Anti-CD73 antibodies and uses thereof

Abstract

The present invention addresses the problem of developing an antibody which targets a CD73 antigen and has a higher function, more specifically, an antibody which is for treating cancer and has a higher activity.　The present invention has shown that the problem can be solved by finding an antibody or a human antibody derivative which includes a heavy or light chain complementarity-determining region having a specific amino acid sequence, has binding properties to a CD73 antigen, and activates T cells having the property of damaging cancer cells.

Description

[Technical Field]

[0001] The present invention relates to an antibody having cytotoxic activity specific to cancers expressing the CD73 antigen, and a composition and method for cancer treatment and cancer testing, which comprise the antibody. [Background technology]

[0002] CD73 is an enzyme that catalyzes the degradation of AMP to adenosine in vivo, and is known to be expressed on the cell surface of cancer cells, regulatory T cells (Treg cells), and vascular endothelial cells in particular. Adenosine generated by CD73 on these cells is released into the microenvironment, binds to adenosine receptors on immune cells, and induces immune cell exhaustion.

[0003] The CD73 antigen is highly expressed in multiple cancers, and it has been reported that cases with high CD73 antigen expression have a poor prognosis. This is thought to occur because an increase in adenosine in the microenvironment surrounding cancer cells suppresses anti-tumor immunity, and the released adenosine promotes the suppression of immune activity by Treg cells.

[0004] On the other hand, it has been reported in non-clinical models that antibodies against the CD73 antigen have tumor growth-inhibitory effects (Oncoimmunology. 2016 Aug; 5(8): e1208875). Based on these findings, clinical development (Phase I to II) of cancer therapeutic antibodies targeting the CD73 antigen is underway. However, no confirmatory clinical trials have yet begun, and the development of antibodies with higher functionality is needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication 2018-501197 [Non-patent literature]

[0006] [Non-Patent Document 1] Oncoimmunology. 2016 Aug; 5(8): e1208875 [Non-patent document 2] Bioinformatics. 2015 Feb 1;31(3):434-5 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to develop an antibody that targets the CD73 antigen and has higher functionality, more specifically, an antibody for cancer treatment that has higher activity. [Means for solving the problem]

[0008] The present invention has demonstrated that the above-mentioned problems can be solved by discovering an antibody or humanized antibody derivative that contains heavy and light chain complementarity determining regions having specific amino acid sequences, has binding affinity to the CD73 antigen, and activates T cells that are toxic to cancer cells. Specifically, the present invention is a monoclonal antibody that specifically binds to the CD73 antigen and has the following functions: - It showed a stronger inhibitory effect on the CD73-mediated degradation of AMP to adenosine than the benchmark antibody (an antibody with the same sequence as the published variable region of MEDI9447); -Relieves adenosine-induced T cell exhaustion and activates T cells.

[0009] More specifically, the present application provides the following aspects to solve the above-mentioned problems: [1]: (1) heavy chain complementarity determining regions, CDR1 (DX1NMD (X1 is C or S), SEQ ID No.: 1), CDR2 (DINPNNGGTIYNQKFKG, SEQ ID No.: 2), and CDR3 (TNWDYAMDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (KASQDINSX2LS (X2 is N, D, or Q), SEQ ID No.: 4), CDR2 (RANRLID, SEQ ID No.: 5), and CDR3 (X3QYDVFPRT (X3 is L or Q), SEQ ID No.: 6); (2) heavy chain complementarity determining regions, CDR1 (SFGMH, SEQ ID No.: 9), CDR2 (YISSGSRTIYYADTVRG, SEQ ID No.: 10), and CDR3 (DFGSSSPNYFDY, SEQ ID No.: 11); and light chain complementarity-determining regions, CDR1 (RASESVDNYGISFMN, SEQ ID No.: 12), CDR2 (AASNQGS, SEQ ID No.: 13), and CDR3 (QQSKEVPWT, SEQ ID No.: 14); (3) heavy chain complementarity determining regions, CDR1 (GYWMN, SEQ ID No.: 17), CDR2 (RIDPYDSETHYSQKFKD, SEQ ID No.: 18), and CDR3 (SSPITTAPFDY, SEQ ID No.: 19); and light chain complementarity-determining regions, CDR1 (RASESVDYYGFSFMN, SEQ ID No.: 20), CDR2 (AASTQGS, SEQ ID No.: 21), and CDR3 (QQSKEVPYT, SEQ ID No.: 22); (4) heavy chain complementarity determining regions, CDR1 (SYGVS, SEQ ID No.: 25), CDR2 (VIWGDGSTNYHSALIS, SEQ ID No.: 26), and CDR3 (TNIFYDYDWYLDV, SEQ ID No.: 27); and light chain complementarity-determining regions, CDR1 (RSSQSLVHSNGNTYLH, SEQ ID No.: 28), CDR2 (KVSNRFS, SEQ ID No.: 29), and CDR3 (SHSTHVPWT, SEQ ID No.: 30); (5) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 33), CDR2 (EINPSNARTNYNENFKS, SEQ ID No.: 34), and CDR3 (RGTSGNYFDF, SEQ ID No.: 35); and light chain complementarity-determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 36), CDR2 (RANRLVD, SEQ ID No.: 37), and CDR3 (LQYDEFPYT, SEQ ID No.: 38); (6) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 41), CDR2 (KIDPYDSETHYNQKFKD, SEQ ID No.: 42), and CDR3 (IRYGTFDY, SEQ ID No.: 43); and light chain complementarity-determining regions, CDR1 (KASQDINNYLS, SEQ ID No.: 44), CDR2 (RANILVD, SEQ ID No.: 45), and CDR3 (LQYDEFPYT, SEQ ID No.: 46); (7) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 49), CDR2 (EINPSNGRTNYNEKFKN, SEQ ID No.: 50), and CDR3 (RGTSGNYFDY, SEQ ID No.: 51); and light chain complementarity-determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 52), CDR2 (RANRLVD, SEQ ID No.: 53), and CDR3 (LQYDEFPYT, SEQ ID No.: 54); (8) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 57), CDR2 (RIDPYDSEAHYNQKFKD, SEQ ID No.: 58), and CDR3 (IRYGTFDY, SEQ ID No.: 59); and light chain complementarity-determining regions, CDR1 (KASQDINSYLS, SEQ ID No.: 60), CDR2 (RSNSLVD, SEQ ID No.: 61), and CDR3 (LQYDEFPYT, SEQ ID No.: 62); an antibody or humanized antibody derivative which binds to the CD73 antigen and activates T cells that are cytotoxic to cancer cells, the antibody or humanized antibody derivative comprising a heavy chain or light chain complementarity determining region selected from the group consisting of: [2]: the antibody or human antibody derivative according to [1], wherein the T cell activation is selected from the group consisting of T cell proliferation, increased cytotoxicity of T cells against cancer cells, and promotion of cytokine secretion from T cells; [3]: The antibody or human antibody derivative according to [1] or [2], wherein the human antibody derivative is a human antibody variant or a functional fragment thereof selected from humanized antibodies, chimeric antibodies, multivalent antibodies, and multispecific antibodies; [4]: The antibody or human antibody derivative according to any one of [1] to [3], wherein the functional fragment is F(ab')2; [5]: The antibody or humanized antibody derivative according to any one of [1] to [4], wherein the amino acid sequence of the heavy chain variable region VH domain of the antibody or humanized antibody derivative is selected from (1) SEQ ID No.: 7, (2) SEQ ID No.: 15, (3) SEQ ID No.: 23, (4) SEQ ID No.: 31, (5) SEQ ID No.: 39, (6) SEQ ID No.: 47, (7) SEQ ID No.: 55, and (8) SEQ ID No.: 63; [6]: The antibody or humanized antibody derivative according to any one of [1] to [5], wherein the amino acid sequence of the light chain variable region VL domain of the antibody or humanized antibody derivative is selected from (1) SEQ ID No.: 8, (2) SEQ ID No.: 16, (3) SEQ ID No.: 24, (4) SEQ ID No.: 32, (5) SEQ ID No.: 40, (6) SEQ ID No.: 48, (7) SEQ ID No.: 56, and (8) SEQ ID No.: 64; [7]: The antibody or human antibody derivative according to any one of [1] to [6], which induces cytotoxicity against cancer cells but not against normal cells; [8]: The antibody or human antibody derivative according to any one of [1] to [7], wherein the cancer cells are selected from the group consisting of melanoma, breast cancer, lung cancer, and colon cancer; [9]: The antibody or human antibody derivative according to any one of [1] to [8], which is conjugated with a drug to form an antibody-drug conjugate (ADC);

