Anti-human CD73 antibody and its applications

Abstract

The present invention provides a novel humanized anti-human CD73 antibody that can specifically bind to the CD73 antigen and has higher affinity and biological activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to anti-human CD73 antibodies and their applications.

Background Art

[0002] CD73, also known as cluster of differentiation 73 and 5'-nucleotide, is anchored to the cell membrane via GPI (glycosylphosphatidylinositol), and its structure is a dimer. CD73 plays an important role in the tumor microenvironment. It can convert AMP to adenosine, which binds to specific G protein-coupled receptors, leading to abnormal infiltration of immune cells at the tumor site, regulating the growth and metastasis of various cancers, mainly inhibiting the functions of the immune system related to T cells, and indicating poor prognosis of tumors.

[0003] A number of past studies have shown that inhibiting CD73 can prevent the occurrence and metastasis of tumors. Whether it is a small molecule enzyme inhibitor of CD73 or a high molecular antibody of CD73, it shows an antitumor effect. CD73 is expressed on the surfaces of various tumor cells, including tumor cells such as lung cancer, melanoma, colon cancer, and pancreatic cancer. In addition, various immune cells are also affected by CD73. CD73 is expressed on the surface of Tregs and participates in the inhibitory regulation of Treg immune activation. The secreted adenosine binds to adenosine receptors on DC cells, inhibiting the functions of DCs. CD73 is highly expressed on M2 macrophages and enhances tumor inheritance.

[0004] The CD73 target holds great potential in tumor treatment, and antibodies against CD73 can further enhance the effects of PD1 antibodies and CTLA4 antibodies. However, currently, CD73-specific target antibodies do not exist in the market. Furthermore, mouse monoclonal antibodies have limitations in clinical treatment because they may induce a human anti-mouse antibody (HAMA) reaction during clinical treatment. Antibody humanization technology can significantly reduce the immunogenicity of mouse monoclonal antibodies. Therefore, considering the role and function of CD73 in various related diseases, there is a need in the art to develop anti-CD73 humanized antibodies suitable for the treatment of patients.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of the present invention is to provide an anti-human CD73 antibody having high affinity and high biological activity and its application.

Means for Solving the Problems

[0006] The first aspect of the present invention provides a heavy chain variable region of an anti-CD73 antibody, and the heavy chain variable region has (1) VH-CDR1 shown in SEQ ID NO.: 15 or 21, (2) VH-CDR2 shown in SEQ ID NO.: 16, and (3) a VH-CDR selected from the group consisting of VH-CDR3 independently selected from the group consisting of SEQ ID NO.: 17, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27.

[0007] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 2.

[0008] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 5.

[0009] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 7.

[0010] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 9.

[0011] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 34.

[0012] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 36.

[0013] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 37.

[0014] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 38.

[0015] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 39.

[0016] In another preferred example, any amino acid sequence in the above amino acid sequence further includes an induced sequence that can retain CD73 binding affinity and is optionally accompanied by addition, deletion, modification, and / or substitution of at least one amino acid.

[0017] The second aspect of the present invention provides a heavy chain of an anti-CD73 antibody, and the heavy chain has the heavy chain variable region described in the first aspect of the present invention.

[0018] In another preferred example, the heavy chain of the antibody further includes a heavy chain constant region.

[0019] In another preferred example, the heavy chain constant region is derived from human, mouse or rabbit, and preferably, it is derived from human.

[0020] In another preferred example, the heavy chain constant region is the human antibody heavy chain IgG1 constant region.

[0021] The third aspect of the present invention provides a light chain variable region of an anti-CD73 antibody, and the light chain variable region (1) VL-CDR1 shown in SEQ ID NO.:18, (2) VL-CDR2 shown in SEQ ID NO.:19, and (3) a VL-CDR selected from the group consisting of VL-CDR3s independently selected from the group consisting of SEQ ID NO.:20, SEQ ID NO.:28, SEQ ID NO.:29, SEQ ID NO.:30, SEQ ID NO.:31, SEQ ID NO.:32, SEQ ID NO:33.

[0022] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:1.

[0023] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:6.

[0024] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:8.

[0025] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:10.

[0026] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:40.

[0027] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 41.

[0028] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 42.

[0029] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 43.

[0030] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 44.

[0031] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 45.

[0032] In another preferred example, any amino acid sequence in the above amino acid sequence further includes an induced sequence capable of retaining CD73 binding affinity, optionally with at least one amino acid addition, deletion, modification, and / or substitution.

[0033] The fourth aspect of the present invention provides a light chain of an anti-CD73 antibody, and the light chain has the light chain variable region described in the third aspect of the present invention.

[0034] In another preferred example, the light chain of the antibody further includes a light chain constant region.

[0035] In another preferred example, the light chain constant region is derived from human, mouse or rabbit, preferably from human.

[0036] In another preferred example, the light chain constant region is a human antibody light chain kappa constant region.

[0037] The fifth aspect of the present invention provides an anti-CD73 antibody, and the antibody is (1) The heavy chain variable region described in the first aspect of the present invention, and / or (2) having the light chain variable region described in the third aspect of the present invention, or alternatively, the antibody has the heavy chain described in the second aspect of the present invention and / or the light chain described in the fourth aspect of the present invention, wherein any amino acid sequence in the above amino acid sequence further includes an induced sequence capable of retaining CD73 binding affinity, which optionally involves addition, deletion, modification, and / or substitution of at least one amino acid.

[0038] In another preferred example, the number of amino acids added, deleted, modified, and / or substituted is 1 to 5 (for example, 1 to 3, preferably 1 to 2, more preferably 1).

[0039] In another preferred example, the antibody is a humanized antibody.

[0040] In another preferred example, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

[0041] In another preferred example, the antibody is a double-stranded antibody or a single-stranded antibody.

[0042] In another preferred example, the antibody is a monoclonal antibody.

[0043] In another preferred example, the antibody includes a monospecific, bispecific, or trispecific antibody.

[0044] In another preferred example, the heavy chain variable region of the antibody includes a human-derived framework region and / or the light chain variable region of the antibody includes a human-derived framework region.

[0045] In another preferred example, the heavy chain variable region of the antibody includes a mouse-derived framework region and / or the light chain variable region of the antibody includes a mouse-derived framework region.

[0046] In another preferred example, the antibody comprises the heavy chain variable region described in the first aspect of the present invention and the light chain variable region described in the third aspect of the present invention, wherein the heavy chain variable region (1) VH-CDR1 shown in SEQ ID NO.: 15 or 21, (2) VH-CDR2 shown in SEQ ID NO.: 16, and (3) a VH-CDR selected from the group consisting of VH-CDR3 independently selected from the group consisting of SEQ ID NO.: 17, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26 or SEQ ID NO.: 27, and the light chain variable region (1) VL-CDR1 shown in SEQ ID NO.: 18, (2) VL-CDR2 shown in SEQ ID NO.: 19, and (3) a VL-CDR selected from the group consisting of VL-CDR3 independently selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 28, SEQ ID NO.: 29, SEQ ID NO.: 30, SEQ ID NO.: 31, SEQ ID NO.: 32 or SEQ ID NO: 33.

[0047] In another preferred example, the antibody comprises the heavy chain variable region described in the first aspect of the present invention and the light chain variable region described in the third aspect of the present invention, where the three light chain CDRs of the light chain variable region and the three heavy chain CDRs of the heavy chain variable region (Z1) VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 20, and VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 17, (Z2) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 20, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 21, 16, 17 (Z3) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 28, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 22 (Z4) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 29, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 23 (Z5) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 30, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 24 (Z6) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 31, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 25 (Z7) The VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 32, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 26, or (Z8) It is selected from the group consisting of the VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO: 18, 19, 33, and the VH-CDR1, VH-CDR2 and VH-CDR3 described in SEQ ID NO: 15, 16, 27. In another preferred example, the amino acid sequence of the light chain variable region (VL) of the anti-human CD73 antibody is as shown in SEQ ID NO: 1, 6, 8, 10, 40, 41, 42, 43, 44 or 45, and the amino acid sequence of the heavy chain variable region (VH) of the anti-human CD73 antibody is as shown in SEQ ID NO: 2, 5, 7, 9, 34, 35, 36, 37, 38 or 39.

