Anti-cd73 antibodies and uses thereof

Abstract

The application belongs to the field of biological medicine, and provides preparation and use of an anti-CD73 antibody or antigen binding fragment thereof, the antibody comprising H CDR1, H CDR2 and H CDR3, and L CDR1, L CDR2 and L CDR3; the sequence of H CDR1 is as shown in SEQ ID NO:1, SEQ ID NO:9 or SEQ ID NO:17, the sequence of H CDR2 is as shown in SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:18, the sequence of H CDR3 is as shown in SEQ ID NO:3, SEQ ID NO:11 or SEQ ID NO:19, the sequence of L CDR1 is as shown in SEQ ID NO:5, SEQ ID NO:13 or SEQ ID NO:21, the sequence of L CDR2 is as shown in SEQ ID NO:6, SEQ ID NO:14 or SEQ ID NO:22, and the sequence of L CDR3 is as shown in SEQ ID NO:7, SEQ ID NO:15 or SEQ ID NO:23. The anti-CD73 antibody of the application can specifically bind to CD73 and inhibit the enzymatic activity of CD73.

Description

[0001] This application is a divisional application of Chinese Invention Patent Application No. 201910532994.4, filed on June 19, 2019, entitled "Anti-CD73 Antibodies and Uses Thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and relates to the preparation and use of antibodies. BACKGROUND

[0003] CD73, also known as ecto-5'-nucleotidase, is a glycoprotein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) and is widely expressed in human endothelial cells and lymphocytes, which helps to convert ATP with immune activation into adenosine with immune suppression. Adenosine, as a signal transduction molecule, can mediate its biological effects through a variety of receptors, including A1, A2A, A2B and A3. For example, adenosine binding to adenosine receptor A2AR (adenosine receptor AR) can inhibit the immune function of T cells, thereby helping cancer cells escape the "pursuit" of T cells. Tumor cells can also express CD73 and release adenosine, thereby reducing anti-tumor activity. In addition to inhibiting immune cell function, more and more studies have shown that CD73 can also directly stimulate tumor cell proliferation, migration and invasion, and tumor angiogenesis. Therefore, compared with other targets that regulate immune responses, targeting CD73 has the advantage of "one arrow with two targets".

[0004] Preclinical studies have shown that targeting CD73 can produce good anti-tumor effects, can change the tumor immune microenvironment by increasing the total number of T lymphocytes and tumor leukocytes, and can also reduce myeloid-derived suppressor cells (MDSC) in the tumor, thereby causing the macrophages of the responding rats to shift towards an activated (anti-tumor) phenotype. Moreover, the combination of CD73 blocking therapy with other immune molecule modulators (such as PD-(L)1 antibodies) can significantly enhance the anti-tumor effect of CD73 monoclonal antibodies, which is also an extremely attractive choice for enhancing the company's anti-tumor product line.

[0005] Analysis of clinical tumor samples has shown that CD73 overexpression is a potential biomarker that is expressed on a variety of different tumors and is closely related to the poor prognosis of various types of tumors, including breast cancer, lung cancer, ovarian cancer, renal cancer, gastric cancer, head and neck cancer, etc. In addition, treatment regimens such as PD-1 / CD73 monoclonal antibodies, radiotherapy and chemotherapy can cause an increase in CD73 expression in the tumor microenvironment, which in turn inhibits the immune response through adenosine signaling, thereby greatly reducing the treatment, which is commonly known as acquired resistance. Therefore, CD73-targeted therapeutic strategies have the potential to be used as single agents or combination therapies in clinical applications.

[0006] In view of the still pressing need for improvements in treatments targeting diseases such as cancer, methods to modulate tumor progression by various mechanisms, as well as methods to modulate CD73 activity and related therapeutic agents are highly desirable. SUMMARY

[0007] It is an object of the present application to provide an anti-CD73 antibody or antigen binding fragment thereof that specifically recognizes and binds CD73 and has desirable functional properties, including the ability to bind to CD73 with high affinity and to inhibit CD73 enzymatic activity. The antibodies described herein can be used to inhibit tumor growth, reduce adenosine production, stimulate immune responses, and detect CD73 protein in a sample.

[0008] In a first aspect, the present application provides an anti-CD73 antibody or antigen binding fragment thereof, said antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a H CDR1, a H CDR2 and a H CDR3, and the light chain variable region comprises a L CDR1, a L CDR2 and a L CDR3;

[0009] the sequence of the H CDR1 is set forth in SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 17, or the sequence of the H CDR1 has more than 95%, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 17;

[0010] the sequence of the H CDR2 is set forth in SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 18, or the sequence of the H CDR2 has more than 95%, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 18;

[0011] the sequence of the H CDR3 is set forth in SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO: 19, or the sequence of the H CDR3 has more than 95%, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO: 19;

[0012] the sequence of the L CDR1 is set forth in SEQ ID NO:5, SEQ ID NO: 13, or SEQ ID NO:21, or the sequence of the L CDR1 has 95% or more, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5, SEQ ID NO: 13, or SEQ ID NO:21;

[0013] the sequence of the L CDR2 is set forth in SEQ ID NO:6, SEQ ID NO: 14, or SEQ ID NO:22, or the sequence of the L CDR2 has 95% or more, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, SEQ ID NO: 14, or SEQ ID NO:22;

[0014] the sequence of the L CDR3 is set forth in SEQ ID NO:7, SEQ ID NO: 15, or SEQ ID NO:23, or the sequence of the L CDR3 has 95% or more, such as 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7, SEQ ID NO: 15, or SEQ ID NO:23.

[0015] In one embodiment, the heavy chain variable region of the antibody comprises:

[0016] a H CDR1 having the amino acid sequence set forth in SEQ ID NO: 1,

[0017] a H CDR2 having the amino acid sequence set forth in SEQ ID NO:2, and

[0018] a H CDR3 having the amino acid sequence set forth in SEQ ID NO:3.

[0019] In one embodiment, the heavy chain variable region of the antibody comprises:

[0020] a H CDR1 having the amino acid sequence set forth in SEQ ID NO:9,

[0021] a H CDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and

[0022] a H CDR3 having the amino acid sequence set forth in SEQ ID NO: 11.

[0023] In one embodiment, the heavy chain variable region of the antibody comprises:

[0024] a H CDR1 having the amino acid sequence set forth in SEQ ID NO: 17,

[0025] H CDR2 whose amino acid sequence is shown in SEQ ID NO: 18, and

[0026] H CDR3 whose amino acid sequence is shown in SEQ ID NO: 19.

[0027] In one embodiment, the light chain variable region of the antibody comprises:

[0028] L CDR1 whose amino acid sequence is shown in SEQ ID NO: 5,

[0029] L CDR2 whose amino acid sequence is shown in SEQ ID NO: 6, and

[0030] L CDR3 whose amino acid sequence is shown in SEQ ID NO: 7.

[0031] In one embodiment, the light chain variable region of the antibody comprises:

[0032] L CDR1 whose amino acid sequence is shown in SEQ ID NO: 13,

[0033] L CDR2 whose amino acid sequence is shown in SEQ ID NO: 14, and

[0034] L CDR3 whose amino acid sequence is shown in SEQ ID NO: 15.

