Anti-CD73 antibody and use thereof

Abstract

An anti-CD73 antibody and a use thereof. The provided antibody can specifically bind to CD73. The provided antibody exhibits high affinity and specificity with an antigen, has a tumor killing effect, and can be applied to treatment of tumors.

Description

Anti-CD73 antibodies and their applications Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to an anti-CD73 antibody and its application. Background Art

[0002] CD73, also known as ecto-5'-nucleotidase (ecto-5'NT, EC 3.1.3.5), has a protein molecular weight of approximately 70 kDa and is a glucosylphosphatidylinositol (GPI)-anchored enzyme on the cell surface. CD73 is present in most tissues, particularly in endothelial cells and hematopoietic cell subsets (Resta et al., Immunol Rev 1998;161:95-109 and Colgan et al., Prinergic Signal 2006;2:351-60). CD39, upstream of CD73, catalyzes the conversion of ATP to adenosine monophosphate (AMP), which is then hydrolyzed by CD73 to adenosine. Adenosine is a widely studied signaling molecule whose biological effects are mediated by several receptors (including A1, A2A, A2B, and A3). In the tumor microenvironment, adenosine not only enhances tumor proliferation, angiogenesis, and metastasis (Zhang et al., Cancer Res 2010; 70: 6407-11), but also significantly inhibits anti-tumor immune responses (F. Ghiringhelli et al., Biomed. Res, Int. 2012). Therefore, CD73 is considered a powerful target for cancer therapy.

[0003] CD73 is expressed on many different cancers, including colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, leukemia, glioma, glioblastoma, melanoma, thyroid cancer, esophageal cancer, prostate cancer and breast cancer (Jin et al., Cancer Res 2010; 70: 2245-55 and Stagg et al., PNAS 2010; 107: 1547-52). CD73 also affects multiple aspects of tumorigenesis, such as proliferation, differentiation, adhesion / migration, angiogenesis and metastasis. It promotes tumor cell proliferation by regulating cell cycle, apoptosis and signaling pathways such as EGFR, β-catenin / cyclin D1, VEGF and AKT / ERK (Zhi X et al., Clin Exp Metastasis 2007, 24(6):439-448; Turcotte M et al., Cancer Res 2015, 75(21):4494-4503; Wu R et al., Oncol Rep 2016, 35(3):1750-1756; Gao ZW et al., BMC cancer 2017, 17(1):135). Moreover, high expression of CD73 as a biomarker is closely associated with poor prognosis in many types of tumors, including breast cancer, lung cancer, ovarian cancer, gastric cancer, kidney cancer, head and neck cancer, etc. In addition to cancer cells, various immune cells such as B cells, T cells, Th cells, and CD8+ T cells also express CD73 (Ghasem G et al., 2019, 23:2, 127-142). Given the key role of CD73 in tumor growth and metastasis, it has become one of the hot targets in the field of tumor immunotherapy. Several companies are already in Phase II or Phase I clinical trials, but no drug targeting this target has yet been approved for marketing.

[0004] Summary of the Invention

[0005] The present invention provides anti-CD73 antibodies and their use in disease treatment. The provided antibodies can specifically bind to CD73 and exhibit anti-tumor effects.

[0006] The first aspect of the present invention provides an anti-CD73 antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a HCDR sequence as set forth in SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence as set forth in SEQ ID NOs: 1, 2 and 3; or a HCDR sequence as set forth in SEQ ID NOs: 4, 5 and 6, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence as set forth in SEQ ID NOs: 4, 5 and 6; or a HCDR sequence as set forth in SEQ ID NOs: 7, 8 and 9, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence as set forth in SEQ ID NOs: 7, 8 and 9; or a HCDR sequence as set forth in SEQ ID NOs: 10, 11 and 12, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence as set forth in SEQ ID NOs: 10, 11 and 12; or a HCDR sequence as set forth in SEQ ID NOs: 13, 14 and 15, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence as set forth in SEQ ID NOs: NO: 13, 14 and 15 HCDR sequences with one, two or three conservative amino acid mutations; or comprising the HCDR sequences of SEQ ID NO: 16, 17 and 18, or a sequence with one, two or three conservative amino acid mutations compared to the HCDR sequences of SEQ ID NO: 16, 17 and 18; or comprising the HCDR sequences of SEQ ID NO: 19, 20 and 21, or a sequence with one, two or three conservative amino acid mutations compared to the HCDR sequences of SEQ ID NO: 19, 20 and 21; the light chain variable region comprising the LCDR sequences of SEQ ID NO: 22, 23 and 24, or a sequence with one, two or three conservative amino acid mutations compared to the LCDR sequences of SEQ ID NO: 22, 23 and 24; or comprising the LCDR sequences of SEQ ID NO: 22, 23 and 25, or a sequence with one, two or three conservative amino acid mutations compared to the LCDR sequences of SEQ ID NO: 22, 23 and 25; or comprising the HCDR sequences of SEQ ID NO: 19, 20 and 21. The LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28, or sequences having one, two, or three conservative amino acid mutations compared to the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28; or sequences comprising the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31, or sequences having one, two, or three conservative amino acid mutations compared to the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31;or comprising the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32; or comprising the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35; or comprising the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38;

[0007] A second aspect of the present invention provides an anti-CD73 antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 is selected from the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequence shown in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequence shown in SEQ ID NO: 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequence shown in SEQ ID NO: 23 or 27 or 30 or 34 or 37, and LCDR3 is selected from the sequence shown in SEQ ID NO: 24 or 25 or 28 or 31 or 32 or 35 or 38.

[0008] The third aspect of the present invention provides an anti-CD73 antibody or antigen-binding fragment comprising: the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52.

[0009] A fourth aspect of the present invention provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion specifically binds to a first epitope, the first antigen-binding portion comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3; or comprising the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6; or comprising the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9; or comprising the HCDR sequence shown in SEQ ID NOs: 10, 11 and 12, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 10, 11 and 12; or comprising the HCDR sequence shown in SEQ ID NOs: NO: 13, 14 and 15, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NO: 13, 14 and 15; or a sequence comprising the HCDR sequences of SEQ ID NO: 16, 17 and 18, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NO: 16, 17 and 18; or a sequence comprising the HCDR sequences of SEQ ID NO: 19, 20 and 21, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequences of SEQ ID NO: 19, 20 and 21; the light chain variable region comprises the LCDR sequences of SEQ ID NO: 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences of SEQ ID NO: 22, 23 and 24; or a sequence comprising the LCDR sequences of SEQ ID NO: 22, 23 and 25, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequences of SEQ ID NO: The LCDR sequences shown in SEQ ID NOs: 22, 23 and 25 have one, two or three conservative amino acid mutations; or the LCDR sequences shown in SEQ ID NOs: 26, 27 and 28 have one, two or three conservative amino acid mutations compared to the LCDR sequences shown in SEQ ID NOs: 26, 27 and 28; or the LCDR sequences shown in SEQ ID NOs: 29, 30 and 31 have one, two or three conservative amino acid mutations compared to the LCDR sequences shown in SEQ ID NOs: 29, 30 and 31;or comprising the LCDR sequence set forth in SEQ ID NOs: 22, 23, and 32, or a sequence having one, two, or three conservative amino acid mutations compared to the LCDR sequence set forth in SEQ ID NOs: 22, 23, and 32; or comprising the LCDR sequence set forth in SEQ ID NOs: 33, 34, and 35, or a sequence having one, two, or three conservative amino acid mutations compared to the LCDR sequence set forth in SEQ ID NOs: 33, 34, and 35; or comprising the LCDR sequence set forth in SEQ ID NOs: 36, 37, and 38, or a sequence having one, two, or three conservative amino acid mutations compared to the LCDR sequence set forth in SEQ ID NOs: 36, 37, and 38;

[0010] The second antigen binding moiety specifically binds to a second epitope, and the second antigen binding moiety is fused to the first antigen binding moiety.

[0011] A fifth aspect of the present invention provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion specifically binds to a first epitope, the first antigen-binding portion comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, the light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3; wherein HCDR1 is selected from the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequence shown in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequence shown in SEQ ID NO: 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequence shown in SEQ ID NO: 23 or 27 or 30 or 34 or 37, and LCDR3 is selected from the sequence shown in SEQ ID NO: NO: the sequence shown in 24 or 25 or 28 or 31 or 32 or 35 or 38; the second antigen binding portion specifically binds to the second epitope, and the second antigen binding portion is fused to the first antigen binding portion.

[0012] The sixth aspect of the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment of any one of the first to third aspects, or encoding the bispecific antibody of any one of the fourth to fifth aspects.

[0013] The seventh aspect of the present invention provides a construct comprising the polynucleotide described in the sixth aspect.

[0014] The eighth aspect of the present invention provides a host cell comprising the polynucleotide described in the sixth aspect or the construct described in the seventh aspect.

[0015] The ninth aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment described in any one of the first to third aspects, or the bispecific antibody described in the fourth to fifth aspects; and a pharmaceutically acceptable carrier.

[0016] The tenth aspect of the present invention provides an antibody conjugate, comprising the antibody or antigen-binding fragment described in any one of the first to third aspects, or the bispecific antibody described in the fourth to fifth aspects; and a functional small molecule connected to the antibody or antigen-binding fragment or the bispecific antibody.

[0017] The eleventh aspect of the present invention provides a kit, comprising the antibody or antigen-binding fragment described in any one of the first to third aspects, or the bispecific antibody described in any one of the embodiments of the fourth to fifth aspects.

[0018] The twelfth aspect of the present invention provides a method for producing the above-mentioned antibody or antigen-binding fragment or producing the above-mentioned bispecific antibody, comprising: culturing the host cell described in the eighth aspect, and collecting the antibody or antigen-binding fragment or the bispecific antibody from the culture.

[0019] The thirteenth aspect of the present invention provides a method for preventing and / or treating a disease, comprising: administering to a subject in need an effective amount of the antibody or antigen-binding fragment described in any one of the first to third aspects above, or the bispecific antibody described in any one of the fourth to fifth aspects above, or the pharmaceutical composition described in the ninth aspect, or the antibody conjugate described in the tenth aspect.

[0020] The fourteenth aspect of the present invention provides a use of the antibody or antigen-binding fragment described in any one of the first to third aspects, or the bispecific antibody described in any one of the fourth to fifth aspects, in the preparation of a drug or a kit or in the preparation of an antibody conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.

[0022] FIG2 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.

[0023] FIG3 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.

[0024] FIG4 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.

[0025] FIG5 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention.

[0026] Figure 6 is a diagram showing the endocytosis results of different anti-CD73 antibodies provided according to an embodiment of the present invention.

[0027] Figure 7 is a diagram showing the anti-tumor effects of different anti-CD73 antibodies combined with anti-PD-L1 antibodies according to an embodiment of the present invention.

[0028] FIG8 shows the enzyme activity blocking results of different bispecific antibodies provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described in detail below with reference to specific examples. At the same time, in order to facilitate understanding by those skilled in the art, some terms of the present invention are explained and illustrated. It should be noted that these explanations and illustrations are only used to facilitate understanding by those skilled in the art and should not be regarded as limiting the scope of protection of the present invention.

[0030] As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide that contains an antigen-binding site, antigen-binding fragment, or antigen-binding portion, encompassing natural and artificial antibodies of various structures, including but not limited to intact antibodies or antigen-binding fragments of antibodies. According to a specific embodiment, the antibody referred to is a monoclonal antibody.

[0032] As used herein, the term "bispecific antibody" encompasses antigen-binding moieties that specifically bind to different epitopes of at least two different biomolecules, as well as antigen-binding moieties that specifically bind to at least two epitopes of the same biomolecule. For purposes of distinction, the portions of a bispecific antibody that bind to different epitopes of the same antigen or different antigens are described as "first antigen-binding moiety" and "second antigen-binding moiety," respectively. The first antigen-binding moiety and the second antigen-binding moiety do not indicate importance or the order of antigen binding; they are merely used to distinguish between different epitopes of an antigen or binding to different antigens. The terms "first antigen-binding moiety" and "second antigen-binding moiety" will be explained and illustrated below. Unless otherwise specified, the order of antigen binding in the listed bispecific antibodies is arbitrary. Of course, when a bispecific antibody specifically binds to different biomolecules, it may bind to more than one antigenic epitope of that particular biomolecule. According to a preferred embodiment of the present invention, a bispecific antibody comprises antigen-binding moieties that specifically bind to different epitopes of two different target antigens.

[0033] As used herein, "intact antibody" or "complete antibody" are used synonymously to refer to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region includes the heavy chain constant domain CHI, the heavy chain constant domain CHI2, and the heavy chain constant domain CHI3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant domain CL. The VH and VL regions can be further divided into complementarity determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDRs), which are separated by conserved framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, starting from the amino terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, starting from the amino terminus.