[0048] In another preferred example, the antibody is selected from the group consisting of the following.

Table 0

[0049] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 2, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 1.

[0050] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 5, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 6.

[0051] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 7, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 8.

[0052] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 9, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 10.

[0053] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 34, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 40.

[0054] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 35, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 41.

[0055] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 36, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 42.

[0056] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 37, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 43.

[0057] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 38, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 44.

[0058] In another preferred example, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 39, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO.: 45.

[0059] In another preferred example, any amino acid sequence in the above amino acid sequence further includes an induced sequence in which at least one (for example, 1 to 3, preferably 1 to 2, more preferably 1) amino acid is optionally added, deleted, modified, and / or substituted and the CD73 binding affinity can be maintained.

[0060] In another preferred example, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO.: 2, 5, 7, 9, 34, 35, 36, 37, 38 or 39 in the sequence listing.

[0061] In another preferred example, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO.: 1, 6, 8, 10, 40, 41, 42, 43, 44 or 45 in the sequence listing.

[0062] In another preferred example, the anti-CD73 antibody comprises a light chain and a heavy chain, and the light chain variable region of the light chain is VL-CDR1 shown in SEQ ID NO.: 18, VL-CDR2 shown in SEQ ID NO.: 19, and three light chain CDRs of VL-CDR3 shown in SEQ ID NO.: 20, 28, 29, 30, 31, 32 or 33, wherein the heavy chain variable region of the heavy chain is VH-CDR1 shown in SEQ ID NO.: 15 or 21, VH-CDR2 shown in SEQ ID NO.: 16, and three heavy chain CDRs of VH-CDR3 shown in SEQ ID NO.: 17, 22, 23, 24, 25, 26 or 27.

[0063] In another preferred example, the light chain of the antibody comprises the three light chain CDRs and a light chain framework region for linking the light chain CDRs, and the heavy chain of the antibody comprises the three heavy chain CDRs and a heavy chain framework region for linking the heavy chain CDRs.

[0064] In another preferred example, the antibody is a humanized antibody.

[0065] In another preferred example, the antibody specifically binds to CD73.

[0066] In another preferred example, the KD value (M) of the affinity of the antibody for human CD73 is from 1.0E-10 to 2.0E-12.

[0067] In another preferred example, the antibody is a diabody or a single-chain antibody.

[0068] In another preferred example, the antibody is a monoclonal antibody.

[0069] In another preferred example, the antibody comprises a monospecific, bispecific, or trispecific antibody.

[0070] The sixth aspect of the present invention provides a recombinant protein, wherein the recombinant protein (i) the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, and (ii) optionally a tag sequence that aids in expression and / or purification.

[0071] In another preferred example, the tag sequence comprises a 6His tag.

[0072] In another preferred example, the recombinant protein (or polypeptide) comprises a fusion protein.

[0073] In another preferred example, the recombinant protein is a monomer, dimer, or multimer.

[0074] In another preferred example, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

[0075] The seventh aspect of the present invention provides an antibody preparation, which (a) an antibody according to the fifth aspect of the present invention, and (b) a vector containing a buffer, sterile water, and an optional surfactant.

[0076] The eighth aspect of the present invention provides a kit, which includes the antibody preparation according to the seventh aspect of the present invention and a container containing the antibody preparation.

[0077] The ninth aspect of the present invention provides a CAR construct. The scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to CD73, and the scFv segment has the heavy-chain variable region according to the first aspect of the present invention and the light-chain variable region according to the third aspect of the present invention. The tenth aspect of the present invention provides a recombinant immune cell, which expresses the exogenous CAR construct according to the fifth aspect of the present invention.

[0078] In another preferred example, the immune cell is selected from the group consisting of NK cells and T cells.

[0079] In another preferred example, the immune cell is derived from a human or non-human mammal (e.g., a mouse).

[0080] The eleventh aspect of the present invention provides an antibody-drug conjugate, which (a) an antibody portion selected from the group consisting of the heavy-chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light-chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or a combination thereof, and (b) a coupling portion coupled to the antibody portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0081] The twelfth aspect of the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody according to the fifth aspect of the present invention, or the recombinant protein according to the sixth aspect of the present invention, the CAR construct according to the seventh aspect of the present invention, the immune cell according to the tenth aspect of the present invention, the antibody-drug conjugate according to the eleventh aspect of the present invention, or a combination thereof, and the active ingredient is (a) for the preparation of a detection reagent or kit, and (b) for the preparation of a drug or formulation for preventing and / or treating CD73-related diseases, and / or (c) for the preparation of a drug or formulation for preventing and / or treating cancer or tumor related to CD73-related diseases.

[0082] In another preferred example, the cancer or tumor is selected from the group consisting of lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

[0083] The thirteenth aspect of the present invention provides a pharmaceutical composition, which comprises (i) an active ingredient selected from the group consisting of the antibody according to the fifth aspect of the present invention, or the recombinant protein according to the sixth aspect of the present invention, the CAR construct according to the seventh aspect of the present invention, the immune cell according to the tenth aspect of the present invention, the antibody-drug conjugate according to the eleventh aspect of the present invention, or a combination thereof, and (ii) a pharmaceutically acceptable carrier.

[0084] In another preferred example, the pharmaceutical composition is a liquid formulation.

[0085] In another preferred example, the pharmaceutical composition is an injection.

[0086] In another preferred example, the pharmaceutical composition is used for the treatment of tumors.

[0087] In another preferred example, the tumor is a tumor that highly expresses CD73.

[0088] The 14th aspect of the present invention provides a polynucleotide, and the polynucleotide code is (1) The heavy chain variable region described in the 1st aspect of the present invention, the heavy chain described in the 2nd aspect of the present invention, the light chain variable region described in the 3rd aspect of the present invention, the light chain described in the 4th aspect of the present invention, or the antibody described in the 5th aspect of the present invention, or (2) The recombinant protein described in the 6th aspect of the present invention, (3) Selected from the group of polypeptides such as the CAR construct described in the 9th aspect of the present invention.

[0089] The 15th aspect of the present invention provides a vector, and the vector contains the polynucleotide described in the 14th aspect of the present invention.

[0090] In another preferred example, the vector includes a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, such as a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

[0091] The 16th aspect of the present invention provides a genetically engineered host cell, and the host cell contains the vector described in the 15th aspect of the present invention or has the polynucleotide described in the 14th aspect of the present invention integrated into its genome.

[0092] The 17th aspect of the present invention provides a method for non-diagnostically (including diagnostically or non-diagnostically) detecting CD73 protein in a sample in vitro, and the method includes (1) In vitro, contacting the sample with the antibody described in the 5th aspect of the present invention, and (2) Detecting whether an antigen-antibody conjugate is formed, where the formation of the conjugate indicates the presence of CD73 protein in the sample.

[0093] The 18th aspect of the present invention provides a method for in vitro detection of CD73 protein in a (diagnostic or non-diagnostic) sample, the method comprising: (1) contacting the sample with the antibody according to the 5th aspect of the present invention in vitro; (2) detecting whether an antigen-antibody conjugate is formed, wherein the formation of the conjugate indicates the presence of CD73 protein in the sample.

[0094] The 19th aspect of the present invention provides a method for treating CD73-related diseases, the method comprising: administering to a subject in need thereof the antibody according to the 5th aspect of the present invention, a drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof.