[10] : A pharmaceutical composition for cancer treatment, comprising the antibody or human antibody derivative according to any one of [1] to [9];

[11] : The pharmaceutical composition according to

[10] , wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, and colorectal cancer;

[12] : contacting cancer cells collected from a subject in vitro with the antibody or human antibody derivative according to any one of [1] to [9]; a step of measuring whether AMP metabolism of cancer cells is reduced under culture conditions, or whether cell viability is reduced, or whether secretion of immune activating substances is enhanced; a method for measuring cytotoxicity against cancer cells, comprising:

[13] : A method for measuring cytotoxicity against cancer cells according to

[12] , which measures whether the cell viability of cancer cells is reduced or whether immune cells derived from peripheral blood lymphocytes are activated in the presence of peripheral blood lymphocytes from the same subject;

[14] : A method for measuring cytotoxicity against cancer cells according to

[12] or

[13] , which measures the enhancement of cytotoxicity against cancer cells when the antibody or human antibody derivative according to any one of [1] to [9] is administered to a subject based on the in vitro AMP metabolism inhibition or cytotoxicity of cancer cells collected from the subject;

[15] : A method for measuring cytotoxicity against cancer cells according to

[12] or

[13] , which measures the enhancement of cytotoxicity against cancer cells when the antibody or human antibody derivative according to any one of [1] to [9] is administered to a subject based on the enhancement of secretion of immune activators in vitro;

[16] : A measurement kit comprising the antibody or human antibody derivative according to any one of [1] to [9], for measuring in vitro the AMP metabolism, cytotoxicity, secretion of immunostimulatory substances, or activation of immune cells in cancer cells collected from a subject. [Effects of the Invention]

[0010] The antibody or human antibody derivative obtained by the present invention suppresses the function of CD73 expressed in cancer cells or immune cells, and relieves exhaustion of immune cells (especially T cells), thereby activating the immune cells and exerting tumor growth inhibitory effects and cancer therapeutic effects.

