Monoclonal antibodies and antigen binding fragments thereof for suppressing CD73 immune checkpoint and uses thereof

Abstract

To provide monoclonal antibodies and antigen binding fragments thereof for suppressing a CD73 immune checkpoint, and uses thereof.SOLUTION: Provided are antibodies or antigen binding fragments thereof for suppressing a CD73 immune checkpoint, having a specific sequence, wherein the antibodies or antigen binding fragments thereof can bind to CD73, reduce enzymatic activity of CD73 protein, and inhibit cancer metastasis or cancer growth, thus enabling the uses of the antibodies or antigen binding fragments thereof as therapeutic agents for cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a continuation of Korean Application No. 10-2020-0043 filed on April 9, 2020. No. 607, and Korean Application No. 10-2020-01 filed on November 2, 2020 No. 44595, which is hereby incorporated by reference in its entirety, is hereby incorporated by reference in its entirety. The contents of which are incorporated herein by reference in their entirety.

[0002] [Reference to electronically submitted sequence listing] The ASCII text file submitted with this application (name: 2662-0003WO0 1-SequenceListing_ST25.txt; Size: 48KB; and Created on: April 7th, The contents of the electronically submitted sequence listing in the 2021 issue of this application are incorporated herein by reference in their entirety. .

[0003] The present invention relates to a monoclonal antibody for inhibiting the CD73 immune checkpoint and and antigen-binding fragments thereof, as well as uses thereof. [Background technology]

[0004] Traditional cancer treatment methods include radiation therapy, chemotherapy, and anticancer drug therapy. The method can treat cancer by directly inhibiting the growth of the cancer. Cancer cells become resistant to anticancer drugs, and the use of anticancer drugs also attacks other cells that are not cancer cells. In order to overcome such problems, new cancer treatment methods are being developed. In response, immunotherapy has been developed that targets immune cells rather than cancer cells, It induces the cells to attack cancer cells.

[0005] Among immunotherapies, immune checkpoint blockade has recently become Immune checkpoints are a system that promotes immune responses. Immune checkpoints are receptors that regulate or inhibit immune responses. The immune checkpoint is essential for the development of cancer. In cancer, the immune checkpoint is also activated, and cancer is thought to be caused by the immune checkpoint. Recently, the immune checkpoint has been used to treat cancer Many studies have been reported to treat CTLA-4 (cytotoxic T-lyase inhibitor). mphocyte-associated antigen 4) and PD-1 (programmed cell death protein 1) Antibodies against immune checkpoints have been developed and shown to increase survival rates in melanoma cancer patients Blockade of immune checkpoints, CTLA-4 and PD-1, has been used in a variety of cancers. However, in some patients, CTLA-4 and P Treatment with immune checkpoint blockade of D-1 has not been shown to be effective. Many studies have been conducted on new immune checkpoints, and mechanisms of immune evasion in cancer have been identified. One of these is known to be due to high levels of the immunosuppressant adenosine.

[0006] "CD73 (Cluster of Differentiation 73)" is a 5' nucleotide deoxyribonuclease An ecto-nucleotidase that catalyzes the phosphorylation of AMP (adenosine monophosphate). CD73 converts GPI-acetylglucosamine (GPI) to adenosine. It exists in the form of homodimer on the cell membrane via an anchor, and The monomer is 65 kDa, and the N- and C-terminal domains are flexible helices. In other words, the CD73 is linked via an adenosine triphosphate (ATO)-binding protein. It is involved in the production of phosphoinositide, is overexpressed in cancer cells, and induces immunosuppressive effects. There is a need to develop new immune checkpoint blockers that focus on controlling adenosine levels. do. Summary of the Invention [Means for solving the problem]

[0007] The present specification relates to a heavy chain phase comprising the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 14 or 17. Complementarity determining domain 1 (CDRH1), amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15 or 18 CDRH2 containing the amino acid sequence of SEQ ID NO: 3, 6, 9, 12, 13, 16 or 19 a heavy chain variable region comprising CDRH3 comprising the amino acid sequence of SEQ ID NO: 20, 23, 26 or light chain complementarity determining domain 1 (CDRL1), comprising a 29 amino acid sequence, SEQ ID NO:21, CDRL2 comprising the amino acid sequence of 24, 27 or 30, and SEQ ID NOs: 22, 25, 28 or a monoclonal antibody comprising a light chain variable region comprising a CDRL3 comprising a sequence of 31 amino acids. An antibody or antigen-binding fragment thereof is disclosed.

[0008] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:1. CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; CDRL1 comprising the amino acid sequence of SEQ ID NO:21, CDRL2 comprising the amino acid sequence of SEQ ID NO:22, and the sequence a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO:22; CDRH1 comprising the amino acid sequence of SEQ ID NO:5, CDRH2 comprising the amino acid sequence of SEQ ID NO:6, a heavy chain variable region comprising CDRH3 comprising the amino acid sequence of SEQ ID NO:23; CDRL1 comprising the amino acid sequence of SEQ ID NO:24, and CDRL2 comprising the amino acid sequence of SEQ ID NO:25. a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:7; CDRH1 comprising the amino acid sequence of SEQ ID NO:8, and CDRH2 comprising the amino acid sequence of SEQ ID NO:9. a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:26; 1. CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and the amino acid sequence of SEQ ID NO:28 a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 10; CDRH2 comprising the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12 or 13 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:20; RL1, CDRL2 comprising the amino acid sequence of SEQ ID NO:21, and CDRL3 comprising the amino acid sequence of SEQ ID NO:22. a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO:14; 15, CDRH2 comprising the amino acid sequence of SEQ ID NO: 16, and a heavy chain variable region of CDRH3 comprising the amino acid sequence of SEQ ID NO:20; CDRL2 comprising the amino acid sequence of SEQ ID NO:21, and C comprising the amino acid sequence of SEQ ID NO:22. a light chain variable region comprising DRL3; or a CDRH1 comprising the amino acid sequence of SEQ ID NO:17 18, CDRH2 comprising the amino acid sequence of SEQ ID NO: 19, a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:29; and a CDRL1 having the amino acid sequence of SEQ ID NO:29. , CDRL2 comprising the amino acid sequence of SEQ ID NO: 30, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 31. The light chain variable region may comprise a light chain variable region comprising a CDRL3 that includes

[0009] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 32, 33, 34, ,35,36,37, or 38, at least 80%, 85%, or 90% , 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 1 a heavy chain variable region comprising amino acids having 0.000% identity to SEQ ID NOs: 39, 40, 41, or or 42, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96 % or more, 97% or more, 98% or more, 99% or more, or 100% identity In one embodiment, the antibody or its derivatives may comprise a light chain variable region comprising The original binding fragment has the amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38. a heavy chain variable region comprising SEQ ID NO: 39, 40, 41 or 42; and a light chain variable region comprising SEQ ID NO: 39, 40, 41 or 42. It may include a variable region.

[0010] Also, CDRH1 comprising the amino acid sequence of SEQ ID NO: 66, 69, 72, 75 or 78; CDRH2 comprising the amino acid sequence of SEQ ID NO: 67, 70, 73, 76 or 79; A heavy chain variable region comprising CDRH3 containing an amino acid sequence of 68, 71, 74, 77 or 80 and CDRL1 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90 or 93, CDRL2 comprising the amino acid sequence of SEQ ID NO: 82, 85, 88, 91 or 94, and SEQ ID NO: A light chain variable region comprising CDRL3 containing the amino acid sequence of No. 83, 86, 89, 90 or 95. The present invention provides a monoclonal antibody or antigen-binding fragment thereof comprising a region thereof.

[0011] In one embodiment, the antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:66. CDRH1 comprising the amino acid sequence of SEQ ID NO:67, CDRH2 comprising the amino acid sequence of SEQ ID NO:68 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO: 81; CDRL1 comprising the amino acid sequence of SEQ ID NO:82, CDRL2 comprising the amino acid sequence of SEQ ID NO:83 a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:69; CDRH1 containing the amino acid sequence of SEQ ID NO:70, CDRH2 containing the amino acid sequence of SEQ ID NO:71, a heavy chain variable region comprising CDRH3 comprising the amino acid sequence of SEQ ID NO: 84; CDRL1 comprising the amino acid sequence of SEQ ID NO:85, CDRL2 comprising the amino acid sequence of SEQ ID NO:86, a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 72; RH1, CDRH2 comprising the amino acid sequence of SEQ ID NO:73, and CDRH3 comprising the amino acid sequence of SEQ ID NO:74. a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO: 87; RL1, CDRL2 comprising the amino acid sequence of SEQ ID NO:88, and CDRL3 comprising the amino acid sequence of SEQ ID NO:89. a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 75; 76, CDRH2 comprising the amino acid sequence of SEQ ID NO: 77, a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:90; and a CDRL1 having the amino acid sequence of SEQ ID NO:90. 91, CDRL2 comprising the amino acid sequence of SEQ ID NO: 92, a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:78; H1, CDRH2 comprising the amino acid sequence of SEQ ID NO:79, and the amino acid sequence of SEQ ID NO:80 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:93; L1, CDRL2 comprising the amino acid sequence of SEQ ID NO:94, and the amino acid sequence of SEQ ID NO:95 and a light chain variable region comprising a CDRL3 comprising the antibody or antigen-binding fragment thereof. It's possible.

[0012] Other aspects include nucleic acids encoding the antibodies or antigen-binding fragments thereof, and expression vectors comprising the nucleic acids. A vector and a cell transformed with the expression vector are provided.

[0013] The present disclosure relates to at least SEQ ID NOs: 32, 33, 34, 35, 36, 37, or 38. All are 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, and 97% or more. %, 98%, 99% or 100% identity. Nucleic acids encoding the variable regions, and sequences that are 80% or more similar to SEQ ID NO: 39, 40, 41, or 42 , 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, and encoding a light chain variable region having amino acids with 99% or more or 100% identity The present specification also provides nucleic acids represented by SEQ ID NOs: 32, 33, 34, 35, 36, 37, and 39. or a nucleic acid encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39,4 Nucleic acids encoding light chain variable regions comprising SEQ ID NO: 0, 41 or 42 are provided.

[0014] The present specification describes a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 32 and a light chain variable region of SEQ ID NO: 39. a nucleic acid encoding the variable region, or a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 33; and Nucleic acids encoding the light chain variable region of SEQ ID NO: 40 are provided. For example, a heavy chain variable region of SEQ ID NO: 32 The nucleic acid encoding the variable region can be represented by SEQ ID NO: 43, and the light chain of SEQ ID NO: 39. The nucleic acid encoding the variable region may be represented by SEQ ID NO: 44. A nucleic acid encoding the heavy chain variable region of SEQ ID NO: 32 or SEQ ID NO: 33, and a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 39 or Nucleic acids encoding the light chain variable region of SEQ ID NO: 40 are also provided. The light chain variable region of SEQ ID NO: 40 is represented by SEQ ID NO: 45, and the light chain variable region of SEQ ID NO: 40 is represented by SEQ ID NO: 46. Ugh.

[0015] Also, at least 80% or more of SEQ ID NO: 96, 98, 100, 102 or 104 Above, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more %, 99% or more, or 100% identity with the heavy chain variable region. and at least one nucleic acid sequence corresponding to SEQ ID NO: 97, 99, 101, 103 or 105. 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more a light chain variable region having an amino acid identity of 98% or more, 99% or more, or 100% Nucleic acids encoding the region are also provided. 4, and a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 97, 99, 101, 103, or Nucleic acids encoding the light chain variable regions of 105 are provided.

[0016] A nucleic acid encoding the heavy chain variable region of SEQ ID NO: 96 and a nucleic acid encoding the light chain variable region of SEQ ID NO: 97 a nucleic acid encoding the heavy chain variable region of SEQ ID NO:98, and a nucleic acid encoding the light chain variable region of SEQ ID NO:99 a nucleic acid encoding the variable region; a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 100; and a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 100. A nucleic acid encoding the light chain variable region of SEQ ID NO:101; a nucleic acid encoding the heavy chain variable region of SEQ ID NO:102 and a nucleic acid encoding the light chain variable region of SEQ ID NO: 103; or a nucleic acid encoding SEQ ID NO: 104 and a nucleic acid encoding the light chain variable region of SEQ ID NO: 105. For example, a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 96 is provided by SEQ ID NO: 106. and the nucleic acid encoding the light chain variable region of SEQ ID NO: 97 is represented by SEQ ID NO: 107. The nucleic acid encoding the heavy chain variable region of SEQ ID NO: 98 is represented by SEQ ID NO: 108. The nucleic acid encoding the light chain variable region of SEQ ID NO:99 can be represented as SEQ ID NO:109. The nucleic acid encoding the heavy chain variable region of SEQ ID NO: 110 and the light chain variable region of SEQ ID NO: 101 are represented by SEQ ID NO: 112. The nucleic acid encoding the variable region may be represented by SEQ ID NO: 111. The nucleic acid encoding the region is represented by SEQ ID NO: 112 and encodes the light chain variable region of SEQ ID NO: 103. The nucleic acid encoding the heavy chain variable region of SEQ ID NO: 104 can be represented by SEQ ID NO: 113. The nucleic acid encoding the light chain variable region of SEQ ID NO: 114 is represented by SEQ ID NO: 105. may be represented by SEQ ID NO:115.

[0017] Also, a sequence similar to SEQ ID NO: 106, 108, 110, 112 or 114 may be used. % or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, A heavy chain variable region having amino acids with 98% or more, 99% or more, or 100% identity thereto. and SEQ ID NO: 107, 109, 111, 113, or 115. 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more a light chain variable region having an amino acid identity of 98% or more, 99% or more, or 100% Nucleic acid encoding the region is provided.

[0018] The present specification also relates to a composition for preventing or treating cancer, comprising the antibody or its In one embodiment, the composition comprises an antigen-binding fragment of an immune control antibody. The therapeutic agent may further comprise a checkpoint blocker or a chemotherapeutic agent.

[0019] The present invention also relates to a method for treating a subject's chronic obstructive pulmonary disease by administering the monoclonal antibody or antigen-binding fragment thereof disclosed herein to a subject. In one embodiment, a method of treating cancer in a subject in need thereof is disclosed, comprising administering In one embodiment, the cancer is a cancer that overexpresses CD74. Triple-negative breast cancer (TBNC), pancreatic and colorectal cancer, ovarian cancer, and gastric cancer (gastric cancer), bladder cancer, leukemia, prostate cancer, malignant melanoma, cancer, esophageal cancer, gastric cancer (stom ach cancer, head and neck cancer, lung cancer or kidney cancer.

[0020] Also provided herein is a method for treating cancer in a subject in need thereof. The present invention also discloses the use of local antibodies and antigen-binding fragments thereof in subjects in need thereof. The monoclonal antibodies and antigen-binding fragments thereof disclosed herein for use in the treatment of cancer. Also disclosed is a method for the manufacture of a medicament for the treatment of cancer in an individual in need thereof. The present invention discloses uses of the monoclonal antibodies and antigen-binding fragments thereof disclosed in.

[0021] These and other aspects, features, and advantages of particular embodiments of the present disclosure may be best understood from the following detailed description, taken in conjunction with the accompanying drawings, in which: This will become more clear from the following explanation. [Brief description of the drawings]

[0022] [Figure 1] 1 is a graph showing the binding ability of anti-CD73 antibodies selected through biopanning to CD73. CD73 binding of phage antibodies was confirmed by measuring absorbance at 450 nm wavelength, and the standard deviation of the data is represented by error bars.

[0023] [Figure 2A] 1 shows a human IgG1 expression vector produced by cloning the VH and VL gene sequences of an anti-CD73 phage antibody into a pdCMV-dhfr vector containing the CH and CL gene sequences.

[0024] [Figure 2B] This shows the results of SDS-PAGE analysis of human IgG1 antibody. Lanes 1 to 12, in order, are L2-3, L2-47, L4-1, L4-4, L4-5, L4-46, L4-66, L6-1, L6-7, L6-30, L6-37, and L8-24.