[0035] In one embodiment, the light chain variable region of the antibody comprises:

[0036] L CDR1 whose amino acid sequence is shown in SEQ ID NO: 21,

[0037] L CDR2 whose amino acid sequence is shown in SEQ ID NO: 22, and

[0038] L CDR3 whose amino acid sequence is shown in SEQ ID NO: 23.

[0039] In one embodiment, the antibody is A7, whose heavy chain variable region comprises:

[0040] H CDR1 whose amino acid sequence is shown in SEQ ID NO: 1,

[0041] H CDR2 whose amino acid sequence is shown in SEQ ID NO: 2, and

[0042] H CDR3 whose amino acid sequence is shown in SEQ ID NO: 3;

[0043] whose light chain variable region comprises:

[0044] L CDR1, the amino acid sequence of which is shown in SEQ ID NO: 5,

[0045] L CDR2, the amino acid sequence of which is shown in SEQ ID NO: 6, and

[0046] L CDR3, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0047] In one embodiment, the antibody is B11, which heavy chain variable region comprises:

[0048] H CDR1, the amino acid sequence of which is shown in SEQ ID NO: 9,

[0049] H CDR2, the amino acid sequence of which is shown in SEQ ID NO: 10, and

[0050] H CDR3, the amino acid sequence of which is shown in SEQ ID NO: 11 ;

[0051] which light chain variable region comprises:

[0052] L CDR1, the amino acid sequence of which is shown in SEQ ID NO: 13,

[0053] L CDR2, the amino acid sequence of which is shown in SEQ ID NO: 14, and

[0054] L CDR3, the amino acid sequence of which is shown in SEQ ID NO: 15.

[0055] In one embodiment, the antibody is B22, which heavy chain variable region comprises:

[0056] H CDR1, the amino acid sequence of which is shown in SEQ ID NO: 17,

[0057] H CDR2, the amino acid sequence of which is shown in SEQ ID NO: 18, and

[0058] H CDR3, the amino acid sequence of which is shown in SEQ ID NO: 19;

[0059] which light chain variable region comprises:

[0060] L CDR1, the amino acid sequence of which is shown in SEQ ID NO: 21,

[0061] L CDR2, the amino acid sequence of which is shown in SEQ ID NO: 22, and

[0062] L CDR3, the amino acid sequence of which is shown in SEQ ID NO: 23.

[0063] In one embodiment, the anti-CD73 antibody A7 comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 4.

[0064] In one embodiment, the anti-CD73 antibody A7 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 8.

[0065] In one embodiment, the anti-CD73 antibody B11 comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 12, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 12.

[0066] In one embodiment, the anti-CD73 antibody B11 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 16.

[0067] In one embodiment, the anti-CD73 antibody B22 comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 20.

[0068] In one embodiment, the anti-CD73 antibody B22 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 24, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 24.

[0069] In one embodiment, the anti-CD73 antibody comprises a heavy chain constant region (CH), wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 26, or has at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 26.

[0070] In an embodiment, the anti-CD73 antibody comprises a light chain constant region (CL), wherein the amino acid sequence of the light chain constant region is set forth in SEQ ID NO: 25, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 25.

[0071] In an embodiment, the anti-CD73 antibody or antigen binding fragment thereof is capable of specifically recognizing and binding to CD73 with an affinity of 1 x 10 -9 M or less, preferably 5 x 10 -10 M or less, more preferably 1 x 10 -10 M or less.

[0072] In an embodiment, the anti-CD73 antibody or antigen binding fragment thereof binds to human CD73 with a KD of about 0.1 nm to 10 nm or less as measured by fortes bio octet analysis.

[0073] In an embodiment, the anti-CD73 antibody or antigen binding fragment thereof binds to human CD73 with an EC 50 of 0.1 nm to 10 nm or less as measured by FACS.

[0074] In another aspect, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the antibody or antigen binding fragment thereof of any one of claims 1-4.

[0075] In an embodiment, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.

[0076] In a further preferred embodiment, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 32 and SEQ ID NO: 36, or SEQ ID NO: 40 and SEQ ID NO: 44, or SEQ ID NO: 48 and 52.

[0077] In another aspect, the present application also provides an expression vector comprising the nucleic acid molecule of the present application.

[0078] In another aspect, the present application also provides a host cell comprising the nucleic acid molecule of the present application or the expression vector of the present application.

[0079] In another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present application and a pharmaceutically acceptable carrier.

[0080] In one embodiment, the antibody in the pharmaceutical composition is A7, B11, B22 or a combination thereof.

[0081] In another aspect, a method of preparing an anti-CD73 antibody is provided, comprising: culturing the host cell of the present application, and isolating the antibody from the host cell.

[0082] In another aspect, the use of the anti-CD73 antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application in the preparation of a medicament for treating a neoplastic disease is provided.

[0083] In another aspect, the present application provides a method of treating a neoplastic disease in a subject, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application.

[0084] In another aspect, the present application relates to the antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application for use in treating a neoplastic disease.

[0085] In one embodiment, the neoplastic disease described herein is a CD73-positive tumor. In another embodiment, the CD73-positive tumor is lung cancer (such as non-small cell lung cancer), ovarian cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, breast cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, hematologic malignancy, head and neck cancer, melanoma, myeloma, gastric cancer, sarcoma, and virus-associated cancer.

[0086] In another aspect, the present application provides a method of reducing the level of adenosine in a CD73-expressing tumor cell, comprising contacting the cell with the anti-CD73 antibody or antigen-binding fragment thereof of the present application.

[0087] In another aspect, the present application provides a method of inhibiting the growth of a CD73-expressing tumor cell, comprising contacting the cell with the anti-CD73 antibody or antigen-binding fragment thereof of the present application.

[0088] In another aspect, the present application provides a method of stimulating an antigen-specific T cell response, comprising administering to a subject in need thereof the anti-CD73 antibody or antigen-binding fragment thereof of the present application.

[0089] In another aspect, the present application provides a method of stimulating an immune response in an individual, comprising administering to an individual in need thereof an anti-CD73 antibody or antigen-binding fragment thereof of the present application.

[0090] In some embodiments, the above method or use further comprises administering an additional anti-tumor therapeutic, such as a chemotherapeutic agent, an antibody targeting another tumor-specific antigen, or radiation therapy.

[0091] In the present application, the CD73 is either human or murine CD73, the sequences of which are well known. For example, the sequence of human CD73 isoform 1 is shown as SEQ ID NO: 27, and the sequence of human CD73 isoform 2 is shown as SEQ ID NO: 28.

[0092] The anti-CD73 antibodies of the present application are capable of specifically binding to CD73, endocytosing CD73 into cells, such as tumor cells, through antibody-mediated CD73 internalization, thereby inhibiting the enzymatic activity of CD73, activating T cells, and further inhibiting tumor growth. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 Flow cytometry (FACS) binding curves of A7, B11 and B22 antibodies to human CD73 positive Calu-6 cells (human lung adenocarcinoma cell line) are shown.

[0094] Figure 2 CD73 enzyme activity inhibition curves are shown. After treatment with A7, B11 and B22 antibodies, Calu-6 cells treated with ATP produced less AMP, and the surface CD73 enzyme activity was inhibited, wherein the inhibition effect of A7 and B11 antibodies was better than that of B22 antibody.

