phe Asn Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Met Thr Val Glu Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Tyr Tyr Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser 115 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 7002-04-N56Q Heavy Chain Variable Region <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Thr Tyr 20 25 30 Pro Ile Glu Trp Met Lys Gln Ala His Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Gln Asp Thr Lys Phe Asn Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ile Asn Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Tyr Tyr Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Leu Thr Val Ser Ser 115 <210> 14 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence of the heavy chain variable region of 72G8 <400> 14 gaggtgcggc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatg 60 tcctgcaagg cttctggcta ctccctcacc acctacccca tcgaatggat gaagcagaac 120 catggcaaga gccttgagtg gattggaaac tttcatcctt ataataatga tacaaagttc 180 aacgagaagt ttaggggcaa ggccaccatg accgtagaaa agtccagcaa cacagtgtac 240 ctggagctga gcaggctgac ctctgacgac tcggccgtgt attactgtac gagaggggat 300 tactacggca atagtgacta ctggggccaa gggaccacgc tcaccgtctc c 351 <210> 15 <211> 321 <212> DNA <213> Artificial sequence <220> <223> 72G8 light chain variable region nucleic acid sequence <400> 15 gacatccaga tgacccagac tacctcctcc ctgtctgcat ctctgggaga cagagtcacc 60 atctcctgcc gggcaagtca ggacattagc aactatttaa attggtatca gcagaaacca 120 gacggaaccg ttaagctcct gatctattat acatccaggt tgcacagtgg ggtcccagcc 180 agattccgtg gcagtggatc tgggacagat tactccctca ccatcagcaa cctgcaacag 240 gaagattttg caacttactt ctgtcaacag ggtaacactc tcccttacac tttcggcgga 300 gggaccaagc tggagatcaa a 321 <210> 16 <211> 354 <212> DNA <213> Artificial sequence <220> <223> 7002-04 Heavy Chain Variable Region Nucleic Acid Sequence <400> 16 caggtgcagc tggtgcagag cggcgccgag gtggtgaagc ccggagcttc cgtgaagatg 60 agctgcaagg ccagcggcta cagcctgaca acataccccca tcgagtggat gaagcaggcc 120 cacggccagg gactggagtg gatcggaat tttcatccct fathercga caccaagttc aacgagaagt tcagaggcaa ggccaccatg accgtggaca agagcatcaa caccgtgtat ctggagctga gcagactgag aagcgacgac accgccgtgt actactgcac cagaggcgac tactacggca acagcgacta ctggggccag ggcacactgc tgaccgtgag ctcc 354 <210> 17 <211> 321 <212> DNA <213> The snowstorm <220> <223> 7002‑04‑‑‑‑                       <400> 17 60. gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc atcacctgca gagccagcca ggacatctcc aactacctga actggtacca gcagaaaccc 180. ggcaaagccg tgaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc agttcagcg gaagcggcag cggcaccgac tacaccctga ctatcagctc cctgcagccc gaagatttcg ccacctactt ctgccagcag ggcaacaccc tgccctatac attcggacag 300 ggcaccaagc tggagatcaa a <210> 18 <211> 354 <212> DNA <213> The snowstorm <220> <223> 7002‐04‐N56Q Small-Scale-Range <400> 18 60. caggtgcagc tggtgcagag cggcgccgag gtggtgaagc ccggagcttc cgtgaagatg agctgcaagg ccagcggcta cagcctgaca acatacccca tcgagtggat gaagcaggcc cacggccagg gactggagtg gatcggaat tttcatccct father caccaagttc aacgagaagt tcagaggcaa ggccaccatg accgtggaca agagcatcaa caccgtgtat ctggagctga gcagactgag aagcgacgac accgccgtgt actactgcac cagaggcgac tactacggca acagcgacta ctggggccag ggcacactgc tgaccgtgag ctcc 354 <210> 19 <211> 330 <212> PRT <213> The snowstorm <220> <223> specific IgG1‐TM antibodies <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human κ Light Chain Constant Region <400> 20 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 21 <211> 574 <212> PRT <213> Artificial Sequence <220> <223> Human CD73 <400> 21 Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu 1 5 10 15 Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu 20 25 30 His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser 35 40 45 Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu 50 55 60 Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu 65 70 75 80 Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr 85 90 95 Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala 100 105 110 Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile 115 120 125 Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile 130 135 140 Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro 145 150 155 160 Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr 165 170 175 Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val 180 185 190 Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys 195 200 205 Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu 210 215 220 Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val 225 230 235 240 Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys 245 250 255 Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly 260 265 270 Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly 275 280 285 Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His 290 295 300 Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile 305 310 315 320 Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr 325 330 335 Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser 340 345 350 Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met 355 360 365 Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val 370 375 380 Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu 385 390 395 400 Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro 405 410 415 Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys 420 425 430 Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu 435 440 445 Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys 450 455 460 Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg 465 470 475 480 Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile 485 490 495 Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys 500 505 510 Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val 515 520 525 Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly 530 535 540 Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu 545 550 555 560 Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln 565 570

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CD73, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO: 3; (ii) VH CDR2, which consists of the following sequence: SEQ ID NO: 4 or SEQ ID NO: 11; (iii) VH CDR3, which consists of the following sequence: SEQ ID NO: 5; and, (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO: 6; (v) VL CDR2, which consists of the following sequence: SEQ ID NO: 7; and (vi) VL CDR3, which consists of the following sequence: SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:12 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment contains a framework region sequence derived from human immunoglobulin.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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.