[0025] [Figure 3A] The results of ELISA performed to analyze the binding of 12 purified human IgG1 isotype antibodies to CD73 are shown.

[0026] [Figure 3B] In vitro assays were performed in MDA-MB-231 cells to compare 12 purified human IgG1 isotype antibodies by inhibition of CD73 enzyme activity, which was determined by measuring phosphate cleaved by CD73 using a malachite green assay.

[0027] [Figure 3C] The cross-linking ability of antibodies to human, monkey, rat and mouse CD73 was analyzed by ELISA. Antibody binding to CD73 was confirmed by measuring absorbance at 450 nm wavelength.

[0028] [Figure 4] 4A to 4D are graphs showing a comparison of the binding ability of the APBA2-01 and APBA2-02 antibodies to water-soluble CD73, and show the ELISA results for human, monkey, rat, and mouse CD73, respectively.

[0029] [Figure 5A] 1 is a flow cytometry graph showing a comparison of membrane CD73 binding ability of APBA2-01 and APBA2-02 antibodies in MDA-MB-231 cells expressing human CD73.

[0030] [Figure 5B] 1 is a flow cytometry graph showing a comparison of membrane CD73 binding ability of APBA2-01 and APBA2-02 antibodies in 4T1 cells expressing mouse CD73.

[0031] [Figure 6A] 1 is a graph showing the results of biolayer interferometry to analyze the competitive binding of APBA2-01 and APBA2-02 antibodies to the epitope of CPI-006.

[0032] [Figure 6B] 1 is a graph showing the results of biolayer interferometry to analyze the competitive binding of APBA2-01 and APBA2-02 antibodies to the epitope of MEDI-9447.

[0033] [Figure 7]1 is a graph showing the results of a malachite green assay to compare the inhibition of soluble CD73 enzyme activity by APBA2-01 and APBA2-02 antibodies.

[0034] [Figure 8A] 1 is a graph showing the results of a malachite green assay to compare the inhibition of membrane CD73 enzyme activity by APBA2-01 and APBA2-02 antibodies in MDA-MB-231 cells.

[0035] [Figure 8B] 1 is a graph showing the results of a CellTiter-Glo® assay to compare the inhibition of membrane CD73 enzyme activity by the APBA2-01 and APBA2-02 antibodies in 4T1 cells.

[0036] [Figure 8C] 1 is a graph showing the results of a malachite green assay to compare the inhibition of membrane CD73 enzyme activity by APBA2-01 and APBA2-02 antibodies in MDA-MB-231 cells. Standard deviations are shown as error bars, and the vertical axis shows relative light units (RLU).

[0037] [Figure 8D] 1 is a graph showing the results of a CellTiter-Glo® assay to compare the inhibition of membrane CD73 enzyme activity by APBA2-01 and APBA2-02 antibodies in 4T1 cells. Standard deviation is shown as error bars, and the vertical axis shows RLU.

[0038] [Figure 9] The results show that the APBA2-01 and APBA2-02 antibodies were transferred into the CD73 membrane of cells, as confirmed by scanning microscopy. CD73, which corresponds to the FITC signal, was expressed as shown in white, and the scale bar is also shown.

[0039] [Figure 10A] This is a graph showing the results of evaluating the anti-cancer efficacy of APBA2-01 antibody and APBA2-02 antibody by measuring the average lung weight in a metastatic breast cancer mouse model; *: P<0.05 (one-way ANOVA).

[0040] [Figure 10B] This is a graph showing the results of evaluating the anti-cancer efficacy of APBA2-01 antibody and APBA2-02 antibody by measuring the number of lung metastatic colonies in a metastatic breast cancer mouse model; *: P<0.05 (one-way ANOVA).

[0041] [Figure 11A] This is a graph showing the results of evaluating the tumor growth inhibitory efficacy of APBA2-01 antibody and APBA2-02mIgG antibody by measuring tumor volume in a breast cancer mouse model; *: P<0.05; **: P<0.01, ns (not significant: P>0.05 (two-way ANOVA)).

[0042] [Figure 11B] 1 is a graph showing the results of evaluating the tumor growth inhibitory efficacy of APBA2-01 antibody and APBA2-02mIgG antibody by comparing the tumor growth inhibitory ability between antibody samples in a breast cancer mouse model.

[0043] [Figure 11C] This is a graph showing the results of evaluating the tumor growth inhibitory efficacy of APBA2-01 antibody and APBA2-02mIgG antibody in a breast cancer mouse model by measuring mouse weight; *: P<0.05; **: P<0.01, ns (not significant: P>0.05 (two-way ANOVA)).

[0044] [Figure 12A]1 is a graph showing the results of a malachite green assay to compare the ability of anti-CD73 antibodies to inhibit the enzymatic activity of membrane CD73 in MDA-MB-231 cells.

[0045] [Figure 12B] 1 is a graph showing the results of a CellTiter-Glo® assay to compare the inhibition of CD73 enzyme activity by anti-CD73 antibodies in MDA-MB-231 cells.

[0046] [Figure 13A] 1 is a graph confirming the binding ability of Myxengo 3, 4, 6, 7 and 15 antibodies to membrane CD73 protein in 4T1 cells.

[0047] [Figure 13B] 1 is a graph confirming the binding ability of Myxengo 3, 4, 6, 7 and 15 antibodies to membrane CD73 protein in 4T1.2 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present specification describes a monoclonal antibody that binds to CD73 (cluster of differentiation 73). An antibody or antigen-binding fragment thereof is disclosed.

[0049] Also, a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof, Also disclosed are expression vectors comprising the vector and cells transformed with the vector.

[0050] Also, there is provided a composition for preventing or treating cancer, comprising the monoclonal antibody or Compositions comprising the antigen-binding fragments are disclosed.

[0051] Further aspects are described in part in and will be apparent from the following description. or may be learned by practice of the embodiments presented in this disclosure.

[0052] A group of sacs composed of the amino acid sequences of SEQ ID NO: 1, 4, 7, 10, 14 or 17 CDRH1 selected from the amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15 or 18. CDRH2 selected from the group consisting of SEQ ID NOs: 3, 6, 9, 1 C selected from the group consisting of 2, 13, 16 or 19 amino acid sequences A heavy chain variable region comprising DRH3; and the amino acid sequence of SEQ ID NO: 20, 23, 26, or 29. CDRL1 selected from the group consisting of SEQ ID NO: 21, 24, 27 or a CDRL2 selected from the group consisting of 30 amino acid sequences, and a sequence Among the group consisting of the amino acid sequences represented by numbers 22, 25, 28, or 31, The present invention discloses an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a CDRL3 selected from the group consisting of: do.

[0053] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:1. CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; CDRL1 comprising the amino acid sequence of SEQ ID NO:21, CDRL2 comprising the amino acid sequence of SEQ ID NO:22, and the sequence a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO:22; CDRH1 comprising the amino acid sequence of SEQ ID NO:5, CDRH2 comprising the amino acid sequence of SEQ ID NO:6, a heavy chain variable region comprising CDRH3 comprising the amino acid sequence of SEQ ID NO:23; CDRL1 comprising the amino acid sequence of SEQ ID NO:24, and CDRL2 comprising the amino acid sequence of SEQ ID NO:25. a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:7; CDRH1 comprising the amino acid sequence of SEQ ID NO:8, and CDRH2 comprising the amino acid sequence of SEQ ID NO:9. a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:26; 1. CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and the amino acid sequence of SEQ ID NO:28 a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 10; CDRH2 comprising the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12 or 13 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:20; RL1, CDRL2 comprising the amino acid sequence of SEQ ID NO:21, and CDRL3 comprising the amino acid sequence of SEQ ID NO:22. a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO:14; 15, CDRH2 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:20; 21, CDRL2 comprising the amino acid sequence of SEQ ID NO: 22, a light chain variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; H1, CDRH2 comprising the amino acid sequence of SEQ ID NO:18, and the amino acid sequence of SEQ ID NO:19 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:29; L1, CDRL2 comprising the amino acid sequence of SEQ ID NO: 30, and the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising a CDRL3 comprising:

[0054] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 32, 33, 34, ,35,36,37, or 38, at least 80%, 85%, or 90% , 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 1 39, 40, 41 or 42. or 42, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 96 % or more, 97% or more, 98% or more, 99% or more, or 100% identity In one embodiment, the antibody or its derivatives may comprise a light chain variable region comprising The original binding fragment has the amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38. and a light chain variable region comprising SEQ ID NO: 39, 40, 41 or 42. It may include a variable region.

[0055] Also, CDRH1 comprising the amino acid sequence of SEQ ID NO: 66, 69, 72, 75 or 78; CDRH2 comprising the amino acid sequence of SEQ ID NO: 67, 70, 73, 76 or 79, and the sequence A heavy chain variable region comprising a CDRH3 having an amino acid sequence of no. 68, 71, 74, 77, or 80 region, and CDRL1 comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90 or 93 82, 85, 88, 91 or 94, and A monoclonal antibody containing CDRL3 containing the amino acid sequence of SEQ ID NO: 83, 86, 89, 90, or 95 The present invention provides a monoclonal antibody or an antigen-binding fragment thereof.

[0056] In one embodiment, the antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:66. CDRH1 comprising the amino acid sequence of SEQ ID NO:67, and CDRH2 comprising the amino acid sequence of SEQ ID NO:68. a heavy chain variable region comprising CDRH3 comprising the amino acid sequence of SEQ ID NO: 81; CDRL1 comprising the amino acid sequence of SEQ ID NO: 82; and CDRL2 comprising the amino acid sequence of SEQ ID NO: 83. a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:69; CDRH1, CDRH2 comprising the amino acid sequence of SEQ ID NO:70, and CDRH3 comprising the amino acid sequence of SEQ ID NO:71. a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO: 84; CDRL1 comprises the amino acid sequence of SEQ ID NO:85, and CDRL2 comprises the amino acid sequence of SEQ ID NO:86. a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO: 72; H1, CDRH2 comprising the amino acid sequence of SEQ ID NO:73, and the amino acid sequence of SEQ ID NO:74 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO: 87; L1, CDRL2 comprising the amino acid sequence of SEQ ID NO:88, and the amino acid sequence of SEQ ID NO:89 a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO: 75; CDRH2 comprising the amino acid sequence of SEQ ID NO:76, and CDRH3 comprising the amino acid sequence of SEQ ID NO:77 a heavy chain variable region comprising a CDRH3; and a CDRL1 comprising the amino acid sequence of SEQ ID NO:90; CDRL2 comprising the amino acid sequence of SEQ ID NO:91, and CDRL3 comprising the amino acid sequence of SEQ ID NO:92. a light chain variable region comprising a CDRL3; or a CDRH1 comprising the amino acid sequence of SEQ ID NO:78 79, CDRH2 comprising the amino acid sequence of SEQ ID NO: 80, a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:93; and a CDRL1 having the amino acid sequence of SEQ ID NO:94. 94, CDRL2 comprising the amino acid sequence of SEQ ID NO: 95, The light chain variable region can be a light chain variable region comprising a CDRL3 that includes

[0057] The heavy chain constant region and the light chain constant region of the antibody may be any of IgG, IgM, IgE, and IgA. The constant region can be an antibody constant region of, for example, IgD, IgE, or a combination thereof. For example, it may be derived from the antibody constant region of IgG1.

[0058] In one embodiment, the antibody or antigen-binding fragment thereof exhibits species cross-reactivity in the individual. The individual or subject may have a spinal cord It can be an animal, such as a mammal, amphibian, reptile, or bird, and the species can be, for example, , human (Homo sapiens), monkey, rat, mouse, etc. For example, the anti-CD73 The antibodies either inhibit the conversion of CD73 to its active conformation or bind to CD73. It can inhibit the enzymatic activity of CD73 by competitively binding with a corresponding inhibitor. In addition, the anti-CD73 antibody was also used as a positive control, MEDI9447 or CPI-006. By binding to an epitope similar to that of The antibody or antigen-binding fragment thereof specifically binds to CD73 while exhibiting cross-species reactivity. It may have a responsiveness.

[0059] As used herein, the term "epitope" refers to a protein to which an antibody can specifically bind. The term epitope means a determinant of a gene. The epitope is usually chemically active. They are made up of surface groups of molecules, e.g., amino acids or sugar side chains, and generally have a specific tertiary structure. They have the original structural features as well as specific charge characteristics. The researchers found that the binding to the former was lost in the presence of denaturing solvents, but the binding to the latter was not lost. The epitope is, for example, a sequence of consecutive amino acids (a line) of a polypeptide. epitopes) or, for example, bipartite epitopes of a polypeptide(s). These can be brought about by the above discontinuous regions (three-dimensional, nonlinear, discontinuous, etc.). In certain embodiments, the epitope to which the antibody binds is, for example, a continuous or discontinuous epitope. For example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA analysis, mass spectrometry (e.g., liquid chromatography) Electrospray mass spectrometry), array-based oligo-peptide scanning sequencing analysis, and / or mutation mapping (e.g., site-directed mutagenesis mapping) In the case of X-ray crystallography, the determination may be made by any one of the methods known in the art. Crystallization can be performed using a method such as Acta Crystallogr D (see, for example, Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 63 00-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, X-PLO. R (Yale University, 1992, distributed by Molecular Simulations, Inc.; for example, See Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., US 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crysta llogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316 The sequence can be refined using computer software such as mutagenesis programs (Mutagenesis Matrix). Mapping studies can be accomplished using any method known to those of skill in the art. Alanine scanning For details of mutagenesis techniques, including sequencing mutation techniques, see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC, and Wells JA (1989) Scienc. e 244:1081-1085. In one embodiment, the epitope of the antibody is alanine. This is determined using scanning mutagenesis studies.

[0060] As used herein, the term "antibody" refers to an antibody that binds to an antigen, such as a virus, bacteria, etc. It refers to a glycoprotein that inactivates and induces extracellular stimulation against microorganisms that have invaded the body. The antibody disclosed herein is a monoclonal antibody. , multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies Antibody, Fab fragment, F(ab') fragment, disulfide-linked Fvs (sdFv) and anti-antibody The antibody may comprise an epitope-binding fragment of said antibody, or an anti-Id antibody. Specifically, the antibody is a monoclonal antibody, but is a fully human antibody or a human antibody. A monoclonal antibody is a substantially homogenous antibody population (a human-mouse chimeric antibody). That is, each antibody in the population is able to detect possible naturally occurring mutations that may be present in minute amounts. The term "monoclonal antibody" refers to an antibody obtained from a highly specific The present disclosure relates to anti-CD73 antibodies that are specific and directed against a single antigenic epitope. Therefore, unless otherwise specified, the term "antibody" is used without modification. The term may refer to an anti-CD73 antibody that binds to an epitope of CD73. The antibody molecules are not only required to specifically bind to CD73, but also to bind to the antigen. Intact antibodies have a structure that contains two full-length light chains and two full-length heavy chains. In this structure, each light chain is linked to a heavy chain by a disulfide bond. The bands are gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε ) type, with subclasses Gamma 1 (γ1), Gamma 2 (γ2), and Gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the chains are of the kappa (κ) and lambda (λ) types.

[0061] "Binding affinity" generally refers to the ability of a molecule (e.g., an antibody) to bind to a single It refers to the strength of the sum of non-covalent interactions between a binding site and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the degree of affinity of a member of a binding pair. The term "specific binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a target molecule (e.g., an antibody and an antigen). The affinity of molecule X for partner Y is generally expressed as the dissociation constant (K D (dissociation The affinity can be expressed as the equilibrium dissociation constant (K D ) and equilibrium binding constant (K A (association constant) and can be measured and determined by various methods known in the art. and / or expression may be made. D is k off / k on It is calculated by the quotient of The KA stands for k on / k off It is calculated by the quotient of k on For example, k stands for the binding rate constant of the antibody. off means, for example, dissociation of an antibody against an antigen. The aforementioned k on and k off The methods are well known to those skilled in the art, such as BIAcore® or KinExA. This can be determined by techniques already in place.