Advantages of the Invention

[0095] It should be understood that within the scope of the present invention, new or preferred technical solutions can be constituted by combining each of the above technical features of the present invention with each technical feature specifically described below (for example, in the examples). Due to space limitations, it will not be repeated here.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0097] After extensive and detailed research and a large amount of screening, the inventors unexpectedly obtained anti-CD73 humanized antibodies with excellent affinity. Specifically, the present invention utilizes phage display technology to obtain highly specific CD73 antibodies by screening and humanizes them while considering similarity and human usage frequency. Through binding experiments and affinity detection of the humanized antibodies, the antibodies specifically bind to human and monkey CD73 proteins and CD73-positive cells and have the effect of inhibiting CD73 protease activity. The antibodies of the present invention can also induce the internalization of CD73 in tumor cells. In mouse experiments, the antibodies of the present invention exhibit antitumor activity superior to that of AstraZeneca (AZ) / Medimmune's MEDI-9447. Based on this, the present invention was completed.

[0098] Terms To more readily understand the present invention, certain technical and chemical terms are specifically defined below. Unless explicitly defined herein, all other technical and chemical terms used herein have the meanings commonly understood by those skilled in the art to which the present invention pertains.

[0099] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0100] As used herein, the terms "administer" and "treat" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or body fluid. "Administer" and "treat" can refer to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of a cell includes contact of a reagent with the cell, contact of a reagent with a liquid, and contact of a liquid with the cell. "Administer" and "treat" also mean in vitro and ex vivo treatment with a reagent, diagnostic, binding composition, or another cell. When applied to a human, animal, or research subject, "treat" refers to therapeutic treatment, prophylactic or preventive measures, research, and diagnosis, and includes contact of an anti-CD73 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0101] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent to a patient, including any anti-CD73 antibody and compositions thereof of the present invention, wherein the patient has one or more disease symptoms, and the known therapeutic agent has a therapeutic effect on these symptoms. Usually, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) that alleviates one or more disease symptoms.

[0102] As used herein, the term "optionally" or "optionally" means that the events or situations described below may occur, but need not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be present, but need not necessarily be present, and may be 1, 2, or 3.

[0103] "Sequence identity" as described in the present invention refers to the degree of identity between two nucleic acid or two amino acid sequences when compared to an optimal one under circumstances having mutations such as appropriate exchanges, insertions or deletions. The sequence identity between the sequences described in the present invention and the sequences having the same identity therewith can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0104] Antibody As used herein, the term "antibody" refers to an immunoglobulin having a tetrameric chain structure of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Since the amino acid composition and the order of arrangement of the immunoglobulin heavy chain constant regions are different, their antigenicity is also different. Therefore, immunoglobulins can be classified into five categories: IgM, IgD, IgG, IgA, and IgE, or different isotypes of immunoglobulins. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, and due to differences in chemical structure and biological function, it can be classified into four subclasses: IgG1, IgG2, IgG3, and IgG4. The light chain is classified into a κ chain or a λ chain due to differences in the constant region. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those skilled in the art.

[0105] The approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of an antibody are quite different and are variable regions (V regions), while the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C regions). The variable region contains three hypervariable regions (HVRs) and four FR regions (FRs) with relatively conserved sequences. The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining regions (CDRs). Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) is composed of three CDR regions and four FR regions (framework regions), and the order from the amino terminus to the carboxyl terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain, namely the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment described in the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the Kabat and Chothia numbering rules (HCDR1). The four FR regions in the variable regions of natural heavy and light chains generally show a β-folding structure and are connected by three CDRs that form connecting loops, and in some cases, can also form a partial β-folding structure. The CDRs of each chain are closely bound through the FR regions and together with the CDRs of another chain form the antigen-binding site of the antibody. By comparing the amino acid sequences of antibodies of the same type, the amino acids constituting the FR region or CDR region can be determined. The constant region does not directly participate in the binding of the antibody to the antigen, but shows different effector functions such as its involvement in antibody-dependent cell cytotoxicity.

[0106] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but does not contain the constant region, and has the smallest antibody fragment of all antigen-binding sites. Generally, an Fv antibody contains a polypeptide linker between the VH domain and the VL domain and can form a structure necessary for antigen binding.

[0107] As used herein, the term "epitope" refers to a three-dimensional spatial site that is not continuous on the antigen and is recognized by the antibody or antigen-binding fragment of the present invention.

[0108] The present invention includes not only complete antibodies but also antibody fragments having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention further includes fragments, derivatives, and analogs of the above antibodies.

[0109] In the present invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies such as chimeric and humanized monoclonal antibodies containing human and non-human parts can be prepared using DNA recombination techniques well known in the art.

[0110] As used herein, the term "monoclonal antibody" refers to an antibody secreted from clones derived from a single cell. Monoclonal antibodies are highly specific and target a single antigen epitope. The cells can be eukaryotic cell lines, prokaryotic cell lines, or phage clone cell lines.

[0111] As used herein, the term "chimeric antibody" refers to an antibody molecule that is expressed by splicing the V region gene of a mouse-derived antibody and the C region gene of a human antibody into a chimeric gene, inserting it into a vector, and transfecting it with a host cell. It not only retains the high specificity and affinity of the parental mouse antibody but also enables its human Fc segment to effectively mediate biological effector functions. As used herein, the term "humanized antibody" refers to a modified form of the variable region of a murine antibody of the invention that has CDR regions derived from (or substantially derived from) a non-human antibody (preferably, a murine monoclonal antibody), as well as FR regions and constant regions substantially derived from human antibody sequences, i.e., the CDR region sequences of the murine antibody are transplanted into various types of human germline antibody framework sequences. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic certain natural antibody properties can be expressed by constructing expression vectors.

[0112] In the present invention, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0113] In the present invention, the antibodies of the present invention further include conservative variants thereof, which refers to the formation of a polypeptide in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are substituted by similar or analogous amino acids as compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably generated by substitution with the amino acids according to Table A. [Table A]

[0114] Anti-CD73 antibody As used herein, the term "CD73" generally refers to native or recombinant human CD73, as well as non-human homologs of human CD73.

[0115] The present invention provides an antibody having high specificity and high affinity for CD73, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable region (VH), and the light chain comprises the amino acid sequence of the light chain variable region (VL).

[0116] In the present invention, a high-quality human CD73 antigen is selected to immunize mice, immune cells of the mice are collected to construct a phage library, and by using a special phage display technology, single-chain antibodies (scfv) are displayed on the phage surface. Through multiple CD73 antigen screenings, an antibody sequence is obtained, and through recombinant construction, it is constructed into a eukaryotic expression vector of the hIgG1 framework, so as to be expressed in mammalian cells to obtain an anti-CD73 full-length antibody (that is, to obtain a human-mouse chimeric antibody).

[0117] In a preferred embodiment of the present invention, the obtained human-mouse chimeric anti-CD73 full-length antibody is the CQ137 antibody protein. The obtained CQ137 antibody binds to the tumor-expressed CD73 molecule and inhibits the CD73 activity against human ovarian cancer cells. It has the potential to treat various tumors overexpressing CD73.

[0118] Preferably, the CDRs of the heavy chain variable region (VH) are VH-CDR1 shown in SEQ ID NO.:15, VH-CDR2 shown in SEQ ID NO.:16, and selected from the group consisting of VH-CDR3 shown in SEQ ID NO.:17, and / or the CDRs of the light chain variable region (VL) are VL-CDR1 shown in SEQ ID NO.:18, VL-CDR2 shown in SEQ ID NO.:19, and selected from the group consisting of VL-CDR3 shown in SEQ ID NO.:20.

[0119] Here, any amino acid sequence in the above amino acid sequence further includes an induced sequence having CD73 binding affinity through the addition, deletion, modification and / or substitution of at least one (for example, 1 to 5, 1 to 3, preferably 1 to 2, more preferably 1) amino acid.