[0034] "Antigen-binding fragment," or "antigen-binding portion" (e.g., first antigen-binding portion, second antigen-binding portion) or "antigen-binding site" refers to a portion of a full-length antibody that exhibits the property or function of binding to an antigen. The term "variable region" or "variable domain" refers to the domain involved in antigen binding to the heavy or light chain of an antibody. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen-binding fragments or antigen-binding portions include, but are not limited to, Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments (variable regions), linear antibodies, single-chain antibodies, single-domain antibodies, and the like. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called Fab fragments, each containing the heavy and light chain variable regions and the light chain constant domain and the heavy chain constant domain CH1. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. Pepsin digestion of an intact antibody produces a F(ab')2 fragment. F(ab')2 fragments contain two antigen-binding F(ab) moieties linked by disulfide bonds and are bivalent antibodies. Single-chain antibodies are fusion proteins composed of the variable regions of the heavy and light chains of antibodies, linked by a flexible peptide of approximately 10-25 amino acids. Single-domain antibodies are antibody fragments composed of the variable regions of a single monomer. Because single-domain antibodies are typically derived from the variable regions of the heavy chains of camelid or shark antibodies, they are often referred to as nanobodies. Nanobodies contain only the heavy chain CDR region and are the smallest fully functional antigen-binding fragment. Nanobodies consist of three CDR regions (CDR1-CDR3) and four framework regions (FR1-FR4). The framework regions FR1, FR2, FR3, and FR4 are separated by the complementarity determining regions (CDR1, CDR2, and CDR3), respectively. Antigen-binding fragments can be obtained recombinantly. For example, a nucleic acid encoding the target antigen-binding fragment can be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen-binding fragment.

[0035] When the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0036] The term "comprising" or "including" means including the elements or steps mentioned, but does not exclude other elements or steps. Of course, unless otherwise specified, "comprising" or "including" also covers the situation where it consists of the elements or steps mentioned. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0037] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or antigen-binding fragment.

[0038] "Specific binding" or "specifically binds to", "binds to", "specifically targets" a specific antigen or epitope, or "has specificity" or "can bind to" a specific antigen or epitope means to distinguish it from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying an antigen can be detected by flow cytometry, and the competitive binding between the test antibody and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation. In combination with the specific embodiments of the present invention, the anti-CD73 antibodies provided have EC50 values ​​of ≤10nM, ≤5nM, ≤1nM, ≤0.5nM, ≤0.1nM, ≤0.09nM, ≤0.08nM, ≤0.07nM, ≤0.06nM, ≤0.05nM, ≤0.04nM, ≤0.03nM, ≤0.02nM, and ≤0.01nM. In combination with the specific embodiments of the present invention, the provided bispecific antibodies have EC50 values ​​of ≤10nM, ≤5nM, ≤1nM, ≤0.5nM, ≤0.1nM, ≤0.09nM, ≤0.08nM, ≤0.07nM, ≤0.06nM, ≤0.05nM, ≤0.04nM, ≤0.03nM, ≤0.02nM, ≤0.01nM with the CD73 antigen, and EC50 values ​​of ≤10nM, ≤5nM, ≤1nM, ≤0.5nM, ≤0.1nM, ≤0.09nM, ≤0.08nM, ≤0.07nM, ≤0.06nM, ≤0.05nM, ≤0.04nM, ≤0.03nM, ≤0.02nM, ≤0.01nM with the PD-L1 antigen. In addition, the binding activity of antibodies and antigens can also be measured by surface plasmon resonance (SPR) or bio-film interferometry (BLI).

[0039] Whether provided herein is an antibody or Fab, or a bispecific antibody, or a polynucleotide, generally separable or recombinant. "Separable" refers to being able to identify and separate and / or recycle from cells or cell cultures expressing a polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to being substantially free of other antibodies or Fabs thereof that have different antigenic specificities. "Recombinant" means that antibodies can be produced in exogenous host cells using genetic recombination techniques.

[0040] "Humanized" antibodies generally refer to antibodies whose antigen-binding portion is derived from a non-human species and whose structure and sequence are based on a portion of a human immunoglobulin molecule. For example, in a humanized antibody, the entire antibody, excluding the CDRs, is encoded by a polynucleotide of human origin, which retains antigen-binding activity while reducing immunogenicity.

[0041] The CDR sequences of the antibodies or bispecific antibodies shown herein can be obtained by combining existing database analysis. The CDR sequences of the first antigen-binding portion of the anti-CD73 antibody or bispecific antibody provided, as well as the CDR sequences of the second antigen-binding portion, are obtained by combining the IMGT definition scheme (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6: 737-749 (2011)). They can also be obtained by definition schemes such as Kabat (see, for example, US Patent No. 20 ... It is known to those skilled in the art that the differences in CDRs caused by differences in definition methods are also included in the scope of protection of the present invention.

[0042] Antibodies or antigen-binding fragments

[0043] The present invention provides an anti-CD73 antibody or antigen-binding fragment comprising the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52. The provided antibody or antigen-binding fragment comprises the HCDR1, HCDR2 and HCDR3 sequences, and the LCDR1, LCDR2 and LCDR3 sequences, which can be obtained by combining different definition schemes of the heavy chain variable region as shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and by combining different definition schemes of the light chain variable region as shown in SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52. It should be noted that the CDR sequences obtained from these heavy chain variable regions and light chain variable regions, when forming heavy chain variable region or light chain variable region sequences as part of an anti-CD73 antibody, can also form different heavy chain variable region or light chain variable region sequences with framework region sequences from different sources. Such variations are all within the scope of protection of the present invention.

[0044] According to an embodiment of the present invention, the present invention provides an anti-CD73 antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3; or comprises the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6; or comprises the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9; or comprises the HCDR sequence shown in SEQ ID NOs: 10, 11 and 12, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 10, 11 and 12; or comprises the HCDR sequence shown in SEQ ID NOs: 13, 14 and 15, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: NOs: 13, 14 and 15 have one, two or three conservative amino acid mutations in the HCDR sequences; or SEQ ID NOs: 16, 17 and 18 have one, two or three conservative amino acid mutations in the HCDR sequences; or SEQ ID NOs: 19, 20 and 21 have one, two or three conservative amino acid mutations in the HCDR sequences; the light chain variable region comprises the LCDR sequences of SEQ ID NOs: 22, 23 and 24, or have one, two or three conservative amino acid mutations in the LCDR sequences of SEQ ID NOs: 22, 23 and 24; or SEQ ID NOs: 22, 23 and 25 have one, two or three conservative amino acid mutations in the LCDR sequences of SEQ ID NOs: 22, 23 and 25; or SEQ ID NOs: 22, 23 and 25 have one, two or three conservative amino acid mutations in the LCDR sequences of SEQ ID NOs: 22, 23 and 25. The LCDR sequences shown in SEQ ID NOs: 26, 27, and 28, or sequences having one, two, or three conservative amino acid mutations compared to the LCDR sequences shown in SEQ ID NOs: 26, 27, and 28; or sequences comprising the LCDR sequences shown in SEQ ID NOs: 29, 30, and 31, or sequences having one, two, or three conservative amino acid mutations compared to the LCDR sequences shown in SEQ ID NOs: 29, 30, and 31;or comprising the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32; or comprising the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35; or comprising the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38;

[0045] Herein, "conservative amino acid mutation" means that the amino acid mutation does not cause a significant change in the conformational structure of the protein or polypeptide and retains its biological activity. The conservative amino acid mutation mentioned can be a mutation between different amino acids with similar physiological and biochemical properties. For example, after conservative amino acid mutation, the provided antibody still retains specific binding activity to CD73. According to an embodiment, the conservative amino acid mutation mentioned can be a substitution, deletion or addition of a conservative amino acid. According to a specific embodiment, the deletion of a conservative amino acid is based on the listed CDR sequence, and one, two or three amino acids are deleted. According to a specific embodiment, the addition of a conservative amino acid is based on the listed CDR sequence, and one, two or three amino acids are added. According to a specific embodiment, "conservative amino acid substitution" means replacing another amino acid residue with a different amino acid residue having a side chain with similar physiological and chemical properties. For example, conservative amino acid substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative amino acid substitutions generally do not cause significant changes in the conformational structure of the protein, and thus can retain the biological activity of the protein. The conservative amino acid substitutions mentioned can be one conservative amino acid substitution, two conservative amino acid substitutions, three conservative amino acid substitutions, etc. The names of the amino acids used herein are represented by standard single-letter or three-letter codes commonly used in the art. In some embodiments, the HCDR sequences provided have one conservative amino acid substitution compared to the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid deletion from the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid addition from the HCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid substitution from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid deletion from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid addition from the LCDR sequences shown.

[0046] The present invention also provides an anti-CD73 antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 is selected from the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequence shown in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequence shown in SEQ ID NO: 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequence shown in SEQ ID NO: 23 or 27 or 30 or 34 or 37, and LCDR3 is selected from the sequence shown in SEQ ID NO: 24 or 25 or 28 or 31 or 32 or 35 or 38.

[0047] According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 25. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, and the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35. According to a specific embodiment, the provided antibodies or antigen-binding fragments thereof comprise the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21, and the LCDR sequences shown in SEQ ID NOs: 36, 37 and 38.

[0048] In some embodiments, the antibodies or antigen-binding fragments provided include a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and the light chain variable region is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: The sequences shown in NO:46 or 47 or 48 or 49 or 50 or 51 or 52 have a sequence identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0049] The "sequence identity", "sequence homology" and "sequence similarity" mentioned herein refer to the degree of sequence identity when two polypeptides, proteins or nucleotide sequences are compared. In order to determine the ratio of sequence identity, it can be achieved in a variety of ways known in the art. For example, it can be obtained using public software such as BLAST, ALIGN, BLAST-2 and the like. In some specific embodiments, the sequence identity is due to conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region may be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations or 10 conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region differs in the framework region.

[0050] According to a specific embodiment, the antibody provided is mab309, which comprises the heavy chain variable region set forth in SEQ ID NO:39 and the light chain variable region set forth in SEQ ID NO:46. According to a specific embodiment, the antibody provided is mab307, which comprises the heavy chain variable region set forth in SEQ ID NO:40 and the light chain variable region set forth in SEQ ID NO:47. According to a specific embodiment, the antibody provided is mab144, which comprises the heavy chain variable region set forth in SEQ ID NO:41 and the light chain variable region set forth in SEQ ID NO:48. According to a specific embodiment, the antibody provided is mab235, which comprises the heavy chain variable region set forth in SEQ ID NO:42 and the light chain variable region set forth in SEQ ID NO:49. According to a specific embodiment, the antibody provided is mab451, which comprises the heavy chain variable region set forth in SEQ ID NO:43 and the light chain variable region set forth in SEQ ID NO:50. According to a specific embodiment, the antibody provided is mab540, which comprises the heavy chain variable region set forth in SEQ ID NO:44 and the light chain variable region set forth in SEQ ID NO:51. According to a specific embodiment, the antibody provided is mab449, which comprises a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:52. The antibody exhibits specific binding to CD73, for example, exhibiting strong specific binding activity to mammalian CD73. The heavy chain constant domain of these antibodies is set forth in SEQ ID NO:100, and the light chain constant domain sequence is set forth in SEQ ID NO:101.

[0051] The provided antibodies or antigen-binding fragments may also be humanized. By humanization, the immunogenicity of the antibody or antigen-binding fragment may be reduced while maintaining binding activity to the target antigen. In some embodiments, the provided antibodies or antigen-binding fragments include a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79 or have a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. According to a specific embodiment, the antibody provided is mab309hu1, which comprises a heavy chain variable region set forth in SEQ ID NO: 53 and a light chain variable region set forth in SEQ ID NO: 66. According to a specific embodiment, the antibody provided is mab309hu3, which comprises a heavy chain variable region set forth in SEQ ID NO: 54 and a light chain variable region set forth in SEQ ID NO: 67. According to a specific embodiment, the antibody provided is mab144hu1, which comprises the heavy chain variable region set forth in SEQ ID NO:55 and the light chain variable region set forth in SEQ ID NO:68. According to a specific embodiment, the antibody provided is mab144hu2, which comprises the heavy chain variable region set forth in SEQ ID NO:56 and the light chain variable region set forth in SEQ ID NO:69. According to a specific embodiment, the antibody provided is mab451hu1, which comprises the heavy chain variable region set forth in SEQ ID NO:57 and the light chain variable region set forth in SEQ ID NO:70. According to a specific embodiment, the antibody provided is mab451hu3, which comprises the heavy chain variable region set forth in SEQ ID NO:58 and the light chain variable region set forth in SEQ ID NO:71. According to a specific embodiment, the antibody provided is mab451hu4, which comprises the heavy chain variable region set forth in SEQ ID NO:59 and the light chain variable region set forth in SEQ ID NO:72. According to a specific embodiment, the antibody provided is mab451hu5, which comprises a heavy chain variable region shown in SEQ ID NO:60 and a light chain variable region shown in SEQ ID NO:73.According to a specific embodiment, the antibody provided is mab451hu6, which comprises the heavy chain variable region set forth in SEQ ID NO:60 and the light chain variable region set forth in SEQ ID NO:74. According to a specific embodiment, the antibody provided is mab540hu1, which comprises the heavy chain variable region set forth in SEQ ID NO:61 and the light chain variable region set forth in SEQ ID NO:75. According to a specific embodiment, the antibody provided is mab540hu3, which comprises the heavy chain variable region set forth in SEQ ID NO:62 and the light chain variable region set forth in SEQ ID NO:76. According to a specific embodiment, the antibody provided is mab449hu1, which comprises the heavy chain variable region set forth in SEQ ID NO:63 and the light chain variable region set forth in SEQ ID NO:77. According to a specific embodiment, the antibody provided is mab449hu3, which comprises the heavy chain variable region set forth in SEQ ID NO:64 and the light chain variable region set forth in SEQ ID NO:78. According to a specific embodiment, the antibody provided is mab449hu4, which comprises the heavy chain variable region set forth in SEQ ID NO:65 and the light chain variable region set forth in SEQ ID NO:79. The heavy chain constant domain of these antibodies is shown in SEQ ID NO: 100, and the light chain constant domain sequence is shown in SEQ ID NO: 101.