[0062] As used herein, the term "immunospecifically binds" "immunospecifically recognizes" and "specifically binds" "Specifically binds" and "specifically recognizes" refer to an antibody. and similar terms in the context of, as understood by those of skill in the art, a molecule that binds to an antigen. (e.g., an epitope, an immune complex, or a binding partner of an antigen-binding site). For example, molecules that specifically bind to an antigen can be detected using, for example, immunoassays, BIAcore®, Kinase ExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other instrument known in the art. Other peptides or polypeptides were identified with generally lower affinity as determined by assay. In one embodiment, a molecule that specifically binds to an antigen is one in which the molecule is K when binding to other antigens A Therefore, at least 2 log, 2.5 log, 3 log, 4 logs or greater K A binds to the antigen.

[0063] In other embodiments, molecules that immunospecifically bind to an antigen exhibit similar binding properties under similar binding conditions. In one embodiment, the antibody is immunospecific for an antigen and does not cross-react with other proteins. In one embodiment, the molecule binds to the IL-11 protein and does not cross-react with other proteins. bind to a specific antigen, such as CD73, with higher affinity than to other unrelated antigens. In certain embodiments, the present application provides monoclonal antibodies and antigen-binding fragments thereof. The method can be carried out, for example, by radioimmunoassay, surface plasmon resonance or kinetic exclusion. Affinity for unrelated antigens, as measured by the fusion assay More than 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 7 CD7 with 0%, 75%, 80%, 85%, 90%, 95% or higher affinity The present invention provides monoclonal antibodies and antigen-binding fragments thereof that bind to specific antigens such as 3. In one embodiment, the monoclonal antibodies and antigen-binding fragments thereof described herein The extent of binding to unrelated proteins is measured, for example, by radioimmunoassay. However, less than 10%, less than 15%, or less than 20% of the antibody binding to a specific antigen is be.

[0064] In one embodiment, the present application provides a method for detecting human CD7 with higher affinity than for other species of antigen. 3. In certain embodiments, the present invention provides monoclonal antibodies and antigen-binding fragments thereof that bind to The present application relates to a method for detecting the presence of a marker, for example, by radioimmunoassay, surface plasmon resonance or equilibrium binding exclusion. 20%, 30%, 35%, 40% higher than the affinity to other species, as measured by , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% Monoclonal antibodies that bind to human CD73 with 95% or higher affinity and antigen-binding fragments thereof.

[0065] In one embodiment, the antibody is in Fv format (e.g., scFv) or in whole antibody format. The heavy chain constant region is gamma (γ), mu (μ), alpha ( α), delta (δ) or epsilon (ε) isotypes. For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4) constant region. The light chain constant region can be of the kappa or lambda type.

[0066] As used herein, the term "heavy chain (HC or CH)" refers to a chain specific to an antigen. Amino acids having sufficient variable region (VR) sequences to confer isomerism A variable region domain VH containing the amino acid sequence, and three constant region domains CH1, CH2 and C The term "light chain (LC or CL)" refers to both the full-length heavy chain including H3 and its fragments. "Light chain" refers to an antigen having sufficient variable region sequence to confer specificity to an antigen. A full-length light chain comprising a variable region domain VL and a constant region domain CL, the variable region domain VL comprising the amino acid sequence It means a fragment of it.

[0067] "Humanized" forms of non-human (e.g., murine) antibodies are the minimally derived components of non-human immunoglobulins. For the most part, humanized antibodies are chimeric antibodies that contain the same sequence as the recipient's Residues from the hypervariable regions were then cloned into mouse, rat or mouse strains that possess the desired specificity, affinity and potency. , residues from the hypervariable region of a non-human species (donor antibody), such as rabbit or non-human primate It is a human immunoglobulin (recipient antibody) replaced by

[0068] A "human antibody" is a molecule derived from human immunoglobulin and has a complementary determining region, structure, The entire amino acid sequence that constitutes an antibody, including the region, is composed of human immunoglobulins. In one embodiment, the present invention provides a method for minimizing unintended immune responses. The present invention relates to fully human antibodies that can be synthesized.

[0069] A "chimeric antibody" is an antibody in which a portion of the heavy and / or light chain is derived from a particular species or or While homologous, the remaining chain(s) may be from another species or belong to another antibody class. or a subclass of antibody that is identical or homologous to a corresponding sequence in an antibody of a desired biological "chimeric" antibodies (immunoglobulins) exhibiting biological activity, as well as fragments of such antibodies. .

[0070] The term "antibody variable region" refers to a complementary determining region (CDR). ), CDRH or CDRL, i.e., CDRH1, CDRH2 and CDRH3), and The light chain portion and the heavy chain portion of an antibody molecule including the amino acid sequence of the framework region (FR) are VH refers to the variable region of the heavy chain. VL refers to the variable region of the heavy chain. , refers to the variable region of the light chain.

[0071] "Complementarity determining region (CDR, CDRH or "CDRLs" (i.e., CDRL1, CDRL2, and CDRL3) are used for antigen binding. Each variable region typically comprises: has three CDR regions identified as CDR1, CDR2 and CDR3.

[0072] As used herein, the term "framework region (FR)" refers to a This refers to variable region residues other than CDR residues. Each variable region typically includes FR1, FR2, , FR3 and FR4.

[0073] In the present invention, the term "antigen-binding fragment" refers to Fab, F(ab'), F(ab')2, Fv, The term "Fv" refers to a fragment of an antibody that has antigen-binding ability, including the fragments of a complete antibody. An antibody fragment that contains the recognition and binding sites. Such a region consists of one heavy chain variable region and and one light chain variable region tightly covalently linked to form a dimer (e.g., scFv). A "Fab" fragment contains the variable and constant regions of the light chain and the variable and first constant region of the heavy chain. F(ab')2 antibody fragments generally contain a hinge region between them. The stain contains a pair of Fab fragments covalently linked near their carboxy termini. "Single-chain Fv," or "scFv," antibody fragments are composed of the VH and VL domains of an antibody. Fv polypeptides contain multiple domains, but the domains are present in a single polypeptide chain. The VH domain further comprises a polypeptide linker between the VL domain, The scFv is allowed to form the desired structure for antigen binding.

[0074] Fv is the smallest antibody segment that contains only the heavy chain variable region and the light chain variable region. Recombinant techniques for producing Fv fragments are described in WO88 / 10649, WO88 / 106630 , WO88 / 07085, WO88 / 07086 and WO88 / 09344. In two-chain Fv, the heavy chain variable region and the light chain variable region are non-covalently bound. In single-chain Fv (scFv), the peptide is generally The heavy chain variable region and the light chain variable region are linked by a covalent bond via a linker. or directly linked to the C-terminus, forming a dimeric structure, such as a two-chain Fv. Such antibody fragments can also be obtained by using proteolytic enzymes (e.g., If you digest a full-length antibody with papain, you can get Fab, and if you digest an antibody with pepsin, you can get For example, F(ab')2 fragments can be obtained. It is also possible.

[0075] As used herein, the term "phage display" refers to the display of mutant polypeptides using phage. phage, e.g., a fusion protein with at least a portion of a coat protein on the surface of a filamentous phage particle. The usefulness of the phage display is shown in the following. A large library of mutant dam proteins was targeted to identify sequences that bind to the target antigen with high affinity. The advantage of this method is that it allows rapid and efficient classification of peptide and protein sequences. Displaying the library on phage allows the production of polypeptides with specific binding properties. The method was used to screen millions of polypeptides to identify polypeptides that are

[0076] Phage display technology allows the generation of novel proteins that bind to specific ligands (e.g., antigens). The phage display technology provides a powerful tool for the generation and selection of Generate large libraries of protein variants and rapidly identify sequences that bind with high affinity to target antigens. The nucleic acid encoding the mutant polypeptide can be rapidly categorized into fused to a nucleic acid sequence encoding a protein, such as the gene III protein or the gene VIII protein. The nucleic acid sequence encoding the protein or polypeptide is referred to as a gene III protein coding portion. A monovalent phage display system has been developed in which the phage is fused to a nucleic acid sequence that encodes the phage. In the phage display system, the gene fusion is expressed at low levels and the wild-type gene is expressed. The gene III protein is also expressed, thereby maintaining the infectivity of the particle.

[0077] Expression of peptides on the surface of filamentous phages and their expression in the periplasm of E. coli The expression of functional antibody fragments in human cytoplasmic cells is a promising approach to antibody phage differentiation. It is important to develop a spray library. Libraries of DNA can be generated in a number of ways, e.g., by inserting random DNA sequences. Prepared by modifying a single gene or cloning a series of related genes Libraries have been used to identify antibodies or antigen-binding proteins with desired characteristics. It is possible to screen for:

[0078] Phage display technology is a general method for producing antibodies with desired characteristics. It has several advantages over hybridoma and recombinant methods. This method allows the generation of large antibody libraries with diverse sequences in a short time without using animals. The production of hybridomas and humanized antibodies takes several months. Production time may be required. Also, phage antibody libraries do not require any immunization. Therefore, antibodies can be generated even against antigens that are toxic or have low antigenicity. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.

[0079] Phage display libraries can be used to identify human germline sequences or immunization or It is possible to use techniques to generate human antibodies from non-immune naive B cell Ig repertoires. Various lymphoid tissues can be used to generate naive or non-immune antigen-binding libraries. can be prepared.

[0080] Techniques for identifying and isolating high affinity antibodies from phage display libraries is important for isolating novel therapeutic antibodies. Isolating high affinity antibodies from libraries The size of the library, the efficiency of production in bacterial cells, and the diversity of the library are important considerations. The size of the library also depends on the suitability of the antibody or antigen-binding protein. This is reduced by inefficient production due to folding and the presence of stop codons. or if the antigen-binding domain is not folded properly, Expression can be suppressed by targeting residues on the surface of the variable / constant interface or by targeting selected CDR residues. This can be improved by alternatively mutating residues in the frame. The sequence of the work region is suitable for generating an antibody phage library in bacterial cells. This is one element in providing the correct folding.

[0081] Generate diverse libraries of antibodies or antigen-binding proteins for high affinity antibody isolation It is important to ensure that the CDR3 region is fully expressed and that it is often involved in antigen binding. The CDR3 region on the heavy chain is similar in size, sequence, and structural conformation to the following: Because it is highly diverse, the CDR3 region can be used to prepare diverse libraries. Cut.

[0082] In addition, all 20 amino acids at each position are used to determine the C sequence of the variable heavy and variable light chains. Diversity can be generated by randomizing the DR region. The use of these amino acids allows for the generation of highly diverse variant antibody sequences, making it possible to identify novel antibodies. This could increase the opportunities for

[0083] The antibodies or antibody fragments of the present disclosure are capable of specifically recognizing CD73. In addition to the sequences of the anti-CD73 antibodies described herein, biological equivalents thereof are also provided. For example, the binding affinity and / or other biological properties of the antibody may be further improved. In order to achieve this, further modifications can be made to the amino acid sequence of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. Amino acid mutations are based on the relative similarity of amino acid side chain substitutions, e.g., hydrophobicity, hydrophilicity, charge, size, etc. The classification is based on the size, shape and type of the amino acid side chain substitution product. Analysis revealed that arginine, lysine, and histidine are all positively charged residues, Alanine, glycine and serine have similar sizes, while phenylalanine, tryptophan, It can be seen that phane and tyrosine have similar morphologies. Based on considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine, respectively, are biologically functionally equivalent. It is found to be equal.

[0084] When making mutations, the hydropathic index of amino acids is taken into consideration. Each amino acid is assigned a hydrophobic index based on its hydrophobicity and charge. Given: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8) ;Phenylalanine(+2.8);Cysteine / Cysteine(+2.5);Methionine( +1.9);Alanine(+1.8);Glycine(-0.4);Threonine(-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline ( -1.6);histidine (-3.2);glutamate (-3.5);glutamine (-3. 5);Aspartate (-3.5);Asparagine (-3.5);Lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in conferring a specific functional group. Substitution with amino acids having similar hydrophobicity indexes may result in similar biological activity. It is well known that mutations can be introduced based on the hydrophobicity index. When the difference is made, it is preferably within ±2, more preferably within ±1, and even more preferably within ± Substitutions are made between amino acids exhibiting hydrophobicity index differences within 0.5.

[0085] It should be noted that substitutions between amino acids with similar hydrophilicity values ​​are equivalent. It is also well known that the resulting protein has a biological activity. As disclosed in US Pat. No. 5,554,101, the following hydrophilicity values ​​are obtained for each amino acid residue: Attached to the group: arginine (+3.0); lysine (+3.0); aspartate ( +3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine ( +0.2);Glutamine(+0.2);Glycine(0);Threonine(-0.4);Pro Phosphorus (-0.5±1); Alanine (-0.5); Histidine (-0.5); Cysteine ​​( -1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); Soloeusine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tri ptophan (-3.4). Amino acid exchanges in proteins that do not globally alter the activity of the molecule. Conversion is known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, , New York, 1979). The most commonly occurring substitutions are the amino acid residues Ala / Ser,Val. / Ile,Asp / Glu,Thr / Ser,Ala / Gly,Ala / Thr,Ser / Asn,Ala / Val,Ser / Gly,Thr / Phe,Ala / Pro,Lys / Arg,Asp / Asn,Leu / Ile,Leu / Val,Ala / Glu and As It is a p / Gly substitution.

[0086] In one embodiment, the VH (e.g., CDR1, CDR2 or or CDR3) region and / or VL (e.g., CDR1, CDR2 or CDR3) region One or more CDR positions along the Maintained, for example, at least 50%, 60%, 70%, 80%, 9 As long as the amino acid sequence is 0% or more and 95% or more, it can be 2, 3, 4, 5, or 6 amino acid positions. For example, the positions defining the CDRs of the antibodies described herein may vary by antigen(s). ) is maintained (e.g., substantially maintained, e.g., at least 5 0% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) For the CDR positions of the monoclonal antibodies or antigen-binding fragments described herein, The N-terminal and / or C-terminal boundaries of R can be 1, 2, 3, 4, 5 or 6 In other embodiments, the amino acid sequence of the present invention may be varied by moving only one amino acid. The VH (e.g., CDR1, CDR2, or CDR3) regions and / or or one or more along the VL (e.g., CDR1, CDR2, or CDR3) region. The length of the CDRs of the polypeptides is such that immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained). For example, at least 50%, 60%, 70%, 80%, 90% % or more than 95% (1, 2, 3, 4, 5 or more nets) Only the amino acids may differ (e.g., they may be shorter or longer).