[0120] In another preferred example, the sequence formed by adding, deleting, modifying and / or substituting the at least one amino acid sequence preferably has an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology.

[0121] The antibody of the present invention may be a double-stranded antibody or a single-stranded antibody, and may be an antibody selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0122] The antibody derivative described in the present invention can be, for example, Fab, Fab’, (Fab’)2 or other antibody derivatives known in the art, as well as single-stranded antibodies such as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies, and / or antibody fragments. Here, the animal is preferably a mammal such as a mouse.

[0123] The antibody of the present invention can be a mouse antibody, a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody that targets human CD73.

[0124] In a preferred embodiment of the present invention, the sequence having CD73 binding affinity by any one or more of the sequences of SEQ ID NO.:15, SEQ ID NO.:16 and SEQ ID NO.:17, or by addition, deletion, modification and / or substitution of at least one amino acid thereof is located in the CDR region of the heavy chain variable region (VH).

[0125] In a preferred embodiment of the present invention, the sequence having CD73 binding affinity by any one or more of the sequences of SEQ ID NO.:18, SEQ ID NO.:19 and SEQ ID NO.:20, or by addition, deletion, modification and / or substitution of at least one amino acid thereof is located in the CDR region of the light chain variable region (VL).

[0126] In a more preferred embodiment of the present invention, VH CDR1, CDR2, and CDR3 are each independently selected from any one or more of the sequences of SEQ ID NO.:15, SEQ ID NO.:16, and SEQ ID NO.:17, or a sequence having CD73 binding affinity by addition, deletion, modification, and / or substitution of at least one amino acid thereof; VL CDR1, CDR2, and CDR3 are each independently selected from any one or more of the sequences of SEQ ID NO.:18, SEQ ID NO.:19, and SEQ ID NO.:20, or a sequence having CD73 binding affinity by addition, deletion, modification, and / or substitution of at least one amino acid thereof.

[0127] In the above content of the present invention, the number of amino acids added, deleted, modified, and / or substituted is preferably less than 40% of the total number of amino acids in the initial amino acid sequence, more preferably less than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and most preferably 15 to 20%. In the present invention, the number of amino acids added, deleted, modified, and / or substituted is usually 1, 2, 3, 4, or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

[0128] In a preferred embodiment of the present invention, an antibody against human CD73 (CQ137) is provided. CQ137 light chain variable region (SEQ ID NO.:1): DILMTQSPSSMYASLGERVTITCKAS QDINTY LTWFQQKPGKSPKTLIY RAN ILVDGVPSRFSGSGSGQDYSLTISSLEFEDMGIYYC LQYDEFPLT FGAGTKLELKR CQ137 heavy chain variable region (SEQ ID NO.:2): EVQLQQSGPELVKPGASVKISCKAS GYSFTGYYIHWVKQSHVKSLEWIGR INPYNGAT TYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYC ARFHYGAPDY WGQGTTLTVSS hIgG1 constant region amino acid sequence (SEQ ID NO.: 3): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK kappa chain constant region amino acid sequence (SEQ ID NO.: 4): TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The present invention provides a nucleotide sequence encoding the above amino acids. CQ137 VH nucleotide sequence (SEQ ID NO.: 11): GAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATCCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGGTGCTACTACCTACAACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATTCCACTACGGTGCCCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG CQ137 VL nucleotide sequence (SEQ ID NO.:12): GATATTCTGATGACCCAATCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATACCTATTTAACTTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACATATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTTTGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGT The selected hIgG1 constant region nucleotide sequence is (SEQ ID NO.:13). GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA The selected kappa chain constant region nucleotide sequence is (SEQ ID NO.:14). ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0129] Humanized anti-CD73 antibody In 1986, Jones et al. first transplanted mouse monoclonal antibody heavy chain CDRs into the human antibody heavy chain framework region and then assembled them with the light chain of the mouse monoclonal antibody to form a complete antibody and retain the same affinity as the original mouse monoclonal antibody, providing an idea for the development of antibody humanization technology. In 1989, Queen et al. successfully constructed a humanized anti-CD25 antibody by the CDR transplantation method, in which the human antibody Eu framework region was used for humanization and mouse antibody amino acids were retained at some sites of the framework region to maintain affinity. In 1992, Presta et al. reported a method that was successful in humanized construction by CDR transplantation using the consensus sequence of the human antibody subgroup as a template. In 1994, Pedersen et al. reported a method using surface resurfacing for antibody humanization. In 1994, Hsiao et al. reported a humanization method of CDR transplantation using the framework region of the human antibody Germline sequence. In 1994, Jespers et al. successfully constructed a humanization method using the phage library shuffling library method.

[0130] The present invention provides a humanized antibody having high specificity and high affinity for CD73.

[0131] In another preferred example, the sequence further comprises a sequence formed by addition, deletion, modification and / or substitution of at least one amino acid sequence, preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% homology.

[0132] The antibody of the present invention can be a double-stranded antibody or a single-stranded antibody, and preferably can be a fully humanized antibody.

[0133] The antibody derivative described in the present invention is a single-stranded antibody and / or antibody fragments such as Fab, Fab', (Fab') 2 etc., or other antibody derivatives known in the art, and can be any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0134] The antibody of the present invention can be a humanized antibody targeting CD73, a CDR-grafted and / or modified antibody.

[0135] In the above content of the present invention, the number of amino acids added, deleted, modified and / or substituted is preferably 40% or less, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids of the initial amino acid sequence.

[0136] Preparation of Antibodies Any method suitable for generating monoclonal antibodies can be used for the generation of the CD73 antibodies of the present invention. For example, animals can be immunized using a linked or naturally occurring CD73 protein or a fragment thereof. Appropriate immunization methods including adjuvants, immunostimulants, and repeated booster immunizations can be used, and one or more routes can be employed.

[0137] Any suitable form of CD73 can be used as an immunogen (antigen) for generating non-human antibodies specific for CD73 and screening for the biological activity of said antibodies. The immunogen can be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen can be generated from a natural source or from genetically modified cells. The DNA encoding the immunogen can be of genomic or non-genomic origin (e.g., cDNA). Appropriate gene vectors can be used to express the DNA encoding the immunogen, and said vectors include, but are not limited to, adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0138] The humanized antibody can be selected from any class of immunoglobulins including IgM, IgD, IgG, IgA, and IgE. Similarly, any type of light chain can be used in the compounds and methods herein. Specifically, a κ chain, a λ chain, or variants thereof can be useful in the compounds and methods of the present invention.

[0139] An exemplary method for preparing the CD73 antibodies of the present invention is described in Example 1.

[0140] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained using conventional techniques such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused to form a single-chain antibody.

[0141] Once the relevant array is obtained, the recombinant method can be used to obtain a large amount of the relevant array. Usually, this is achieved by cloning it into a vector, then transforming cells, and then isolating the relevant array from the host cells grown by conventional methods.

[0142] Furthermore, especially when the length of the fragment is relatively short, the relevant array can also be synthesized by the artificial synthesis method. Usually, a very long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them. Next, the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art.

[0143] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked". For example, when a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

[0144] The term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is ligated. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA ring to which additional DNA segments can be ligated.

[0145] The present invention further relates to vectors containing the appropriate DNA sequence and the appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so as to be able to express proteins.

[0146] The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be a prokaryotic cell such as a bacterial cell, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a plant cell or an animal cell (for example, a mammalian cell).

[0147] The step of transforming a host cell with the recombinant DNA described in the present invention can be carried out using techniques well-known in the art. The obtained transformant can be cultured according to conventional methods, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.