[0011] The present invention can be widely used as a therapeutic and diagnostic agent for cancer. Because the antibody according to the present invention has the effect of relieving exhaustion of immune cells, it is expected to be particularly effective in combination with other immunotherapies. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the AMP degradation inhibitory function (H322 cells) of the 002_m antibody produced in Example 1. [Figure 2] FIG. 2 shows the amino acid sequences of the heavy chain variable region and light chain variable region of the 002_m antibody. [Figure 3] FIG. 3 shows the binding affinity of the resulting chimeric antibodies 002_1_c to 002_6_c to the human lung cancer cell line H322 cells expressing CD73. [Figure 4] FIG. 4 shows the AMP degradation inhibitory activity of antibodies 002_1_c to 002_6_c in human lung cancer H322 cells. [Figure 5-1] FIG. 5-1 shows the amino acid sequences of the heavy chain variable regions of the obtained humanized antibodies of 002_6_c (002_6_h1 to 002_6_h8). [Figure 5-2]FIG. 5-2 shows the amino acid sequences of the light chain variable regions of the obtained humanized antibodies of 002_6_c (002_6_h1 to 002_6_h8). [Figure 6-1] FIG. 6-1 shows the binding of humanized antibodies (002_6_h1 to 002_6_h8) to the human lung cancer cell line H322 cells expressing CD73. [Figure 6-2] FIG. 6-2 shows the binding of humanized antibodies (002_6_h1 to 002_6_h8) to the human lung cancer cell line H322 cells expressing CD73. [Figure 7] FIG. 7 shows the AMP degradation inhibitory function of three types of humanized antibodies (002_6_h2, 002_6_h4, 002_6_h8) using human lung cancer H322 cells. [Figure 8-1] FIG. 8-1 shows the binding ability of humanized antibody 002_6_h4 to various cells, in comparison with existing antibodies and the base chimeric antibody 002_6_c. [Figure 8-2] FIG. 8-2 shows the binding ability of humanized antibody 002_6_h4 to various cells, in comparison with existing antibodies and the base chimeric antibody 002_6_c. [Figure 9] FIG. 9 shows the AMP degradation inhibitory function of humanized antibody 002_6_h4 in human breast cancer MDA-MB-231 cells, compared with that of an existing antibody, the base chimeric antibody 002_6_c. [Figure 10] FIG. 10 shows the function of 002_6_h4 in inhibiting AMP degradation of recombinant CD73 antigen. [Figure 11] FIG. 11 shows the effect of 002_6_h4 on the division (proliferation) of human peripheral blood T cells. [Figure 12] FIG. 12 shows the AMP degradation inhibitory function (H322 cells) of the 003_m antibody to 009_m antibody prepared in Example 1. [Figure 13]FIG. 13 shows the AMP degradation inhibitory function (MDA-MB-231 cells) of four antibodies (004_m, 006_m, 007_m, 008_m) that were determined to be highly functional in FIG. [Figure 14-1] FIG. 14-1 shows the amino acid sequences of the heavy chain variable regions of the obtained antibodies (003_m antibody to 009_m antibody). [Figure 14-2] FIG. 14-2 shows the amino acid sequences of the light chain variable regions of the obtained antibodies (003_m antibody to 009_m antibody). [Figure 15] FIG. 15 shows the binding of the 003_m to 009_m antibodies to target-expressing cells (H322 cells). [Figure 16] FIG. 16 shows the binding of the 003_m to 009_m antibodies to target-expressing cells (MDA-MB-231 cells). [Figure 17] FIG. 17 shows the binding of the 003_m to 009_m antibodies to target-expressing cells (CD73-forced expressing HEK-293T cells). [Figure 18] FIG. 18 shows the results of comparing the percentage of dividing cells when the antibody concentration was set to 1 nM. [Figure 19] FIG. 19 shows the binding activity of antibodies to cells in which the human CD73 gene has been expressed. [Figure 20-1] FIG. 20-1 shows the binding activity of antibodies to cells in which the human CD73 gene has been expressed. [Figure 20-2] FIG. 20-2 shows the binding activity of antibodies to cells in which the human CD73 gene has been expressed. [Figure 21] FIG. 21 shows the binding activity of antibodies to cells expressing cynomolgus monkey CD73. [Figure 22] FIG. 22 shows that the antibody of the present invention has inhibitory activity against the AMP degradation action of cell surface CD73. [Figure 23]FIG. 23 shows that the antibodies of the present invention have a mitogenic effect on CD4 T cells and CD8 T cells, and have the effect of enhancing the ability of these cells to produce IFNγ, TNF, and IL-2. [Figure 24] FIG. 24 shows fluorescence microscopic images showing the uptake of 002_6_h4 antibody and 006_hKB antibody into cells. [Figure 25] FIG. 25 shows that the antibodies of the present invention bound to CD73 on the surface of T cells decrease over time when these cells are cultured (are internalized into T cells). [Figure 26] FIG. 26 shows that the antibodies of the present invention that bind to CD73 on the surface of cancer cells decrease over time when these cells are cultured (are internalized into the cancer cells). [Figure 27] FIG. 27 shows the results of measuring the number of tumor-infiltrating T cells in mice on day 35 after administration of 002_6_h4 antibody or an isotype control antibody. [Figure 28-1] Figure 28-1 shows whether the developed antibodies of the present invention enhance the cytotoxic activity of T cells to cancer cells. [Figure 28-2] Figure 28-2 shows whether the developed antibodies of the present invention enhance the cytotoxic activity of T cells to cancer cells. [Figure 29] FIG. 29 shows the results of an animal experiment investigating whether 002_6_h4 can enhance the antitumor effect of a human immune system simulated in mice. [Figure 30] FIG. 30 shows the results of changes in tumor mass volume in the bodies of immunodeficient mice. [Figure 31] FIG. 31 shows the antitumor effect of the anti-CD73 antibody of the present invention confirmed in vivo in knock-in mice expressing human CD73. DETAILED DESCRIPTION OF THE INVENTION

[0013] In one aspect, the present invention provides a binding substance for the CD73 antigen, particularly an antibody or human antibody derivative, which has binding affinity to the CD73 antigen on cancer cells or immune cells and activates T cells that are toxic to cancer cells. The binding substance of the present invention, particularly an antibody or human antibody derivative, can be used to treat cancer cells that express the CD73 antigen, inhibit cancer cell growth, and shrink or eliminate tumors, and can also be used to activate T cells that are toxic to cancer cells when they have become exhausted.

[0014] Antibodies and human antibody derivatives In one aspect, the present invention provides an antibody or human antibody derivative that has binding activity to the CD73 antigen. This antibody or human antibody derivative is characterized by activating T cells that have cytotoxicity against cancer cells.

[0015] The antibody or human antibody derivative of the present invention may be one that suppresses the function of CD73 expressed in cancer cells and relieves the exhaustion of immune cells (particularly T cells) caused by the accumulation of AMP metabolites (adenosine) around cancer cells, thereby activating the immune cells and exerting a tumor growth inhibitory effect and a cancer therapeutic effect; one that suppresses the function of CD73 expressed in immune cells (particularly T cells), activates the immune cells, and exerts a tumor growth inhibitory effect and a cancer therapeutic effect; or one that exerts a cancer therapeutic effect by controlling the environment around the tumor other than immune cells, or by directly controlling the tumor.

[0016] In the present invention, when simply referring to an "antibody," the antibody may be derived from any mammalian species, and the species from which the antibody is derived is not limited to humans, but may also be mice, rats, guinea pigs, hamsters, rabbits, etc.

[0017] The present invention also includes humanized antibody derivatives of the above-mentioned antibodies, so long as they have binding affinity to the CD73 antigen and the functional characteristic of suppressing cancer. In one embodiment, the humanized antibody derivative of the present invention refers to an antibody derivative that has the amino acid sequences of the six CDRs of the above-mentioned antibody (heavy chain complementarity-determining regions (CDRs) 1 to 3 and light chain CDRs 1 to 3) and the amino acid sequences of the constant regions derived from a human antibody, with the remaining amino acid sequences being a combination of the amino acid sequences derived from the original antibody and the amino acid sequences derived from a human antibody. Examples of such antibody derivatives include humanized antibodies in which the regions other than the complementarity-determining regions (CDRs) of the above-mentioned "antibody" are replaced with amino acid sequences derived from a human antibody, chimeric antibodies in which the variable regions of the above-mentioned antibodies are linked to the constant regions of a human antibody, multivalent antibodies in which one type of antibody has multiple antigen-binding sites, and multispecific antibodies (bispecific antibodies) in which one type of antibody has multiple specificities, but other antibodies are also included.