[0087] In one embodiment, the CDRL1, CDRL2, CDRL3, CDR H1, CDRH2 and / or CDRH3 are capable of immunospecific binding to an antigen(s). Maintained (e.g., substantially maintained, e.g., at least 50% or more, 60% or more, 70% or more % or more, 80% or more, 90% or more, or 95% or more) of the CDRs described herein. It is 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the In another embodiment, the CDRL1, CDRL2, CDRL3 , CDRH1, CDRH2 and / or CDRH3 are immunospecific for an antigen(s). The binding is maintained (e.g., substantially maintained, e.g., at least 50% or more, 60% or more) As long as the C described in this application is Only 1, 2, 3, 4, 5 or more amino acids from one or more of the DRs In yet another embodiment, the CDRL1, CDRL2 , CDRL3, CDRH1, CDRH2 and / or CDRH3 are amino-terminal to the antigen ( immunospecific binding to the antibody or antibodies (e.g., a plurality of antibodies) is maintained (e.g., substantially maintained, e.g., at least (50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) As long as the CDRs are not different from one or more of the CDRs described herein, one, two, three, four, five, or more of the CDRs may be different from one or more of the CDRs described herein. In yet another embodiment, the nucleic acid sequence described herein may be extended by 1 or more amino acids. CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and / or C The carboxy terminus of DRH3 maintains immunospecific binding to an antigen(s) (e.g., Substantially maintained, for example, at least 50%, 60%, 70%, or 80% 90% or more, 95% or more) compared to one or more of the CDRs described herein. In comparison, a nucleotide sequence may be extended by 1, 2, 3, 4, 5, or more amino acids. In yet another embodiment, the CDRL1, CDRL2, CDRL3, CDRL4, CDRL5, CDRL6, CDRL7, CDRL8, CDRL9, CDRL10, CDRL11, CDRL12, CDRL13, CDRL14, CDRL15, CDRL16, CDRL17, CDRL18, CDRL19, CDRL110, CDRL111, CDRL12, CDRL13, CDRL14, CDRL15, CDRL1 The amino terminus of DRH1, CDRH2 and / or CDRH3 is a ligand for the antigen(s). Immunospecific binding is maintained (e.g., substantially maintained, e.g., at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) as long as 1, 2, 3, 4, 5 or more compared to one or more of the CDRs In yet another embodiment, the CDRLs described herein may be truncated by 1 or 2 amino acids. 1, CDRL2, CDRL3, CDRH1, CDRH2 and / or CDRH3 The oxy terminus is a nucleotide that maintains (e.g., substantially maintains) immunospecific binding to the antigen(s). For example, at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more 95% or more) compared to one or more of the CDRs described herein, The amino acid sequence may be truncated by 3, 4, 5, or more amino acids. Any method can be used to determine whether immunospecific binding to the antigen(s) is maintained. For example, the binding assays and methods described in the "Examples" section of this specification can be used to identify Conditions may be used.

[0088] The percent identity between two base sequences (e.g., amino acid base sequences or nucleic acid base sequences) Determination of gender can also be accomplished using a mathematical algorithm. Specific, non-limiting examples of mathematical algorithms are given in Karlin S & Altschul SF (1993) PN Karlin S & Altschul SF (1990) PNAS 87: 226, as amended, as in AS 90: 5873-5877 4-2268. The algorithm is based on the method described in Altschul SF et al., (1990) J Mol Integrated into the NBLAST and XBLAST programs in Biol 215:403 BLAST nucleotide searches identify nucleotide sequences homologous to the nucleic acid molecules described herein. To obtain a sequence, the NBLAST nucleotide program parameters are set, e.g., BLAST protein searches can be performed using the methods described herein. To obtain amino acid sequences homologous to the protein molecules listed in Run the hit program parameters set, e.g., score=50, word length=3. To obtain gapped alignments for comparison purposes, the method of Altschul SF et al. Using Gapped BLAST as described in al., (1997) Nuc Acids Res 25: 3389 3402 Alternatively, PSI BLAST can be used to perform iterative searches to detect distant relationships between molecules. Searches can be performed using the BLAST, Gapped BLAST, and PSI Blast programs. When using the BLAST and NBLAST programs, use the default parameters. (For example, the National Center for Biotechnology Information (NCBI) on (See the NCBI worldwide web at ncbi.nlm.nih.gov.) The mathematical algorithm used for sequence comparison Other specific, non-limiting examples of algorithms are given in Myers and Miller, 1988, CABIOS 4: 11 17 Such an algorithm is implemented in the GCG sequence alignment software. It is integrated into the ALIGN program (version 2.0), which is part of the software package. When using the ALIGN program to compare amino acid sequences, use PAM120 A weight residue table, cap length penalty of 12 and gap penalty of 4 can be used. do.

[0089] The percent identity between two sequences can be determined using techniques similar to those described above. Percent identity can be determined using a quantification method, with or without allowing gaps. When making the calculations, typically only exact matches are counted.

[0090] Other aspects include nucleic acids encoding the antibodies or antigen-binding fragments thereof, and expression vectors comprising the nucleic acids. A vector and a cell transformed with the expression vector are provided.

[0091] At least 80% of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38 Above, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more %, 99% or more, or 100% identity with the heavy chain variable region. and 80% or more, 85% or more of the nucleic acid sequence corresponding to SEQ ID NO: 39, 40, 41 or 42. , 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% The present invention provides a nucleic acid encoding a light chain variable region having amino acids having at least 100% identity with the nucleic acid. The amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38 is also provided. and a nucleic acid encoding a heavy chain variable region comprising the sequence of SEQ ID NO: 39, 40, 41 or 42. Nucleic acids encoding the light chain variable regions including the numbers are provided.

[0092] In one embodiment, the heavy chain variable region of SEQ ID NO: 32 and the light chain variable region of SEQ ID NO: 39. or a nucleic acid encoding a heavy chain variable region of SEQ ID NO: 33 and a light chain variable region of SEQ ID NO: 40 For example, a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 32 is provided. The nucleic acid is also represented by SEQ ID NO: 43, which encodes the light chain variable region of SEQ ID NO: 39. The nucleic acid to be encoded can be that represented by SEQ ID NO: 44. In one embodiment, the nucleic acid to be encoded can be that represented by SEQ ID NO: 32 or SEQ ID NO: 33, and a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 39 or sequence The present invention also provides a nucleic acid encoding the light chain variable region of SEQ ID NO: 40. The nucleic acid encoding the variable region is also represented by SEQ ID NO: 45, The nucleic acid encoding the light chain variable region can be that represented by SEQ ID NO:46.

[0093] Also, at least 80% or more of SEQ ID NO: 96, 98, 100, 102 or 104 Above, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more %, 99% or more, or 100% identity with the heavy chain variable region. 85% to the nucleic acid encoding SEQ ID NO: 97, 99, 101, 103 or 105 or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 9 The present invention provides a nucleic acid encoding a light chain variable region having an identity of 9% or more or 100%. and a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 96, 98, 100, 102 or 104. and a nucleic acid encoding the light chain variable region of SEQ ID NO: 97, 99, 101, 103 or 105. Provide the acid.

[0094] In one embodiment, the heavy chain variable region of SEQ ID NO: 96 and the light chain variable region of SEQ ID NO: 97. a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 98, and a nucleic acid encoding the light chain variable region of SEQ ID NO: 99; a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 100 and the light chain variable region of SEQ ID NO: 101; a nucleic acid encoding the heavy chain variable region of SEQ ID NO: 102 and the light chain variable region of SEQ ID NO: 103; or a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105 For example, a nucleic acid encoding the heavy chain variable region of SEQ ID NO:96 is provided. is also represented by SEQ ID NO: 106, and encodes the light chain variable region of SEQ ID NO: 97. The nucleic acid for the heavy chain variable region of SEQ ID NO: 98 can be represented by SEQ ID NO: 107. The nucleic acid encoding the light chain variant of SEQ ID NO: 99 is also represented by SEQ ID NO: 108. The nucleic acid encoding the variable region can be represented by SEQ ID NO: 109. The nucleic acid encoding the heavy chain variable region of the sequence is also represented by SEQ ID NO: 110, The nucleic acid encoding the light chain variable region of SEQ ID NO:101 may be represented by SEQ ID NO:111. The nucleic acid encoding the heavy chain variable region of SEQ ID NO: 102 is represented by SEQ ID NO: 112. The nucleic acid encoding the light chain variable region of SEQ ID NO: 103 is represented by SEQ ID NO: 113. The nucleic acid encoding the heavy chain variable region of SEQ ID NO: 104 can be 14, and the nucleic acid encoding the light chain variable region of SEQ ID NO: 105 is It may be represented by SEQ ID NO:115.

[0095] Also, a sequence similar to SEQ ID NO: 106, 108, 110, 112 or 114 may be used. % or more, 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, A heavy chain variable region comprising an amino acid sequence having 98% or more, 99% or more, or 100% identity Nucleic acids encoding the region, and SEQ ID NO: 107, 109, 111, 113 or 115 85% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% The present invention provides a nucleic acid encoding a light chain variable region having 99% or more or 100% identity to the above. do.

[0096] Nucleic acids encoding the antibodies or antigen-binding fragments of the invention are isolated and used to produce monoclonal antibodies. or an antigen-binding fragment thereof can be recombinantly produced. Insert it into a replicable vector for cloning (amplifying the DNA) or expression In one embodiment, one or more of the nucleic acids disclosed herein are contained in a vector. Alternatively, each of the nucleic acids disclosed herein may be inserted into a separate vector. Based on this, the present disclosure in another aspect relates to the The present invention relates to a vector comprising the nucleic acid.

[0097] As used herein, the term "nucleic acid" refers to DNA (gDNA and cDNA) molecules, as well as It has a comprehensive meaning including RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, are In addition to natural nucleotides, there are also analogs with modified sugar or base moieties. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modification may be an addition, deletion, or non-conservative or conservative substitution of nucleotides. Contains optional substitutions.

[0098] The DNA encoding the antibody may be prepared using conventional procedures (e.g., the heavy and light chains of the antibody may be synthesized using conventional methods). An oligonucleotide probe capable of specifically binding to DNA encoding the Vectors can be easily isolated or synthesized. Many vectors are available. The actuator components typically include, but are not limited to, one or more of the following: is not: a signal sequence, an origin of replication, one or more marker genes, or an enhancer element , a promoter, and a transcription termination sequence.

[0099] As used herein, the term "vector" refers to a vector that is capable of expressing a gene of interest in a host cell. means for transfecting a plasmid vector; cosmid vector; bacteriophage vector vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors In the vector, a nucleic acid encoding an antibody is is operably linked to a promoter.

[0100] "Operably linked" means a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or means a functional linkage between a set of transcriptional regulatory factor binding sites) and another nucleic acid sequence, It is regulated by transcription and / or translation of the sequence.

[0101] When a prokaryotic cell is used as a host, the vector contains a strong promoter that can drive transcription. Low motor (e.g., tac promoter, lac promoter, lacUV5 promoter) Low promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter promoter, trp promoter, and T7 promoter), and Typically, the nucleic acid sequence contains a nucleic acid sequence that encodes a nucleic acid sequence that encodes a nucleic acid fragment, such as a nucleic acid fragment that encodes a nucleic acid fragment containing a nucleic acid fragment, a nucleic acid fragment containing a nucleic acid fragment that encodes ... In the case of eukaryotic hosts, a promoter derived from the genome of a mammalian cell (e.g. : Metallothionine promoter, β-actin promoter, human hemoglobin promoter human creatine promoter and human muscle creatine promoter), or derived from mammalian viruses promoters (e.g., adenovirus late promoter, vaccinia virus 7.5 K promoter, SV40 promoter, cytomegalovirus (CMV) promoter promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter , HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus promoter EBV promoter and Rous sarcoma virus (RSV) promoter A polyadenylation sequence is generally used as a transcription termination sequence. Optionally, the vector may contain other sequences to facilitate purification of the antibody expressed therefrom. The sequence to be fused may be, for example, glutathione S-transferase. β-lactamase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, (Qiagen, USA) and 6xHis(hexahistidine) (Qiagen, USA). As a selection marker, antibiotic resistance genes commonly used in the art are included, for example These include ampicillin, gentamicin, carbenicillin, chloramphenicol, and sucralose. Treptomycin, Kanamycin, Geneticin, Neomycin and Tetracycline There are resistance genes against

[0102] In another aspect, the present invention provides a cell transformed with the above-mentioned vector. The cells used to produce the antibodies or antigen-binding fragments thereof disclosed in The cell may be, but is not limited to, a bacterial cell, a yeast cell or a higher eukaryotic cell. (Escherichia coli), Bacillus subtilis and Bacillus thuringiensis Bacillus species, Streptomyces, Pseudomonas nas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus aureus Prokaryotic host cells such as Staphylococcus (e.g., Staphylococcus carnosus) can be used. However, most interest has been in animal cells, and an example of a useful host cell line is COS-7. , BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV 1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BR L3A, W138, HepG2, SK-Hep, MMT, TRI, MRC5, FS4, 3 T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, Including but not limited to U20S or HT1080.

[0103] The present invention further comprises the steps of (a) culturing the cells containing the expression vector, and (b) culturing the cells. and recovering the antibody or antigen-binding fragment thereof from the cultured cells. The cells can be cultured in a variety of media, and methods for producing the cells or antigen-binding fragments thereof are also provided. Any commercially available medium can be used as the culture medium without limitation. All other necessary supplements may be included at appropriate concentrations. Culture conditions, e.g., temperature, pH, etc. These have already been used with the host cells selected for expression and are obvious to those skilled in the art. The antibody or antigen-binding fragment thereof may be recovered, for example, by centrifugation or ultrafiltration. and subjecting the resultant to, for example, affinity chromatography or the like. Additional purification techniques, such as shaping, Ion exchange chromatography or cation exchange chromatography, hydrophobic interactions For example, chromatography using silica gel, hydroxylapatite chromatography, etc. may be used.

[0104] Another aspect is a method for preventing, improving, or treating cancer comprising the antibody or an antigen-binding fragment thereof. A composition is provided, which may be a pharmaceutical composition or a health functional food. In some embodiments, the composition further comprises an immune checkpoint blockade or a chemotherapeutic agent. For example, the immune checkpoint blockade agent may be an anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, etc., and the chemotherapeutic agent may be, for example, a chemotherapy Chemotherapy drugs, tyrosine kinase inhibitors, etc. In another embodiment, the composition is administered in combination with a radiotherapeutic agent. Thus, the composition according to one embodiment may be capable of inhibiting CD73 expression in various types of cancer. It has significant anti-cancer activity and can therefore be used as a therapeutic agent for cancer.

[0105] For example, the composition may comprise: (a) a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof against CD73; and (b) a pharma- ceutical composition for preventing or treating cancer, comprising: The present invention also relates to a method for preventing or treating cancer in a subject in need thereof. The present invention also provides a method of treating a subject with a rheumatoid arthritis, the method comprising administering to a subject an antibody against CD73 according to the present disclosure. or an antigen-binding fragment thereof.

[0106] The composition comprises a monoclonal antibody or an antigen-binding fragment thereof disclosed herein. Since the two will be used interchangeably, the details common to both will be omitted.

[0107] As demonstrated in the Examples below, the antibodies or antigen-binding fragments thereof of the present invention can be used to inhibit CD4+ It binds to CD73 with high affinity and inhibits the migration of cancer cells that overexpress CD73. Therefore, the composition can be used for the prevention and treatment of cancer. "Treatment" means any action that inhibits or delays the progression of cancer. This means inhibiting the onset, reducing cancer, or eliminating cancer.

[0108] "CD73-overexpressing cancer" refers to cancers that overexpress CD73, but not CD73-overexpressing cancers. Hay, CM et al., ncoImmunol. 5: e1208875, 10 pages (2016), and Gao, Zw. et al., BioMed Res. Intl . 2014: 460654, 9 pages (2014). The disease to which the composition is applied is cancer, Cancers that overexpress CD73, such as breast cancer, colon cancer, glioblastoma, brain and spinal cord tumors, head and neck cancer, etc. Neck cancer, lung cancer, thymus cancer, esophageal cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer Round cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia In one embodiment, the cancer may be multiple myeloma, sarcoma, malignant melanoma, or skin cancer. The cancer is breast cancer, triple-negative breast cancer (TBNC), pancreatic cancer, Colorectal cancer, ovarian cancer, gastric cancer, bladder cancer, leukemia, prostate cancer, malignant melanoma, cancer , esophageal cancer, stomach cancer, head and neck cancer, lung cancer or kidney cancer.