[0052] According to a specific embodiment, the provided antibodies bind to human CD73 or monkey (e.g., cynomolgus) CD73 with an EC50 of 10 nM or less, for example, the affinity EC50 value for CD73 is less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.8 nM, less than 0.5 nM, less than 0.3 nM, or less than 0.1 nM, less than 0.05 nM. The provided antibodies can also promote CD73 endocytosis and exhibit anti-tumor activity.

[0053] The anti-CD73 antibodies provided may be from any of the commonly known isotypes, including but not limited to IgA, IgG, IgM and secretory IgA. IgG isotypes can be divided into different subclasses in different species, for example, IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. According to an embodiment, the provided antibody further comprises an Fc region, and the Fc region mentioned may be derived from a natural Fc region or a modified Fc region. The natural Fc region may be of any origin, including but not limited to murine, primate, or human, preferably a human Fc region. The human Fc region mentioned can be obtained from a public database.

[0054] Bispecific antibodies

[0055] Bispecific antibodies (BsAbs) are antibodies that can specifically bind to two antigens or antigenic epitopes simultaneously. Since Nisonoff and his collaborators first proposed the concept of BsAbs in 1960, bispecific antibodies have developed rapidly with the gradual maturity of genetic engineering antibody technology. Because BsAbs can target multiple antigens or antigenic epitopes, they have more advantages than monoclonal antibodies and are gradually showing higher efficacy than monoclonal antibody combination therapies. For example, specific immune effector cells can be redirected to adjacent tumor cells to enhance tumor killing; and the specificity of binding can be increased through the interaction of two different cell surface antigens. At the same time, compared with the combination of single antibodies, development costs can be reduced, and there is no need to balance the different half-lives of the two antibodies resulting in different dosing times, resulting in low toxicity. Bispecific antibodies targeting CD73 have been developed and have a wide range of applications in fields such as tumor treatment and autoimmune diseases. Bispecific antibodies are used to treat diseases and have shown excellent therapeutic effects.

[0056] The present invention provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion and the second antigen-binding portion specifically bind to different epitopes, respectively. The different epitopes mentioned may be different epitopes from the same antigen or different epitopes from different antigens. According to a specific embodiment of the present invention, the bispecific antibody provided comprises a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion specifically binds to a first epitope and the second antigen-binding portion specifically binds to a second epitope, and the first antigen-binding portion and the second antigen-binding portion are fused. The first epitope and the second epitope may be from the same antigen or from different antigens. According to a preferred embodiment of the present invention, the first antigen-binding portion specifically binds to the first epitope and the second antigen-binding portion specifically binds to a second epitope from a different antigen. As indicated herein, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated, nor are they to be used to indicate a sequential order. The terms "first antigen-binding portion" and "second antigen-binding portion" refer to portions that can bind to different antigens or different epitopes of the same antigen, respectively, and can be an intact antibody or a portion of an intact antibody, such as a heavy chain variable region, a light chain variable region, Fab, Fab', F(ab')2, a single-chain antibody (ScFv), or a single-domain antibody (sdAb), etc.

[0057] The present invention provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion specifically binds to a first epitope, and the second antigen-binding portion specifically binds to a second epitope; the first antigen-binding portion comprises the HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52. It should be noted that the CDR sequences mentioned can be obtained by combining the heavy chain variable region and light chain variable region sequences shown in different definition schemes. Those skilled in the art should also understand that the CDR sequences of the heavy chain variable region and light chain variable region sequences shown may vary slightly due to different antibody sequence definition schemes, such as the classic Kabat amino acid sequence numbering system, the IMGT system, the Chothia system, etc. mentioned above. The different CDR sequences analyzed are all included within the scope of protection of the present invention. These CDR sequences can form different heavy chain variable region sequences or light chain variable region sequences with framework region sequences from different sources, and these changes and differences are also included in the scope of protection of the present invention.

[0058] According to an embodiment of the present invention, the present invention also provides a bispecific antibody, comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion specifically binds to a first epitope, the second antigen-binding portion specifically binds to a second epitope, and the second antigen-binding portion is fused to the first antigen-binding portion; the first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 1, 2 and 3; or comprising the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6; or comprising the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9; or comprising the HCDR sequence shown in SEQ ID NOs: 10, 11 and 12, or a sequence having one, two or three conservative amino acid mutations with the HCDR sequence shown in SEQ ID NOs: NOs: 10, 11 and 12 have one, two or three conservative amino acid mutations in the HCDR sequences; or SEQ ID NOs: 13, 14 and 15, or SEQ ID NOs: 13, 14 and 15 have one, two or three conservative amino acid mutations in the HCDR sequences; or SEQ ID NOs: 16, 17 and 18, or SEQ ID NOs: 16, 17 and 18 have one, two or three conservative amino acid mutations in the HCDR sequences; or SEQ ID NOs: 19, 20 and 21, or SEQ ID NOs: 19, 20 and 21 have one, two or three conservative amino acid mutations in the HCDR sequences; the light chain variable region comprises SEQ ID NOs: 22, 23 and 24, or SEQ ID NOs: 22, 23 and 24 have one, two or three conservative amino acid mutations in the LCDR sequences; or SEQ ID NOs: The LCDR sequences set forth in SEQ ID NOs: 22, 23, and 25, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 25; or sequences comprising the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28;or comprising the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31; or comprising the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32; or comprising the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35; or comprising the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38, or sequences having one, two, or three conservative amino acid mutations relative to the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38;

[0059] The first antigen-binding portion or the second antigen-binding portion of the bispecific antibody can be selected from a complete antibody or a fragment thereof as needed, as long as they can target different target antigens. The first antigen-binding portion or the second antigen-binding portion can be solely a heavy chain variable region, Fab, Fab', F(ab')2, single-chain antibody (ScFv), trap, or single-domain antibody (sdAb).

[0060] According to a specific embodiment, the bispecific antibodies provided comprise the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24. According to a specific embodiment, the bispecific antibodies provided comprise the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 25. According to a specific embodiment, the bispecific antibodies provided comprise the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28. According to a specific embodiment, the bispecific antibodies provided comprise the HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31. According to a specific embodiment, the bispecific antibodies provided comprise the HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 32. According to a specific embodiment, the provided bispecific antibodies comprise the HCDR sequences shown in SEQ ID NOs: 16, 17, and 18, and the LCDR sequences shown in SEQ ID NOs: 33, 34, and 35. According to a specific embodiment of the present invention, the provided bispecific antibodies comprise the HCDR sequences shown in SEQ ID NOs: 19, 20, and 21, and the LCDR sequences shown in SEQ ID NOs: 36, 37, and 38. The HCDR sequences and LCDR sequences shown serve as the first antigen-binding portion of the bispecific antibodies and specifically bind to the target antigen.

[0061] In some embodiments, the bispecific antibodies provided include the following heavy chain variable region and light chain variable region: a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45 or 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79 have a light chain variable region with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 are humanized heavy chain variable region sequences, respectively. SEQ ID NO: 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 and 79 are humanized light chain variable region sequences, respectively. The heavy chain variable region and light chain variable region sequences shown serve as the first antigen-binding portion of the bispecific antibody, specifically binding to the target antigen. After humanization, the heavy chain variable region and light chain variable region sequences of the first antigen-binding portion have lower immunogenicity and retain binding activity to the target antigen (such as CD73). Humanized antibody sequences can be obtained by methods commonly used in the art (e.g., complementary determining region grafting). In some embodiments, the humanized antibody sequence provided retains the CDR region sequence and replaces the framework region sequence with a human framework region sequence. The human framework region sequence can be obtained from a published human sequence. For example, by database comparison and computer homology modeling, a human FR region template with the greatest homology is found, and a comprehensive consideration is taken to determine whether the FR region needs to be backmutated and the key residues for the backmutation to obtain a high-affinity humanized antibody. These sequences are usually recorded in commonly used databases, such as the PDB protein structure database, IMGT, Genebank and other databases.

[0062] In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:39 and the light chain variable region set forth in SEQ ID NO:46. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:40 and the light chain variable region set forth in SEQ ID NO:47. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:41 and the light chain variable region set forth in SEQ ID NO:48. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:42 and the light chain variable region set forth in SEQ ID NO:49. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:43 and the light chain variable region set forth in SEQ ID NO:50. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:44 and the light chain variable region set forth in SEQ ID NO:51. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:45 and the light chain variable region set forth in SEQ ID NO:52. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:53 and the light chain variable region set forth in SEQ ID NO:66. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:54 and the light chain variable region set forth in SEQ ID NO:67. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:55 and the light chain variable region set forth in SEQ ID NO:68. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:56 and the light chain variable region set forth in SEQ ID NO:69. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:57 and the light chain variable region set forth in SEQ ID NO:70. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:58 and the light chain variable region set forth in SEQ ID NO:71. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:59 and the light chain variable region set forth in SEQ ID NO:72. In some embodiments, the first antigen binding portion comprises the heavy chain variable region set forth in SEQ ID NO:60 and the light chain variable region set forth in SEQ ID NO:73.In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:60 and the light chain variable region set forth in SEQ ID NO:74. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:61 and the light chain variable region set forth in SEQ ID NO:75. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:62 and the light chain variable region set forth in SEQ ID NO:76. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:63 and the light chain variable region set forth in SEQ ID NO:77. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:64 and the light chain variable region set forth in SEQ ID NO:78. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO:65 and the light chain variable region set forth in SEQ ID NO:79.

[0063] According to an embodiment of the present invention, the heavy chain variable region and the heavy chain constant domain of the first antigen binding moiety are connected, and the light chain variable region and the light chain constant domain of the first antigen binding moiety are connected to form an IgG type structure; the second antigen binding moiety is a single domain antibody. According to a specific embodiment of the present invention, the second antigen binding moiety is directly connected to the carboxyl terminus (C-terminus) of the Fc region or is connected through a connector. According to a specific embodiment of the present invention, the second antigen binding moiety is directly connected to the amino acid (N-terminus) of the heavy chain variable region or light chain variable region of the first antigen binding moiety or is connected through a connector. According to a specific embodiment of the present invention, the second antigen binding moiety is directly connected to the carboxyl terminus of the light chain constant domain CL of the first antigen binding moiety or is connected through a connector. Of course, when the second antigen binding moiety and the first antigen binding moiety are fused, they may not be limited to the above-mentioned methods, they may also be combined in these fusion methods, or they may be connected in other ways.

[0064] According to a preferred embodiment of the present invention, as shown in Figure 1, the heavy chain variable region and the heavy chain constant domain of the first antigen-binding portion of the bispecific antibody are connected, and the light chain variable region and the light chain constant domain of the first antigen-binding portion are connected to form an IgG-type structure. The second antigen-binding portion is a single domain antibody, and the second antigen-binding portion is directly connected to the carboxyl terminus of the Fc region through a linker, and the two single domain antibodies are respectively connected to the two ends of the carboxyl terminus of the Fc region through a linker. The two single domain antibodies contained in the second antigen-binding portion can be the same or different. Of course, as needed, one single domain antibody or more single domain antibodies can be connected to only one end of the carboxyl terminus of the Fc region. As shown in Figure 2, the second antigen-binding portion can also contain four single domain antibodies as needed, with two single domain antibodies respectively connected to the two ends of the carboxyl terminus of the Fc region. The connected single domain antibodies can be the same or different. The single domain antibodies can be connected by a linker.