[0148] Generally, the obtained host cells can be cultured and transformed under conditions suitable for the expression of the antibody of the present invention. Next, the antibody of the present invention is generated and obtained using conventional separation and purification means well-known to those skilled in the art, such as conventional immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

[0149] The obtained monoclonal antibody can be identified by conventional means. For example, the binding specificity of the monoclonal antibody can be measured using immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0150] Antibody-drug conjugate (ADC) The present invention further provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0151] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, and the antibody is coupled to the effector molecule, preferably by chemical coupling. Here, the effector molecule is preferably a drug having therapeutic activity. Further, the effector molecule can be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug or a radionuclide.

[0152] The antibody of the present invention can be coupled to the effector molecule via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that covalently bonds an effector molecule and an antibody, such as glutaraldehyde. The coupling agent using a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (for example, cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0153] Specific residues on the antibody (such as Cys or Lys, etc.) are used to bind to various functional groups, where imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as glycol polymers), and therapeutic agents are included. The antibody can be coupled to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is coupled (covalently bonded) to the antibody. The functional agent can be directly or indirectly bound to the antibody via a linker.

[0154] Antibodies can form antibody-drug conjugates (ADCs) by being coupled to drugs. Typically, an ADC contains a linker positioned between the drug and the antibody. The linker can be a cleavable or non-cleavable linker. Cleavable linkers are usually susceptible to cleavage in the intracellular environment, for example, at the site intended to cleave the linker, thereby releasing the drug from the antibody. Suitable cleavable linkers include, for example, enzymatically cleavable linkers containing peptidyl-containing linkers that can be cleaved by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be cleaved by glucuronidase. The peptidyl linker can contain, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable cleavable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that can be cleaved under reducing conditions (such as disulfide bond linkers). Non-cleavable linkers usually release the drug under conditions where the antibody is hydrolyzed by proteases.

[0155] Before binding to the antibody, the linker has reactive groups that are active and can react with specific amino acid residues, and the binding is achieved via the reactive groups. Sulfhydryl-specific reactive groups are preferred and include maleimide compounds, halogenated amides (such as iodinated, brominated or chlorinated), haloesters (such as iodinated, brominated or chlorinated), halomethyl ketones (such as iodinated, brominated or chlorinated), halogenated benzyls (such as iodinated, brominated or chlorinated), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3,6-bis-(mercury methyl) dioxane where the counter ion is acetate, chloride ion or nitrate, and polymethylene dimethyl sulfide thiosulfonate. The linker can contain, for example, maleimide bound to the antibody via thiosuccinimide.

[0156] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In embodiments, the linker binds to the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a keto group capable of forming a bond with the linker. When the drug is directly bound to the linker, the drug has a reactive active group before being bound to the antibody.

[0157] Classes of useful drugs include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, the drug-linker can form an ADC in one simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step method. For example, a cysteine residue is reacted with the reactive moiety of the linker in the first step, and then in a subsequent step, the functional group on the linker is reacted with the drug to form an ADC.

[0158] Typically, the functional groups on the linker are selected to facilitate a specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group of the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates, and activated esters such as N-hydroxysuccinimidyl esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, as described in “Bioconjugation Techniques”, Second Edition (Elsevier), are well known to those skilled in the art. It should be understood by those skilled in the art that for the selective reactions of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.

[0159] Antibody formulation Antibodies have different stabilities in various formulation buffers, which are manifested as changes in charge heterogeneity, degradation and aggregation of antibody molecules, etc. Such quality changes are related to the physical and chemical properties of the antibody itself. Therefore, in the development process of antibody drugs, it is necessary to screen a formulation buffer suitable for the antibody according to the physical and chemical properties of different antibodies. Currently, commonly used antibody formulation buffer systems include phosphate buffer, citrate buffer, histidine buffer, etc. At the same time, according to the properties of the antibody, various concentrations of salt ions or excipients such as sorbitol, trehalose, sucrose, etc., as well as an appropriate amount of surfactants such as Tween20 or Tween80, are added to maintain the stability of the antibody.

[0160] The antibody-drug conjugate formulation of the present invention can effectively inhibit side reactions such as aggregation, precipitation, hydrolysis, oxidation, and deamidation of the humanized antibody of the present invention, and at the same time, can effectively improve the stability of the product under pressurization (such as high temperature, strong light irradiation, and freeze-thaw), acceleration, and long-term refrigeration conditions.

[0161] Pharmaceutical composition The present invention further provides a composition. In a preferred example, the composition is a pharmaceutical composition comprising the above antibody or its active fragment or its fusion protein or its ADC or the corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Usually, these substances can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous vector medium, where the pH is usually about 5 to 8, preferably about 6 to 8, and the pH value varies depending on the nature of the substance being formulated and the disease condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.

[0162] The antibody described in the present invention can also be expressed intracellularly by a nucleotide sequence used for cell therapy. For example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0163] The pharmaceutical composition of the present invention can be directly used for binding to the CD73 protein molecule, and thus can be used for the prevention and treatment of CD73-related diseases. Furthermore, other therapeutic agents can also be used simultaneously.

[0164] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to, physiological saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation needs to be consistent with the administration method. The pharmaceutical composition of the present invention can be prepared in the form of an injection by a conventional method using, for example, an aqueous solution containing physiological saline or glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions need to be prepared under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0165] When using the pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, where the safe and effective amount is usually at least about 10 μg / kg body weight and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dosage is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage needs to consider factors such as the administration route and the health status of the patient, and these are all within the skill range of a skilled physician.

[0166] Detection uses and kits The antibody of the present invention can be used for detection applications, for example, the detection of samples for providing diagnostic information.

[0167] In the present invention, the sample (samples) used includes cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present invention should include any type of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.

[0168] The samples used in the present invention include fixed or preserved cell or tissue samples.

[0169] The present invention further provides a kit containing the antibody (or a fragment thereof) of the present invention. In a preferred example of the present invention, the kit further includes a container, an instruction manual, a buffer, etc. In a preferred example, the antibody of the present invention can be immobilized on a detection plate.

[0170] The main advantages of the present invention are as follows. (1) The humanized antibody of the present invention has better safety compared with mouse-derived antibodies and chimeric antibodies. (2) The antibody of the present invention has higher affinity by competitive screening. (3) The antibody of the present invention is derived from phage display technology and is more diverse than hybridoma technology.

[0171] Hereinafter, the present invention will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without indicating detailed conditions usually follow conventional conditions such as those described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0172] Example 1. Preparation of anti-human CD73 monoclonal antibody In this example, the acquisition of anti-human CD73 single-chain antibody sequences mainly by mouse immunization and phage display will be described. Balb / C mice were immunized with recombinant human CD73 protein (C446, Novoprotein). After the first immunization, a total of 4 booster immunizations were performed every 14 days, and mouse sera were collected to evaluate antibody titers. B cells were collected from effectively evaluated mice, RNA was extracted and reverse transcribed to construct a phage display library. Recombinant human CD73 protein was plated at 1 - 5 μg / ml, the displayed phages were added, and the phage library was screened. After collecting the phages that were not washed after screening, they were infected into host bacteria to obtain the first screening library, and the second and third screenings were performed according to this method. After the screening was completed, those identified by phage-ELISA and identified as positive were sequenced to obtain Anti-CD73 candidate sequences. CQ137 VH and VL were constructed into eukaryotic expression vectors containing the hIgG1-kappa constant region respectively, freestyle 293F cells were transfected, the supernatant was collected after culturing for 3 - 7 days, and purified by a protein A column to obtain CQ137 antibody protein. CQ137 light chain variable region (SEQ ID NO.: 1) DILMTQSPSSMYASLGERVTITCKAS QDINTY LTWFQQKPGKSPKTLIY RAN ILVDGVPSRFSGSGSGQDYSLTISSLEFEDMGIYYC LQYDEFPLT FGAGTKLELKR CQ137 heavy chain variable region (SEQ ID NO.: 2) EVQLQQSGPELVKPGASVKISCKAS GYSFTGYY IHWVKQSHVKSLEWIGR INPYNGAT TYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYC ARFHYGAPDY WGQGTTLTVSS hIgG1 constant region amino acid sequence (SEQ ID NO.: 3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK kappa constant region amino acid sequence (SEQ ID NO.: 4) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0173] Example 2. Detection of the affinity of CQ137 antibody In this example, the detection of the affinity between CQ137 and human and monkey recombinant CD73 proteins, as well as the detection of the affinity between human CD73-positive cells (SK-OV-3, human ovarian cancer cells) and the monkey CD73 cell line (CHOK1-cynoCD73), will be described.