[0095] Figure 3 The percentage of endocytosis of CD73 on the surface of Calu-6 cells treated with A7, B11 and B22 antibodies is shown, which shows antibody-mediated CD73 endocytosis in Calu-6 cells over time.

[0096] Figure 4 Antibody charge isoform maps are shown, wherein A is antibody A7, B is antibody B11, and C is antibody B22.

[0097] Figure 5 SEC maps of antibodies are shown, wherein A is antibody A7, B is antibody B11, and C is antibody B22. DETAILED DESCRIPTION

[0098] The following detailed description and examples illustrate other features and advantages of the present application, but are not to be construed as limiting. The content of the application is defined solely by the range of claims.

[0099] Definitions

[0100] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques employed herein of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology are those commonly used by those skilled in the corresponding field. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0101] As used herein, "at least one" or "one or more" can mean 1, 2, 3, 4, 5, 6, 7, 8, or more.

[0102] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or produced in whole or in part synthetically (e.g., recombinantly), including any fragment thereof that comprises at least a portion of the variable region of an immunoglobulin molecule that retains the ability to bind specifically to a full-length immunoglobulin. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies encompass antibody fragments. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b). In preferred embodiments, the antibodies of the present application are human antibodies.

[0103] As used herein, an "antibody fragment" or "antigen binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length, but which retains at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody combining sites) of the antibody that binds antigen, and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen binding fragment refers to an antibody fragment that comprises an antigen binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments can include multiple chains linked together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), acquires an antibody that immunospecifically binds an antigen.

[0104] As used herein, a "conventional antibody" refers to an antibody comprising two heavy chains (which can be designated H and H') and two light chains (which can be designated L and L') and two antigen binding sites, wherein each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding ability (e.g., heavy chains include but are not limited to VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or any functional region thereof (e.g., light chains include but are not limited to VL chains and VL-CL chains). Each heavy chain (H and H') pairs with a light chain (L and L', respectively).

[0105] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, e.g., an antibody naturally produced by an antibody secreting B cell, as well as synthetically produced antibodies having the same domains.

[0106] As used herein, dsFv refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

[0107] As used herein, a Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment having the same structure produced synthetically, e.g., by recombinant methods. A Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising a variable domain of a heavy chain (VH) and one constant region domain of a heavy chain (CH1).

[0108] As used herein, a F(ab')2 fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, e.g., by recombinant methods. A F(ab')2 fragment essentially comprises two Fab fragments, with each heavy chain portion comprising an additional few amino acids, including a cysteine that forms a disulfide bond linking the two fragments. 2 As used herein, a F(ab')2 fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, e.g., by recombinant methods. A F(ab')2 fragment essentially comprises two Fab fragments, with each heavy chain portion comprising an additional few amino acids, including a cysteine that forms a disulfide bond linking the two fragments. 2 As used herein, a F(ab')2 fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, e.g., by recombinant methods. A F(ab')2 fragment essentially comprises two Fab fragments, with each heavy chain portion comprising an additional few amino acids, including a cysteine that forms a disulfide bond linking the two fragments.

[0109] As used herein, a Fab' fragment is a fragment comprising one half (one heavy and one light chain) of a F(ab')2 fragment. 2 As used herein, a Fab' fragment is a fragment comprising one half (one heavy and one light chain) of a F(ab')2 fragment.

[0110] As used herein, a scFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker is of a length such that the two variable domains are essentially unimpeded in their bridging. An exemplary linker is a (Gly-Ser)n residue with some Glu or Lys residues interspersed to increase solubility.

[0111] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies among different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

[0112] As used herein, "antigen binding domain" and "antigen binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A native, conventional full-length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. A conventional antigen binding site comprises loops connecting pairs of the reverse-parallel beta strands within the variable region domains. An antigen binding site can comprise other portions of the variable region domains. Each conventional antigen binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).

[0113] As used herein, “hypervariable region,” “HV,” “complementarity determining region,” and “CDR,” and “antibody CDR,” are used interchangeably, and refer to one of multiple portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain contains 3 CDRs, designated CDR1, CDR2, and CDR3. For example, a light chain variable region domain contains 3 CDRs, designated L CDR1 (or VL CDR1), L CDR2 (or VL CDR2), and L CDR3 (or VL CDR3); a heavy chain variable region domain contains 3 CDRs, designated H CDR1 (or VH CDR1), H CDR2 (or VH CDR2), and H CDR3 (or VH CDR3). The 3 CDRs in a variable region are not contiguous along the linear amino acid sequence but are proximate in a folded polypeptide. The CDRs are located within loops connecting the beta sheets of the variable domain. As described herein, one of skill in the art knows and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0114] As used herein, a framework region (FR) is a domain within an antibody variable region domain that is located within the beta sheets; FR regions are relatively more conserved in terms of amino acid sequence than hypervariable regions.

[0115] As used herein, a “constant region” domain is a domain in an antibody heavy or light chain that comprises a relatively more conserved amino acid sequence than the amino acid sequence of a variable region domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (CL) domain, while each heavy chain comprises one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and CH4. Full-length IgA, IgD, and IgG isotypes comprise CH1, CH2, CH3, and a hinge region, while IgE and IgM comprise CH1, CH2, CH3, and CH4. The CH1 and CL domains extend the Fab arm of an antibody molecule, thus contributing to interaction with an antigen as well as to swiveling of the antibody arm. Antibody constant regions can serve effector functions, such as but not limited to clearing antigens, pathogens, and toxins to which the antibody specifically binds, for example, by interacting with various cells, biomolecules, and tissues.

[0116] As used herein, a functional region of a VH domain is at least a portion of a complete VH domain that retains at least part of the binding specificity of the complete VH domain, e.g., by retaining one or more CDRs of the complete VH domain, such that the functional region of the VH domain binds an antigen, either alone or in combination with another antibody domain, e.g., a VL domain, or region thereof. An exemplary functional region of a VH domain is a region comprising CDR1, CDR2, and / or CDR3 of a VH domain.

[0117] As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least part of the binding specificity of the complete VL domain, e.g., by retaining one or more CDRs of the complete VL domain, such that the functional region of the VL domain binds an antigen, either alone or in combination with another antibody domain, e.g., a VH domain, or region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of a VL domain.

[0118] "Affinity" or "binding affinity" KD is often determined by measuring the equilibrium association constant (ka) and the equilibrium dissociation constant (kd) and calculating the quotient of kd divided by ka (KD = kd / ka). As used herein, "specific binding" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with the same antigen via noncovalent interactions between the antibody combining site of the antibody and the antigen. The antigen can be an isolated antigen or present in, e.g., a tumor cell.

[0119] In an embodiment of the application, the antibody or antigen-binding fragment thereof of the application can bind to a target antigen (e.g., CD73, such as human CD73) with an affinity of 2 x 10 -9 M or less, 1 x 10 -9 M or less, 9 x 10 -10 M or less, 8 x 10 -10 M or less, 7 x 10 -10 M or less, 6 x 10 -10 M or less, 5 x 10 -10 M or less, 4 x 10 -10 M or less, 3 x 10 -10 M or less, 2 x 10 -10 M or less, 1 x 10 -10 M or less.

[0120] Affinity can be readily determined using routine techniques, for example, by equilibrium dialysis; by using a BIAcore 2000 instrument, using the general methods set forth by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to the skilled artisan.