[0065] According to a preferred embodiment of the present invention, the heavy chain variable region and the heavy chain constant domain of the first antigen-binding portion of the bispecific antibody are connected, and the light chain variable region and the light chain constant domain of the first antigen-binding portion are connected to form an IgG-type structure. The second antigen-binding portion is a single domain antibody, and the second antigen-binding portion is connected to the carboxyl terminus of the light chain constant domain via a linker. In at least some embodiments, the second antigen-binding portion comprises four single domain antibodies, two of which are connected to the carboxyl terminus of the light chain constant domain via a linker, as shown in Figure 3. The four single domain antibodies contained in the second antigen-binding portion may be the same or different. The second antigen-binding portion may also, as needed, comprise only two single domain antibodies, one of which is directly connected to the carboxyl terminus of the light chain constant domain or connected via a linker, as shown in Figure 5. The second antigen-binding portion may also, as needed, comprise four single domain antibodies, one of which is connected to both ends of the carboxyl terminus of the Fc region via a linker, and one of which is connected to the carboxyl terminus of the light chain constant domain via a linker, as shown in Figure 4. The connected single domain antibodies may be the same or different. Single domain antibodies can be connected by linkers. It should be noted that Figures 1 to 5 are only schematic drawings, for example, the interchain disulfide bonds between the light chain and the heavy chain are not shown in the figures.

[0066] According to a preferred embodiment of the present invention, the first antigen-binding moiety, in addition to the heavy chain variable region and light chain variable region mentioned above, further comprises a heavy chain constant domain CH1 and a light chain constant domain, forming a (Fab')2 structure, the bispecific antibody further comprises an Fc region, and the second antigen-binding moiety comprises a single domain antibody, which is connected to the carboxyl terminus of the light chain constant domain and / or to the carboxyl terminus of the Fc region. There is no special requirement for the number of single domain antibodies, and the connected single domain antibodies can be the same or different.

[0067] Herein, "linker" and "linker fragment" have the same meaning. The linker can be some oligopeptides or polypeptides, and can be any amino acid sequence that can provide flexibility. Available linkers are linkers commonly used in the art, including but not limited to the following group: GS, SG, GGS, GSG, SGG, GGG, GGGS, GGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGS and the like.

[0068] The second antigen-binding portion mentioned above specifically binds to an antigen that is different from the first epitope, including but not limited to: PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, 4-1BB, OX40, OX40L, VEGF, VEGF trap, VEGFR, EGFR, GARP, TGF-β, CD47, c-Met, CD3, CD33, TIGIT, PVRIG, CD69, CD96, LEFA1, CD226, FAP2, GITR, CEACAM-1, CD48, TIM-1, VISTA, CD39, CD70, CD27, CD40, Claudin18.2, ICOS, BLTA, B7-H3, BCMA, MUC1 / 16, etc. For example, a commercially available or researched antibody molecule sequence can be combined with the first antigen-binding portion mentioned herein to form a bispecific antibody to exert a disease therapeutic effect. Taking PD-1 antibodies as an example, partial or complete sequences of antibody molecules such as nivolumab, toripalimab, camrelizumab, and tislelizumab can be fused with the first antigen-binding moiety mentioned herein to form a bispecific antibody. Taking PD-L1 antibodies as an example, partial or complete sequences of antibody molecules such as durvalumab, atezolizumab, and avelumab can be fused with the first antigen-binding moiety mentioned herein to form a bispecific antibody. For another example, the second antigen-binding moiety can be a VEGF trap structure. Aflibercept (VEGF-trap) can specifically target VEGFA, VEGFB, and PIGF, and it inhibits angiogenesis by specifically binding to and capturing these proteins. The VEGF-trap molecule can be combined with the first antigen-binding moiety to form a bispecific antibody to exert a therapeutic effect on the disease. Bevacizumab is a recombinant humanized immunoglobulin G1 (IgG1) monoclonal antibody that binds to VEGFA, inhibiting its binding to VEGFR2, and subsequently inhibiting the biological effects of VEGF. Partial or complete sequences of bevacizumab or its biosimilar antibody molecules can be fused to the first antigen-binding moiety mentioned herein to form a bispecific antibody.

[0069] According to a specific embodiment, the present invention provides a bispecific antibody comprising a first antigen binding portion and a second antigen binding portion, the first antigen binding portion specifically binding to CD73, and the second antigen binding portion specifically binding to PD-L1. As mentioned above, the second antigen binding portion is not limited in form as long as it can specifically target PD-L1. For example, it can be a complete antibody or a part of a complete antibody, including but not limited to heavy chain variable region, Fab, Fab', F(ab')2, single-chain antibody or nanobody, etc. The second antigen binding portion can be obtained by antibody library screening as needed, or it can be derived from commercially available or existing (for example, disclosed in the literature or clinically studied) antibody sequences. In some specific embodiments, the second antigen binding portion is a single domain antibody. According to a preferred embodiment of the present invention, the bispecific antibody further comprises an Fc region, the Fc region is fused to the first antigen binding portion to form an IgG-type structure, and the second antigen binding portion is directly connected to the carboxyl terminus (also called the C-terminus) of the Fc region or connected through a linker.

[0070] In some embodiments, the second antigen-binding portion provided comprises CDR1, CDR2, and CDR3, wherein the CDR1 is selected from the sequence set forth in SEQ ID NO: 80, 83, or 86, or a sequence having one or two conservative amino acid mutations compared to SEQ ID NO: 80, 83, and 86; the CDR2 is selected from the sequence set forth in SEQ ID NO: 81, 84, or 87, or a sequence having one or two conservative amino acid mutations compared to SEQ ID NO: 81, 84, and 87; and the CDR3 is selected from the sequence set forth in SEQ ID NO: 82, 85, 88, or 89, or a sequence having one, two, or three conservative amino acid mutations compared to SEQ ID NO: 82, 85, 88, and 89. As mentioned above, it will be appreciated by those skilled in the art that when CDR regions are characterized using different databases, the characterized CDR region sequences may differ, and such differences are also intended to be encompassed within the scope of protection of the present invention.

[0071] According to specific embodiments, the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NO:80, the CDR2 sequence set forth in SEQ ID NO:81, and the CDR3 sequence set forth in SEQ ID NO:82. According to specific embodiments, the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NO:83, the CDR2 sequence set forth in SEQ ID NO:84, and the CDR3 sequence set forth in SEQ ID NO:85. According to specific embodiments, the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NO:86, the CDR2 sequence set forth in SEQ ID NO:87, and the CDR3 sequence set forth in SEQ ID NO:88. According to specific embodiments, the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NO:80, the CDR2 sequence set forth in SEQ ID NO:81, and the CDR3 sequence set forth in SEQ ID NO:89. The CDR1, CDR2, and CDR3 sequences shown can be combined with framework regions (FRs) of different origins to form single-domain antibodies. The framework region FR sequences are not particularly limited and can be of murine or human origin, or partially murine and partially human origin.

[0072] In some embodiments, the second antigen-binding portion has a sequence as set forth in SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the sequence of the second antigen-binding portion has a sequence identity of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% to the amino acid sequence set forth in SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. The sequences set forth in SEQ ID NO: 95, 96, 97, 98, 99 are humanized antibody sequences. The humanized sequences provided have low immunogenicity and high affinity for the target protein.

[0073] The sequence of the aforementioned second antigen-binding portion is described in PCT patent application number PCT / CN2022 / 110423 and international publication number WO2023 / 011614A. The patent describes obtaining an anti-PD-L1 nanobody library by immunizing alpacas, and screening and identifying nanobodies with good affinity, specific binding, and tumor-killing effects. The contents of PCT patent application number PCT / CN2022 / 110423 and international publication number WO2023 / 011614A are incorporated herein in whole or in part as needed.

[0074] The present invention also provides a bispecific antibody, comprising: a first antigen binding portion and a second antigen binding portion, wherein the first antigen binding portion can bind to CD73, and the second antigen binding portion can bind to PD-L1.

[0075] According to a specific embodiment, the first antigen-binding portion comprises the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24; the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen-binding portion comprises the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 25; the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen-binding portion comprises the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR sequences set forth in SEQ ID NOs: 26, 27, and 28; the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen-binding portion comprises the HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and the LCDR sequences set forth in SEQ ID NOs: 29, 30, and 31; the second antigen-binding portion comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen binding portion comprises the HCDR sequence shown in SEQ ID NO: 13, 14 and 15, and the LCDR sequence shown in SEQ ID NO: 22, 23 and 32; the second antigen binding portion comprises the CDR1 sequence shown in SEQ ID NO: 80 or 83 or 86, the CDR2 sequence shown in SEQ ID NO: 81 or 84 or 87, and the CDR3 sequence shown in SEQ ID NO: 82 or 85 or 88 or 89.According to a specific embodiment, the first antigen-binding moiety comprises the HCDR sequences set forth in SEQ ID NOs: 16, 17, and 18, and the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35; the second antigen-binding moiety comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen-binding moiety comprises the HCDR sequences set forth in SEQ ID NOs: 19, 20, and 21, and the LCDR sequences set forth in SEQ ID NOs: 36, 37, and 38; the second antigen-binding moiety comprises the CDR1 sequence set forth in SEQ ID NOs: 80, 83, or 86, the CDR2 sequence set forth in SEQ ID NOs: 81, 84, or 87, and the CDR3 sequence set forth in SEQ ID NOs: 82, 85, 88, or 89. According to a specific embodiment, the first antigen-binding moiety and the second antigen-binding moiety are coupled by (G4S). 3-5 The linker connects.

[0076] In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 39 and the light chain variable region of SEQ ID NO: 46; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 40 and the light chain variable region of SEQ ID NO: 47; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 41 and the light chain variable region of SEQ ID NO: 48; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 42 and the light chain variable region of SEQ ID NO: 49; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 50; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 51; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 45 and the light chain variable region of SEQ ID NO: 52; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 53 and the light chain variable region of SEQ ID NO: 66; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99.In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 54 and the light chain variable region of SEQ ID NO: 67; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO: 68; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 56 and the light chain variable region of SEQ ID NO: 69; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 57 and the light chain variable region of SEQ ID NO: 70; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 58 and the light chain variable region of SEQ ID NO: 71; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 59 and the light chain variable region of SEQ ID NO: 72; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 60 and the light chain variable region of SEQ ID NO: 73; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 60 and the light chain variable region of SEQ ID NO: 74; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99.In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 61 and the light chain variable region of SEQ ID NO: 75; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 62 and the light chain variable region of SEQ ID NO: 76; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding portion comprises the heavy chain variable region of SEQ ID NO: 63 and the light chain variable region of SEQ ID NO: 77; the second antigen-binding portion comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding moiety comprises the heavy chain variable region of SEQ ID NO: 64 and the light chain variable region of SEQ ID NO: 78; the second antigen-binding moiety comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. In some embodiments, the first antigen-binding moiety comprises the heavy chain variable region of SEQ ID NO: 65 and the light chain variable region of SEQ ID NO: 79; the second antigen-binding moiety comprises the sequence of SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99. Depending on the embodiment, the first antigen-binding moiety and the second antigen-binding moiety are separated by (G4S). 3-5 The linker connects.

[0077] According to a specific embodiment, the heavy chain variable region and the heavy chain constant domain of the first antigen binding portion are connected, and the light chain variable region and the light chain constant domain of the first antigen binding portion are connected to form an IgG type structure. In addition to the heavy chain variable region and light chain variable region sequences mentioned above, the other sequences of the IgG type structure formed can adopt natural sequences of mammalian origin (for example, natural sequences of human origin). According to a specific embodiment, the second antigen binding portion is connected to the carboxyl terminus (C-terminus) of the Fc region; or the second antigen binding portion is connected to the amino terminus (N-terminus) of the heavy chain variable region or the light chain variable region of the first antigen binding portion; or the second antigen binding portion is connected to the carboxyl terminus (C-terminus) of the light chain constant domain CL.