[0174] (1) Affinity for human CD73 protein Using Biacore, the affinity between CQ137 and recombinant hCD73 protein was measured. CQ137 was immobilized, and the CD73 protein was diluted with a two-fold gradient (2.5 nM to 40 nM). The obtained affinity diagram is as shown in Figure 1.

[0175] The affinity data is as shown in Table 1. [Table 1] The results show that the affinity of the measured CQ137 for rhCD73 is 8.653×10 -11 M.

[0176] (2) Affinity for cynomolgus CD73 protein Recombinant cynomolgus CD73 protein was plated at 3 μg / ml, gradient-diluted CQ137 was added, and the binding of CQ137 to recombinant cynomolgus CD73 was detected using the ELISA method. The results are shown in Figure 2. The results show that the EC 50 calculated for the binding of CQ137 to recombinant cynomolgus CD73 is 0.217 μg / ml.

[0177] (3) Binding to human CD73-positive cells 4×10 5 cells of SK-OV-3 were taken, CQ137 protein diluted in a triple gradient (0.009 - 20 μg / ml) was added, incubated for 1 hour, washed 3 times with PBS, Anti-hFC-APC (purchased from Jackson immunology) was added, and flow cytometry detection was performed. The resulting S curve is shown in Figure 3.

[0178] According to the results, the EC 50 for the binding of CQ137 to SK-OV-3 cells is 0.5883 ng / ml, and the EC 50 for the binding of MEDI-9447 to SK-OV-3 cells is 9.627 ng / ml. Therefore, the binding of CQ137 to SK-OV-3 cells is superior to that of MEDI-9447.

[0179] (4) Binding to cynomolgus CD73-positive cells A eukaryotic expression vector containing full-length cynomolgus CD73 and NEO resistance was constructed, transfected into CHO-K1 cells, and after screening with G418, the CHOK1-cynoCD73 cell line was obtained. 4×10 5Individual CHOK1-cynoCD73 cells were collected, gradient-diluted CQ137 protein was added, and after incubation for 1 hour, they were washed three times with PBS, and Anti-hFC-APC (purchased from Jackson immunology) was added and detected by flow cytometry. The results show that the EC of CQ137 50 = 0.09991 ng / ml, and the EC of MEDI-9447 50 = 4.119 ng / ml. Therefore, the binding of CQ137 to CHOK1-CynoCD73 cells is superior to that of MEDI-9447.

[0180] Example 3. Detection of CD73 enzyme activity mediated by CQ137 In this example, it mainly shows that CQ137 inhibits CD73 protein and cell enzyme activity. Specifically, (1) Inhibition of CD73 protease activity The CD73 protein was diluted to a working concentration of 5 μg / ml, CD73 antibody diluted in a three-fold gradient (0.001 - 10 μg / ml) was added, incubated at 37°C for 15 minutes, a mixture of 1 mM ATP and AMP was added, incubated at 37°C for 30 minutes, an equal volume of CellTiter-Glo detection reagent (purchased from promega) was added, and the chemiluminescence value was read with a microplate reader. When calculating with the enzyme activity without adding the antibody as 100%, the change in rhCD73 activity is shown in Figure 5. The results show that in the inhibition of CD73 protease activity, CQ137 is superior to the control molecule MEDI-9447.

[0181] (2) Inhibition of CD73 enzyme activity in human SK-OV-3 cells 5×10 4Take individual SK-OV-3 cells, add the antibody diluted in a three-fold gradient (0.009 - 20 μg / ml), incubate at 37 °C for 15 minutes, add 1 mM of AMP and incubate at 37 °C for 2 hours, add 1 mM of ATP, immediately add the CellTiter-Glo detection reagent, read the luminescence value with a microplate reader, calculate with the enzyme activity without adding the antibody as 100%, and the change in CD73 activity on the obtained cells is shown in Figure 6. The results show that CQ137 can inhibit the CD73 enzyme activity on SK-OV-3 cells, and the IC 50 of CQ137 is 0.3708 μg / ml.

[0182] (3) Inhibition of CD73 enzyme activity in CHOK1-cynoCD73 cells Take 5×10 4 individual CHOK1-cynoCD73 cells, add the antibody diluted in a three-fold gradient (0.003 - 20 μg / ml), incubate at 37 °C for 15 minutes, add 1 mM of AMP and incubate at 37 °C for 2 hours, add 1 mM of ATP, immediately add the CellTiter-Glo detection reagent, read the luminescence value with a microplate reader, calculate with the enzyme activity without adding the antibody as 100%, and the change in CD73 activity on the obtained cells is shown in Figure 7. The results show that CQ137 can inhibit the CD73 enzyme activity on CHOK1-cynoCD73 cells, and the IC 50 of CQ137 is 0.9899 μg / ml.

[0183] Example 4. Humanization of CQ137 Using structural simulation and rational design, analyze the human framework closest to CQ137 to obtain a series of humanized antibodies. Construct the VH and VL of the humanized sequences into vectors containing the hIgG1 and kappa light chain constant regions respectively, transfect into Freestyle 293F cells, collect the supernatant, and obtain the desired antibody protein after purification by a protein A column.

[0184] The sequences of the nine humanized CQ137 monoclonal antibodies obtained (137-1, 137-2, 137-3, 137-4, 137-5, 137-6, 137-7, 137-8, 137-9) are as follows: The heavy chain VH amino acid sequence (SEQ ID NO.:5) of the humanized CQ137 monoclonal antibody (137-1) is as follows. QVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYC ARFHYGAPDY WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.:6) of the humanized CQ137 monoclonal antibody (137-1) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWFQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYC LQYDEFPLT FGGGTKVEIKR The heavy chain VH amino acid sequence (SEQ ID NO.:7) of the humanized CQ137 monoclonal antibody (137-2) is as follows. QVQLVQSGAEVKKPGASVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYC ARFHYGAPDY WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.:8) of the humanized CQ137 monoclonal antibody (137-2) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWFQQKPGKAPKSLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDEFPLT FGGGTKVEIKR The VH amino acid sequence (SEQ ID NO.:9) of the heavy chain of the humanized CQ137 monoclonal antibody (137-3) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAPDY WGQGTLVTVSS The VL amino acid sequence (SEQ ID NO.:10) of the light chain of the humanized CQ137 monoclonal antibody (137-3) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDEFPLT FGGGTKVEIKR The VH amino acid sequence (SEQ ID NO.:34) of the heavy chain of the humanized CQ137 monoclonal antibody (137-4) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAVDY WGQGTLVTVSS The VL amino acid sequence (SEQ ID NO.:40) of the light chain of the humanized CQ137 monoclonal antibody (137-4) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYEEFPLT FGGGTKVEIKR The VH amino acid sequence (SEQ ID NO.:35) of the heavy chain of the humanized CQ137 monoclonal antibody (137-5) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGRINPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAADY WGQGTLVTVSS The light chain VL amino acid sequence of the humanized CQ137 monoclonal antibody (137-5) is as follows. (SEQ ID NO.:41) DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYNEFPLT FGGGTKVEIKR The heavy chain VH amino acid sequence (SEQ ID NO.:36) of the humanized CQ137 monoclonal antibody (137-6) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAPEY WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.:42) of the humanized CQ137 monoclonal antibody (137-6) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYAEFPLT FGGGTKVEIKR The heavy chain VH amino acid sequence (SEQ ID NO.:37) of the humanized CQ137 monoclonal antibody (137-7) is as follows. QVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYYIHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAPNY WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.: 43) of the humanized CQ137 monoclonal antibody (137-7) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDDFPLT FGGGTKVEIKR The heavy chain VH amino acid sequence (SEQ ID NO.: 38) of the humanized CQ137 monoclonal antibody (137-8) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAPDF WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.: 44) of the humanized CQ137 monoclonal antibody (137-8) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIY RAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDQFPLT FGGGTKVEIKR The heavy chain VH amino acid sequence (SEQ ID NO.: 39) of the humanized CQ137 monoclonal antibody (137-9) is as follows. QVQLVQSGAEVKKPGSSVKVSCKAS GYSFTGYY IHWVRQAPGQGLEWMGR INPYNGAT TYNQNFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARFHYGAPDL WGQGTLVTVSS The light chain VL amino acid sequence (SEQ ID NO.: 45) of the humanized CQ137 monoclonal antibody (137-9) is as follows. DIQMTQSPSSLSASVGDRVTITCKAS QDINTY LTWYQQKPGKAPKTLIYRAN ILVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQYDAFPLT FGGGTKVEIKR