[0018] The antibody or human antibody derivative of the present invention is characterized by having the same amino acid sequences of six positions, namely, heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3, as those of an antibody that has binding ability to the CD73 antigen. Examples of the amino acid sequences of the six CDRs of such an antibody that has binding ability to the CD73 antigen include the following (1) to (8): (1) heavy chain complementarity determining regions, CDR1 (DX1NMD (X1 is C or S), SEQ ID No.: 1), CDR2 (DINPNNGGTIYNQKFKG, SEQ ID No.: 2), and CDR3 (TNWDYAMDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (KASQDINSX2LS (X2 is N, D, or Q), SEQ ID No.: 4), CDR2 (RANRLID, SEQ ID No.: 5), and CDR3 (X3QYDVFPRT (X3 is L or Q), SEQ ID No.: 6); (2) heavy chain complementarity determining regions, CDR1 (SFGMH, SEQ ID No.: 9), CDR2 (YISSGSRTIYYADTVRG, SEQ ID No.: 10), and CDR3 (DFGSSSPNYFDY, SEQ ID No.: 11); and light chain complementarity-determining regions, CDR1 (RASESVDNYGISFMN, SEQ ID No.: 12), CDR2 (AASNQGS, SEQ ID No.: 13), and CDR3 (QQSKEVPWT, SEQ ID No.: 14); (3) heavy chain complementarity determining regions, CDR1 (GYWMN, SEQ ID No.: 17), CDR2 (RIDPYDSETHYSQKFKD, SEQ ID No.: 18), and CDR3 (SSPITTAPFDY, SEQ ID No.: 19); and light chain complementarity-determining regions, CDR1 (RASESVDYYGFSFMN, SEQ ID No.: 20), CDR2 (AASTQGS, SEQ ID No.: 21), and CDR3 (QQSKEVPYT, SEQ ID No.: 22); (4) heavy chain complementarity determining regions, CDR1 (SYGVS, SEQ ID No.: 25), CDR2 (VIWGDGSTNYHSALIS, SEQ ID No.: 26), and CDR3 (TNIFYDYDWYLDV, SEQ ID No.: 27); and light chain complementarity-determining regions, CDR1 (RSSQSLVHSNGNTYLH, SEQ ID No.: 28), CDR2 (KVSNRFS, SEQ ID No.: 29), and CDR3 (SHSTHVPWT, SEQ ID No.: 30); (5) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 33), CDR2 (EINPSNARTNYNENFKS, SEQ ID No.: 34), and CDR3 (RGTSGNYFDF, SEQ ID No.: 35); and light chain complementarity-determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 36), CDR2 (RANRLVD, SEQ ID No.: 37), and CDR3 (LQYDEFPYT, SEQ ID No.: 38); (6) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 41), CDR2 (KIDPYDSETHYNQKFKD, SEQ ID No.: 42), and CDR3 (IRYGTFDY, SEQ ID No.: 43); and light chain complementarity-determining regions, CDR1 (KASQDINNYLS, SEQ ID No.: 44), CDR2 (RANILVD, SEQ ID No.: 45), and CDR3 (LQYDEFPYT, SEQ ID No.: 46); (7) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 49), CDR2 (EINPSNGRTNYNEKFKN, SEQ ID No.: 50), and CDR3 (RGTSGNYFDY, SEQ ID No.: 51); and light chain complementarity-determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 52), CDR2 (RANRLVD, SEQ ID No.: 53), and CDR3 (LQYDEFPYT, SEQ ID No.: 54); (8) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 57), CDR2 (RIDPYDSEAHYNQKFKD, SEQ ID No.: 58), and CDR3 (IRYGTFDY, SEQ ID No.: 59); and light chain complementarity-determining regions, CDR1 (KASQDINSYLS, SEQ ID No.: 60), CDR2 (RSNSLVD, SEQ ID No.: 61), and CDR3 (LQYDEFPYT, SEQ ID No.: 62); However, antibodies or human antibody derivatives specified by CDR combinations other than the above-mentioned CDR combinations are also included, as long as they have the characteristic of binding to the CD73 antigen.

[0019] The antibody or human antibody derivative of the present invention is also characterized by its ability to activate T cells. In particular, it is preferable that the antibody or human antibody derivative suppresses the function of CD73 expressed on cancer cells or immune cells, thereby relieving exhaustion of immune cells (especially T cells), thereby activating immune cells and exerting cancer growth inhibitory and therapeutic effects. Such T cell activation may occur in vitro or in vivo. Whether an antibody has the ability to activate T cells that are toxic to cancer cells can be determined by screening antibodies that bind to the CD73 antigen to determine whether they actually activate T cells.

[0020] In the present invention, exhaustion of immune cells (especially T cells) refers to a state in which immune cells are dysfunctional. Specifically, this refers to a decrease in the cytokine secretion and cytotoxicity of T cells, a decrease in the cytokine secretion and cytotoxicity of NK cells, a decrease in the antigen presentation ability of dendritic cells, and activation of regulatory T cells. In this state, an individual's cancer immunity against cancer cells is either non-functional or impaired, resulting in a decrease in the ability to eliminate cancer cells. Such immune cell exhaustion is known to occur when checkpoint proteins such as PD-1 and CTLA-4 increase on the surface of T cells, or in effector T cells when the immune system is forced to remain active for a long period of time due to continuously proliferating cancer cells. However, this is a reversible state, and it is believed that the exhausted state can be resolved by activating the same cells.

[0021] In the present invention, T cell activation can be understood using indicators such as T cell proliferation, increased cytotoxicity of T cells against cancer cells, cytokine secretion from T cells, etc. Observation of such T cell activation indicates that the antibody or human antibody derivative of the present invention has inhibited CD73 activity in T cells, thereby suppressing the reaction that decomposes AMP to adenosine.

[0022] Screening of the ability of the antibody or human antibody derivative of the present invention to activate T cells can be performed in vivo or ex vivo. In vivo screening can be performed by transplanting target cancer cells into animals such as wild-type mice or immunodeficient mice (e.g., nude mice or SCID mice) into which immune cells have been transplanted, and measuring the change in tumor mass size in vivo after administration of the antibody or human antibody derivative of the present invention, or by anatomically examining T cell infiltration around the tumor mass in vivo after administration of the antibody or human antibody derivative of the present invention. Ex vivo screening can be performed by contacting peripheral blood T cells with the antibody or human antibody derivative of the present invention under culture conditions and measuring the proliferation of the T cells, measuring cytokine secretion by the T cells, or examining whether the T cells cause cell death in cancer cells.

[0023] In one embodiment, the antibodies or humanized antibody derivatives of the present invention can be identified as having the following heavy chain variable region VH domain amino acid sequences: (1) 002_m heavy chain variable region (SEQ ID No.: 7), (2) 003_m heavy chain variable region (SEQ ID No.: 15), (3) 004_m heavy chain variable region (SEQ ID No.: 23), (4) 005_m heavy chain variable region (SEQ ID No.: 31), (5) 006_m heavy chain variable region (SEQ ID No.: 39), (6) 007_m heavy chain variable region (SEQ ID No.: 47), (7) 008_m heavy chain variable region (SEQ ID No.: 55), and (8) 009_m heavy chain variable region (SEQ ID No.: 63). Here, the numbers (1) to (8) correspond to the numbers of the combinations of the heavy chain CDR1 to CDR3 sequences described above. For example, the amino acid sequence of SEQ ID NO: 7 is a sequence that includes the amino acid sequences of heavy chain CDR1 to CDR3 identified by SEQ ID NO: 1 to SEQ ID NO: 3.