[0078] The Fc region mentioned can be a natural Fc region sequence or a modified Fc region sequence. For example, the sequence of the Fc region can be mutated. Mutations in the Fc region can regulate the antibody's ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis), or CDC (complement-mediated cytotoxicity). ADCC refers to the binding of the Fab segment of an antibody to the antigenic epitope of virus-infected cells or tumor cells, and the binding of its Fc segment to the Fc receptor (FcR) on the surface of killer cells (such as NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells. ADCP is mainly mediated by monocytes, macrophages, neutrophils, and dendritic cells to phagocytose target cells. CDC is the binding of antibodies to corresponding antigens on the cell membrane surface, forming a complex that activates the classical complement pathway and exerts a lytic effect on target cells. Fc region sequence mutations can be mutations of one, two, three, four, or five amino acids. Of course, in addition to amino acid mutations in the Fc region, mutations may also occur in other regions, such as the heavy chain constant domain CH1, hinge region, etc. The amino acid mutations mentioned are known to those skilled in the art, and include but are not limited to L234F, L234A, L235E, G236R, G237A, D270A, K322A, P329A, P331S, P331A, N297A, N297G, A330S, V309L, P238S, G236R, L328R, etc. The amino acid mutation mentioned can also be L235V, R292P, K326W, E333S, S267E, H268F, S324T, F243L, R292P, Y300L, V305L, V305I, P396L, S239D, I332E, A330L, S298A, E333A, K334A etc. The amino acid mutation mentioned can be selected from these mutation sites, or the combination of several mutation sites. In some embodiments, A330S and P331S replace in the bispecific antibody provided. In some embodiments, D270A, K322A, P329A and P331A replace in the bispecific antibody provided. In some embodiments, L234F, L235E, P331S and N297A replace in the bispecific antibody provided. In some embodiments, the provided bispecific antibodies have S267E, H268F, and S324T mutations. In some embodiments, the provided bispecific antibodies have K214R, L234F, L235E, P331S, D356E, and L358M mutations. Of course, the half-life of antibody drugs can also be improved by modifying the Fc region sequence, and suitable amino acid mutation sites are also known to those skilled in the art.The numbering of the mutation sites in this article is based on the EU index proposed by Kabat (Kabat, EA et al., Sequences of Proteins of Immunological Interest. 5). th Edition-US Department of Health and human services, NIH publication n 91-3242, pp662, 680, 689 (1991)).

[0079] polynucleotides

[0080] The present invention also provides a polynucleotide encoding an antibody, antigen-binding fragment, or bispecific antibody. The polynucleotide is isolatable and includes, but is not limited to, DNA, RNA, or cDNA. Conventional methods in the art can be used to obtain an isolated polynucleotide sequence encoding a bispecific antibody, antibody, or antigen-binding fragment.

[0081] Construct

[0082] To prepare the antibodies described herein, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or cell-free expression system. The present invention also provides a construct comprising the polynucleotide described above. Constructs can be obtained using a variety of methods commonly used in the art, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. For example, the polynucleotide can be inserted into the multiple cloning site of an expression vector to form a construct. The construct can contain various operators, such as promoters, terminators, and marker genes, as needed, operably linked to the polynucleotide. Promoters typically provide a signal to initiate transcription; promoters can include the lactose promoter (Lac), Trp promoter, Tac promoter, or phage PL and PR promoters. Terminators provide a signal to terminate transcription during the transcription process, and marker genes in the construct are often used for screening. Enhancers can also be included as needed to enhance protein expression. The expression vector is not particularly limited and can be commercially available or artificially modified, such as plasmids, phages, or viruses. Viruses can include plant cell viruses and mammalian cell viruses. The construct can express antibodies or proteins in vitro or be transferred into cells to express antibodies or proteins.

[0083] host cells

[0084] The present invention also provides a host cell containing the aforementioned polynucleotide or construct. Any cell suitable for expressing an antibody or protein using a polynucleotide or construct can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell or a mammalian cell. Commonly used host cells include yeast cells, CHO cells, HEK-293 cells, COS cells, and Drosophila S2 or Sf9 insect cells. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, and viral-mediated transformation.

[0085] Pharmaceutical composition

[0086] The present invention also provides a pharmaceutical composition comprising: the above-mentioned antibody or antigen-binding fragment, or the above-mentioned bispecific antibody, and a pharmaceutically acceptable carrier.

[0087] The pharmaceutical composition also includes a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (e.g., as described in Berge, S. M. et al (1977) J. Pharma. Sci. 66: 1-19). The pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dose or concentration used. Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; carbohydrates, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtration through a sterile filtration membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as lyophilized dosage forms, injections, and other dosage forms. The prepared different pharmaceutical dosage forms can be formulated and administered to a subject by any suitable route of administration, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, and the like.

[0088] In addition to the above, the pharmaceutical composition may further include other therapeutic agents. The therapeutic agent mentioned is a substance that can exert a therapeutic effect. For example, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-VEGF antibodies, anti-EGFR antibodies, anti-GARP antibodies, anti-CD3 antibodies, anti-TIGIT antibodies, anti-OX40 antibodies, anti-TGF-β antibodies, etc. By combining with these therapeutic antibodies, the therapeutic effect of the disease can be further improved, thereby effectively treating a variety of diseases.

[0089] Antibody conjugates

[0090] The present invention also provides an antibody conjugate, the antibody conjugate includes the bispecific antibody or antibody or antigen binding fragment, and a functional small molecule or protease connected to the bispecific antibody or antibody or antigen binding fragment. The functional small molecule mentioned can be a developed or undeveloped small molecule drug, which is chemically coupled with the bispecific antibody or monoclonal antibody or antigen binding fragment to enhance the therapeutic effect of anti-tumor drugs and reduce adverse reactions. Small molecules such as toxins, chemotherapeutics, photosensitizers, etc. can also be coupled to the antibody mentioned by a linker. Of course, an antibody Protac conjugate can also be obtained by coupling an antibody with a protac molecule, such as connecting an antibody and a protease targeting ligand (such as an E3 ligase ligand) by a linker, closing the distance between the target protein and the E3 ubiquitin ligase in the cell, and utilizing the ubiquitin-proteasome pathway to specifically degrade the target protein.

[0091] Reagent kit or medicine box

[0092] The present invention also provides a kit comprising the above-mentioned bispecific antibody or the above-mentioned monoclonal antibody or antigen-binding fragment. The kit may also include containers, buffer reagents, and controls such as positive controls and negative controls as needed. Those skilled in the art can make corresponding choices as needed. Accordingly, the kit may also include instructions for use to facilitate operation and use by those skilled in the art. In some embodiments, the present invention also provides a drug kit comprising the above-mentioned anti-CD73 antibody or antigen-binding fragment, or the above-mentioned bispecific antibody. The drug kit may further include an immunotherapeutic agent, wherein the immunotherapeutic agent is one or more of a variety of therapeutic antibodies such as anti-PD-L1 antibodies, anti-PD-1 antibodies, or anti-CTLA-4 antibodies. The anti-PD-L1 antibodies, anti-PD-1 antibodies, or CTLA-4 antibodies mentioned are drugs that are already on the market, disclosed in the literature, or will be on the market in the future.

[0093] Methods for producing antibodies

[0094] The present invention further provides a method for producing the above-mentioned bispecific antibody, comprising: culturing the above-mentioned host cell, and collecting the bispecific antibody from the culture.

[0095] The present invention also provides a method for producing an antibody or antigen-binding fragment, comprising: culturing the above host cell, and collecting the antibody or antigen-binding fragment from the culture.

[0096] The bispecific antibody, monoclonal antibody, or antigen-binding fragment collected from the culture can be purified to obtain a substantially pure product. "Substantially pure" means that the purity of the bispecific antibody, monoclonal antibody, or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, or 99.8%.

[0097] Methods for preventing and / or treating disease

[0098] In addition, the present invention provides a method for preventing and / or treating a disease, comprising: administering an effective amount of the above-mentioned bispecific antibody, or the above-mentioned antibody or antigen-binding fragment to a subject in need.

[0099] Reference to "treating" means reducing the severity of disease symptoms, increasing the frequency and duration of symptom-free periods, or preventing a decrease in suffering caused by the disease. The effective amount of an antibody used for disease prevention is generally lower than the effective amount used for disease treatment. An "effective amount" is an amount sufficient to achieve, or at least partially achieve, the desired effect. Following treatment with the bispecific antibody, monoclonal antibody, or antigen-binding fragment, the inhibition rate of cells in the subject is greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or even greater than 90% or 95%, compared to a subject not treated with the antibody. The subject can be an animal or a human. For example, it can be a mammal, including cattle, sheep, mice, horses, etc.

[0100] The provided bispecific antibodies or monoclonal antibodies or antigen-binding fragments thereof can be used to treat diseases including but not limited to cancer, inflammatory diseases, chronic infections, cardiovascular and cerebrovascular diseases, fibrosis, neurodegenerative diseases, immunomodulatory diseases, central nervous system diseases and diabetes. In some embodiments, the methods provided by the present invention can be used to treat CD73 target-related diseases, and can also be used to treat PD-1 / PD-L1-related diseases. The cancers mentioned include but are not limited to non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, glioma, colon cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastrointestinal cancer, esophageal cancer, prostate cancer, cervical cancer, uterine cancer, head and neck cancer, adrenal cancer, urethral cancer, vaginal cancer, bladder cancer, melanoma and blood cancer, etc. In some embodiments, the diseases mentioned are non-small cell lung cancer and melanoma.

[0101] The aforementioned antibodies, antigen-binding fragments, or bispecific antibodies can also be combined with other therapeutic approaches to treat diseases. Such other therapeutic approaches may include chemotherapy or radiotherapy. Clinically combining chemotherapy or radiotherapy with other therapeutic approaches can further enhance the therapeutic efficacy of the drug and improve patient outcomes.

[0102] The present invention also provides the use of bispecific antibodies, monoclonal antibodies, or antigen-binding fragments in the preparation of medicaments or kits. The medicaments are used to treat cancer. The provided bispecific antibodies, monoclonal antibodies, or antigen-binding fragments can be used to prepare medicaments for treating various diseases. They can also be used to prepare kits for use as immunodiagnostic reagents.

[0103] The technical solutions of the present invention are described in detail below with reference to the examples. It should be noted that these examples are intended only to facilitate the understanding of those skilled in the art and should not be construed as limiting the scope of protection of the present invention. The embodiments of the present invention are described in detail below. The described embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. The PD-L1 binding portion derived from the second antigen-binding portion is a nanobody obtained by immunizing alpacas. Its sequence preparation and method of obtaining are described in the PCT International Patent Application No. PCT / CN2022 / 110423 and International Publication No. WO2023 / 011614A. The contents of the application are incorporated herein in whole or in part as needed. First, an anti-PD-L1 nanolibrary is obtained by immunizing alpacas, and screening and identification are performed to obtain candidate nanobodies. Specifically, a fusion protein consisting of the extracellular domain sequence of the human PD-L1 protein and the Fc region sequence of a human immunoglobulin is mixed with Freund's adjuvant, emulsified, and used to immunize healthy alpacas to stimulate B cells to express antigen-specific nanobodies. Alpaca blood was then collected, lymphocytes isolated, and total RNA extracted using the Trizol method. Reverse transcription was performed to obtain cDNA, and VHH antibody gene fragments were amplified from the reverse-transcribed cDNA by PCR. The VHH gene fragments were co-transformed with a yeast display vector into competent yeast. Yeast homologous recombination enzymes ligated the fragments to the vector to form a complete plasmid, creating a library of yeast transformants with high insertion rates and excellent diversity. The library was then maintained stably in nutrient-deficient medium. Antibody-expressing yeast cells were incubated with magnetic beads enriched with the target protein antigen and cultured multiple times. The magnetic bead-enriched products were identified by flow cytometry and sorted. After multiple rounds of screening, five high-expressing positive clones were obtained, designated Antibody A through Antibody E. The amino acid sequence of Antibody A is shown in SEQ ID NO:90, the amino acid sequence of Antibody B is shown in SEQ ID NO:91, the amino acid sequence of Antibody C is shown in SEQ ID NO:92, the amino acid sequence of Antibody D is shown in SEQ ID NO:93, and the amino acid sequence of Antibody E is shown in SEQ ID NO:94. The humanized antibodies are antibody Ahu, whose amino acid sequence is shown in SEQ ID NO:95; antibody Bhu, whose amino acid sequence is shown in SEQ ID NO:96; antibody Chu, whose amino acid sequence is shown in SEQ ID NO:97; antibody D1hu, whose amino acid sequence is shown in SEQ ID NO:98; and antibody D2hu, whose amino acid sequence is shown in SEQ ID NO:99.

[0104] These antibodies and humanized antibodies have been confirmed to have high binding activity to hPD-L1 and cynomolgus monkey PD-L1 by FACS and ELISA experiments. They also exhibit blocking activity against PD-1 / PD-L1 and CD80 / PD-L1.

[0105] For example, the binding activity of antibodies and human PD-L1 was detected by FACS. The experimental process is as follows: the affinity of different antibodies and antigens was detected by flow cytometry. MDA-MB-231 cells endogenously expressing PD-L1 antigen were cultured at 1×10 5 Each well was added to a 96-well plate. Samples of varying concentrations were then added and incubated at 4 degrees Celsius for 30 minutes. A fluorescently labeled goat anti-human IgG secondary antibody (manufacturer: Abcam) was then added to detect antibodies bound to the cell surface. Antibody-antigen binding dose-response curves were generated using geometric values, and the four-parameter raw data were plotted using Graphpad Prism V6.0 software to determine the EC50 results of antibody binding to antigen. The experimental results showed that the EC50 values ​​of Antibody A, Antibody B, Antibody C, Antibody D, and Antibody E were 0.089nM, 0.031nM, 0.042nM, 0.039nM, and 0.053nM, respectively, which were similar to the positive control (atezolizumab, 0.059nM), demonstrating high binding activity. A similar method was used to determine the binding activity of humanized antibodies, and the EC50 values ​​of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu, and antibody D2hu were 0.051 nM, 0.18 nM, 0.023 M, 0.18 nM, and 0.29 nM, respectively.