[0121] An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is associated with other natively related components less than in its natural state, (2) is free of other proteins from the same species or from the same source, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cell system that differs from the cell from which the polypeptide is naturally derived will be "isolated" from its naturally related components. A protein can also be isolated by substantially removing naturally occurring components using protein purification techniques well known in the art.

[0122] In peptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those of skill in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0123] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer of at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds.

[0124] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode the provided antibodies or antigen binding fragments.

[0125] The sequence "identity" has the conventionally accepted meaning in the art and can be calculated using published techniques to determine the percent sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to one of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0126] As used herein, "expression" refers to the process by which a polynucleotide is transcribed and translated into a polypeptide. The level of expression of a polypeptide can be evaluated using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying a polypeptide in a cell lysate by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0127] As used herein, "host cell" is a cell that is used to accept, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. As a host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells, such as HEK 293 cells.

[0128] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors with respect include those into which a nucleic acid encoding a polypeptide or fragment thereof can be inserted by standard techniques, such as restriction enzyme digestion and ligation. Vectors with respect also include those that comprise a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those skilled in the art.

[0129] As used herein, a vector also includes a "viral vector" or "viral vector." A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0130] As used herein, an "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, capable of affecting expression of such DNA fragments. Such additional fragments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain episomal or integrate into the host cell genome.

[0131] As used herein, "treatment" of an individual having a disease or condition means partial or complete alleviation of symptoms of the individual, or remaining unchanged after treatment. Thus, treatment includes prevention, therapy, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the symptoms from worsening or the disease from progressing. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and compositions provided herein.

[0132] As used herein, "therapeutic effect" means an effect resulting from treatment of an individual that alters, typically improves or ameliorates, symptoms of a disease or condition, or cures the disease or condition.

[0133] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, after administration to the subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder. Also, as used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, upon administration to the subject, will have the intended prophylactic effect, e.g., prevent or delay the onset of a disease or symptoms, reduce the likelihood of the onset of a disease or symptoms. A fully prophylactically effective dose does not necessarily occur with the administration of one dose, and can only occur after the administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0134] As used herein, the term "individual" refers to a mammal, such as a human.

[0135] Antibodies of the Invention

[0136] The present invention provides an antibody against CD73, i.e., an anti-CD73 antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically recognizes and binds to CD73.

[0137] In one embodiment, the antibody or antigen binding fragment thereof is capable of specifically recognizing and binding to CD73 with an affinity (KD) of 1 x 10 -9 M or lower, preferably 5 x 10 -10 M or lower, more preferably 1 x 10 -10 M or lower.

[0138] In another embodiment, the antibody or antigen binding fragment thereof has at least one of the following characteristics:

[0139] 1) inhibits CD73 enzymatic activity; 2) internalizes CD73 into a cell, e.g., a tumor cell, by antibody-mediated CD73 internalization; 3) activates T cells; 4) inhibits tumor growth.

[0140] In some embodiments, the antibody or antigen binding fragment thereof of the present invention is capable of specifically binding to CD73 (e.g., human CD73) and inhibiting CD73 enzymatic activity, reducing the production of adenosine.

[0141] In some embodiments, the tumor against which the antibody or antigen binding fragment thereof of the present invention is capable of inhibiting tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%.

[0142] In particular embodiments, the anti-CD73 antibody comprises anti-CD73 antibody A7, anti-CD73 antibody B11, anti-CD73 antibody B22, or a combination thereof.

[0143] Antibody A7

[0144] In one aspect, the present application provides an antibody A7 or an antigen binding fragment thereof against CD73,

[0145] wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein

[0146] the heavy chain variable region comprises:

[0147] a H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1,

[0148] a H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and

[0149] a H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3;

[0150] the light chain variable region comprises:

[0151] a L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5,

[0152] a L CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and

[0153] a L CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0154] In another embodiment, the anti-CD73 antibody A7 comprises a heavy chain variable region (VH),

[0155] wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 4.

[0156] In another embodiment, the anti-CD73 antibody A7 comprises a light chain variable region (VL),

[0157] wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 8.

[0158] In yet another embodiment, the anti-CD73 antibody A7 comprises a heavy chain constant region (CH),

[0159] wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 26.

[0160] In yet another embodiment, the CD73 antibody A7 comprises a heavy chain variable region (VH),

[0161] wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 26.

[0162] Antibody B11

[0163] In another aspect, the present application provides an antibody B11 or an antigen binding fragment thereof against CD73,

[0164] wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein

[0165] the heavy chain variable region comprises:

[0166] a H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9,

[0167] a H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and

[0168] a H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 ;

[0169] the light chain variable region comprises:

[0170] a L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,

[0171] a L CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and

[0172] a L CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0173] In one embodiment, the anti-CD73 antibody B11 comprises a heavy chain variable region (VH),

[0174] wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 12.

[0175] In one embodiment, the anti-CD73 antibody B11 comprises a heavy chain variable region (VH),

[0176] The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 16.

[0177] In a preferred embodiment, the anti-CD73 antibody B11 comprises a heavy chain constant region (CH),

[0178] The heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 26.

[0179] In yet another embodiment, the anti-CD73 antibody B11 comprises a light chain constant region (CL),

[0180] The light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 25.

[0181] Antibody B22

[0182] In yet another aspect, the present application provides an antibody B22 or an antigen binding fragment thereof against CD73,

[0183] The antibody comprises a heavy chain variable region and a light chain variable region, wherein

[0184] The heavy chain variable region comprises:

[0185] a H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17,

[0186] a H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and

[0187] a H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19;

[0188] The light chain variable region comprises:

[0189] a L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21,

[0190] a L CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and

[0191] LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 23.

[0192] In another embodiment, the anti-CD73 antibody B22 comprises a heavy chain variable region,

[0193] wherein the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 20.

[0194] In another embodiment, the anti-CD73 antibody B22 comprises a light chain variable region,

[0195] wherein the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 24.

[0196] In yet another embodiment, the anti-CD73 antibody B22 comprises a heavy chain constant region (CH),

[0197] wherein the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 26.

[0198] In yet another embodiment, the anti-CD73 antibody B22 comprises a light chain constant region (CL),

[0199] wherein the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 25 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 25.

[0200] Neoplastic disease

[0201] The blockade of CD73 by the antibodies of the present application can enhance the immune response to cancer cells in a patient. CD73 converts ATP, which has immune-activating effects, to adenosine, which has immune-suppressive effects. Adenosine can mediate its biological effects as a signaling molecule by a variety of receptors, including A1, A2A, A2B, and A3. For example, adenosine binding to the adenosine receptor A2ARcan suppress the immune effects of T cells, thereby helping cancer cells to escape the “takedown” by T cells. Tumor cells can also express CD73 and release adenosine, thereby reducing anti-tumor activity. Targeting CD73 can produce a good anti-tumor effect by changing the tumor immune microenvironment by increasing the total number of T lymphocytes and tumor leukocytes, and by reducing tumor myeloid-derived suppressor cells (MDSCs).