[0185] Here, the underlined parts are CDRs (defined by IMGT), and 137-1, 137-2, 137-3, 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 are the protein numbers of nine humanized CQ137 antibodies respectively. The third amino acid (Thr) of the heavy chain VH-CDR1:GYTFTGYY (SEQ ID NO.:21) of the humanized antibody 137-1 is different from the third amino acid (Ser) of the heavy chain VH-CDR1:GYSFTGYY (SEQ ID NO.:15) of the CQ137 antibody. The CDR regions of the humanized antibodies 137-2 and 137-3 are the same as those of the CQ137 antibody. The FR regions of the humanized antibodies are replaced based on the CQ137 antibody. A part of the CDRs of the humanized antibodies is replaced based on the CQ137 antibody.

[0186] Example 5. Affinity evaluation after CQ137 humanization In this example, mainly the affinity of the humanized CD73 antibody with recombinant human and monkey CD73 proteins respectively, as well as the affinity situation with CD73-positive cells SK-OV-3 cells and CHOK1-cynoCD73 cells are shown. Specifically, (1) Binding with recombinant human CD73 protein The recombinant human CD73 protein was plated at 3 μg / ml, and serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3) were added, and the binding situation between the antibody and the recombinant human CD73 was detected by ELISA method, and the results are shown in Figure 8A.

[0187] The results are as shown in Table 2. [Table 2]

[0188] The results regarding the binding of humanized antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to recombinant human CD73 are as shown in Table 3 and Figure 15. [Table 3]

[0189] (2) Binding to recombinant monkey CD73 protein Recombinant monkey CD73 protein was plated at 3 μg / ml, and serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3) were added. The binding of the antibody to recombinant monkey CD73 was detected by ELISA, and the results are shown in Figure 8B.

[0190] The results are as shown in Figure 8B and Table 4. [Table 4]

[0191] The binding of humanized antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to recombinant monkey CD73 protein is as shown in Table 5 and Figure 16. [Table 5]

[0192] (3) Binding to human CD73-positive cells 4×10 5 cells of SK-OV-3 were taken, serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3) were added, incubated for 1 hour, washed 3 times with PBS, Anti-hFC-APC (purchased from Jackson immunology) was added, and detected by flow cytometry. The S curve of the results is shown in Figure 9.

[0193] The results are as shown in Table 6 and Figure 9. [Table 6]

[0194] The results show that the binding of the humanized CD73 antibodies (137-1, 137-2, 137-3) to SK-OV-3 cells is superior to that of MEDI-9447. The results regarding the binding of the humanized antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to SK-OV-3 cells are as shown in Table 7 and Figure 17.

Table 7

[0195] (4) Binding to cynomolgus CD73-positive cells A eukaryotic expression vector containing full-length cynomolgus CD73 and NEO resistance was constructed, CHO-K1 cells were transfected, and after screening with G418, the CHOK1-cynoCD73 cell line was obtained. 4×10 5 individual CHOK1-cynoCD73 cells were taken, and serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3) were taken, incubated for 1 hour, then washed three times with PBS, Anti-hFC-APC (purchased from Jackson immunology) was added, and detected by flow cytometry. The resulting S curve is shown in Figure 10.

[0196] The results are as shown in Table 8.

Table 8

[0197] The results show that the humanized CD73 antibodies have high binding activity to cynomolgus CD73-positive cells. The results regarding the binding of the humanized antibodies 137-4, 137-5, 137-6, 137-7, 137-8, 137-9 to cynomolgus CD73-positive cells are as shown in Table 9 and Figure 18.

Table 9

[0198] Example 6. Evaluation of the inhibition of enzyme activity after CQ137 humanization In this example, it is mainly shown that the humanized 137 antibody inhibits CD73 protein and cellular enzyme activity. Specifically, (1) Inhibition of CD73 protease activity Dilute CD73 protein to a working concentration of 5 μg / ml, add serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3), incubate at 37°C for 15 minutes, add a mixture of 1 mM ATP and AMP, incubate at 37°C for 30 minutes, add an equal volume of Cell Titer-Glo detection reagent (purchased from Promega), read the luminescence value with a microplate reader, calculate the enzyme activity without adding the antibody as 1, and obtain the change diagram of rhCD73 activity in Figure 11.

[0199] The results in Figure 19 show that all six humanized CD73 antibodies can inhibit the enzyme activity of recombinant human CD73.

[0200] (2) Inhibition of CD73 enzyme activity in human SK-OV-3 cells Take 5×10 4 individual SK-OV-3 cells, add serially diluted humanized CD73 antibodies (137-1, 137-2, 137-3, 137-4, 137-5, 137-6, 137-7, 137-8, 137-9), incubate at 37°C for 15 minutes, add 1 mM AMP and incubate at 37°C for 2 hours, add 1 mM ATP, immediately add CellTiter-Glo detection reagent, read the luminescence value with a microplate reader, calculate the enzyme activity without adding the antibody as 1, and the change in CD73 activity on the obtained cells, k, is as shown in Figures 12 and 20.

[0201] The experimental results show that nine humanized 137 antibodies can inhibit CD73 protein and CD73 enzyme activity on the cells.

[0202] Example 7. Cell Internalization Mediated by Humanized 137 Antibody 3×10 5 Take 3×10 5 SK-OV-3 cells, add 0.2 μg of CD73 antibody, and incubate at 37 °C for 0, 1, 2, 3 hours and overnight. After the incubation, add an equal amount of the corresponding humanized CD73 antibody (137-1, 137-2, 137-3, 137-4, 137-5, 137-6, 137-7, 137-8, 137-9). After 1 hour of incubation, add Anti-Hfc-APC flow cytometry antibody. After incubation and elution, perform flow cytometry detection. The results calculated with the relative fluorescence intensity (MFI) at 0 hour as 1 are shown in Figures 13 and 21. The experimental results show that the nine humanized CD73 antibodies of the present invention can also induce the internalization of CD73 in tumor cells.