[0106] Another example is to measure the blocking activity of antibodies against PD-1 / PD-L1 by FACS. The experimental process is: using a competitive flow cytometry method to detect the blocking effect of antibodies on PD-1 and its ligand PD-L1. Resuscitate the 293T-PD-1 cell line overexpressing PD-1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), and the cells are plated at 1×10 5Cells were plated in a 96-well plate, and different concentrations of antibody dilutions (starting working concentration was 200nM, 4-fold dilution) and PD-L1-Biotin dilutions (2ug / ml, 50ul / well) were mixed and incubated at room temperature for 30 minutes. The mixture was then added to the cells at 100ul per well, mixed, incubated at 4 degrees Celsius for 60 minutes, then washed with FACS buffer (PBS containing 2% FBS), centrifuged at 1200rpm for 4 minutes, and the supernatant discarded. PE-labeled streptavidin was added at 100ul / well, incubated in the dark for 30 minutes, and washed with FACS buffer after the incubation. 120ul of FACS buffer was added to resuspend the cells and tested on the machine. Live cells were gated according to FSC / SSC, and their average fluorescence values ​​were measured. The antibody-antigen blocking reaction curve was generated using geometric values, and a four-parameter graph was drawn using Graphpad Prism V6.0 software to determine the IC50 value. The experimental results showed that the IC50 values ​​of Antibody A, Antibody B, Antibody C, Antibody D, and Antibody E were 1.0nM, 2.1nM, 1.7nM, 2.1nM, and 2.5nM, respectively, indicating that all antibodies were able to block the binding of the antigen and its ligand. A similar method was used to determine the blocking activity of the humanized antibodies, and the IC50 values ​​of Antibody Ahu, Antibody Bhu, Antibody Chu, Antibody D1hu, and Antibody D2hu were 4.0nM, 3.4nM, 1.0nM, 2.6nM, and 12nM, respectively.

[0107] The blocking activity of the antibody against CD-80 / PD-L1 was determined by FACS. The experimental process was as follows: a competitive flow cytometry-based method was used to detect the blocking effect of the antibody on PD-L1 and CD-80. The 293T-PD-L1 cell line overexpressing PD-L1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) was revived and the cells were plated at 1×10 5Each well of a 96-well plate was plated with different concentrations of antibody dilutions (starting at 200 nM, diluted 4-fold) and incubated at 4°C for 60 minutes. Following incubation, the cells were washed with FACS buffer, centrifuged at 1200 rpm for 4 minutes, and the supernatant discarded. Human-CD80-mFc (2 μg / ml) was then added at 100 μl / well and incubated at 4°C in the dark for 30 minutes. Following incubation, the cells were washed with FACS buffer. A 1:200 diluted fluorescently labeled goat anti-mouse secondary antibody (manufacturer: Abcam) was added to each well and incubated at 4°C in the dark for 30 minutes. Following incubation, the cells were washed with FACS buffer and analyzed by flow cytometry. Mean fluorescence values ​​were measured. Antibody-antigen blocking curves were generated using geometric values, and four-parameter graphs were plotted using Graphpad Prism V6.0 software to determine IC50 values. The experimental results showed that the IC50 values ​​of Antibody A, Antibody B, Antibody C, Antibody D, and Antibody E were 4.0nM, 3.4nM, 4.1nM, 4.8nM, and 3.0nM, respectively, indicating that all antibodies were able to block the binding of the antigen and its ligand. A similar method was used to determine the blocking activity of the humanized antibodies, and the IC50 values ​​of Antibody Ahu, Antibody Bhu, Antibody Chu, and Antibody D1hu were approximately 1.8nM, 1.7nM, 3.2nM, and 1.5nM, respectively.

[0108] Example 1 Preparation of Monoclonal Antibodies

[0109] Mice were immunized with the CD73 antigen and monoclonal antibodies were obtained through hybridoma screening. Mouse myeloma cells were fused with mouse spleen cells and the fused cells were plated. Then, HAT selective medium was added for screening. Myeloma cells could not survive, and spleen cells had a very short survival time. Ultimately, only myeloma cells fused with lymphocytes survived, thus obtaining hybridoma cells through screening.

[0110] Hybridoma cells that produce specific antibodies are then selected through limiting dilution cloning. Hybridoma cells are diluted multiple times and seeded onto multi-well cell culture plates, ensuring no more than one cell per well. Culture and proliferation are performed. Once the clones reach a certain size, the supernatant from each well is assayed for secreted antibodies using ELISA. Wells that bind to the specific antigen (CD73) are considered positive, and dilutions are continued until a single clone is obtained.

[0111] Using the above method, multiple positive clones were screened and differentially screened, resulting in seven highly active clones, numbered clone 309, clone 307, clone 144, clone 235, clone 451, clone 540, and clone 449. All seven clones exhibited high affinity for the human CD73 antigen and high enzyme blocking activity.

[0112] The selected clones were sequenced, and the variable regions of the selected clones were fused with human constant region sequences and constructed into the PCDNA3.1 mammalian expression vector to create chimeric antibodies. The resulting antibodies were then expressed and purified using a mammalian cell system to obtain antibodies with a purity of at least 90%. The expressed antibodies were numbered mab309, mab307, mab144, mab235, mab451, mab540, and mab449.

[0113] Example 2 Identification of Antibody Binding Activity to CD73

[0114] Coat the microtiter plate with human CD73 (hCD73, manufacturer: ACRO) at 100 ng / well and incubate overnight at 4°C. Wash with PBST, add 200 μL of 2% BSA to each well, and block at 37°C for 1 hour. Wash with PBST, and add 100 μL / well of the test antibody at varying concentrations (the initial concentration of each antibody is 31.25 nM, with a 4-fold serial dilution) and incubate at 37°C for 2 hours. Wash with PBST, and add 100 μL / well of goat anti-human secondary antibody (1% BSA dilution, 1:10,000, manufacturer: Jackson) and incubate at 37°C for 1 hour. Wash with PBST, add 100 μL of TMB colorimetric solution, and develop for 10-15 minutes. Stop the reaction by adding 50 μL of 2M sulfuric acid. The OD450 value was read using a microplate reader, and the EC50 value of the test antibody was calculated using the data processing software Graphpad Prism 8.0, as shown in Table 1 below, where the positive control was the anti-CD73 antibody olecumab (MEDI9447) from Medimune, a subsidiary of AstraZeneca. The same positive control was used in the following examples.

[0115] Table 1

[0116] The experimental results showed that all the antibodies shown were able to specifically bind to CD73.

[0117] Example 2 Antibody Binding Activity to MDA-MB-231 Cells

[0118] CD73 antigen is highly expressed on the surface of MDA-MB-231 cells. FACS was used to detect the binding activity of the antibody to MDA-MB-231 cells. MDA-MB-231 cells were cultured and the cell concentration was adjusted to 1-2×10 6Cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and then different test antibodies were added at 100 ul / well (initial antibody concentration was 200 nM, 4-fold serial dilution), and incubated at 4°C for 30 minutes. The cells were washed with buffer, and fluorescently labeled goat anti-human secondary antibody (manufacturer: Abcam) was added and incubated at 4°C for 30 minutes. The cells were washed twice with buffer, resuspended in buffer, and the fluorescence signal was detected by flow cytometry. The EC50 values ​​of the test samples were calculated using the data processing software Graphpad Prism 8.0, as shown in Table 2 below.

[0119] Table 2

[0120] As can be seen from the results in Table 2, all antibodies can bind to human CD73 and exhibit properties similar to those of the positive control.

[0121] Example 3 Antibodies Cross-React with Monkey CD73

[0122] Use monkey CD73 (CynoCD73, manufacturer: ACRO) to coat the ELISA plate, 100 ng / well, incubate at 4°C overnight. Then wash with PBST, add 200ul 2% BSA to each well, and block at 37°C for 1h. Wash with PBST, and add different concentrations of the test antibody at 100uL / well (the initial concentration of the antibody is 31.25nM, 4-fold gradient dilution), and incubate at 37°C for 2h. Wash with PBST, and add goat anti-human secondary antibody (1% BSA dilution, manufacturer: Jackson) at 100uL / well, and incubate at 37°C for 1h. Wash with PBST, add 100ul TMB colorimetric solution for color development for 10-15min, and then add 50ul 2M sulfuric acid to terminate the reaction. The OD450 value was read using a microplate reader, and the EC50 value of the test antibody was calculated using the data processing software Graphpad Prism8.0, as shown in Table 3 below.

[0123] Table 3

[0124] The experimental results showed that all the listed antibodies showed binding activity to cynoCD73.

[0125] Example 4 Antibody enzyme activity blocking experiment

[0126] MDA-MB-231 tumor cells were cultured in DMEM + 10% FBS + 1% PS. The cells were digested and resuspended for counting, and the cell density was adjusted to 4.5 × 10 5Plate 100 μL of the test antibody per well in a 96-well cell culture plate. Dilute the test antibody in culture medium (initial concentration: 200 nM, 4-fold serial dilution). Mix thoroughly, then add 50 μL / well of the plate to the cell culture plate. Incubate in a 37°C, 5% CO2 incubator for 1 hour. Add 50 μL of 0.8 mM AMP (Sigma) to each well. Mix thoroughly, then incubate in a 37°C, 5% CO2 incubator for 2 hours. Centrifuge at 1000 rpm for 5 minutes, aspirate 50 μL of the supernatant, and transfer to a new 96-well plate. Add 50 μL of 130 μM ATP (Sigma) to each well. Mix thoroughly, then add 100 μL of Cell titer glo detection reagent (Promega) to each well. Incubate in the dark for 5-10 minutes. Read the fluorescence signal from the 96-well plate using a microplate reader. Plot a four-parameter curve using relative fluorescence as the y-axis and antibody sample concentration as the x-axis. The curves were analyzed using GraphPad Prism 8.0 software and the IC50 values ​​of the antibodies were calculated.

[0127] Table 4

[0128] The experimental results showed that Mab144 antibody, Mab235 antibody, Mab451 antibody, Mab540 antibody, and Mab449 antibody all exhibited enzyme blocking activity, which was similar to that of the positive control.

[0129] Example 5 Antibody endocytosis experiment

[0130] MDA-MB-231 tumor cells were cultured, digested with 0.25% trypsin, resuspended and counted, and the cell density was adjusted to 1×10 6 / mL, spread 100ul / well in a 96-well culture plate. Centrifuge at 1000rpm for 5 minutes and discard the supernatant. Dilute the antibody to be tested to 20nM with FACS buffer, then add 100uL / well to the 96-well plate, mix with the cells and place in a 4℃ refrigerator for 30 minutes. Then centrifuge at 1000rpm for 10min and wash with FACS buffer. One of the samples is marked as 0h, placed on ice, and temporarily stored in a 4℃ refrigerator. The other samples are placed in a 37℃ constant temperature incubator and incubated for 4h. Then take out the 0h sample on ice and the 37℃ 4h sample, centrifuge and discard the supernatant, add 100ul goat anti-human fluorescent secondary antibody (1:200 dilution, Abcam), mix well, and place in a 4℃ refrigerator for 30min. Then wash with FACS buffer. Add buffer to resuspend the cells and detect the fluorescence signal on a flow cytometer. Calculate the antibody internalization ratio based on the read fluorescence signal. The calculation formula is:

[0131] Internalization ratio (%) = (0 h fluorescence signal - 4 h fluorescence signal) / 0 h fluorescence signal * 100%.

[0132] The experimental results are shown in Figure 6. The results showed that anti-CD73 antibodies can promote the endocytosis of CD73.