[0109] Another embodiment is a method for treating metastasis of cancer cells comprising the antibody against CD73 or an antigen-binding fragment thereof. Alternatively, the present invention may be a composition for suppressing invasion of a subject in need thereof. The present invention provides a method for inhibiting metastasis or invasion of cancer cells, the method comprising: The method includes administering to a subject a monoclonal antibody or antigen-binding fragment thereof against

[0110] The pharma- ceutically acceptable carriers contained in the compositions of the present invention are not limited to the following: However, lactose, dextrose, sucrose, sorbitol, mannitol, Starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methylhyd. Hydroxybenzoate, Propyl Hydroxybenzoate, Talc, Magnesium Stearate The compositions of the present invention include, but are not limited to, ethyl alcohol and mineral oil. In addition to the above ingredients, the product may contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may additionally include:

[0111] The pharmaceutical composition of the present invention can be administered orally or parenterally. These include intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, topical, intranasal, and pulmonary In one embodiment, the composition can be administered intravenously, intramuscularly, or rectally. When administered orally, the protein or peptide is digested and Therefore, oral compositions should coat or otherwise protect the active agent from degradation in the stomach. The pharmaceutical composition should be formulated so as to deliver the active substance to the target cells. The composition may be administered by any device capable of administering the composition.

[0112] The appropriate dosage of the composition according to the present disclosure depends on the formulation method, the mode of administration, and the age, weight, and sex of the patient. The drug may be administered depending on factors such as the patient's condition, pathological condition, food and drink, time of administration, route of administration, rate of excretion, and sensitivity to certain drugs. Therefore, the dosage will vary and a skilled physician will generally determine an effective dose for the desired treatment or prophylaxis. For example, the daily dosage of the pharmaceutical composition of the present invention can be determined and formulated easily. 0.0001 to 100 mg / kg of elephant body weight, 0.001 mg to 50 mg / kg of elephant body weight, 0.01 mg to 25 mg, 0.1 mg to 10 mg, 1 mg to 5 mg, or As used herein, the term "therapeutically effective amount" refers to an amount that is effective to prevent or treat cancer. means an amount sufficient to treat.

[0113] The pharmaceutical composition of the present invention can be easily produced by a person skilled in the art in the technical field to which the present disclosure pertains. The pharmaceutical composition may be formulated using pharma- ceutically acceptable carriers and / or excipients by the method described above. It may be prepared in unit dose form or in multi-dose containers. In this case, the dosage form is in the form of a solution, suspension or emulsion in an oily or aqueous medium. or in the form of an extract, powder, suppository, powder, granule, tablet or capsule. The composition may further comprise a dispersing agent or a stabilizing agent.

[0114] The compositions of the invention may be administered as individual therapeutic agents or in combination with other therapeutic agents. The therapeutic agent may be administered sequentially or simultaneously with an existing therapeutic agent.

[0115] In the health functional food for preventing or improving cancer according to the present disclosure, When using the original binding fragments as additives in health functional foods, should they be added as they are? or together with other foods or food additives, and may be added by a conventional method. The amount of the active ingredients mixed may be adjusted according to the purpose of prevention, health or treatment. It can be appropriately determined depending on the purpose.

[0116] The dosage form of health functional foods is not only powder, granule, pill, tablet, and capsule, Any of the common food or drink forms are possible.

[0117] The type of food is not particularly limited, and examples of foods to which the substance can be added include These include meat, sausages, bread, chocolate, candy, snacks, confectionery, Pizza, ramen and other noodles, gum, dairy products including ice cream, various soups, Beverages, teas, energy drinks, alcoholic beverages and vitamin complexes are some of the general meanings. It can include any food in the world.

[0118] In general, when producing a food or beverage, the above-mentioned antibody or antigen-binding fragment thereof is added to the raw material 1. 00 parts by weight, it should be added in an amount of 15 parts by weight or less, specifically, 10 parts by weight or less. However, long-term measures for health and hygiene purposes or for health regulation purposes may be In the case of ingestion over a period of time, the amount may be less than the ranges mentioned above, and the present invention is also directed to the use of natural products. Since there is no problem in terms of safety in using these fractions, It can be used in any amount.

[0119] The beverage in the health functional food according to the present invention can be flavored with various flavoring agents or flavorings, just like a normal beverage. The natural carbohydrates may be, for example, bromine, glycerol, sorbitol, sorbitol derivatives ... Monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; Polysaccharides such as dextrin, cyclodextrin; or xylitol, sol Sweeteners can be sugar alcohols such as sorbitol and erythritol. Natural sweeteners such as stevia extract and synthetic sweeteners such as saccharin and aspartame The proportion of natural carbohydrates in the beverage of the present invention is preferably 100%. It can be about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per mL. .

[0120] In addition to the above-mentioned ingredients, the health functional food for preventing or improving cancer according to the present invention contains various Nutrients, vitamins, electrolytes, flavorings, colorings, pectinic acid and its salts, alginic acid and and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin In addition, the present invention may include carbonation agents used in alcoholic and carbonated beverages. The cancer improving composition disclosed herein is for the production of natural fruit juices, fruit juice drinks and vegetable drinks. Such ingredients may be used individually or in admixture. The ratio of such additives is not limited, but may be any ratio other than that of the functional health food of the present invention. In general, the amount is selected in the range of 0.01 to 0.1 parts by weight relative to 00 parts by weight. do.

[0121] Other aspects include a composition for diagnosing cancer comprising the above-mentioned antibody or its antigen-binding fragment, and a method for diagnosing cancer comprising the same. The present disclosure also provides a kit for diagnosing cancer, comprising an antibody or a kit for diagnosing cancer according to the present disclosure. The present invention relates to a method of diagnosing cancer by treatment with the antigen-binding fragment.

[0122] The expression level of CD73 in a sample can be determined by an antibody against CD73 according to the present disclosure. The expression level can be measured by a general immunoassay method to diagnose cancer. The CD73 antibody can be used for radioimmunoassay, Radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent) ent assay), capture ELISA, inhibition or competitive assay, sandwich assay , which can be measured via flow cytometry, immunofluorescence staining, and immunoaffinity purification. The present invention is not limited to the above. By analyzing the intensity of the final signal, cancer can be diagnosed. In the biological sample, the proteins of the markers of the present disclosure are highly expressed, and the signal is normal biological If the sample shows a stronger positive result than the sample (e.g., normal stomach tissue, blood, plasma, or serum), it is considered to be cancer. Diagnosed.

[0123] Another aspect provides a cancer diagnostic kit comprising the cancer diagnostic composition. The antibody against CD73 according to the disclosure is indicated by the reaction of the antibody with the sample. The signal can be analyzed to diagnose cancer. Enzymes such as alkaline phosphatase, β-galactosidase, horseradish Also contains peroxidase, luciferase or cytochrome P450 However, the present invention is not limited to this. In this case, alkaline phosphatase is used as the enzyme. When used, the substrates are bromochloroindolyl phosphate (BCIP), NBT, naphthol-AS-B1-phosphate ol-AS-B1-phosphate) and colorimetric reaction substrates such as ECF (enhanced chemifluorescence) is used, and when horseradish peroxidase is used, chloronaphtho carbazole, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (Bis-N-methylacridinium nitrate), Resorufin benzyl ether, Lumi Nal, Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine ), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenz idine), ABTS(2,2'-azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylene OPD and naphthol / pyronine, glucose oxidase, t-nitrilo Neoblue tetrazolium (NBT) and m-PMS (phenzaine methosulfate) Substrates may be utilized, but are not limited to these.

[0124] The kit may also include a label that produces a detectable signal, the label being a chemical Chemical substances (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase and cytochrome P450), radioactive materials (e.g. C14, I125, P32 and S35), fluorescent substances (e.g., fluorescein), luminescent substances chemiluminescent and FRET (fluorescence resonance energy These may include, but are not limited to, immunological transfer. The measurement of the enzyme activity or signal can be performed by a variety of methods known in the art. Through such measurement, CD73 expression can be analyzed qualitatively or quantitatively.

[0125] Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings, in which: The reference numerals denote similar elements. In this regard, the present embodiment may have different forms. The description given in this specification should not be construed as being limited to the above. For purposes of illustrating aspects of the present disclosure, embodiments will be briefly described with reference to the following. As used herein, the term "and / or" refers to the optional association of one or more of the associated listed items. Any combination of the above is included. Such an expression modifies the entire component list and not any individual element of the list. EXAMPLES

[0126] In the following, exemplary embodiments are presented to aid in understanding the present disclosure. The following exemplary embodiments are merely provided to make the present disclosure more readily understandable. However, the contents of the present disclosure are not limited to the following exemplary embodiments.

[0127] [Preparation Example 1. Selection of anti-CD73 antibodies by biopanning]

[0128] 1-1.Selection of phage antibodies that specifically bind to CD73

[0129] In order to select antibody clones that specifically bind to human and mouse CD73 proteins, Biopanning was performed using the HuDVFab-8L phage antibody library. First, recombinant human CD73 protein (Sino Biological, China) and recombinant mouse CD7 The three proteins (Sino Biological) were each purified using Dynabeads M-280 Tosylactivated beads. The reaction was performed at 4°C for 48 hours. The beads to which each CD73 protein was bound were DVFab-8L (HuDVFab-L1~L6, L8, L12) recombinant antibody (pharmaceutical Blocking factor containing Fab library displayed on the coat protein pIII Blocking buffer: 3% skim milk, 0.1% Tween-20 The cells were then treated with phosphate-buffered saline (PBS) and incubated at 4°C for 16 hours. Then, the recombinant phages nonspecifically bound to the CD73 protein were isolated by the Ki The phages that specifically bind to the CD73 protein were removed using an ngFisher 1 mL device. After treatment with 0.2 M glycine (pH 2.2) buffer and elution, The antibody library was neutralized by treatment with is-HCl (pH 9.0) buffer. TG1 cells (Amersham Pharmacia Biotech, Sweden) transfected with the siRNA gene were The cells were treated with the purified phages and infected at 37°C for 1 hour. LB / ACT solid medium (50 μg / ml ampicillin, 10 μg / ml Carbenicillin and 10 μg / ml tetracycline e) was smeared on LB medium containing TG and incubated at 27°C for 16 hours. 1. Remove the cells. Add 10 ml of liquid medium to the plate containing the cultured colony. After that, 4.8×10 8 The cells were suspended and diluted to a concentration of 37 cells / ml. Ex-phage was added and the cells were incubated for 37 The TG1 cells were infected at 4°C for 1 hour. The infected TG1 cells were then centrifuged. The cells were allowed to settle, and the supernatant was removed. The cells were then cultured in 5 ml of 2xYT / ACTKA medium (50 μg / ml Ampicillin, 10 μg / ml carbenicillin, 1 0 μg / ml tetracycline, 50 μg / ml kanamycin TG1 cells were cultured in 2xYT medium containing 0.001% arabinose. The cells were resuspended and incubated at 27°C for 6 hours. Then, the cells were centrifuged to separate the fibronectin. The supernatant containing the CD73 protein was collected and then transferred to Dynabeads M-280 T The mixture was reacted with osylactivated beads at 4°C for about 20 hours. The process was repeated three times to select phage antibodies that specifically bind to CD73. As a result, the HuDVFab-8L phage antibody library was used to identify L2, L4, L6, and L8 light chains. A polyclonal antibody was selected that binds to CD73.

[0130] 1-2. Selection of monoclonal phage antibodies that specifically bind to CD73

[0131] Among the polyclonal antibodies selected in 1-2 above, Monoclonal antibodies with different heavy chain CDR (HCDR) sequences were isolated from each other using phage EL Selection was carried out by performing phage enzyme-linked immunosorbent assay (ISA). Recombinant human CD73 protein and recombinant mouse CD73 protein in coating buffer (Coating buffer) (0.1M NaHCO3, pH 9.6) diluted to 2μg / ml Then, the wells were placed in a 96-well Maxisorp ELISA plate (Nunc, Denmark). 50 μL was dispensed per plate and incubated at 4°C for 16 hours to infect the CD73 protein onto the plate. The uncoated proteins and buffer were completely removed, and the blocking solution was applied. The mixing buffer was dispensed at 200 μL per well and reacted at 37° C. for 1 hour. After 1 h, the buffer was completely removed and the cells were resuspended in PBS containing 0.1% Tween-20 (PBS-T). 200 μL of buffer was dispensed into all wells, and such washing process was repeated three times. The phage-containing supernatant was diluted 1 / 10 with blocking buffer and added to each well. The mixture was then dispensed in 50 μL portions and reacted at 37°C for 1 hour. The cells were washed using the method described above, and then goat anti-M13 HRP (horseradish peroxidase) antibody (GE Healthcare (Chicago, Illinois) and incubated at 37°C for 1 hour. After washing, all wells of the plate were filled with TMB (3,3',5,5'-tetramethylbenzidine). (BD Biosciences, Franklin Lakes, NJ) Substrate was dispensed in 50 μL aliquots. Then, the plates were read using a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, Calif.). The CD73-specific monoclonal antibody was detected by measuring the absorbance at 450 nm using a NIA instrument. Fourteen types of general phage antibodies were selected.

[0132] FIG. 1 shows the CD73 binding ability of anti-CD73 antibodies selected via biopanning. This is a graph. As shown in Figure 1, all phage antibodies except L2-20 bound to human CD73. In addition, L2-47, L4-66, L6-1, L6-30, and L6-37 phages The antibody binds to mouse CD73 with binding activity that is 50% or more of that of human CD73. The L8-34 phage antibody bound to CD32B protein, not CD73. Among 14 antibodies with different HCDR sequences, The 12 anti-CD7 antibodies were selected from the 12 antibodies except for L2-20, which showed weak binding, and L8-34, which showed nonspecific binding. The three phage antibodies were used in subsequent experiments.

[0133] [Preparation Example 2. Production and selection of human anti-CD73 IgG1 antibodies]

[0134] 2-1. Preparation of anti-CD73 human IgG1 isotype expression vector

[0135] The 12 anti-CD73 phage antibodies selected in Preparation Example 1 above were cloned and Specifically, the phage antibodies selected in Preparation Example 1 were used to prepare IgG1 antibodies. Body V L (Light chain variable region) gene sequence and V H (heavy chain variable region) gene sequence and human Ig G1 (immunoglobulin G1) isotype C L (Light chain constant region) gene sequence and CH1- CH2-CH3 (constant heavy chain, light chain constant region) gene sequence and The pdCMV-dhfr expression vector (provided by Professor Hyo Jeong Hong of Kangwon University) was cloned into The polymerase chain reaction was performed by scanning the IgG1 fragments, thereby preparing the intact IgG1 form (Figure 2A). The reaction (PCR: polymerase chain reaction) was performed using Pyrobest DNA polymerase (Takara The reaction was carried out using a thermal cycler (T100, T100, Japan) and a 30-μL PBS. In order to clone each light chain sequence of the phage antibody selected in Preparation Example 1, Using SEQ ID NO: 47 and SEQ ID NO: 48, 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 20 seconds PCR was carried out under the conditions described above to amplify the light chain leader sequence. V of the phage antibody selected in Preparation Example 1 L From the gene, sequence numbers 49 and 50 were used. By performing PCR using L Each gene in the sequence was amplified. The PCR products were assembled at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 60 seconds. - Polymerase chain reaction (assembly PCR) was used to fuse the PCR product and pd The CMV-dhfr vector was transformed with the vector cloning at 37°C and 55°C using HindIII and BsiWI (Takara ) and reacted with each other. - Array V L The gene fragment and the pdCMV-dhfr vector were ligated using T4 DNA ligase. 1. Igation and heat shock to CaCl2-treated competent cells Next, the heavy chain sequence of the phage antibody selected in the above Preparation Example 1 was cloned. To perform scanning, SEQ ID NO: 51 and SEQ ID NO: 52 were used to identify overlapping regions containing the EcoRI sequence. The leader sequence of the ApG strand was amplified by PCR using SEQ ID NO:53 and SEQ ID NO:54. V containing aI sequence H Each gene in the sequence was amplified by PCR. The two amplified PCs The R products were fused via assembly PCR reaction (leader-sequence V H ), EcoRI system The DNA was digested with restriction enzymes ApaI and ApaI in the same manner as above. Leader sequence-V H The gene fragment was cloned into the pdCMV-dhfr vector. , and there is light chain (V L -C L ) gene and CH1-CH2-CH3 genes to create a human An IgG1 expression vector was prepared.