[0202] The antibodies or antigen-binding fragments thereof of the application can be used to treat neoplastic diseases. Preferred neoplastic diseases (or cancers) that can be prevented and / or treated using the antibodies or antigen-binding fragments thereof of the application include cancers that are generally responsive to immunotherapy. Non-limiting examples of cancers that can be treated include, but are not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, and osteosarcoma. Examples of other cancers include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, including

[0203] Nucleic acid, vector and antibody production methods

[0204] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the aforementioned antibody or antigen binding fragment thereof of the present application. In some embodiments, the nucleotide sequence of the nucleotide sequence region can be codon optimized for the host cell used for expression. For example, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51; further preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 32 and SEQ ID NO: 36, or SEQ ID NO: 40 and SEQ ID NO: 44, or SEQ ID NO: 48 and 52.

[0205] The present application also provides an expression vector comprising at least one of the aforementioned nucleic acid molecules of the present application.

[0206] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the aforementioned antibody or antigen binding fragment thereof of the present application. In some embodiments, the nucleotide sequence of the nucleotide sequence region can be codon optimized for the host cell used for expression. For example, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51; further preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 32 and SEQ ID NO: 36, or SEQ ID NO: 40 and SEQ ID NO: 44, or SEQ ID NO: 48 and 52.

[0207] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the aforementioned antibody or antigen binding fragment thereof of the present application. In some embodiments, the nucleotide sequence of the nucleotide sequence region can be codon optimized for the host cell used for expression. For example, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51; further preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 32 and SEQ ID NO: 36, or SEQ ID NO: 40 and SEQ ID NO: 44, or SEQ ID NO: 48 and 52.

[0208] Pharmaceutical composition

[0209] The present application also provides a pharmaceutical composition comprising the antibody of the present application and a pharmaceutically acceptable carrier. In one embodiment, the aforementioned antibody comprises anti-CD73 antibody A7, anti-CD73 antibody B11, anti-CD73 antibody B22 or a combination thereof, in particular anti-CD73 antibody B11.

[0210] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, active compounds, such as antibody molecules or immunoconjugates, may be encapsulated in a material to protect them from acids and other natural conditions that could inactivate them.

[0211] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0212] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants.

[0213] The presence of microorganisms can be prevented through sterilization procedures or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenolic sorbic acid. In many cases, the composition preferably contains isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium oxide. Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.

[0214] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the provisional preparation of sterile injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any ranges incompatible with the active compound, may be used in the pharmaceutical compositions of the present invention. Additional active compounds may also be incorporated into the compositions.

[0215] Therapeutic compositions must generally be sterile and stable under preparation and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, by using coatings, such as lecithin, appropriate flowability can be maintained in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0216] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0217] The amount of active ingredient that can be combined with the carrier materials to produce a single dose form will vary depending upon the host and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material for a single dose unit will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 to about 99 percent of the active ingredient, preferably from about 0.1 to about 70 percent, most preferably from about 1 to about 30 percent of the active ingredient, in combination with a pharmaceutically acceptable carrier.

[0218] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the application are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0219] For administration of an antibody molecule, a dose range is about 0.0001 to 100 mg / kg, more usually 0.01 to 20 mg / kg, of the recipient's body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg or 20 mg / kg of body weight, or within the range of 1-20 mg / kg. An exemplary treatment regimen calls for administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or a short initial dosing interval followed by longer intervals between dosing. In one embodiment, the dose used can be 1200 mg administered every three weeks. The mode of administration can be intravenous infusion.

[0220] Alternatively, the antibody molecule directed against a tumor can be administered as a sustained release formulation, in which case less frequent administration results. Dosage and frequency vary depending on the half-life of the antibody molecule in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosages and frequencies of administration are also dependent on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or eliminated. Thereafter, the patient can be administered a prophylactic regime.

[0221] Actual dosage levels of the active ingredients in the pharmaceutical compositions can vary so as to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the present application employed, the

[0222] An "effective amount" of an antibody of the present application, or antigen-binding fragment thereof, preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of a tumor, an "effective amount" of an antibody of the present application, or antigen-binding fragment thereof, preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received treatment. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in humans. Alternatively, the ability to inhibit cell growth can also be evaluated, such inhibition can be determined in vitro by assays known to the skilled practitioner. An effective amount of an antibody of the present application, or antigen-binding fragment thereof, is capable of reducing tumor size, or otherwise ameliorating a subject's symptoms, such as preventing and / or treating metastasis or recurrence. Such amounts can be determined by one of skill in the art, based upon factors such as the size of the subject, the severity and stage of the subject's symptoms, and the particular composition or route of administration chosen.

[0223] The antibodies of the application, or antigen-binding fragments thereof, or the pharmaceutical compositions of the application, can be administered by one or more routes of administration using one or more methods known in the art. One of skill in the art will appreciate that the route and / or mode of administration depends on the desired results. Preferred routes of administration of the antibodies of the application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0224] Alternatively, the antibodies of the application, or antigen-binding fragments thereof, or the pharmaceutical compositions of the application, can also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

[0225] The active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0226] The antibodies of the application, or antigen-binding fragments thereof, in the pharmaceutical compositions can also be conjugated to therapeutic moieties, such as cytotoxins, radioisotopes or biologically active proteins.

[0227] Cytotoxins include any agent that is detrimental to cells, e.g., kills cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, actinomycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0228] Therapeutic agents that can be conjugated include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and aminopterin), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, plicamycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0229] Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the application include duocarmycins, calicheamicins, maytansinoids, auristatins, and derivatives thereof.

[0230] Cytotoxins can be conjugated to the antibodies of the application using linker technology used in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. Linkers can be selected, for example, for ease of cleavage within the lysosomal compartment, at low pH or by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsin B, C, D).

[0231] The antibodies of the application can also be conjugated to a radioisotope, resulting in a cytotoxic radiopharmaceutical, also known as a radioimmunoconjugate. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods of making radioimmunoconjugates are well known in the art.

[0232] The antibodies of the application can also be conjugated to proteins with desirable biological activities that can be used to modify a particular biological response. Such biologically active proteins include, for example: toxins or active fragments thereof such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers such as lymphokins, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), interleukin-10 ("IL-10"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other immunological factors such as IFN, etc.

[0233] Combination therapy

[0234] The antibody or pharmaceutical composition of the present application can be administered in combination with a chemotherapeutic agent or an antibody targeting other tumor antigens. The antibody of the present application and the chemotherapeutic agent or the antibody targeting other tumor antigens can be all administered at once or administered separately. When administered separately (in the case of different administration schedules from each other), they can be administered consecutively without interruption or at predetermined intervals. The antibody of the present application or the pharmaceutical composition of the present application can also be combined with radiotherapy, for example, including administration of ionizing radiation to the patient, which is earlier than, during, and / or later than the administration of the antibody or pharmaceutical composition of the present application.

[0235] Kit

[0236] Also included within the scope of the present application are kits comprising an antibody or antigen-binding fragment thereof of the present application, and instructions for use. A kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material that is provided with the kit or is otherwise available to one of ordinary skill in the art to which the kit is directed to to understand that the kit is intended to be used in connection with the methods of the present application.

[0237] Beneficial effects

[0238] The antibody of the present application has very strong affinity to the target antigen, inhibits the enzyme activity of the target antigen, reduces the production of adenosine, has potential T cell activation and anti-tumor activity, excellent stability, and lower side effects.