[0133] Example 6 Antitumor efficacy of antibodies

[0134] CT26-hPDL1-hCD73 cells were cultured to the logarithmic growth phase, the cells were collected, the culture medium was removed, and the cells were washed with PBS. The cells were then inoculated into BALB / C-hPD1 / hPDL1 / hCD73 triple transgenic mice (humanized mouse model expressing human PD1, human PDL1 extracellular domain protein, and human CD73 protein, manufactured by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) with an inoculum size of 1×10 6 cells / 100μL / mouse. When the average tumor volume reaches 80-120mm 3 When the mice were about 6 hours old, they were randomly divided into 10 groups, with 6 mice in each group. The day of grouping was defined as D0 day, and anti-PD-L1 antibody (antibody number D2hu, dosing concentration was 1.5mpk (mg / kg)) and anti-CD73 antibody (dosing concentration was 10mpk) were co-administered starting on D0 day. The drug was administered twice a week for two weeks by intraperitoneal injection, and the coefficient of variation CV of tumor volume did not exceed 1 / 3. After administration, the tumor volume and mouse weight were measured 2-3 times a week according to the tumor growth. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental results are shown in Figure 7. G1 is PBS, G2 is anti-PD-L1 antibody administration (dosage concentration is 1.5 mpk), G3 is anti-PD-L1 antibody and mab307 antibody co-administration, G4 is anti-PD-L1 antibody and mab309 antibody co-administration, G5 is anti-PD-L1 antibody and mab235 antibody co-administration, G6 is anti-PD-L1 antibody and mab540 antibody co-administration, G7 is anti-PD-L1 antibody and mab144 antibody co-administration, G8 is anti-PD-L1 antibody and mab449 antibody co-administration, G9 is anti-PD-L1 antibody and mab451 antibody co-administration.

[0135] The results showed that the anti-tumor efficacy of mab309 antibody, mab307 antibody, mab235 antibody, mab540 antibody, mab144 antibody, mab449 antibody, and mab451 antibody combined with PD-L1 antibody in mice was better than that of PD-L1 antibody alone.

[0136] Example 7

[0137] Humanized antibodies are obtained using the complementary determining region grafting (CDR-grafting) method. The amino acid sequences of the antibody and template are uniformly divided into FR and CDR regions. The CDR regions are then transplanted onto the FR regions of the template, allowing the CDR regions, which determine the antibody's specificity, to combine with the framework regions of the humanized antibody, achieving the goal of humanization. To prevent a decrease in antibody affinity, key amino acid residue positions are calculated through computer simulation and other techniques, and back mutations are used to ensure the affinity of the humanized antibody. Humanized antibodies are obtained, and the letters "hu" are added to the end of the antibody number when naming the humanized antibodies, with different numbers to indicate different humanized antibodies. In addition, the humanized Fc region of the antibody is mutated, and the mutated antibodies are named with the letters "mu" as a suffix. One of these mutations includes L234F, L235E, P331S, and N297A. The mutated antibodies are named with the letters "mu1" as a suffix. Another type of mutation includes K214R, L234F, L235E, P331S, D356E, and L358M. Antibodies with these mutations are named with the suffix "mu2." For example, mab309hu1 designates the first humanized version of mab309. mab144hu2 designates the second humanized version of mab144. mab309hu3mu designates the third humanized version of mab309, with additional mutations in the Fc region.

[0138] Example 8 Humanized Antibody Binding Activity to MDA-MB-231 Cells

[0139] MDA-MB-231 cells highly express CD73 antigen on their surface. MDA-MB-231 cells were added into 96-well plates at 100 μl / well, with a cell concentration of 1-2 × 10 6 Cells were washed with FACS buffer (PBS solution containing 2% FBS), and then different test antibodies were added at 100ul / well (initial antibody concentration was 200nM, 4-fold serial dilution), and incubated at 4°C for 30min. Cells were washed with buffer, and fluorescently labeled goat anti-human secondary antibody (manufacturer: Abcam) was added and incubated at 4°C for 30min. Cells were washed twice with buffer, resuspended in buffer, and fluorescence signals were detected on a flow cytometer. The EC50 values ​​of the test samples were calculated using the data processing software Graphpad Prism 8.0, as shown in Table 5 below.

[0140] Table 5

[0141] The humanized antibody showed high binding activity to MDA-MB-231 cells, similar to that of the positive control.

[0142] Example 9 Humanized Antibody Enzyme Blocking Activity

[0143] A375 tumor cells were cultured and digested with 0.25% trypsin to adjust the cell density to 4.5 × 10 5 100 μL / well of a 96-well cell culture plate was plated. Different antibodies to be tested were diluted in culture medium (initial concentration: 200 nM, serially diluted 4-fold). After mixing, 50 μL / well of the diluted solution was added to the plate. After incubation at 37°C, 5% CO₂ incubator for 1 hour, 50 μL of 0.8 mM AMP (Sigma) was added to each well. After mixing, the solution was incubated at 37°C, 5% CO₂ incubator for 2 hours. After centrifugation at 1000 rpm for 5 minutes, 50 μL of the supernatant was transferred to a new 96-well cell culture plate. 50 μL of 130 μM ATP (Sigma) was added to each well. After mixing, 100 μL of Cell titer glo detection reagent (Promega) was added to each well. Incubate in the dark for 5-10 minutes and read the fluorescence signal in the 96-well plate using a microplate reader. A four-parameter curve was plotted with relative fluorescence as the y-axis and antibody sample concentration as the x-axis. The curve was analyzed using GraphPad Prism 8.0 software and the IC50 values ​​of the antibodies were obtained, as shown in Table 6 below.

[0144] Table 6

[0145] The results showed that the humanized antibody exhibited enzyme blocking activity.

[0146] Example 10 Construction of bispecific antibodies

[0147] The anti-CD73 antibody is used as the first antigen binding portion, and the anti-PD-L1 fragment is used as the second antigen binding portion, and genetic engineering is performed to construct a bispecific antibody, in which the anti-PD-L1 fragment and the anti-CD73 antibody are connected together through a (G4S)4 linker. During the connection process, the anti-PD-L1 fragment is connected to the carboxyl end of the heavy chain constant region domain CH3 of the IgG type structure. There is an anti-PD-L1 single domain antibody (the anti-PD-L1 single domain antibody is numbered D2hu) and the carboxyl end of the heavy chain constant region domain CH3 of the IgG structure, as shown in Figure 1. The bispecific antibodies formed are numbered starting with "b". For example, b144mu-D12hu represents the mab144 antibody (and the Fc region is mutated) as the first antigen binding portion, D2hu as the second antigen binding portion, and the bispecific antibody formed by the two. For example, b144hu2mu1-D2hu represents a bispecific antibody formed by the second antibody after humanization of mab144 (with the first mutation in the Fc region) as the first antigen-binding moiety and D2hu as the second antigen-binding moiety. And so on.

[0148] The bispecific antibody protein was then produced by transient transfection of CHO-S cells, the supernatant was collected, and purified using a protein A affinity column. The purity of the purified antibody was determined by HPLC and SDS-PAGE, and the purity of the bispecific antibody was determined to be above 90%.

[0149] Example 11 Bispecific Antibody Binding Activity

[0150] CHO-K1-CD73 overexpressing cells were added to a 96-well plate at a concentration of 1-2 × 10 6 Cells were washed with FACS buffer (PBS solution containing 2% FBS), and then different test antibodies were added at 100ul / well (initial antibody concentration was 200nM, 4-fold serial dilution), and incubated at 4°C for 30min. Cells were washed with buffer, and fluorescently labeled goat anti-human secondary antibody (manufacturer: Abcam) was added, and incubated at 4°C for 30min. Cells were washed with buffer, and the cells were resuspended in buffer, and the fluorescence signal was detected on a flow cytometer. The EC50 values ​​of the test samples were calculated using the data processing software Graphpad Prism 8.0, as shown in Table 7 below.

[0151] Table 7

[0152] Example 12 Determination of Bispecific Antibody Enzyme Blocking Activity

[0153] The enzyme blocking activity of the bispecific antibody was tested using the method of Example 9, and the results are shown in Tables 8-1 and 8-2 and Figure 8. The enzyme blocking activity of the bispecific antibody was stronger than that of the positive control, at least 2 to 5 times that of the positive control.

[0154] Table 8-1

[0155] Table 8-2

[0156] Example 13 Bispecific Antibody Reporter Gene Blocking Activity

[0157] The blocking effect of the bispecific antibody on the PD-L1 / PD-1 pathway was investigated using reporter gene assays. This experiment was conducted using two cell lines: the Jurkat-PD1-CD3zeta-NFAT-Luc2 cell line (manufacturer: Kangyuan Broad Biotechnology (Beijing) Co., Ltd.) and the 293T-hPD-L1 cell line (manufacturer: Kangyuan Broad Biotechnology (Beijing) Co., Ltd.). The Jurkat-PD1-CD3zeta-NFAT-Luc2 cell line stably expresses a fusion protein consisting of the PD-1 ECD and CD3zeta in Jurkat cells, along with an inserted NF-AT-driven luciferase reporter gene. The 293T-hPD-L1 cell line is a 293T cell line expressing human PD-L1. When the two cells are co-cultured, the PD-1 / PD-L1 interaction serves as the first signal, and the intracellular CD3zeta chain serves as the second signal to transmit the activation signal inward, allowing the NFAT-driven luciferase reporter gene to be expressed and emit fluorescence; when PD-L1 antibodies are added to the culture system, the binding of PD-1 / PD-L1 is blocked, inhibiting the transmission of the activation signal, and the luciferase reporter gene cannot be expressed. The experimental process is as follows:

[0158] 293T-hPD-L1 cells were cultured at 2×10 4 Jurkat-PD1-CD3zeta-NFAT-Luc2 effector cells were plated in 96-well plates at a rate of 2×10 4 / well continue to add to the 96-well plate; then add different concentrations of the test antibody (starting concentration is 60ug / ml, 4-fold gradient dilution), incubate at 37℃ for 18-24h. Add 100ul of luciferase substrate ONE-Glo to each well TMLuciferase Assay system detection reagents were incubated in the dark for 5 minutes. Fluorescence signals were read on a microplate reader in a 96-well plate. A four-parameter curve was plotted with relative fluorescence values ​​on the y-axis and antibody sample concentration on the x-axis. This curve was analyzed using GraphPad Prism 8.0 software to determine the IC50 values ​​of the bispecific antibodies, as shown in Tables 9-1 and 9-2 below.

[0159] Table 9-1

[0160] Table 9-2

[0161] In the description of this specification, the reference terms "embodiment", "some embodiments", "example", "specific implementation", or "implementation" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0162] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An anti-CD73 antibody or antigen-binding fragment, characterized in that: comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region: A HCDR sequence comprising SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations compared to the HCDR sequence of SEQ ID NOs: 1, 2 and 3; or comprising the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6; or comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9; or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12; or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15; or comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, or sequences having one, two or three conservative amino acid mutations compared to the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18; or comprising the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or sequences having one, two or three conservative amino acid mutations compared to the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21; The light chain variable region: A sequence comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations compared to the LCDR sequence shown in SEQ ID NOs: 22, 23 and 24; or comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 25, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 22, 23 and 25; or comprising the LCDR sequence shown in SEQ ID NOs: 26, 27 and 28, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 26, 27 and 28; or comprising the LCDR sequence shown in SEQ ID NOs: 29, 30 and 31, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 29, 30 and 31; or comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 32, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 22, 23 and 32; or comprising the LCDR sequence shown in SEQ ID NOs: 33, 34 and 35, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 33, 34 and 35; or comprises the LCDR sequence shown in SEQ ID NO:36, 37 and 38, or is a sequence that is identical to SEQ ID NO:36, 37 and The LCDR sequences shown in 38 have one, two or three conservative amino acid mutations.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that Select at least one of the following: (1) comprising the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24; or (2) comprising the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 25; or (3) comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, and the LCDR sequences shown in SEQ ID NOs: 26, 27 and 28; or (4) comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, and the LCDR sequences shown in SEQ ID NOs: 29, 30 and 31; or (5) comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 32; or (6) comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, and the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35; or (7) comprising the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21, and the LCDR sequences shown in SEQ ID NOs: 36, 37 and 38.

3. An anti-CD73 antibody or antigen-binding fragment, characterized in that: The invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementary determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises complementary determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 is selected from the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequence shown in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequence shown in SEQ ID NO: 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequence shown in SEQ ID NO: 23 or 27 or 30 or 34 or 37, LCDR3 is selected from the sequence shown in SEQ ID NO: 24 or 25 or 28 or 31 or 32 or 35 or 38.