[0024] In one embodiment, the antibodies or humanized antibody derivatives of the present invention can be identified as having the following amino acid sequences of the light chain variable region VL domains: (1) 002_m light chain variable region (SEQ ID No.: 8), (2) 003_m light chain variable region (SEQ ID No.: 16), (3) 004_m light chain variable region (SEQ ID No.: 24), (4) 005_m light chain variable region (SEQ ID No.: 32), (5) 006_m light chain variable region (SEQ ID No.: 40), (6) 007_m light chain variable region (SEQ ID No.: 48), (7) 008_m light chain variable region (SEQ ID No.: 56), and (8) 009_m light chain variable region (SEQ ID No.: 64). Here, the numbers (1) to (8) correspond to the numbers of the combinations of the light chain CDR1 to CDR3 sequences described above. For example, the amino acid sequence of SEQ ID NO: 8 is a sequence that includes the light chain CDR1 to CDR3 amino acid sequences identified by SEQ ID NO: 4 to SEQ ID NO: 6.

[0025] In the present invention, the human antibody derivatives of the present invention also include functional fragments of the above-mentioned antibodies or human antibody derivatives. Functional fragments of antibodies or human antibody derivatives of the present invention include F(ab')2, Fab', Fab, single-chain Fv (scFv), and the like. The functional fragment of the present invention may be any of these fragments as long as it is capable of inducing toxicity to cancer cells. For example, F(ab')2 fragments and the like can be used as such functional fragments.

[0026] Obtaining antibodies or human antibody derivatives The antibody or humanized antibody derivative of the present invention can be obtained by administering a CD73 antigen as an immunogen to an animal of the aforementioned origin species and culturing antibody-producing cells collected from the animal's body. Alternatively, the antibody or humanized antibody derivative can be obtained recombinantly by designing a protein expression vector containing a DNA sequence that can specify the amino acid sequence of the antibody or humanized antibody derivative and introducing the vector into cells for protein production.

[0027] A DNA sequence that can specify the amino acid sequence of an antibody or humanized antibody derivative of the present invention can be prepared by a method of obtaining it from cells that produce the desired antibody or humanized antibody derivative, a method of designing it based on the amino acid sequence using codons optimized for the animal species used in the expression system, or a method that combines these methods.

[0028] The prepared DNA sequence can be obtained using techniques well known to those skilled in the art, such as by incorporating it into an expression vector suitable for the cell type for protein expression (e.g., CHO cells) in which the antibody or humanized antibody derivative is to be expressed, and then introducing it into the cell type for protein expression.

[0029] In the case of antibodies, because they have a structure in which a heavy chain and a light chain are combined, they can be produced by a method in which a vector containing a DNA sequence that specifies the heavy chain amino acid sequence and a vector containing a DNA sequence that specifies the light chain amino acid sequence are introduced into a cell type for protein expression, and both proteins are expressed in the cell, resulting in intracellular production of an antibody; or a method in which a vector containing both a DNA sequence that specifies the heavy chain amino acid sequence and a DNA sequence that specifies the light chain amino acid sequence is introduced into the cell, and both proteins are expressed in the cell, resulting in intracellular production of an antibody.

[0030] An example of a humanized antibody derivative obtained as an embodiment of the present invention is a humanized antibody derivative prepared from the antibody (1) above. an antibody comprising the 002_6_h1 heavy chain variable region (SEQ ID NO: 65) and the 002_6_h1 light chain variable region (SEQ ID NO: 66); an antibody comprising the 002_6_h2 heavy chain variable region (SEQ ID NO: 67) and the 002_6_h2 light chain variable region (SEQ ID NO: 68); an antibody comprising the 002_6_h3 heavy chain variable region (SEQ ID NO: 69) and the 002_6_h3 light chain variable region (SEQ ID NO: 70); an antibody comprising the 002_6_h4 heavy chain variable region (SEQ ID NO: 71) and the 002_6_h4 light chain variable region (SEQ ID NO: 72); an antibody comprising the 002_6_h5 heavy chain variable region (SEQ ID NO: 73) and the 002_6_h5 light chain variable region (SEQ ID NO: 74); an antibody comprising the 002_6_h6 heavy chain variable region (SEQ ID NO: 75) and the 002_6_h6 light chain variable region (SEQ ID NO: 76); an antibody comprising the 002_6_h7 heavy chain variable region (SEQ ID NO: 77) and the 002_6_h7 light chain variable region (SEQ ID NO: 78); an antibody comprising the 002_6_h8 heavy chain variable region (SEQ ID NO: 79) and the 002_6_h8 light chain variable region (SEQ ID NO: 80); an antibody comprising the 004_h3_ heavy chain variable region (SEQ ID NO: 81) and the 004_h3_ light chain variable region (SEQ ID NO: 82); an antibody comprising the 006_hKB_ heavy chain variable region (SEQ ID NO: 83) and the 006_hKB_ light chain variable region (SEQ ID NO: 84); an antibody comprising the 007_h1_ heavy chain variable region (SEQ ID NO: 85) and the 007_h1_ light chain variable region (SEQ ID NO: 86); an antibody comprising the 008_h4_ heavy chain variable region (SEQ ID NO: 87) and the 008_h4_ light chain variable region (SEQ ID NO: 88); These include, but are not limited to, human antibody derivatives prepared from other antibodies, and are also included as aspects of the present invention.

[0031] Uses of antibodies or human antibody derivatives Based on the above-mentioned characteristic of the antibody or human antibody derivative of the present invention that it has the ability to activate immune cells (particularly T cells) that have cytotoxicity against cancer cells, in one aspect, it is possible to provide a pharmaceutical composition comprising the antibody or human antibody derivative of the present invention that activates immune cells (particularly T cells) that have cytotoxicity against cancer cells in a subject in need of cancer treatment or prevention.

[0032] Cancer cells that can be targeted in the present invention include, for example, leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma, diffuse lymphoma, and follicular lymphoma), myeloma (including multiple myeloma), melanoma, lung cancer, breast cancer, colon cancer, kidney cancer, stomach cancer, ovarian cancer, pancreatic cancer, cervical cancer, uterine cancer, endometrial cancer, and eosinophilic lymphoma. The cancer cells include cells derived from cancers selected from the group consisting of esophageal cancer, liver cancer, head and neck cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, thecoma, male embryonal tumor, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, osteoblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms' tumor. In an embodiment of the present invention, the cancer cells are preferably cancer cells that express the CD73 antigen, and are preferably cells derived from melanoma, breast cancer, lung cancer, or colorectal cancer.