6. The antibody or antigen-binding fragment thereof according to claim 4, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity compared to it.

7. The antibody or antigen-binding fragment thereof according to claim 4, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:13 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity with it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10 or a sequence having at least 85%, 90%, 95%, or 99% sequence identity with it.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment further includes a constant region derived from human immunoglobulins.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin, and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region of the human immunoglobulin of the antibody or antigen-binding fragment thereof is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4.

11. The antibody or antigen-binding fragment thereof according to claim 9, wherein the light chain constant region of the human immunoglobulin is a κ or λ light chain constant region.

12. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from: (1) Human IgG1 heavy chain constant region; (2) A variant of the human IgG1 heavy chain constant region, which has the following substitutions compared to the wild-type sequence from which it is derived: L234F, L235E, P331S, the amino acid positions mentioned above being the positions according to the EU numbering system.

13. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) shown in SEQ ID NO:

19.

14. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises a constant region of the human κ light chain.

15. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) shown in SEQ ID NO:

20.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv and scFv; and / or the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antigen-binding fragment is selected from Fv linked by disulfide bonds.

18. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-17, or a variable region of the heavy chain and / or a variable region of the light chain thereof.

19. A vector comprising the isolated nucleic acid molecule of claim 18.

20. The vector of claim 19, wherein the vector is a cloning vector or an expression vector.

21. A host cell comprising the isolated nucleic acid molecule of claim 18 or the vector of claim 19 or 20.

22. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-17, comprising culturing a host cell according to claim 21 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

23. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-17.

24. The bispecific or multispecific molecule of claim 23, wherein the bispecific or multispecific molecule specifically binds to CD73 and additionally specifically binds to one or more other targets.

25. The bispecific or multispecific molecule of claim 23, wherein the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-17 or a bispecific or multispecific molecule as described in any one of claims 23-25, and a pharmaceutically acceptable carrier and / or excipient.

27. The pharmaceutical composition of claim 26, further comprising an additional pharmaceutically active agent.

28. The pharmaceutical composition of claim 27, wherein the additional pharmaceutically active agent is a drug having antitumor activity.

29. The pharmaceutical composition of claim 27, wherein the additional pharmaceutically active agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

30. The pharmaceutical composition of claim 27, wherein the additional pharmaceutically active agent is selected from immune checkpoint inhibitors, anti-CD39 antibodies, anti-A2AR antibodies, or anti-HER2 / ErbB2 antibodies.

31. The pharmaceutical composition of claim 30, wherein the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, or any combination thereof.

32. A kit containing the antibody or antigen-binding fragment thereof as described in any one of claims 1-17.

33. The kit of claim 32, wherein the antibody or its antigen-binding fragment is labeled with a detectable tag.

34. The kit of claim 33, wherein the detectable marker is selected from enzymes, radionuclides, fluorescent dyes, luminescent substances or biotin.

35. The kit of claim 32, further comprising a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of any one of claims 1-17.

36. The kit of claim 35, wherein the second antibody further comprises a detectable marker.

37. The kit of claim 36, wherein the detectable marker is selected from enzymes, radionuclides, fluorescent dyes, luminescent substances or biotin.

38. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-17, the bispecific or multispecific molecule according to any one of claims 23-25, or the pharmaceutical composition according to any one of claims 26-31 in the preparation of a medicament for the prevention and / or treatment of tumors in a subject, said tumor being selected from breast cancer, melanoma, degenerative carcinoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, bladder cancer, thyroid cancer, esophageal cancer, prostate cancer, and glioma.

39. The use of claim 38, wherein the antibody or its antigen-binding fragment, bispecific or multispecific molecule or pharmaceutical composition is administered in combination with another pharmaceutically active agent.

40. The use as described in claim 39, wherein the additional pharmaceutically active agent is a drug having antitumor activity.

41. The use according to claim 40, wherein the antitumor active drug is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.

42. The use as described in claim 39, wherein the additional pharmaceutically active agent is selected from immune checkpoint inhibitors, anti-CD39 antibodies, anti-A2AR antibodies, or anti-HER2 / ErbB2 antibodies.

43. The use according to claim 42, wherein the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, or any combination thereof.

44. The use as described in claim 38, wherein the subject is a human being.

45. The use according to claim 38, wherein the tumor is selected from non-small cell lung cancer, ductal carcinoma of the breast, and glioblastoma.

46. ​​The use as described in claim 45, wherein the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma of the lung.

47. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-17 in the preparation of a detection reagent for determining the presence or amount of CD73 in a sample.

48. The use as described in claim 47, wherein the CD73 is human CD73.

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