[0136] CPI-006 (Corvus Pharmaceuticals, Burlingame, CA) was used as a positive control. game) and MEDI9447 (MedImmune, Gaithersburg, MD) heavy chain genes, light The chain genes (WO2018 / 013611, WO2016 / 075099) were obtained from Cosmogenet The light chain restriction enzymes were HindIII (Takara) and XbaI (Takara), and The heavy chain restriction enzymes were EcoRI (Takara) and NotI (Takara), and the same procedure was repeated as described above. The same method was used to clone into the pdCMV-dhfr vector. [Table 1]

[0137] 2-2. Production and purification of anti-CD73 human IgG1 isotype antibody

[0138] To produce the 12 selected anti-CD73 human IgG1 antibodies, The cloned pdCMV-dhfr vector was transfected with ExpiCHO-S™ (Gibco Specifically, ExpiCHO-S™ cells were transfected with Exp iCHO-S™ Expression Medium (Gibco, ThermoFisher Scientific) was used at 37° C. The cells were incubated in a shaking incubator at 140 rpm, 80% humidity, and 5% CO2. ExpiCHO-S™ cells that had been subcultured at least three times were incubated at 4°C for 1 h. The vectors containing the 12 selected antibody genes were then transfected into the cells using ExpiFectamine CHO Tran. The sfection kit and OptiPRO™ SFM (1x) (Gibco) were used to Afterwards, the cells were incubated for 20 hours in a shaking incubator under the same conditions as above. The incubated cells were then cultured with ExpiFectamine™ CHO feed and Treat with enhancer and incubate for 7 days under the same conditions as above. The culture medium was collected and centrifuged at 4,000 rpm for 15 minutes at 4°C to extract the protein sample. The supernatant was filtered through a 0.2 μm filter paper to remove impurities. The antibody sample produced by the CHO cells was then purified by affinity chromatography. Specifically, the CaptureSelect IgG-CH1 Affinity Matrix resin was used to remove impurities. The resin was incubated at 4°C for 16 hours to allow the antibody sample in the culture medium to bind to the resin. The resin was then washed with 10 column volumes (CV) of TBS buffer (pH 7. 4) and then wash the column with 100 mM citrate buffer (pH 3.0) to elute the antibody sample. Weakly acidic (pH 6.0-6.0) is achieved by adding 1 M Tris-HCl buffer (pH 9.0). The neutralized buffer was filtered through a 0.2 μm filter paper to remove impurities. Afterwards, the proteins were quantitatively analyzed at UV wavelength of 280 nm.

[0139] 2-3. Size analysis of anti-CD73 human IgG1 isotype antibody protein

[0140] The size of the antibody protein produced and purified in 2-2 above was analyzed by SDS-PAGE. First, the antibody protein was diluted with Pierce LDS buffer containing 2-mercaptoethanol. After dilution with sample buffer and non-reducing buffer (4x; ThermoFisher Scientific), The samples were then heated for 10 min. The protein samples were then diluted with 4-15% Mini-PROTEAN® TGX 1 μg per well in a Bio-Rad Precast Protein Gel gradient. After loading, run at 140V for 40 minutes using a PowerPac™ Basic Power Supply. After electrophoresis, the gel was stained with Brilliant Blue R 250 protein staining solution (EL After incubation for 60 min in destaining buffer [30% methanol, 10 % acetic acid] and protein bands were observed.

[0141] FIG. 2B shows the results of SDS-PAGE analysis of human IgG1 antibodies. As shown in FIG. 2B, the heavy and light chains of the 12 purified antibodies were each 50 The protein bands showed sizes of 25 kDa and 25 kDa, both of which corresponded to the theoretical sizes. was observed at the corresponding position.

[0142] 2-3. Confirmation of CD73 binding ability of anti-CD73 human IgG1 isotype antibody

[0143] CD73 of 12 types of human anti-CD73 IgG1 antibodies produced and purified in 2-2 above To compare the binding ability, ELISA experiments were performed. Recombinant human CD73 protein was After diluting to a concentration of 1 μg / ml in carbonate coating buffer, the plate was plated in a 96-well Maxiso Dispense 100 μL per well into an rp ELISA plate and coat at 4°C for 16 hours. The uncoated proteins and buffer were completely removed, and the gel was washed with 3% BSA. PBS buffer (pH 7.4) containing 0.1% Tween-20 was added to each well at 300 ml. After dispensing each aliquot in μL, the reaction was allowed to proceed at room temperature for 2 hours, and blocking was performed. The buffer was removed, and 300 μL of PBS-T buffer was dispensed into each well. The anti-CD73 IgG1 antibody was then washed three times with PBS buffer. The solution was diluted with 100 nM of 100 mM sodium dodecyl sulfate, added to the wells, and incubated at 37°C for 1 hour. Donkey anti-human IgGFcHRP antibody (Jackson ImmunoResearch, West Grove, Baltimore, MD) ke) was added and reacted at 37°C for 1 hour. After carrying out the same washing process as above, 100 μL of TMB substrate was dispensed into each well and reacted for 7 minutes. Model 680 Mi The absorbance at 450 nm was measured using a croplate reader. The positive control (CPI-006, MEDI9447) samples were also The absorbance was measured in the same manner as in the previous example.

[0144] FIG. 3A shows the human CD73 binding ability of 12 purified human anti-CD IgG1 antibodies. This is a graph. As shown in Figure 3A, 9 of the 12 purified antibodies and 2 positive controls The group bound significantly more strongly to human CD73 than to BSA, and the remaining three antibodies (L4-46 , L6-7, and L6-37) bound relatively weakly. The IgG1) antibody did not bind to human CD73.

[0145] 2-4. Confirmation of enzyme activity of membrane CD73 protein

[0146] Membrane CD7 of 12 types of human anti-CD73 IgG1 antibodies produced and purified in 2-2 above To compare the inhibitory effects of the three enzymes on the cell surface, we used MDA-MB- Malachite green assay was performed with 231 cells (Korea Cell Line Bank). MDA-MB -231 cells, 2.0 × 10 4 96-well flat plate with a cell / well concentration of Incubate in a 37℃, 5% CO2 incubator for 20 hours. After incubation, the plate was washed twice with assay buffer and incubated with 12 types of human anti-C D73 IgG1 antibody sample was diluted in assay buffer at a concentration of 500 nM and then added to each well. Then, 250 μM AMP was added to each well and incubated at 37°C for 1 hour. The mixture was incubated at 37°C for 20 minutes. 40 μL of the supernatant was then added to a new 96-well plate. To measure the enzyme activity of CD73, Malachite Green Phosphatase was used. Phosphate was detected in the supernatant using the Epoch Microplate Detection Kit. The absorbance was measured at a wavelength of 620 nm using a 50% spectrophotometer. Regarding the (rituximab) samples and positive control (CPI-006, MEDI9447) samples The absorbance was measured in the same manner as described above.

[0147] Figure 3B shows that 12 purified human anti-CD73 IgG1 isotype antibodies inhibited CD7 These are the results of an investigation into whether or not the enzyme activity is inhibited. As shown in Figure 3B, five of the 12 anti-CD73 human IgG1 antibodies (L2-3, L4-4, L4-5, L4-66 and L6-1) were 5% higher than the negative control. 0% or more of CD73 enzyme activity inhibition, of which L6-1 antibody inhibited more than 90% of CD It showed inhibition of 73 enzyme activities, with high inhibitory potency similar to that of CPI-006 (91%). The remaining four antibodies (L2-3, L4-4, L4-5, and L4-66) accounted for 63% and 61%, respectively. , 58%, and 50% inhibition of CD73 enzyme activity, respectively, which was similar to MEDI9447 (34%). or even greater inhibitory potential.

[0148] 2-5. Confirmation of CD73 cross-binding ability of anti-CD73 human IgG1 isotype antibody

[0149] In the above 2-3, the inhibition of CD73 enzyme activity was as high as or higher than that of the positive control group. Five human anti-CD73 IgG1 antibodies (L2-3, L4-4, L4-5, L4 -66, L6-1) of human, monkey, rat and mouse CD73 proteins (Sino Biologica l) To compare the cross-linking ability of the antibody, an ELISA experiment was performed in the same manner as in 2-3 above. was carried out.

[0150] Figure 3C shows the results of analysis of the cross-linking ability to human, monkey, rat, and mouse CD73. The results are shown. Figure 3C shows that the L4-66, L6-1 and MEDI9447 antibodies were expressed in humans, monkeys, rats and Both antibodies reacted with mouse CD73 and L2-3, L4-5, and CPI-006. The antibody reacted only with human CD73 and monkey CD73, whereas the L4-4 antibody reacted only with human CD73 and monkey C They reacted only with D73 and rat CD73, thus showing different binding patterns. Based on the above results, the antibody cross-reacts with human CD73 and mouse CD73, similar to the MEDI9447 antibody. Two antibodies (L4-66 and L6-1) that bind to the antibody were selected, and the antibodies were compared with APBA2-01 and and named it APBA2-02.

[0151] [Preparation Example 3. Preparation of APBA2-01 antibody and APBA2-02 antibody]

[0152] 3-1.APBA2-01 monoclonal antibody and APBA2-02 monoclonal antibody Preparation of

[0153] The APBA2-01 monoclonal IgG antibody selected in Preparation Example 2-5 above and AP We have established a stable cell pool expressing the BA2-02 monoclonal IgG antibody. First, the APBA2-01 monoclonal antibody and the APBA2-02 monoclonal antibody were produced. The light and heavy chain genes of the null antibody were cloned into pD2539 and pD2535nt( The antibody was cloned into a vector from Horizon Discovery, UK. APBA2-01 antibody and V of APBA2-02 antibody L ,V H Genes and human IgG isotype C L ,C H The gene was then transformed into Chinese hamster ovary (CHO) cells. The light chain gene of the APBA2-01 antibody was synthesized with a codon sequence optimized for , Bbs1 (ThermoFisher Scientific) restriction enzyme and BsrG1 (ThermoFisher Scientific) ic) digested with restriction enzyme and ligated into pD2539 vector digested with the same restriction enzyme The V of the synthesized APBA2-01 antibody was H The gene is SEQ ID NO: 51 in Table 1, and The C of human IgG was amplified by PCR using SEQ ID NO:55 in Table 2. H The genes are listed in Table 2 below. The PCR products were amplified using SEQ ID NO: 56 and SEQ ID NO: 57. The products were fused via assembly PCR. The vector was treated with Bbs1 restriction enzyme and reacted. V L The gene was amplified by PCR using SEQ ID NO: 47 in Table 1 and SEQ ID NO: 58 in Table 2. The CL gene of human IgG was synthesized by PCR using SEQ ID NO: 59 and SEQ ID NO: 60 in Table 2 below. The APBA2-02 antibody was amplified withH is SEQ ID NO: 51 in Table 1 and SEQ ID NO: 6 in Table 2 The CH gene of human IgG was amplified by PCR using SEQ ID NO: 57 in Table 2 below. The PCR products were amplified using column number 62. The fusion was performed via assembly PCR reaction. ng. [Table 2]

[0154] 3-2. Preparation of APBA2-01 and APBA2-02 chimeric antibodies

[0155] The APBA2-01 and APBA2-02 antibodies were L Gene sequence and human V H Gene sequence and mouse antibody sequence C L Sequence and C H Human and mouse IgG with sequences The APBA2 chimeric antibody sequence and the IgG2A chimeric antibody sequence were prepared. -01 and APBA2-02 human V L Mouse C L The chimeric antibody gene fragment was transformed into pD253 The synthesized mouse I was inserted into the 9 vector in the same manner as in 3-1 above and cloned. The CH genes of mouse IgG1 and mouse IgG2A are SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively. The DNA was amplified by PCR using sequence number 64 and sequence number 65. Then, the DNA was amplified in the same manner as in 3-1. The VH gene was fused to the VH gene of the APBA2-01 antibody by assembly PCR reaction. The fused APBA2-01 antibody gene and the synthesized APBA2-02 antibody V H Mau The mouse IgG1 gene fragment and the mouse IgG2A gene fragment were inserted into the pD2535nt vector. , and cloned in the same manner as described above.

[0156] 3-3. Preparation of stable pools of APBA2-01 and APBA2-02 antibody proteins

[0157] A stable pool producing the antibody proteins of the above Preparation Examples 3-1 and 3-2 was prepared. First, GS nullCHO K1 (Horizon Discovery) cells were cultured with CDfortiCHO (T HermoFisher Scientific) was supplemented with 4 mM L-glutamine (Gibco). The cells were then placed in a shaking incubator at 37°C, 125 rpm, 80% humidity, and 5% CO2. The light chain genes of APBA2-01 and APBA2-02 were The vector into which the DNA strand genes were inserted was incubated with Freestyle™ MAX Reagent (Invitrogen). The plasmid pD2539 was used in the culture at a ratio of 1:3 [pD2539 (light chain):pD2535nt (heavy chain)]. A total of 37.6 μg of plasmid vector was used to transfect the cells. The cells were cultured for 48 hours under the same conditions as above. Then, the cultured cells were After transferring to a conical tube (Nunc), the mixture was centrifuged and L-glutamine was extracted. Resuspend the cell pellet in CDfortiCHO medium without added menthol and 50 μM menthol. Methionine sulfoximine (MSX, Sigma-Aldrich, St. Lewis, Missouri) for 48 hours. The cells were treated with puromycin (Gibco) for 48 hours for secondary selection. The cells were cultured at a concentration of 0.5 × 10 6L-glutamine was added to obtain 100 cells / ml. After suspending the cells in a medium containing no MSX, they were treated with MSX and puromycin for 3 weeks. Further selection was carried out. At this time, the cell concentration was 2.0 × 10 6 Maintain below 100 cells / ml The medium was replaced periodically to maintain cell viability at 90% or higher. The cells were cultured until 100% yield and a stable pooled cell stock was prepared. Viability was measured using the COUNTESSII automated cell counter.

[0158] 3-4. Production and purification of APBA2-01 and APBA2-02 chimeric antibody proteins

[0159] From the stable pool prepared in 3-3, APBA2-01 and APBA2-02 The human-mouse chimeric antibody protein was produced. 6 cell / The cells were suspended in CDfortiCHO medium to a total volume of 1 ml, and incubated at 37°C for 125 min. The cells were incubated in a shaking incubator at 37 °C for 20 min at 20 °C, with 80% humidity and 5% CO2. After incubation, the cell concentration was 2.0 × 10 6 When cells / ml was reached, 32°C, 125 rpm The plates were then transferred to a shaking incubator at 80% humidity and 5% CO2 to incubate the antibody samples for 7 days. The supernatant containing the antibody sample was collected by centrifugation and filtered using a 0.2 μm filter. The antibody sample produced from the CHO cells was then filtered through a filter paper to remove impurities. Chromatography steps (affinity chromatography, cation exchange purification, anion exchange purification) First, the parent protein was purified using MabSelect SuReLX resin. A affinity chromatography was performed using 10 CV of tris-buffered saline (TBS). After washing the resin with the saline buffer, the supernatant containing the antibody sample was added at 20 ml / min. The resin was passed through at a flow rate of 5 CV of TBS buffer and 5 CV of 3% D-mannitol. The non-specific binding to the resin was removed by flowing TBS buffer containing 100% ethanol at a flow rate of 25 ml / min. The eluted material was removed. The buffer was run at a flow rate of 20 ml / min to wash the antibody sample specifically bound to the resin. The buffer containing the eluted protein sample was then diluted with 1M tris-HCl. (pH 8.0) buffer to neutralize, and then filter through 0.2 μm filter paper to remove impurities. Next, the column was equilibrated with a buffer of 50 mM tris and 200 mM citric acid (pH 7.0). Cation exchange purification was performed using Capto adhere ImpRes multimodal chromatography resin. The antibody sample obtained from affinity chromatography was diluted to 1 / 4 with sterile distilled water. The antibody sample was then bound to the resin at a flow rate of 5 ml / min, and then diluted with 50 mM t The column was washed with 5 CV of ris, 200 mM citric acid, 1 M NaCl (pH 7.0) buffer. Then, add 5 CV of 50 mM tris, 200 mM citric acid (pH 7.0) buffer and add 25 ml The resin was washed at a flow rate of 50 μl / min to remove non-specifically bound substances. M tris, 200 mM citric acid (pH 3.0) buffer at 20%, 30%, 40%, 50 %, 70%, and 100% were run in sequence to elute the antibody sample that was specifically bound to the resin. The eluate from each step was collected and filtered through 0.2 μm filter paper to remove impurities. Anion exchange purification was performed using POROS™ 50 HQ Strong Anion Exchange resin. First, wash the resin with 2M NaCl buffer at a flow rate of 3 ml / min, then rinse the resin with 20 ml of NaCl buffer. The column was equilibrated with 5 CV of M sodium phosphate buffer (pH 6.5). To bind the purified antibody sample to the resin, the sample was diluted with 20 mM sodium phosphate. The pH and salt concentration were adjusted by dialysis against sodium thorium buffer (pH 6.0). The sample was passed through the resin at a flow rate of 5 ml / min, and the buffer containing the collected proteins was then added at 0.2 ml / min. The mixture was filtered through a .mu.m filter paper to remove impurities, and the protein was quantified and analyzed.