[0239] Further understanding of the present application can be obtained by reference to some specific examples given herein, which are only for the purpose of illustrating the present application, and are not intended to make any limitation on the scope of the present application. Obviously, various modifications and changes can be made to the present application without departing from the essence of the present application, and therefore, these modifications and changes are also within the scope of the present application claimed herein. The proportions used herein include percentages, and if not specifically indicated, are by weight.

[0240] The reagents and instruments used in the examples are all commercially available.

[0241] Example 1: Generation of human anti-CD73 antibodies

[0242] Peripheral blood of 106 adult healthy humans was collected, and PBMC (peripheral blood mononuclear cell) cells were isolated using lymphocyte separation medium, and a total of 2.7 x 1011PBMC cells were collected. 9Total RNA was extracted by Trizol method and reverse transcribed into cDNA. The variable region genes of different antibody subtypes were amplified by routine PCR method according to the method in Recombinant Antibodies (Science Press, 2005), and the antibody variable region genes were cloned into the same enzyme-treated pDF vector by routine molecular biology techniques, and the E. coli XL1-Blue (Agilent Technology) was transformed by electroporation method, and finally a large capacity antibody library of 1E+10 pfu was obtained. After being cultured in 2YT culture solution, the primary phage antibody library was obtained by infection with helper phage VCSM13 (BioVector NTCC Inc.).

[0243] After blocking the immunotubes (Maxisorp immunotubes, Thermo Nunc) coated with recombinant human CD73 (R&D product) with 5% skim milk powder, the phage antibody library was added and incubated at 37°C for 2h; the unbound phage was discarded, and the non-specifically adsorbed phage was washed away by 5 times of PBS-T washing; 1mL elution buffer (0.1mol / L glycine-HCl, pH 2.2) was added to elute the phage and neutralized with 2mol / L Tris solution; logarithmic phase XL1-Blue bacteria (Agilent Technology), 2YT culture medium and helper phage VCSM13 were added for amplification and enrichment; the process was repeated for 3-4 times, and the eluted phage was used to infect fresh logarithmic phase XL1-Blue bacteria to coat the culture plate, which was incubated at 37°C overnight to obtain monoclonal antibodies (for details and reagents, refer to Phage Display, Humana Press).

[0244] Recombinant CD73 was incubated with 96-well plates at 4°C for 16 hours, and then blocked with BSA solution. About 400 (5 96-well plates) single phage clones were selected from the phage library with obvious enrichment after screening, and were cultured in 500μL of 2YT liquid medium supplemented with carbenicillin for 24 hours, and the supernatant was obtained by centrifugation. The supernatant was added to the CD73-coated 96-well plate and incubated for 1h. After PBST washing, it was incubated with HRP-conjugated anti-phage antibody (anti-M13 HRP), and finally the substrate was added for color development to determine the amount of phage bound to the 96-well plate. Eight candidate antibodies with relatively high affinity and specific sequences were preliminarily selected by this experiment for further detection and screening.

[0245] The 8 selected candidate antibodies were expressed in E. coli. The preserved monoclonal bacteria liquid was transferred into 50 ml 2YT-Tet medium at a ratio of 1:100, and cultured overnight for 12-16 h. The bacterial bodies were collected, centrifuged, and resuspended in 10 ml PBS. The bacterial bodies were broken by ultrasonic, and the supernatant was collected for fortebio analysis of the affinity of the CD73 Fab antibody, so as to perform affinity ranking (Table 1).

[0246] Table 1. Fab antibody affinity measured by anti-Fab sensor chip of fortebio

[0247] Antibody clone Ka (1 / Ms) Kd (1 / s) KD (M) A7 3.647 x 10 6 ]]> 1.35 x 10 -3 ]]> 3.7 x 10 -10 ]]> B11 6.517 x 10 6 ]]> 1.16 x 10 -3 ]]> 1.78 x 10 -10 ]]> B22 2.56 x 10 6 ]]> 1.263 x 10 -3 ]]> 4.93 x 10 -10 ]] A5 1.79 x 10 6 ]]> 2.63 x 10 -3 ]] 1.47 x 10 -9 ]]> A10 1.51 x 10 6 ]] 1.34 x 10 -3 ]]> 8.9 x 10 -10 ]] B17 1.49 x 10 6 ]]> 2.37 x 10 -3 ]]> 1.59 x 10 -9 ]]> B53 4.16 x 10 5 ]]> 3.86 x 10 -3 ]] 9.3 x 10 -9 ]] A65 1.8 x 10 5 ]]> 6.19 x 10 -4 ]]> 3.434 x 10 -9 ]]

[0248] Example 2: Antibody expression

[0249] The A7, B11 and B22 clones were selected for sequencing and optimization of the encoding genes. The variable region genes of the A7 (SEQ ID NO: 32, SEQ ID NO: 36), B11 (SEQ ID NO: 40, SEQ ID NO: 44) and B22 (SEQ ID NO: 48, SEQ ID NO: 52) clones were fused with the gene of human IgG constant region (the sequence of light chain constant region is SEQ ID NO: 53; the sequence of heavy chain constant region is SEQ ID NO: 54), and then cloned into the expression vector pCDNA3.4 (Thermo Fisher). The obtained eukaryotic expression vector was used to transfect Expi-CHO cells (Thermo Fisher) by transient transfection. After about 12 days of serum-free medium culture, the culture supernatant was purified by protein A medium to obtain the target antibody. Sequencing showed that the obtained A7, B11 and B22 were consistent with the target sequence.

[0250] Example 3: Binding of anti-CD73 antibody to CD73 positive cells

[0251] Calu-6 (endogenously expressing CD73, human lung adenocarcinoma cell line, purchased from the Chinese Academy of Sciences Cell Library) and DMS114 (CD73 negative, human small cell lung cancer cell line, purchased from Guangzhou Sike Biotechnology Co., Ltd.) were used as the cells to be detected, Alexa Fluor 488 goat anti-human IgG (H+L) (Thermo Fisher) was used as the secondary antibody, and the following flow analysis technique was used to generate the binding curve

[0252] One cell was divided into 6 parts with 5+10E5 cells in each part according to 6 concentrations of each antibody. The initial concentration of anti-CD73 antibody was 5 μg / ml, which was diluted by 6 concentrations gradient with 1640 medium, and added to the cells, 300 μl per part of cells, and incubated at 37°C for 1 h. After washing, the secondary antibody was added for staining at 1:1000 dilution for 45 min. After washing, the fluorescence value was detected using a flow analyzer (Merk Guava).

[0253] Figure 1 The results showed that all anti-CD73 antibodies could bind to CD73 naturally expressed cells (Calu-6). Three antibodies did not bind to cells that did not express CD73 (DMS114) (data not shown). The EC 50 The results are shown in Table 2.

[0254] Table 2: EC values of antibody binding to Calu-6 cells 50 values

[0255]

[0256] Example 4: Antibody affinity analysis

[0257] In this example, Fortebio was used to determine the affinity of the antibodies. In this experiment, AHC (anti-Fc) sensing chip (Fortebio, 18-5060) was used to bind the ligand, i.e. IgG antibody, and then the analyte was bound. The anti-CD73 antibody was diluted with 0.02% PBST buffer, and the AHC chip was used to adsorb antibody A7, B11, B22 as ligand in the same buffer. Recombinant His-tagged CD73 (Coastal Dynamics Technology Co., Ltd., C446) was diluted with PBST to 8 μg / ml, 4 μg / ml, 1.6 μg / ml, 0.64 μg / ml, 0.256 μg / ml and 0.102 μg / ml as analyte, and affinity analysis experiment was carried out. Each concentration of analyte was set to capture time 180 s; ligand and analyte binding time 180 s; dissociation time 2400 s. The raw data was imported into the analysis software, and the zero concentration control was deducted, and the reference channel was deducted to eliminate the volume effect, and the figure was fitted with 1:1 binding mode in Kinetics analysis method, and the affinity data of antigen antibody was calculated by regression analysis software according to the binding curve. The experimental results showed that the candidate antibodies A7, B11, B22 could recognize the target antigen with high affinity (Table 3).