[0033] As described above, the antibody or human antibody derivative of the present invention is characterized by activating immune cells (particularly T cells) that have cytotoxicity against target cancer cells. That is, when administered to a living body, activation of immune cells (particularly T cells) occurs only against immune cells that have cytotoxicity against target cancer cells that express the CD73 antigen, and is required not to induce clinically problematic cytotoxicity against normal cells that express the CD73 antigen.

[0034] In the present invention, the antibody or human antibody derivative of the present invention can be provided as a composition in combination with another antibody or another drug, such as an anti-cancer drug. In one embodiment, the antibody or human antibody derivative of the present invention can be conjugated with a drug to form an antibody-drug conjugate (ADC).

[0035] The present invention also provides formulations comprising the antibody or human antibody derivative of the present invention together with a physiologically acceptable diluent or carrier. Suitable carriers include, but are not limited to, buffers (phosphate buffer, citrate buffer, acetate buffer, etc.), salts (sodium chloride, etc.), sugars (glucose, trehalose, mannitol, sorbitol, etc.), and additives (amino acids such as arginine, surfactants such as polysorbates, etc.). Alternatively, the antibody or human antibody derivative of the present invention can be lyophilized (freeze-dried) and reconstituted when needed by adding an aqueous buffer solution such as those described above. Formulations comprising the antibody or human antibody derivative of the present invention can be administered in various dosage forms, including parenteral administration via injection, infusion, etc.

[0036] The dosage of the antibody or human antibody derivative of the present invention varies depending on symptoms, age, body weight, etc., but typically, for parenteral administration, the dosage is 0.01 mg to 1000 mg per kg of body weight per dose, preferably 0.05 mg to 500 mg, 0.05 mg to 100 mg, 0.05 mg to 50 mg, or 0.05 mg to 20 mg per kg of body weight per day, and more preferably 0.1 mg to 10 mg per kg of body weight per day, and can be administered via an administration route appropriate for the type of cancer, such as intraperitoneal injection, subcutaneous injection, intramuscular injection, intratumoral injection, or intravenous injection.

[0037] Based on the aforementioned characteristic of having the ability to activate immune cells (particularly T cells) that have cytotoxicity against cancer cells, in another aspect, the present invention can also provide a method for treating or preventing cancer in a subject, comprising administering an effective amount of the antibody or human antibody derivative of the present invention to a subject in need of cancer treatment or prevention. Cancer treatment or prevention by the antibody or human antibody derivative of the present invention occurs when the antibody or human antibody derivative activates immune cells (particularly T cells) in the body that have cytotoxicity against cancer cells.

[0038] When the antibody or human antibody derivative of the present invention is administered to a living body for the treatment or prevention of cancer, it can be administered together with an adjuvant (e.g., those described in Clin. Microbiol. Rev., 7:277-289, 1994) to effectively establish cellular or humoral immunity in the body, or it can be administered in a particulate formulation, such as a liposome formulation, a particulate formulation bound to beads with a diameter of several μm, or a formulation bound to lipids.

[0039] In vitro uses of antibodies and human antibody derivatives The antibodies or human antibody derivatives of the present invention can be used to detect CD73 antigen in a sample based on their binding affinity to the CD73 antigen. Specifically, the antibodies or human antibody derivatives of the present invention can be used to perform various antibody-based CD73 antigen detection methods on samples containing cancer cells or immune cells (e.g., T cells) collected from a subject, including antibody-based purification methods such as immunoprecipitation, agglutination reactions, and magnetic bead techniques; immunoassays such as ELISA, Western blotting, and immunohistochemistry; and immunocytochemistry such as flow cytometry. In each case, the antibodies or human antibody derivatives of the present invention can be detected using detection labels (e.g., fluorescent, enzyme, etc.) commonly known to those skilled in the art.

[0040] The antibody or human antibody derivative of the present invention can also be used to measure the cytotoxicity of the antibody or human antibody derivative of the present invention against cancer cells in a subject, based on the characteristic that the antibody or human antibody derivative has binding affinity to the CD73 antigen and activates immune cells (particularly T cells) that are toxic to cancer cells. A method for measuring cytotoxicity against cancer cells in such a subject comprises the following steps: contacting cancer cells collected from a subject with the antibody or human antibody derivative of the present invention under culture conditions (i.e., in vitro); a step of measuring whether AMP metabolism (the reaction that breaks down AMP into adenosine) of cancer cells is reduced under culture conditions, a step of measuring whether cell viability is reduced, or a step of measuring whether secretion of immune activating substances is enhanced; An example of such a method is a method for measuring cytotoxicity against cancer cells using the antibody or human antibody derivative of the present invention, including:

[0041] It is known that a decrease in AMP metabolism (the reaction that breaks down AMP to adenosine) in cancer cells in vivo can activate T cells and shrink tumors, and is thought to activate cancer immunity against cancer cells. Taking advantage of this phenomenon, in one embodiment of the present invention, cancer cells collected from a subject are contacted in vitro with an antibody or human antibody derivative of the present invention, and the cytotoxicity to cancer cells can be measured by measuring whether the AMP metabolism of the cancer cells is reduced under culture conditions. More specifically, by contacting cancer cells collected from a subject with an antibody or human antibody derivative of the present invention and measuring the breakdown of AMP to adenosine under culture conditions (i.e., in vitro), the cytotoxicity to cancer cells in the subject (in vivo) when the antibody or human antibody derivative of the present invention is administered to the subject can be measured.

[0042] In this embodiment, the decomposition reaction of AMP to adenosine can be measured by HPLC, LC-MS, ELISA, or AMP-dependent chemiluminescence (Cell titer glo, etc.).

[0043] In vivo, peripheral blood lymphocytes cause a decrease in the cell viability of cancer cells. Taking advantage of this phenomenon, in one embodiment of the present invention, cancer cells collected from a subject are contacted in vitro with an antibody or human antibody derivative of the present invention, and the cytotoxicity to cancer cells is measured by measuring whether the cell viability is reduced under culture conditions. More specifically, cancer cells collected from a subject are contacted with an antibody or human antibody derivative of the present invention in the presence of peripheral blood lymphocytes from the same subject, and the cytotoxicity to cancer cells in vivo when the antibody or human antibody derivative of the present invention is administered to the subject is measured by measuring whether the cell viability of the cancer cells is reduced under culture conditions (i.e., in vitro).

[0044] It is known that enhanced secretion of immunostimulatory substances from peripheral blood lymphocytes in vivo activates T cells and shrinks tumors, and is thought to activate cancer immunity against cancer cells. Taking advantage of this phenomenon, in one embodiment of the present invention, cancer cells collected from a subject are contacted in vitro with an antibody or human antibody derivative of the present invention, and cytotoxicity against cancer cells is measured by measuring whether secretion of immunostimulatory substances is enhanced under culture conditions. More specifically, cancer cells collected from a subject are contacted with an antibody or human antibody derivative of the present invention in the presence of peripheral blood lymphocytes from the same subject, and measured whether secretion of immunostimulatory substances from peripheral blood lymphocytes is enhanced under culture conditions (i.e., in vitro). This allows for the measurement of activation of immune cells such as cytotoxic lymphocytes in the subject (in vivo) and the resulting enhancement of cytotoxicity against cancer cells when the antibody or human antibody derivative of the present invention is administered to the subject.