[0160] [Example 1. Comparison of binding ability to CD73 protein]

[0161] 1-1. Binding ability to water-soluble CD73 protein

[0162] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 The binding ability of the antibody to the soluble CD73 protein was confirmed through an ELISA experiment. The solution was added in a serial dilution ranging from 1,500 ng / ml to 0.019 ng / ml. The experiment was carried out in the same manner as in 2-3 above, except that the positive control (MEDI944 7, CPI-006) and the negative control (rituximab) were subjected to the same procedure.

[0163] Figures 4A to 4D show the APBA2 binding to human, monkey, rat, and mouse CD73, respectively. 1 is a graph confirming the binding ability of -01 antibody and APBA2-02 antibody. As shown in FIG. 4, for human CD73 protein (A), MEDI9447>A The order of binding ability was PBA2-01>APBA2-02>CPI-006, and monkey CD73 For protein (B), MEDI9447=APBA2-01>CPI-006>AP In addition, in the case of rat CD73 protein (C), The binding ability was highest in the order of MEDI9447>APBA2-02>APBA2-01. In the case of CD73 protein (D), MEDI9447>APBA2-01>APBA2 The binding ability is shown in the order of APBA2-01 to MEDI9447. It showed the second highest CD73 protein (human, monkey and mouse) binding ability after APBA. 2-02 showed relatively low CD73 binding compared to MEDI9447 and APBA2-01. The binding ability of CPI-006 was similar to that of CPI-006.

[0164] 1-2. Binding ability to membrane CD73 protein

[0165] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 We examined whether the CD73 protein binds to the membrane by flow cytometry. First, we investigated the effect of human CD73-overexpressing breast cancer cell line MDA-MB-231 on the expression of CD73. (Korea Cell Line Bank) and 4T1 (ATC C) and 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin RPMI-1640 medium (ThermoFisher) supplemented with rifampicin and streptomycin (Gibco) The plates were then placed in a humidified incubator (Humid Scientific) at 37°C and 5% CO2. The number and viability of the cells were determined using a Vi-cell automized incubator. The measurements were performed using a quantified cell analyzer. MDA-MB-231 cells and 4T1 cells were Put 2.0 x 10 5 Aliquot cells at density and MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) twice. Then, APBA2-01 antibody and APBA2-02 antibody were added at 40 nM concentration in MACS buffer. The mixture was diluted in 100 mL and used to treat DA-MB-231 and 4T1 cells, respectively. The cells were incubated at 4°C for 1 hour. After 1 hour, the cells were washed three times with MACS buffer and incubated with goat Anti-human IgG Fc FITC antibody (Invitrogen) was added and reacted at 4°C for 30 minutes. MDA-MB-231 and 4T1 cells were washed and incubated with 1% hol The cells were fixed by treatment with methylaldehyde and then analyzed using a BD FACSVERSE instrument. Flow cytometry was performed using positive controls (CPI-006, MEDI9447) and The negative control (rituximab) was also analyzed in the same manner.

[0166] FIG. 5A shows the results of the antibody APBA2-01 and APBA2 in MDA-MB-231 cells. FIG. 5B is a graph comparing the binding ability of the -02 antibody to the membrane CD73 protein. 1 is a graph comparing the binding ability in T1 cells. As shown in Figures 5A and 5B, MDA-MB-231 cells and 4T1 cells In the order MEDI9447>APBA2-01>APBA2-02>CPI-006 The MEDI9447 antibody, APBA2-01 antibody and A The PBA2-02 antibody showed stronger binding ability than the negative control (rituximab). However, the positive control CPI-006 antibody did not show any significant differences with the MEDI9447 antibody, APBA2 It showed weaker binding ability compared to the -01 antibody and APBA2-02 antibody.

[0167] Example 2. Comparative analysis of epitopes

[0168] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 and positive controls (CPI-006, MEDI9447), CD73 epitopes were detected by BLI. First, we performed a comparative analysis using anti-human IgGFc Capture (AH C) The chips were hydrated for 15 minutes in sterile distilled water, supplemented with 0.1% BSA. The solution was prepared using PBS (pH 7.4) (0.1% PBA) buffer and 10 mM glycine. ) (pH 1.7) buffer for 20 seconds each, three times. Next, the positive control CPI-006 antibody was used. The antibody and MEDI9447 antibody were diluted to a concentration of 200 nM in 0.1% PBA buffer. Each was loaded onto the AHC chip for 600 seconds. Recombinant human CD73 protein sample diluted to a concentration of 20 nM in PBS was reacted for 900 seconds. Finally, the APBA2-01 and APBA2-02 samples were mixed with 0.1% Recombinant human CD73 protein was bound to the antibody at a concentration of 20 nM in PBA buffer. The reaction was performed for 900 seconds with CPI-006. All experimental procedures were performed at 30°C and 1,000 r The experiment was performed under shaking conditions, and the results were analyzed using ForteBio Data analysis 8 software. The data were analyzed using software.

[0169] FIG. 6A shows that the APBA2-01 and APBA2-02 antibodies bind to the epitope of CPI-006. Figure 6B shows the results of an analysis of whether MEDI94 binds competitively to the peptide. The results of an analysis of whether or not the antibody competitively binds to 47 epitopes are shown. As shown in Figure 6A, no antibodies sharing an epitope with CPI-006 were observed. As shown in FIG. 6B, the positive control (MEDI9447, CPI- 006) and APBA2-01 antibodies did not bind to human CD73 protein, but APB The A2-02 antibody bound.

[0170] As a result, APBA2-0 was detected on the human CD73 protein bound to the MEDI9447 antibody. 1 antibody was not able to bind to MEDI9447. It was suggested that it may be possible for the antibody to competitively bind to an epitope similar to that of the antibody. On the other hand, the CPI-006 antibody showed no binding to the MEDI9447 antibody, unlike the results in Figure 6A. CPI-006 was unable to bind to the human CD73 protein, which is a target of IL-1. , which binds to an epitope at the C-terminus where AMP binds to CD73, and thus competes with AMP. MEDI9447 inhibits the enzyme activity of CD73 by binding to the homodimer C It binds to the N-terminal domain of CD73, preventing it from transitioning to its active conformation and inhibiting CD73 activation. 73 enzyme activity, thus showing a similar inhibitory pattern to MEDI9447. The APBA2-01 antibody is predicted to inhibit the transition of CD73 to its active conformation. The APBA2-02 antibody, which shows a similar inhibitory pattern to PI-006, inhibits AMP It is predicted to competitively inhibit binding to CD73.

[0171] [Example 3. Evaluation of in vitro CD73 enzyme activity inhibitory activity]

[0172] 3-1. Evaluation of inhibitory activity against soluble CD73

[0173] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 In order to confirm whether or not soluble CD73 protein inhibits its enzymatic activity, Malachite green assay was performed to quantify phosphate. Recombinant human CD73 protein was incubated in assay buffer (25 mM tris, 5 mM MgCl2, p The APBA2-01 antibody sample and the APBA2-02 antibody sample were diluted to a concentration of 2 nM with 50 mL of 10 ... The samples were added to the assay buffer at concentrations ranging from 50 nM to 0.7813 nM. The diluted CD73 protein and each antibody sample were incubated at 37°C for 1 hour. Then, AMP was added to a final concentration of 400 μM, and the mixture was reacted at 37° C. for 20 minutes. After that, the reaction was carried out for 20 minutes using the Malachite Green Phosphate Detection Kit. The absorbance was measured at 620 nm wavelength using an Epoch Microplate Spectrophotometer. Positive controls (CPI-006, MEDI9447) and negative controls (human IgG1 isotype (BIO X Cell, West Lebanon, New Hampshire) was also analyzed by the same method as described above. The degree was measured.

[0174] FIG. 7 shows the enzyme activity of soluble CD73 by APBA2-01 and APBA2-02 antibodies. 1 shows the results of the Malachite Green assay method for evaluating activity inhibition. As shown in Figure 7, the enzyme activity inhibitory ability of anti-CD73 antibodies against soluble CD73 The order of the highest is CPI-006>APBA2-01>APBA2-02>MEDI9447. It was.

[0175] 3-2. Evaluation of inhibitory activity against membrane CD73

[0176] The APBA2-01 antibody and the APBA2-02 antibody produced in the above Preparation Example 3 were used in the membrane C To determine whether the enzyme activity of the D73 protein was inhibited, phosphate was measured. Quantification (Malachite Green Assay) and Luciferin Oxidation Measurement (CellTiter-Glo (registered trademark) A target assay was performed for each.

[0177] The enzyme activity of CD73 was analyzed by malachite green assay. To measure the enzyme activity, MDA-MB-231 cells and 4T1 cells were cultured at 2 × 1 0 4 1.0 x 10 cells / well 4 Dispense APBA2-01 and A at a concentration of 100 cells / well. The PBA2-02 antibody was serially diluted to concentrations of 400 nM to 0.0244 nM. The assay was carried out in the same manner as in Preparation Example 2-4 above, except that the negative control (rituximab) and positive control (CPI-006, MEDI9447) samples The absorbance was measured in the same manner as described above.

[0178] Next, the enzyme activity of CD73 was analyzed by CellTiter-Glo (registered trademark) assay. MDA-MB-231 cells and 4T1 cells were cultured at 2.0 × 10 4 Cells / well, 1 .0×10 4 Dispense cells / well into a 96-well flat plate (SPL) at a concentration of 3 The plates were incubated at 7°C in a 5% CO2 incubator for 20 hours. After incubation, the supernatant was removed and the cells were resuspended in RPMI-1640 medium without FBS. Then, the APBA2-01 antibody sample and the APBA2-02 antibody sample were washed with medium for 5 h. Each well was treated with serial dilutions from 0.00 nM to 0.0305 nM at 37°C. After the reaction, AMP was added at a concentration of 400 μM, and the mixture was incubated at 37°C for 3 hours. The supernatant was then diluted with 200 μM of 100% ethanol in a new 96-well white plate. The cells were reacted with 100 μg of ATP, treated with CellTiter-Glo®, and then measured using the standard method provided by the manufacturer. The luminescence was measured using a Synergy M1 reader according to the method. Negative control (rituximab) and positive control (CPI-006, MEDI9447) samples Luminescence was also measured for the same as described above.

[0179] FIG. 8A shows the effect of anti-CD73 antibody against membrane CD73 in MDA-MB-231 cells. FIG. 8C shows the results of the Malachite Green assay method for analyzing enzyme activity inhibition. , the results of the CellTiter-Glo® assay method are shown. As shown in Figure 8A, the inhibitory activity against membrane CD73 was significantly increased by MDA-MB-23. In one cell, APBA2-02>CPI-006>MEDI9447>APBA2- As shown in Figure 8C, MEDI9447=APBA2-01> The measurements were performed in the order of CPI-006>APBA2-02.

[0180] FIG. 8B shows the ability of anti-CD73 antibodies to inhibit enzyme activity against membrane CD73 in 4T1 cells. Figure 8D shows the results of the malachite green assay to analyze the CellTiter- The results of the Glo® assay are shown. As shown in Fig. 8B, in 4T1 cells, the inhibitory potency was APBA2-02>M As shown in Figure 8D, the order of EDI9447>APBA2-01 was measured. The order of measurements was DI9447>APBA2-01>APBA2-02. Differences in inhibitory potency were shown.

[0181] That is, in the malachite green assay, the APBA2-02 antibody was The highest inhibition of activity was observed in the control group (Figures 8A and 8B). The results of the target assay showed the lowest inhibition of activity (Fig. 8C and Fig. 8D). In addition, the APBA2-01 antibody and MEDI9447 inhibited the following in MDA-MB-231 cells: Although the antibody showed only about 50% inhibition of CD73 activity in 4T1 cells (Fig. 8A), The antibody CPI-006 inhibited the expression of mouse CD73 by more than 90% (Fig. 8B). It did not cross-link and therefore showed no reaction in mouse 4T1 cells (Fig. 8B, Fig. 8D ).

[0182] [Example 4. Transfection of anti-CD73 antibody into CD73 cells]

[0183] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 is internalized into cells by binding to CD73 on the cell surface membrane. We confirmed whether the antibody was positive by immunofluorescence microscopy. First, sterile cover glasses (Paul Marienfeld et al.) were placed in a 12-well plate (SPL). , Germany) and place MDA-MB-231 cells (1.0 × 10 5 ) was dispensed into each well, The cells were incubated at 37°C in 5% CO2 for 24 hours. After that, the culture medium was removed. Each well was incubated with APBA2-01 antibody and APBA2-02 antibody at a concentration of 10 μg / ml in the medium. The mixture was treated with ethanol and incubated at 37°C for 20, 40, 60, and 120 minutes, respectively. The positive controls (CPI-006, MEDI9447) were also reacted in the same manner. After the reaction was completed, the culture medium was removed. The attached cells were washed three times with PBS buffer. After washing, 1 ml of 4% formaldehyde was dispensed into each well and incubated at 37°C for 15 min. The cells were fixed and washed in the same manner as above, then resuspended in 0.05% Triton X. 100 was dispensed in 1 ml portions per well and incubated at room temperature for 15 minutes until pores were formed in the cells. The cells were then washed and induced to grow by adding 2% PBA buffer. After washing, FITC-conjugated antibodies were Human Fc antibody was added and reacted at room temperature for 50 minutes. Washing was performed in the same manner as above. After that, the cover slip on which the cells were fixed was treated with 4',6-diamidino-2- VECTASHIELD HardSet Antifade mounting medium containing DAPI The cells were then placed on a slide with mounting medium. were analyzed with an ultrasensitive, high-resolution confocal laser scanning microscope.

[0184] FIG. 9 shows the membrane CD7 3. Images showing the transfer of APBA2-01 and APBA2-02 antibodies into cells. . As shown in FIG. 9, the APBA2-01 antibody or the APBA2-02 antibody, or MDA-MB-231 cells were treated with MEDI9447 or MEDI9447 for 20 min. CD73 signals were observed around the membrane of 231 cells, and CD However, intracellular uptake of CPI-006 was not observed. I couldn't.

[0185] [Example 5. Evaluation of efficacy in an in vivo model]

[0186] 5-1. Confirmation of anticancer effects in a mouse model of metastatic breast cancer

[0187] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 The anticancer efficacy of was confirmed in a metastatic breast cancer mouse model. 10 5 ) was administered into the tail vein of 6-week-old female BALB / c mice to induce lung cancer. 3 Days later, APBA2-01 antibody and APBA2-02 antibody were intraperitoneally administered at 20 mg / kg. The positive control group (MEDI9447 hIgG1) and the negative control group were administered 4 times at 3-day intervals. The same dose and method were administered to both groups (isotype control IgG). After the experiment, the lungs and spleens were removed and weighed. The lungs were then stained with Bouin's solution and the number of colonies was counted. Statistical analysis was performed using the Prism Version 5.0 program. Body weight and tumor size were analyzed by single-factor regression. The results were analyzed by one-way ANOVA, and p<0.05 was considered significant. did.