[0258] Table 3: Affinity determination results of antibodies A7, B11, B22

[0259] Antibody clone Ka (1 / Ms) Kd (1 / s) KD (M) A7 1.435 x 10 6 ]]> 1.23 x 10 -4 ]]> 8.6 x 10 -11 ]] B11 1.361 x 10 6 ]] 1.07 x 10 -4 ]] 7.89 x 10 -11 ]] B22 1.190 x 10 6 ]] 1.063 x 10 -4 ]]> 8.93 x 10 -11 ]]

[0260] Example 5 Inhibition of Calu-6 cell surface CD73 enzymatic activity

[0261] Calu-6 (endogenously expressing CD73, human lung adenocarcinoma cell line) was used as the cell to be tested. A7, B11, B22, IgG control (irrelevant antibody) four mAb solutions were diluted to 10 concentration gradients with 5-fold concentration gradient from the starting concentration of 30 pg / ml, with 3 replicates for each concentration. Calu-6 cells were digested and plated into U-bottom 96-well plates, 10,000 cells / well. Then the antibodies were added to the cell plate, 10 mΐ / well, and incubated on a 96-well plate shaker at 37°C for 30 min, 150 rpm / min. AMP (20 mM) and ATP (100 mM) were diluted according to the ratio of AMP:ATP:1640 medium = 1:1:8, and added to the cell plate, 10 mΐ / well, and incubated on a 96-well plate shaker at 37°C for 2 h. Centrifugation, 30 mΐ of cell culture supernatant was taken to a white-bottom 96-well plate, and then 30 mΐ of celltiter-Glo 2.0 assay (promega / G9241) was added, mixed well, and then the luminescence plate reader was used.

[0262] As shown in Figure 2 , with the increase of antibody concentration, the inhibition of the enzymatic activity of the cell surface CD73 by the antibodies of the application was enhanced. Table 4 calculates the IC 50 .

[0263] Table 4: IC of antibody to inhibition of cell surface CD73 enzymatic activity 50

[0264] Antibody μg / ml A7 B11 B22 IC 50 ]] 0.0537 0.0336 0.08017

[0265] Example 6: CD73 endocytosis activity

[0266] Calu-6 (endogenously expressing CD73, human lung adenocarcinoma cell line) was used as the cell to be tested, Alexa Fluor 488 goat anti-human IgG (H+L) (Thermo Fisher) was used as the secondary antibody, and the following flow cytometry technique was used to detect endocytosis.

[0267] B11, A7, B22 three mAbs were incubated with three groups of equal amounts of Calu-6 cells, respectively, at room temperature for 30 min, and then the different mAb cell groups were divided into three equal parts, centrifuged according to the time of 1 h, 2 h, 4 h, the antibody supernatant was discarded, the cells were washed, and the secondary antibody was added at a dilution of 1:1000 for 30 min. After washing, the fluorescence value was detected using a flow cytometer (Merk).

[0268] Figure 3The endocytosis effect of the three monoclonal antibodies on the surface of Calu-6 cells was close. With the passage of time, endocytosis increased, and endocytosis at 2h and 4h was close. Endocytosis at 4h was saturated.

[0269] Example 7: Detection of antibody molecule charge isomers

[0270] Capillary isoelectric focusing electrophoresis instrument (Protein Simple iCE3), capillary column (Protein Simple FC-coated)

[0271] Sample solution: Take an appropriate amount of system suitability sample, test sample, dilute with ultrapure water to 1 mg / ml, shake well and use.

[0272] Take an appropriate amount of blank solution (dilution ratio is the same as the test sample), sample solution according to table 5, take 150 μl into the inner tube, put the inner tube into the centrifuge tube, centrifuge at 12000 rpm for 1 min, get the blank control solution, system suitability solution and test sample solution.

[0273] Table 5: Preparation method of sample injection solution

[0274] Reagent Volume Final volume percentage 1% methylcellulose 70 μl 35％ Amphoteric electrolyte 3-10 7.0 μl 3.5％ Amphoteric electrolyte 8-10.5 3.0 μl 1.5％ Marker: pi = 7.05, pi = 9.50 0.5 μl each, 1.0 μl total 0.25% each 1 mg / ml sample solution / blank control 50 μl 25％ 40 mM arginine 69 μl 34.5％

[0275] In turn, inject 1 needle of blank control solution, 3 needles of system suitability solution, and 1 needle of test sample solution each, and finally inject 1 needle of system suitability solution. According to the conditions in table 6, operate the experiment. With the instrument self-operating software, correct and analyze the pI value and its corresponding pixel position value of two markers, and automatically calculate the corresponding pI value of the sample.

[0276] Table 6: Isoelectric focusing conditions

[0277] Method parameters State Focus Period 1 Time 1.00 min Focus Period 1 Voltage 1500V Focus Period 2 Time 9.00 min Focus Period 2 Voltage 3000V

[0278] The ratio of basic peak, main peak and acidic peak of the sample is shown in table 7, which shows that the concentration of the main peak of the antibody meets the typical antibody drug analysis results. Figure 4 The isoelectric point and purity determination spectrum of the sample are shown, which also meets the typical antibody drug analysis results.

[0279] Table 7: Ratio of acid-base peak and main peak of sample

[0280] Name Basic peak Main peak Acidic peak A7 11.86 80.89 7.25 B11 1.35 86.77 11.88 B22 50.42 43.65 5.94

[0281] Example 8: Analysis by molecular exclusion chromatography

[0282] Liquid chromatography column: TOSOH TSK-GEL G3000 SWXL 7.8*300mm 5 μm;

[0283] Chromatographic conditions: mobile phase: 0.02M sodium dihydrogen phosphate-0.2M sodium chloride buffer, pH 7.4;

[0284] The sample was diluted with ultrapure water to 5mg / ml, 20μl was injected for detection, wavelength 280nm, flow rate 0.5ml / min, room temperature constant temperature operation for 30min.

[0285] Table 8 lists the proportion of each sample measured by molecular exclusion, monomer, fragment, which meets the typical antibody drug analysis results. Figure 5 The specific SEC chromatogram is listed, which meets the typical antibody drug analysis results.

[0286] Table 8: Antibody molecular exclusion component ratio

[0287] Name Polymer Monomer Fragment A7 0.459 99.310 0.231 B11 0.075 99.842 0.084 B22 0.250 99.640 0.110

[0288] Example 9: Capillary gel electrophoresis method for determining the purity of monoclonal antibody

[0289] Instrument: capillary electrophoresis instrument Agilent Technology 7100, capillary Agilent Technology inner diameter 50μm, running buffer Beckman Coulter SDS-MW Gel Buffer-proprietary formulation, pH 8, 0.2% SDS;

[0290] Solution configuration: sample buffer (100mM Tris-HCl, pH 9.0, 1% SDS); 0.1M hydrochloric acid wash; 800mM iodoacetamide solution. Running solution preparation (Table 9).