[0045] In this embodiment, examples of immune activators measured to assess cytotoxicity to cancer cells include IFN-γ, TNFα, etc. IFN-γ is known to activate immune cells such as NK cells and exert antitumor effects.

[0046] The present invention can also provide an assay kit for in vitro assaying AMP metabolism, cytotoxicity, secretion of immunostimulatory substances, or activation of immune cells against cancer cells collected from a subject, which comprises an antibody or human antibody derivative of the present invention.

[0047] A measurement kit for measuring AMP metabolism can include, in addition to the antibody or human antibody derivative of the present invention, reagents (AMP-dependent chemiluminescent reagent, ELISA reagent) and devices (HPLC system, LC-MS system) for measuring the substrate AMP and its metabolic product adenosine.

[0048] A measurement kit for measuring cytotoxicity can include, in addition to the antibody or human antibody derivative of the present invention, known means for measuring cell proliferation (e.g., measurement of thymidine incorporation, BrdU incorporation, free lactate dehydrogenase (LDH) activity, measurement of substances derived from living cells (reductase activity, esterase activity, ATP, etc.)).

[0049] A measurement kit for measuring the secretion of an immunostimulatory substance can include, in addition to the antibody or human antibody derivative of the present invention, means for detecting the immunostimulatory substance to be measured (e.g., a primary antibody against the immunostimulatory substance and a secondary antibody for detecting the primary antibody).

[0050] A measurement kit for measuring immune cell activation can contain, in addition to the antibody or human antibody derivative of the present invention, a labeling reagent for measuring immune cell division with a flow cytometer.

[0051] The present invention will be specifically illustrated by the following examples, which are not intended to limit the present invention in any way. [Example]

[0052] Example 1: Preparation of antibodies This example was carried out with the aim of obtaining a monoclonal antibody against the CD73 antigen using mice.

[0053] (1-1) Antibody production by B cell single cell sorting Mice (BALB / cAJcl, female, 7 weeks old, CLEA Japan) were immunized with 10 μg of CD73 recombinant antigen (autologous preparation) intraperitoneally every two weeks for 4 to 8 doses. One week after the final dose, blood was collected from the tail vein, and the increase in CD73 antigen-specific antibody titer in the blood was confirmed by conventional antigen-fixed ELISA. Spleens were then collected from mice with significantly elevated CD73 antigen-specific antibody titers.

[0054] Single-cell suspensions of splenocytes were prepared from the spleen according to published methods, and B cells were purified using a MACS-pan B cell isolation kit (Miltenyi 130-104-443). The purified B cells were stained with ZOMBIE-APC-Cy7 (Biolegend), Alexa488 anti-IgM (Biolegend), VB421 anti-IgG (Biolegend), and PE-CD73 (self-prepared). Then, the IgG-positive cell population, which exhibited CD73 antigen binding, was sorted into 96-well plates (single cell per well).

[0055] The genes corresponding to the antibody heavy and light chain variable regions were amplified for each well by RT-PCR. Primers used for amplification included the 5' and 3' ends of the heavy and light chain variable regions, respectively, based on published literature information. Taking into account the diversity of antibody gene sequences, a mix of primers with multiple sequences was used (heavy chain: H1 mix and H2 mix, light chain: k mix) (Lotta von Boehmer et al., Nature Protocols, Vol. 11, pp. 1908-1923 (2016)).

[0056] After amplification of the antibody heavy chain and light chain genes, linker sequences for insertion into an antibody expression vector were added to the ends of each gene by PCR, and the genes were inserted by ligation into a heavy chain expression vector or a light chain expression vector (each containing a CMV promoter, a secretion signal, a heavy chain variable region gene or a light chain variable region gene, and a heavy chain constant region gene linked in tandem).

[0057] The ligation product was transformed into E. coli to recover the antibody expression plasmids, and then the heavy chain gene expression plasmid (derived from the H1 or H2 mixed amplification product) and the light chain gene expression plasmid (derived from the k mixed amplification product) were transfected into CHO cells. The antibodies produced in the culture supernatant were recovered from the CHO cells, and the antigen specificity of each antibody was confirmed by antigen-immobilized ELISA.

[0058] (1-2) Antibody purification For antibodies that were found to bind to the antigen (CD73) by antigen-immobilized ELISA, the CHO cell culture supernatant was collected, and the antibodies in the culture supernatant were purified using a MonospinProG column (GL Science).

[0059] (1-3) Antibody functional screening Human lung cancer cell line H322 (ATCC® CRL-5806 TM ) was seeded onto a 96-well plate, purified antibodies were added, and the plate was incubated for 1 hour at 37°C. AMP (Sigma, A1752), a substrate for CD73, was added to a final concentration of 200 μM, and the plate was incubated overnight at 37°C to allow CD73 to catalyze the degradation of AMP to adenosine.

[0060] The supernatant was collected, and ATP (Sigma, A2383, final concentration 100 μM) and Cell TiterGlo (Promega, G9243) were added and mixed. Chemiluminescence was measured using a microplate reader. In other words, the inhibitory effect of the antibody on enzyme activity was evaluated based on the principle that AMP inhibits the chemiluminescence generated by the reaction of ATP and Cell TiterGlo.

[0061] As a positive control, we used MEDI9447, a previously developed product (based on the sequence published in JP2018-501197, an antibody with the same sequence as the published variable region of MEDI9447 was manufactured using genetic engineering technology; hereafter referred to as the "benchmark antibody").

[0062] As a result of antibody functional screening, eight mouse antibodies, designated 002_m, 003_m, 004_m, 005_m, 006_m, 007_m, 008_m, and 009_m, were selected as antibodies with positive CD73 binding activity.

[0063] Example 2: Evaluation of antibody function (concentration dependency evaluation) This example was carried out with the aim of evaluating the function of the 002_m antibody obtained in Example 1.

[0064] The antibody function evaluation was carried out by the same method as the antibody function screening described in Example 1 (1-3). That is, human lung cancer cell line H322 cells or human breast cancer cell line MDA-MB-231 cells (ATCC® HTB-26 TM In this example, the AMP degradation reaction was measured in the same manner as in Example 1 (1-3), and the antibody concentration dependency of the AMP degradation inhibitory function was analyzed to calculate the IC50 value (50% inhibitory concentration: here, this refers to the antibody concentration that can inhibit 50% of the enzymatic function of CD73) to quantitatively evaluate the antibody function.