[0188] FIG. 10A is a graph measuring mean lung weight in a metastatic breast cancer mouse model. As shown in FIG. 10A, the positive control and the negative control for the APBA2-01 antibody There was no change in the weight of the mouse lungs compared to the control group, and in the case of APBA2-02, the weight of the mouse lungs increased It was found that the reduction was 10.85%.

[0189] FIG. 10B is a graph showing the number of lung metastatic colonies in a metastatic breast cancer mouse model. be. As shown in FIG. 10B, the positive control group, the APBA2-01-administered group, and the APBA2 In the -02-treated group, the number of lung metastatic colonies was 0.0 compared to the negative control group. The decreases were 4%, 13% and 16%, but were not statistically significant.

[0190] On the other hand, the body weight and spleen weight of the mice were measured using the APBA2-01 antibody and the APBA2-0 No significant changes were observed in the antibody and positive control groups compared to the negative control group. There was no one there.

[0191] 5-2. Confirmation of cancer growth inhibition in breast cancer mouse models

[0192] APBA2-01 antibody and APBA2-02 antibody produced and purified in the above Preparation Example 3 The cancer growth inhibitory efficacy of was confirmed in a breast cancer mouse model. 10 5 ) was injected into the mammary fat pad to induce breast cancer. Three days later, APB A2-01 antibody and APBA2-02 antibody were mixed with TBS (pH 7.4) buffer. The mice were intraperitoneally administered with a total dose of 20 mg / kg (6 times at 2-day intervals). EDI9447 mouse IgG1, IgG2A) and negative control group (isotype control mouse Breast cancer serogroup IgG1, IgG2A (Bio X Cell) was also administered at the same dose and by the same method. The size was measured at 2-day intervals using calipers. Six days later, the lungs of the mice were removed and observed for the presence or absence of cancer cell metastasis.

[0193] FIG. 11A shows that APBA2-0 was significantly increased in breast cancer mice by measuring tumor volume. mIgG1 antibody, mIgG2A antibody, and mIgG1 antibody, mI FIG. 11B is a graph showing the tumor growth inhibitory effect of the gG2A antibody. This is a graph showing the efficacy of inhibiting cancer growth by comparison.

[0194] As shown in Figures 11A and 11B, the APBA2-01 antibody inhibits Fc function of the antibody. The APBA2-02 antibody showed approximately 17% inhibition of breast cancer growth compared to the negative control group, regardless of its activity. In breast cancer, 33% of cases are mIgG2A with Fc function, and 18% are mIgG1 without Fc function. The positive control group showed a growth inhibitory effect of 23%. mIgG1, which showed long-term inhibition and lacks Fc function, showed 35% inhibition of activity.

[0195] FIG. 11C is a graph showing the weight measurements of a mouse model of breast cancer. As shown in FIG. 11C, changes in the body weight of the mice were observed during the course of the experiment. Furthermore, no metastasis of cancer cells to the lungs has occurred.

[0196] [Example 6. Efficacy evaluation in an in vitro cell model]

[0197] 6-1. Preparation of monoclonal antibodies

[0198] We asked Myxengo (USA) to select an antibody that specifically binds to the CD73 protein. Go will conduct biopanning using its own naive human scFv library. The selected clones were expressed as IgG4 antibodies. The corresponding culture medium was delivered by Myxengo. Each delivered culture medium was then analyzed using the CaptureSe lect IgG-CH1 Affinity Matrix resin was used for affinity chromatography, and Each IgG4 antibody protein was separated and purified, and the experiment was carried out in the same manner as in Preparation Example 2-2. .

[0199] 6-2. Confirmation of enzyme activity of membrane CD73 protein

[0200] The seven types of human anti-CD73 IgG1 antibodies prepared in Example 6-1 were analyzed using a membrane CD73 enzyme. The inhibitory ability against the activity was confirmed by the same method as in Example 3-2. CPI-006 and MEDI9947 were used as the inhibitors.

[0201] FIG. 12A shows the effect of anti-CD73 antibody against membrane CD73 in MDA-MB-231 cells. The enzyme activity inhibitory ability of each compound was evaluated by the malachite green assay. The results are evaluated using the CellTiter-Glo (registered trademark) assay method. As shown in Figures 12A and 12B, five of the seven antibodies (Nos. 3 and 4) ,6,7,15) showed significantly higher CD73 enzyme activity inhibition ability than the negative control group. He admitted that he

[0202] [Example 7. Confirmation of binding ability to membrane CD73 protein]

[0203] The species of anti-CD73 antibodies (Myxengo antibodies 3, 4, 6, 7, and 15) selected in Example 6 To confirm the cross-reactivity between the antibodies, a mouse plasma membrane CD73-expressing cell line (4T1 (ATCC, 4T1.2 (ATCC, CRL-3406)) were used to bind antibodies. A confluence experiment was performed. First, 4T1 cells were cultured in RPMI-1640 (Gibco, Cat No. A10491-01) medium. The cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat No. 16000-044). 4T1.2 cells were cultured in Alpha MEM (Minimum Essential Medium) (Corning Fisher Science, 10- The cells were cultured in 022-CV medium supplemented with 10% fetal bovine serum. The cells were detached using trypsin-EDTA (Gibco, Cat No. 25200-056) and then incubated for 1 2.0x10 in .5ml tube 5 The cells were then pelleted in a centrifuge. After that, the supernatant was removed. , 100 nM in DPBS buffer (Gibco) supplemented with 2 mM EDTA (Intronbio) The cells were suspended in 100 μl of the antibody sample diluted to the desired concentration and reacted at 4°C for 1 hour. Then, 800 μl of MACS buffer was added, centrifuged, and the supernatant was removed. Secondary antibody Goat anti-human IgG Fc FIT diluted 1:500 in ACS buffer C (Novex, A18830), 50 μl, was added to suspend the cells. The cells were incubated at 4°C for 1 hour. After the reaction, 500 μl of MACS buffer was added, centrifuged, and the supernatant was removed. Then, cells were incubated with 200 μl of 0.4% paraformaldehyde (T&I, diluted in PBS). After suspension and fixation, the prepared samples were run on a flow cytometer (BD, FACSVer. se) was used for the analysis.

[0204] FIG. 13A shows the binding of Myxengo 3, 4, 6, 7 and 15 antibodies to membrane CD73 in 4T1 cells. 1 is a graph confirming the binding ability to proteins. FIG. 13B shows the membrane CD40 of Myxengo 3, 4, 6, 7 and 15 antibodies in 4T1.2 cells. 7 is a graph confirming the binding ability to 73 proteins.

[0205] As shown in Figures 13A and 13B, five types of antibodies (Nos. 3, 4, 6, 7, and 15 ) One of the antibodies (No. 15) bound strongly to mouse membrane CD73 compared to the negative control group. and showed a similar level of antigen-binding ability to the APBA2-02 antibody. Species antibodies (No. 3, 4, 6, and 7) did not bind to mouse membrane CD73. The xengo 15 antibody exhibits a similar level of antigen-binding ability to the APBA2-02 antibody. It is predicted to have similar properties to APBA2-02. Thus, Myxengo 15 antibody is While specifically binding to CD73, it may have species cross-reactivity.

[0206] In summary, the monoclonal antibody or antigen-binding fragment thereof binds to CD73 and inhibits CD It is possible to reduce the enzyme activity of 73 protein and inhibit the metastasis or growth of cancer. The antibody or its derivatives can be used as a therapeutic agent for cancer. The antigen-binding fragments of the antibody show species cross-reactivity and can be used in preclinical animal experiments. The time, cost and other factors involved in testing immunotherapeutic agents for side effects, toxicity and / or stability are also significant. This can save money.

[0207] The foregoing description of the invention is given by way of example only and is intended to provide an understanding to those skilled in the art to which the invention pertains. If there is any, the present invention may be easily modified into another specific embodiment without changing the technical idea or essential features of the present invention. It will be understood that many variations are possible. The described embodiments should be considered in all respects as illustrative and not restrictive. Must be.

[0208] The breadth and scope of the present invention may be further understood by reference to any of the foregoing exemplary embodiments. Not limited, but defined only by the claims and their equivalents. It is.

[0209] The various aspects, embodiments, and options described herein may be combined in any desired variations. It can be done.

[0210] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety. Unless specifically and individually indicated that a patent or patent application is incorporated by reference. To the same extent as herein incorporated by reference.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof, including: A heavy chain complementarity determining domain comprising the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 14 or 17. CDRH1, comprising the amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15 or 18 and the amino acid sequence of SEQ ID NO: 3, 6, 9, 12, 13, 16 or 19. a heavy chain variable region comprising a CDRH3 comprising Light chain complementarity determining domain 1 comprising the amino acid sequence of SEQ ID NO: 20, 23, 26 or 29 (CDRL1), CDRL2 comprising the amino acid sequence of SEQ ID NO: 21, 24, 27 or 30 and a light chain comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO: 22, 25, 28 or 31. Variable region.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDR comprising the amino acid sequence of SEQ ID NO:2 a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:3; and CDRL1 comprising the amino acid sequence of SEQ ID NO:20, CDRL2 comprising the amino acid sequence of SEQ ID NO:21 2, and a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:22; CDRH1 comprising the amino acid sequence of SEQ ID NO:4, CDR comprising the amino acid sequence of SEQ ID NO:5 a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:6; and a sequence CDRL1 comprising the amino acid sequence of SEQ ID NO:23, CDRL2 comprising the amino acid sequence of SEQ ID NO:24 2, and a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:25; CDRH1 comprising the amino acid sequence of SEQ ID NO:7, CDR comprising the amino acid sequence of SEQ ID NO:8 a heavy chain variable region comprising a CDRH3 having the amino acid sequence of SEQ ID NO:9; and a sequence CDRL1 comprising the amino acid sequence of SEQ ID NO:26, CDRL2 comprising the amino acid sequence of SEQ ID NO:27 2, and a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:28; CDRH1 comprising the amino acid sequence of SEQ ID NO: 10, C comprising the amino acid sequence of SEQ ID NO: 11 A heavy chain variable domain comprising a DRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 12 or 13. region; and CDRL1 comprising the amino acid sequence of SEQ ID NO:20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

22. region; CDRH1 comprising the amino acid sequence of SEQ ID NO: 14, C comprising the amino acid sequence of SEQ ID NO: 15 a heavy chain variable region of DRH2, and a heavy chain variable region of CDRH3 comprising the amino acid sequence of SEQ ID NO: 16; and CDRL1 comprising the amino acid sequence of sequence number 20, CDR comprising the amino acid sequence of SEQ ID NO:21 a light chain variable region comprising a CDRL3 comprising the amino acid sequence of SEQ ID NO:22; or CDRH1 comprising the amino acid sequence of SEQ ID NO: 17, C comprising the amino acid sequence of SEQ ID NO: 18 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:19; and CDRL1 comprising the amino acid sequence of SEQ ID NO:29, CRL2 comprising the amino acid sequence of SEQ ID NO:30 A light chain variable region comprising a DRL2, a CDRL3 comprising the amino acid sequence of SEQ ID NO:

31.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising: At least one amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38 heavy chain variable regions comprising amino acids having at least 80% identity to SEQ ID NOs: 39, 40, 41 or 42 amino acid sequence having at least 80% identity to the amino acid sequence. The light chain variable region.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising: A heavy chain containing the amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37 or 38. A light chain variable region comprising the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42.

5. The antibody or antigen-binding fragment thereof of claim 1, comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDR comprising the amino acid sequence of SEQ ID NO:2 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and CDRL1 comprising the amino acid sequence of SEQ ID NO:20, C comprising the amino acid sequence of SEQ ID NO:21 A light chain variable region comprising a DRL2, a CDRL3 comprising the amino acid sequence of SEQ ID NO:

22.

6. The antibody or antigen-binding fragment thereof of claim 1, comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:4, CDR comprising the amino acid sequence of SEQ ID NO:5 a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO:6; and CDRL1 comprising the amino acid sequence of SEQ ID NO:23, C comprising the amino acid sequence of SEQ ID NO:24 A light chain variable region comprising a DRL2, a CDRL3 comprising the amino acid sequence of SEQ ID NO:

25.

7. A monoclonal antibody or antigen-binding fragment thereof, including: A heavy chain complementarity determining domain comprising the amino acid sequence of SEQ ID NO: 66, 69, 72, 75 or 78. CDRH1, comprising the amino acid sequence of SEQ ID NO: 67, 70, 73, 76 or 79 and a C1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 74, 77, or 80. A heavy chain variable region comprising DRH3; and A light chain complementarity determining domain comprising the amino acid sequence of SEQ ID NO: 81, 84, 87, 90 or 93. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, CDRL2 and a CD comprising the amino acid sequence of SEQ ID NO: 83, 86, 89, 90 or 95. The light chain variable region comprises RL3.

8. 8. The monoclonal antibody or antigen-binding fragment thereof of claim 7, comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:66, C comprising the amino acid sequence of SEQ ID NO:67 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:68; and CDRL1 comprising the amino acid sequence of SEQ ID NO: 81, CRL2 comprising the amino acid sequence of SEQ ID NO: 82 a light chain variable region comprising a CDRL2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:83; CDRH1 comprising the amino acid sequence of SEQ ID NO:69, C comprising the amino acid sequence of SEQ ID NO:70 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:71; and CDRL1 comprising the amino acid sequence of SEQ ID NO: 84, CRL2 comprising the amino acid sequence of SEQ ID NO: 85 a light chain variable region comprising a CDRL2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:86; CDRH1 comprising the amino acid sequence of SEQ ID NO:72, C comprising the amino acid sequence of SEQ ID NO:73 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:74; and CDRL1 comprising the amino acid sequence of SEQ ID NO: 87, CRL2 comprising the amino acid sequence of SEQ ID NO: 88 a light chain variable region comprising a CDRL2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:89; CDRH1 comprising the amino acid sequence of SEQ ID NO:75, C comprising the amino acid sequence of SEQ ID NO:76 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:77; and CDRL1 comprising the amino acid sequence of SEQ ID NO: 90; CRL2 comprising the amino acid sequence of SEQ ID NO: 91 a light chain variable region comprising a CDRL2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:92; and teeth CDRH1 comprising the amino acid sequence of SEQ ID NO:78, C comprising the amino acid sequence of SEQ ID NO:79 a heavy chain variable region comprising a DRH2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:80; and CDRL1 comprising the amino acid sequence of SEQ ID NO:93, CRL2 comprising the amino acid sequence of SEQ ID NO:94 A light chain variable region comprising a DRL2, a CDRL3 comprising the amino acid sequence of SEQ ID NO:

95.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, The antibody or the antibody thereof is a monoclonal antibody, which is a fully human antibody or a chimeric antibody. Antigen-binding fragment.

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

11. An expression vector comprising the nucleic acid of claim 10.

12. A cell transformed with the expression vector of claim 11.

13. A method for preventing cancer, comprising administering to a patient an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9. Or a therapeutic composition.

14. The composition of claim 13 further comprising an immune checkpoint blockade or a chemotherapeutic agent. Composition.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 is administered to a subject. The present invention relates to a method for treating cancer in a subject in need thereof, the method comprising:

16. The method of claim 15, wherein the cancer is a cancer that overexpresses CD73.

17. The cancers include breast cancer, triple negative breast cancer (TBNC), pancreatic and colorectal cancer, ovarian cancer, gastric cancer, cancer), bladder cancer, leukemia, prostate cancer, malignant melanoma, cancer, esophageal cancer, stomach cancer 17. The method of claim 15 or 16, wherein the cancer is head and neck cancer, lung cancer or kidney cancer.

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