[0291] Table 9: Running solution preparation table

[0292]

[0293] Note: Place sample bottles 1-12 in positions 1-12 of the capillary electrophoresis instrument sample tray (or positions 15-26 of the sample tray) in order.

[0294] CE-SDS reducing electrophoresis sample preparation: including test solution and blank control solution.

[0295] Take an appropriate amount of sample, add sample buffer and mercaptoethanol to dilute to 1.0mg / ml, where the volume ratio of mercaptoethanol is 5%, mix well, then 70℃ water bath for 15min; remove, cool to room temperature, centrifuge at 6000rpm / min for 1min, transfer 75μl solution to the sample bottle, ensure that no bubbles are generated, then the test solution is obtained, and the sample is replaced with sample buffer to obtain the blank control solution.

[0296] CE-SDS non-reducing electrophoresis sample preparation, i.e. test sample solution: take sample, add sample buffer and 800 mM iodoacetamide solution to dilute to 1.0 mg / ml, wherein the volume ratio of 800 mM iodoacetamide solution is 5%, mix well, then 70°C water bath for 5 min; take out, cool to room temperature, then centrifuge at 6000 rpm for 1 min, transfer 75 μl solution to the sample bottle, ensure no air bubbles are generated. Replace the sample with sample buffer to obtain the blank control solution.

[0297] The detection method is shown in Table 10.

[0298] Table 10: Summary of method parameters

[0299]

[0300] Non-reducing electrophoresis: calculate by area normalization method, report the content of immunoglobulin monomer peak, the sum of the content of low molecular weight fragment peak, and the content of HHL peak.

[0301] Reducing electrophoresis: calculate by area normalization method, report the content of light chain, the content of heavy chain, and the sum of the content of immunoglobulin heavy chain and light chain.

[0302] The results are shown in Table 11: the content of complete antibody in A7 is 82% in non-reducing CE-SDS, the content of complete antibody in B11 is 85%, which are close to each other, and the content of fragment HHL is 14-18%; the content of HC is 64% in reducing CE-SDS.

[0303] Table 11: CE-SDS results table

[0304]

[0305] Sequence Listing

[0306] A7

[0307]

[0308]

[0309] B11

[0310]

[0311] B22

[0312]

[0313]

[0314] A7

[0315]

[0316]

[0317] B11

[0318]

[0319]

[0320] B22

[0321]

[0322]

Claims

1. An anti-CD73 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region of the anti-CD73 antibody or antigen-binding fragment thereof comprises: a H CDR1 having the amino acid sequence of SEQ ID NO: 1, a H CDR2 having the amino acid sequence of SEQ ID NO: 2, and a H CDR3 having the amino acid sequence of SEQ ID NO: 3; the light chain variable region of the anti-CD73 antibody or antigen-binding fragment thereof comprises: a L CDR1 having the amino acid sequence of SEQ ID NO: 5, a L CDR2 having the amino acid sequence of SEQ ID NO: 6, and a L CDR3 having the amino acid sequence of SEQ ID NO: 7; or the heavy chain variable region of the anti-CD73 antibody or antigen-binding fragment thereof comprises: a H CDR1 having the amino acid sequence of SEQ ID NO: 17, a H CDR2 having the amino acid sequence of SEQ ID NO: 18, and a H CDR3 having the amino acid sequence of SEQ ID NO: 19; the light chain variable region of the anti-CD73 antibody or antigen-binding fragment thereof comprises: a L CDR1 having the amino acid sequence of SEQ ID NO: 21, a L CDR2 having the amino acid sequence of SEQ ID NO: 22, and a L CDR3 having the amino acid sequence of SEQ ID NO:

23.

2. The anti-CD73 antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the heavy chain variable region has at least 85% sequence identity to SEQ ID NO: 4; the amino acid sequence of the light chain variable region has at least 85% sequence identity to SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region has at least 85% sequence identity to SEQ ID NO: 20; the amino acid sequence of the light chain variable region has at least 85% sequence identity to SEQ ID NO:

24.

3. The anti-CD73 antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 4; the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 20; the amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO:

24.

4. The anti-CD73 antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 4; the amino acid sequence of the light chain variable region has at least 95% sequence identity to SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 20; the amino acid sequence of the light chain variable region has at least 95% sequence identity to SEQ ID NO:

24. the amino acid sequence of the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 20; and the amino acid sequence of the light chain variable region has at least 95% sequence identity to SEQ ID NO:

24.

5. The anti-CD73 antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20; and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

24.

6. The anti-CD73 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, the anti-CD73 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

7. The anti-CD73 antibody or antigen-binding fragment thereof of claim 6, wherein, the amino acid sequence of the heavy chain constant region has at least 85% sequence identity to SEQ ID NO: 26; and, the amino acid sequence of the light chain constant region has at least 85% sequence identity to SEQ ID NO:

25.

8. The anti-CD73 antibody or antigen-binding fragment thereof of claim 6, wherein, the amino acid sequence of the heavy chain constant region has at least 90% sequence identity to SEQ ID NO: 26; and, the amino acid sequence of the light chain constant region has at least 90% sequence identity to SEQ ID NO:

25.

9. The anti-CD73 antibody or antigen-binding fragment thereof of claim 6, wherein, the amino acid sequence of the heavy chain constant region has at least 95% sequence identity to SEQ ID NO: 26; and, the amino acid sequence of the light chain constant region has at least 95% sequence identity to SEQ ID NO:

25.

10. The anti-CD73 antibody or antigen-binding fragment thereof of claim 6, wherein, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 26; and, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:

25.

11. A nucleic acid molecule comprising a nucleotide sequence region encoding the anti-CD73 antibody or antigen-binding fragment thereof of any one of claims 1-10.

12. The nucleic acid molecule of claim 11, wherein, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:

51.

13. The nucleic acid molecule of claim 12, wherein, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO: 32 and SEQ ID NO: 36, or SEQ ID NO: 48 and 52.

14. An expression vector comprising the nucleic acid molecule of any one of claims 11-13.

15. A host cell comprising the nucleic acid molecule of any one of claims 11-13 or the expression vector of claim 14.

16. A pharmaceutical composition comprising the anti-CD73 antibody or antigen-binding fragment thereof of any one of claims 1-10 and a pharmaceutically acceptable carrier.

17. A method of making an anti-CD73 antibody or antigen binding fragment thereof of any one of claims 1-10, comprising: culturing the host cell of claim 15, and isolating the antibody from the host cell.

18. Use of an anti-CD73 antibody or antigen binding fragment thereof according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment of a neoplastic disease, wherein, The neoplastic disease is a CD73-positive tumor, which is lung cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, esophageal cancer, nasopharyngeal cancer, pancreatic cancer, colon cancer, rectal cancer, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, head and neck cancer, melanoma, myeloma, gastric cancer, thyroid cancer, endometrial cancer or glioma.

19. The use according to claim 18, wherein the lymphoma is Hodgkin's disease or non-Hodgkin's lymphoma.

20. The use according to claim 18, wherein the head and neck cancer is laryngeal cancer.

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