[0203] Example 8. Humanized 137 Antibody that Inhibits the Proliferation of Human Melanoma Cell A375 in Mice Using NSG mice, construct a mouse model NSG-A375 of human melanoma cells (A375), prepare a preparation inoculated with human PBMC and a control mouse not inoculated with human PBMC, administer the drug every other day, and measure the tumor volume. For the experimental mice, set a blank control of solvent PBS, a low dose of MEDI-9447 (the low dose is 0.5 mpk), a high dose (3 mpk), and a low dose of 137-2, a high dose, a medium dose, and a low dose (the medium dose is 1 mpk) of 137-3. The experimental results are shown in Figure 14. Here, mpk (Milligrams Per Kilograms) is mg / kg. The results show that at low doses, the tumor inhibitory effects of 137-2 and 137-3 are significantly higher than that of MEDI-9447.

[0204] Sequence Information of the Present Invention The CDRs of CQ137 and its humanized antibodies are summarized in Table 10 below. [Table 10]

[0205] The sequence information of the present invention is as shown in the following table.

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Table 11-7

[0206] All documents mentioned in the present invention are incorporated by reference in this application as if each document was individually cited as a reference. Further, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms are also included in the scope defined by the appended claims of this application.

Claims

1. An anti-human CD73 antibody, wherein the antibody comprises a light chain and a heavy chain, and the three light chain CDRs of the light chain variable region and the three heavy chain CDRs of the heavy chain variable region are (Z1) the group consisting of VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NOs: 18, 19 and 20, and VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NOs: 15, 16 and 17, (Z2) the group consisting of VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NOs: 18, 19 and 20, and VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NOs: 21, 16 and 17, (Z3) the group consisting of VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NOs: 18, 19 and 28, and VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NOs: 15, 16 and 22, (Z4) the group consisting of VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NOs: 18, 19 and 30, and VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NOs: 15, 16 and 24, or (Z5) the group consisting of VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NOs: 18, 19 and 32, and VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NOs: 15, 16 and 26, and the anti-human CD73 antibody is characterized by being selected from the above.

2. The amino acid sequence of the light chain variable region (VL) of the anti-human CD73 antibody is shown in SEQ ID NO: 1, 6, 8, 10, 40, 42 or 44, and the amino acid sequence of the heavy chain variable region (VH) of the anti-human CD73 antibody is shown in SEQ ID NO: 2, 5, 7, 9, 34, 36 or 38. The antibody according to claim 1, characterized in that.

3. A recombinant protein, wherein the recombinant protein is (i) a light chain and a heavy chain, or an anti-CD73 antibody formed by the light chain and the heavy chain, wherein the three light chain CDRs of the light chain variable region and the three heavy chain CDRs of the heavy chain variable region are The group consisting of VL - CDR1, VL - CDR2, and VL - CDR3 shown in SEQ ID NOs: 18, 19, and 20, and VH - CDR1, VH - CDR2, and VH - CDR3 shown in SEQ ID NOs: 15, 16, and 17, The group consisting of VL - CDR1, VL - CDR2, and VL - CDR3 shown in SEQ ID NOs: 18, 19, and 20, and VH - CDR1, VH - CDR2, and VH - CDR3 shown in SEQ ID NOs: 21, 16, and 17, The group consisting of VL - CDR1, VL - CDR2, and VL - CDR3 shown in SEQ ID NOs: 18, 19, and 28, and VH - CDR1, VH - CDR2, and VH - CDR3 shown in SEQ ID NOs: 15, 16, and 22, The group consisting of VL - CDR1, VL - CDR2, and VL - CDR3 shown in SEQ ID NOs: 18, 19, and 30, and VH - CDR1, VH - CDR2, and VH - CDR3 shown in SEQ ID NOs: 15, 16, and 24, or The group consisting of VL - CDR1, VL - CDR2, and VL - CDR3 shown in SEQ ID NOs: 18, 19, and 32, and VH - CDR1, VH - CDR2, and VH - CDR3 shown in SEQ ID NOs: 15, 16, and 26, selected from, and (ii) a tag sequence that optionally assists in expression and / or purification, characterized in that the recombinant protein has the above.

4. An antibody preparation, wherein the antibody preparation, (a) the antibody according to claim 1, and (b) a buffer, sterile water, and a vector containing an optional surfactant, characterized in that the antibody preparation contains the above.

5. A kit, wherein the kit contains the antibody preparation according to claim 4 and a container containing the antibody preparation, characterized in that the kit has the above.

6. A CAR construct, wherein the scFv segment of the antigen - binding region of the CAR construct is a binding region that specifically binds to CD73, and the scFv segment has a light - chain variable region and a heavy - chain variable region. Here, the three light - chain CDRs of the light - chain variable region and the three heavy - chain CDRs of the heavy - chain variable region are, The group consisting of VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NOs: 18, 19, and 20, and VH-CDR1, VH-CDR2, and VH-CDR3 shown in SEQ ID NOs: 15, 16, and 17, The group consisting of VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NOs: 18, 19, and 20, and VH-CDR1, VH-CDR2, and VH-CDR3 shown in SEQ ID NOs: 21, 16, and 17, The group consisting of VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NOs: 18, 19, and 28, and VH-CDR1, VH-CDR2, and VH-CDR3 shown in SEQ ID NOs: 15, 16, and 22, The group consisting of VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NOs: 18, 19, and 30, and VH-CDR1, VH-CDR2, and VH-CDR3 shown in SEQ ID NOs: 15, 16, and 24, or The group consisting of VL-CDR1, VL-CDR2, and VL-CDR3 shown in SEQ ID NOs: 18, 19, and 32, and VH-CDR1, VH-CDR2, and VH-CDR3 shown in SEQ ID NOs: 15, 16, and 26, The CAR construct, characterized in that it is selected from the above. Claim 7 A recombinant immune cell, wherein the immune cell expresses the exogenous CAR construct according to claim 6. The recombinant immune cell. Claim 8 An antibody-drug conjugate, wherein the antibody-drug conjugate, (a) an antibody moiety selected from the group consisting of the antibody according to claim 1, or the recombinant protein according to claim 3, or a combination thereof, and (b) a coupling moiety coupled to the antibody moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof. The antibody-drug conjugate. Use of an active ingredient for preparing the following (a) to (c), (a) a detection reagent or kit, (b) a drug or formulation for preventing and / or treating CD73-related diseases, and / or (c) A drug or preparation for preventing and / or treating cancer or tumor associated with CD73-related diseases, wherein the active ingredient is selected from the group consisting of the antibody according to claim 1, the recombinant protein according to claim 3, the CAR construct according to claim 6, the immune cell according to claim 7, the antibody-drug conjugate according to claim 8, or a combination thereof, and the use of the active ingredient is characterized in that.

10. A pharmaceutical composition, wherein the pharmaceutical composition, (i) an active ingredient selected from the group consisting of the antibody according to claim 1, the recombinant protein according to claim 3, the CAR construct according to claim 6, the immune cell according to claim 7, the antibody-drug conjugate according to claim 8, or a combination thereof, and (ii) a pharmaceutically acceptable vector, and the pharmaceutical composition is characterized in that it contains the above.

11. A polynucleotide, wherein the polynucleotide code, (1) the antibody according to claim 1, or (2) the recombinant protein according to claim 3, (3) a polypeptide selected from the group consisting of the CAR construct according to claim 6, and the polynucleotide is characterized in that it encodes the polypeptide.

12. A vector, wherein the vector contains the polynucleotide according to claim 11, and the vector is characterized in that.

13. A genetically engineered host cell, wherein the host cell contains the vector according to claim 12, or the polynucleotide according to claim 11 is integrated into the genome, and the genetically engineered host cell is characterized in that.

14. A method for non-diagnostically detecting CD73 protein in a sample in vitro, wherein the method, (1) contacting the sample with the antibody according to claim 1 in vitro, and (2) detecting whether an antigen-antibody conjugate is formed, and the formation of the conjugate indicates the presence of CD73 protein in the sample, and the method for non-diagnostically detecting CD73 protein in the sample in vitro is characterized in that.

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