4. An anti-CD73 antibody or antigen-binding fragment, characterized in that: include: The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region are shown as SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region are shown as SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that Include: The sequence shown in SEQ ID NO:39 or 40 or 41 or 42 or 43 or 44 or 45 or 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and A light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:46 or 47 or 48 or 49 or 50 or 51 or 52 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that Select at least one of the following: having a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:46; or has a heavy chain variable region shown in SEQ ID NO:40 and a light chain variable region shown in SEQ ID NO:47; or having a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:48; or having a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:49; or has a heavy chain variable region shown in SEQ ID NO:43 and a light chain variable region shown in SEQ ID NO:50; or having a heavy chain variable region shown in SEQ ID NO:44 and a light chain variable region shown in SEQ ID NO:51; or having a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:52; or having a heavy chain variable region shown in SEQ ID NO:53 and a light chain variable region shown in SEQ ID NO:66; or having a heavy chain variable region as shown in SEQ ID NO:54 and a light chain variable region as shown in SEQ ID NO:67; or having a heavy chain variable region as shown in SEQ ID NO:55 and a light chain variable region as shown in SEQ ID NO:68; or having a heavy chain variable region shown in SEQ ID NO:56 and a light chain variable region shown in SEQ ID NO:69; or having a heavy chain variable region shown in SEQ ID NO:57 and a light chain variable region shown in SEQ ID NO:70; or having a heavy chain variable region shown in SEQ ID NO:58 and a light chain variable region shown in SEQ ID NO:71; or having a heavy chain variable region as shown in SEQ ID NO:59 and a light chain variable region as shown in SEQ ID NO:72; or having a heavy chain variable region as shown in SEQ ID NO:60 and a light chain variable region as shown in SEQ ID NO:73; or having a heavy chain variable region as shown in SEQ ID NO:60 and a light chain variable region as shown in SEQ ID NO:74; or having a heavy chain variable region as shown in SEQ ID NO:61 and a light chain variable region as shown in SEQ ID NO:75; or having a heavy chain variable region as shown in SEQ ID NO:62 and a light chain variable region as shown in SEQ ID NO:76; or having a heavy chain variable region as shown in SEQ ID NO:63 and a light chain variable region as shown in SEQ ID NO:77; or having a heavy chain variable region shown in SEQ ID NO:64 and a light chain variable region shown in SEQ ID NO:78; or It has a heavy chain variable region shown in SEQ ID NO:65 and a light chain variable region shown in SEQ ID NO:

79.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that: The antibody is selected from at least one of the following groups: IgG1, IgG2, IgG3, IgG4, or variants thereof.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that Have at least one of the following properties: (a) Inhibit CD73 enzyme activity; (b) binds human CD73 with a Kd of 10 nM or less; (c) Binds to monkey CD73 with a Kd of 10 nM or less.

9. A bispecific antibody, characterized in that: comprising a first antigen binding portion and a second antigen binding portion; The first antigen binding portion specifically binds to a first epitope, the first antigen binding portion comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region: A HCDR sequence comprising SEQ ID NOs: 1, 2 and 3, or a sequence having one, two or three conservative amino acid mutations compared to the HCDR sequence of SEQ ID NOs: 1, 2 and 3; or comprising the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6; or comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9; or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12; or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15; or comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18; or sequences having one, two or three conservative amino acid mutations with the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18; or comprising the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or sequences having one, two or three conservative amino acid mutations compared to the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21; The light chain variable region: A sequence comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 24, or a sequence having one, two or three conservative amino acid mutations compared to the LCDR sequence shown in SEQ ID NOs: 22, 23 and 24; or comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 25, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 22, 23 and 25; or comprising the LCDR sequence shown in SEQ ID NOs: 26, 27 and 28, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 26, 27 and 28; or comprising the LCDR sequence shown in SEQ ID NOs: 29, 30 and 31, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 29, 30 and 31; or comprising the LCDR sequence shown in SEQ ID NOs: 22, 23 and 32, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 22, 23 and 32; or comprising the LCDR sequence shown in SEQ ID NOs: 33, 34 and 35, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 33, 34 and 35; or comprising the LCDR sequence shown in SEQ ID NOs: 36, 37 and 38, or a sequence having one, two or three conservative amino acid mutations with the LCDR sequence shown in SEQ ID NOs: 36, 37 and 38; The second antigen binding moiety specifically binds to a second epitope, and the second antigen binding moiety is fused to the first antigen binding moiety.

10. The bispecific antibody according to claim 9, characterized in that The second antigen binding portion specifically binds to at least one of the following: PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, 4-1BB, OX40, VEGF, VEGF trap, VEGFR, EGFR, GARP, TGF-β, CD47, c-Met, CD3, CD33, TIGIT, PVRIG, CD96, LEFA1, CD226, FAP2, GITR, VISITA, Claudin 18.2, ICOS, BTLA, B7-H3, BCMA, MUC1 / 16.

11. The bispecific antibody according to claim 9 or 10, characterized in that: The first antigen binding moiety is selected from at least one of the following: (1) having the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24; or (2) having the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 25; or (3) having the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, and the LCDR sequences shown in SEQ ID NOs: 26, 27 and 28; or (4) having the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, and the LCDR sequences shown in SEQ ID NOs: 29, 30 and 31; or (5) having the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 32; or (6) having the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, and the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35; or (7) having the HCDR sequences shown in SEQ ID NOs: 19, 20 and 21, and the LCDR sequences shown in SEQ ID NOs: 36, 37 and 38.

12. A bispecific antibody, characterized in that: comprising a first antigen binding moiety and a second antigen binding moiety, The first antigen binding moiety specifically binds to a first epitope, the second antigen binding moiety specifically binds to a second epitope, and the second antigen binding moiety is fused to the first antigen binding moiety; The first antigen binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3; in: HCDR1 is selected from the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16 or 19, HCDR2 is selected from the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17 or 20, HCDR3 is selected from the sequence shown in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18 or 21, LCDR1 is selected from the sequence shown in SEQ ID NO: 22 or 26 or 29 or 33 or 36, LCDR2 is selected from the sequence shown in SEQ ID NO: 23 or 27 or 30 or 34 or 37, LCDR3 is selected from the sequence shown in SEQ ID NO: 24 or 25 or 28 or 31 or 32 or 35 or 38.

13. The bispecific antibody according to any one of claims 9 to 12, characterized in that The first antigen binding moiety comprises: a heavy chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:39 or 40 or 41 or 42 or 43 or 44 or 45 or 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and A light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:46 or 47 or 48 or 49 or 50 or 51 or 52 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79.

14. The bispecific antibody according to claim 13, characterized in that The first antigen binding moiety comprises: (1) a heavy chain variable region set forth in SEQ ID NO: 39 or 40 or 41 or 42 or 43 or 44 or 45, and a light chain variable region set forth in SEQ ID NO: 46 or 47 or 48 or 49 or 50 or 51 or 52; or (2) a heavy chain variable region represented by SEQ ID NO: 53 or 54 or 55 or 56 or 57 or 58 or 59 or 60 or 61 or 62 or 63 or 64 or 65, and a light chain variable region represented by SEQ ID NO: 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79.

15. The bispecific antibody according to claim 14, characterized in that The first antigen binding moiety comprises: The heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO:46; or The heavy chain variable region shown in SEQ ID NO:40 and the light chain variable region shown in SEQ ID NO:47; or The heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:48; or The heavy chain variable region shown in SEQ ID NO:42 and the light chain variable region shown in SEQ ID NO:49; or The heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:50; or The heavy chain variable region shown in SEQ ID NO:44 and the light chain variable region shown in SEQ ID NO:51; or The heavy chain variable region shown in SEQ ID NO:45 and the light chain variable region shown in SEQ ID NO:52; or The heavy chain variable region shown in SEQ ID NO:53 and the light chain variable region shown in SEQ ID NO:66; or The heavy chain variable region shown in SEQ ID NO:54 and the light chain variable region shown in SEQ ID NO:67; or The heavy chain variable region shown in SEQ ID NO:55 and the light chain variable region shown in SEQ ID NO:68; or The heavy chain variable region shown in SEQ ID NO:56 and the light chain variable region shown in SEQ ID NO:69; or The heavy chain variable region shown in SEQ ID NO:57 and the light chain variable region shown in SEQ ID NO:70; or The heavy chain variable region shown in SEQ ID NO:58 and the light chain variable region shown in SEQ ID NO:71; or The heavy chain variable region shown in SEQ ID NO:59 and the light chain variable region shown in SEQ ID NO:72; or The heavy chain variable region shown in SEQ ID NO:60 and the light chain variable region shown in SEQ ID NO:73; or The heavy chain variable region shown in SEQ ID NO:60 and the light chain variable region shown in SEQ ID NO:74; or The heavy chain variable region shown in SEQ ID NO:61 and the light chain variable region shown in SEQ ID NO:75; or The heavy chain variable region shown in SEQ ID NO:62 and the light chain variable region shown in SEQ ID NO:76; or The heavy chain variable region shown in SEQ ID NO:63 and the light chain variable region shown in SEQ ID NO:77; or The heavy chain variable region shown in SEQ ID NO:64 and the light chain variable region shown in SEQ ID NO:78; or The heavy chain variable region is shown in SEQ ID NO:65 and the light chain variable region is shown in SEQ ID NO:

79.

16. The bispecific antibody according to any one of claims 9 to 15, characterized in that The heavy chain variable region and the heavy chain constant domain of the first antigen binding moiety are connected, and the light chain variable region and the light chain constant domain of the first antigen binding moiety are connected to form an IgG-like structure; The second antigen binding moiety is linked to the IgG-like structure, and the second antigen binding moiety comprises a single domain antibody that specifically binds to a second epitope.

17. The bispecific antibody according to any one of claims 9 to 15, characterized in that The second antigen binding portion comprises a single domain antibody that specifically binds to a second epitope, and the second epitope is selected from at least one of PD-1, PD-L1, VEGF trap, TGF-β, GARP, TIGIT, PVRIG, and CD96.

18. The bispecific antibody according to any one of claims 9 to 17, characterized in that The second antigen binding portion comprises CDR1, CDR2 and CDR3, and the second antigen binding portion is selected from at least one of the following: (1) having a CDR1 sequence as shown in SEQ ID NO: 80, a CDR2 sequence as shown in SEQ ID NO: 81, and a CDR3 sequence as shown in SEQ ID NO: 82; or (2) having a CDR1 sequence as shown in SEQ ID NO: 83, a CDR2 sequence as shown in SEQ ID NO: 84, and a CDR3 sequence as shown in SEQ ID NO: 85; or (3) having a CDR1 sequence as shown in SEQ ID NO: 86, a CDR2 sequence as shown in SEQ ID NO: 87, and a CDR3 sequence as shown in SEQ ID NO: 88; or (4) having the CDR1 sequence shown in SEQ ID NO:80, the CDR2 sequence shown in SEQ ID NO:81, and the CDR3 sequence shown in SEQ ID NO:

89.

19. The bispecific antibody according to claim 18, characterized in that The second antigen binding moiety is selected from: (a) having a sequence as shown in SEQ ID NO: 90 or 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99; (b) a sequence having a sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to (a).

20. The bispecific antibody according to claim 18 or 19, characterized in that: The heavy chain variable region and the heavy chain constant domain of the first antigen binding moiety are connected, and the light chain variable region and the light chain constant domain of the first antigen binding moiety are connected to form an IgG type structure; The second antigen binding moiety is linked to the carboxyl terminus (C-terminus) of the Fc region; or The second antigen binding moiety is linked to the amino terminus (N-terminus) of the heavy chain variable region or the light chain variable region of the first antigen binding moiety; or The second antigen binding moiety is linked to the carboxyl terminus (C-terminus) of the light chain constant domain CL.

21. The bispecific antibody according to any one of claims 18 to 20, characterized in that Have at least one of the following properties: (a) specifically binds to PD-L1 with an EC50 value of 10 nM or less; (b) specifically binds to CD73 with an EC50 value of 10 nM or less; (c) having anti-tumor activity; (d) Promotes CD73 internalization.

22. A polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment of any one of claims 1 to 8 or encodes the bispecific antibody of any one of claims 9 to 21.

23. A construct, characterized in that Comprising the polynucleotide of claim 22.

24. A host cell, characterized in that Containing the polynucleotide according to claim 22 or the construct according to claim 23.

25. A pharmaceutical composition, characterized in that include: The antibody or antigen-binding fragment of any one of claims 1 to 8, or the bispecific antibody of any one of claims 9 to 21; and A pharmaceutically acceptable carrier.

26. An antibody conjugate, characterized in that: include: The antibody or antigen-binding fragment of any one of claims 1 to 8, or the bispecific antibody of any one of claims 9 to 21; and A functional small molecule connected to the antibody or antigen-binding fragment or the bispecific antibody.

27. A kit, characterized in that The kit comprises the antibody or antigen-binding fragment according to any one of claims 1 to 8 or the bispecific antibody according to any one of claims 9 to 21.

28. A method for producing the antibody or antigen-binding fragment of any one of claims 1 to 8 or the bispecific antibody of any one of claims 9 to 21, characterized in that: include: Cultivating the host cell of claim 24, and The antibody or antigen-binding fragment or the bispecific antibody is collected from the culture.

29. A method for preventing and / or treating a disease, characterized in that: include: Administering an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 8, or the bispecific antibody of any one of claims 9 to 21, or the pharmaceutical composition of claim 25, or the antibody conjugate of claim 26 to a subject in need thereof.

30. The method according to claim 29, characterized in that The disease is selected from cancer or autoimmune disease.

31. Use of the antibody or antigen-binding fragment of any one of claims 1 to 8, or the bispecific antibody of any one of claims 9 to 21 in the preparation of a medicament, a kit or an antibody conjugate.

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