[0065] The AMP degradation inhibitory function (H322 cells) of the 002_m antibody produced in Example 1 is shown in Figure 1. Although this antibody is a mouse antibody, it was found to exhibit an IC50 value close to that of benchmark antibodies (see Table 1).

[0066] [Table 1]

[0067] Example 3: Antibody gene sequence analysis This example was carried out with the aim of determining the amino acid sequence of the 002_m antibody obtained in Example 1.

[0068] For the 002_m antibody clone, which was found to have the ability to inhibit AMP degradation in functional analysis, single colonies were recovered from an inoculated plate of E. coli transfected with the ligation product prepared in Example 1 (1-1), and plasmid DNA was obtained.

[0069] The resulting plasmid DNA was analyzed by Sanger sequencing to determine the gene sequences defining the heavy chain variable region and the light chain variable region, and the amino acid sequence was determined based on the sequences.

[0070] The amino acid sequences of the heavy chain variable region and light chain variable region of the 002_m antibody are shown in Figure 2 (SEQ ID NO: 7 and SEQ ID NO: 8, respectively). In this figure, CDR1, CDR2, and CDR3 of the heavy chain (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively) and CDR1, CDR2, and CDR3 of the light chain (SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are underlined.

[0071] Example 4: Production of human-mouse-human chimeric antibodies This example was carried out with the aim of producing a chimeric antibody having a mouse antibody variable region and a human antibody constant region using the 002_m antibody sequenced in Example 3.

[0072] The gene sequence of the variable region of a mouse antibody was artificially synthesized and incorporated into a vector for expressing a chimeric recombinant antibody with a human constant region (Fc). A plasmid carrying a light chain expression element (EF-1α promoter, secretion signal, DNA sequence encoding the light chain variable region, and DNA sequence encoding the light chain constant region, all linked in tandem) and a heavy chain expression element (EF-1α promoter, secretion signal, DNA sequence encoding the heavy chain variable region, and DNA sequence encoding the heavy chain constant region, all linked in tandem) was constructed as the expression vector. The plasmid was transfected into ExpiCHO cells (Thermo) to secrete the antibody into the culture medium, and the antibody was purified using a Protein A column (GL Science).

[0073] The chimeric antibody thus constructed was designated antibody 002_1_c. The heavy chain variable region of this antibody consists of the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of this antibody consists of the amino acid sequence of SEQ ID NO: 8.

[0074] Example 5: Preparation of CDR-modified antibodies This example was carried out with the aim of producing a modified antibody of the 002_m antibody in which residues in the CDRs that may be subject to post-translational modification were substituted with residues that are not subject to modification.

[0075] Specifically, the 32nd amino acid cysteine ​​(C) in the heavy chain variable region of the 002_m antibody (SEQ ID NO: 7) was substituted with serine (S), and the 32nd amino acid asparagine (N) in the light chain variable region of the 002_m antibody (SEQ ID NO: 8) was substituted with aspartic acid (D) or glutamine (Q) (see Table 2 below).

[0076] [Table 2]

[0077] A gene containing the relevant region was artificially synthesized and inserted into a human Fc chimeric recombinant antibody expression vector by ligation. Antibody expression and purification were carried out in the same manner as for the human chimeric antibody.

[0078] The binding of each of the chimeric antibodies 002_1_c to 002_6_c to target-expressing cells was analyzed by FACS analysis using CD73 native-expressing cells (human lung cancer cell line H322). After reacting the test antibody or isotype control antibody with the cells, they were stained with a fluorescently labeled secondary antibody and subjected to flow cytometry analysis. The antibody binding was evaluated based on the fluorescence intensity.

[0079] The results are shown in Figure 3. It was revealed that both the chimeric antibodies prepared in Example 4 and this Example based on the 002_m antibody exhibited high binding affinity to H322 cells.

[0080] Next, antibody function screening was performed on each chimeric antibody, 002_1_c antibody to 002_6_c antibody, using the same method as in (1-3), to measure the AMP degradation inhibitory function using human lung cancer H322 cells. The results are shown in Figure 4.

[0081] Based on these results, the IC50 values ​​of each antibody were calculated, as shown in Table 3 below. 002_1_c to 002_6_c were determined to have equivalent binding and functionality. Furthermore, the IC50 values ​​were comparable to those of the benchmark antibody. Therefore, further development was focused on 002_6_c, which is unlikely to be post-translationally modified.

[0082] [Table 3]

[0083] Example 6: Production of humanized antibodies This example was carried out with the aim of producing a humanized antibody based on the 002_6_c antibody (SEQ ID NO: 7(C32S) / SEQ ID NO: 8(N32Q)) selected in Example 5.

[0084] Humanized antibodies were created using the publicly available software Tabhu (http: / / circe.med.uniroma1.it / tabhu / ). Using this software, a human antibody database was extracted to find sequences with similar sequence and conformational characteristics to 002_6_c. Four sequences (FJ039788, AF146404, 3SQO_H, and AY686911) were selected as candidate sequences, and the sequences other than the CDR regions of these antibodies were grafted onto the CDR region sequence of 002_6_c.

[0085] Next, some amino acid residues were backmutated to the mouse sequence. The amino acid residues targeted for backmutation were selected based on the structural differences between the humanized and mouse antibodies (high TubHu scores), with a maximum of 10 amino acid residues per sequence. Details were obtained according to the method described in Bioinformatics. 2015 Feb 1;31(3):434-5.

[0086] The amino acid sequences of the variable regions of the resulting 002_6_c humanized antibodies (002_6_h1 to 002_6_h8) are shown in Figure 5 (Figure 5-1 shows the heavy chain variable region, and Figure 5-2 shows the light chain variable region). Each amino acid sequence was assigned a SEQ ID NO: as shown in Table 4 below.

[0087] [Table 4]

[0088] The binding of each of the resulting humanized antibodies (002_6_h1 to 002_6_h8) to target-expressing cells was analyzed by FACS analysis using CD73 native-expressing cells (human lung cancer cell line H322). The results are shown in Figure 6. It was confirmed that all humanized antibodies exhibited binding properties similar to those of the chimeric antibody 002_6_c on which they were based.

[0089] Next, three of the humanized antibodies 002_6_h1 to 002_6_h8 (002_6_h2, 002_6_h4, and 002_6_h8) were subjected to antibody function screening using human lung cancer H322 cells to measure their inhibitory function against AMP degradation, using the same method as in (1-3). The results are shown in Figure 7.

[0090] Based on these results, the IC50 values ​​of each humanized antibody for its ability to inhibit AMP degradation using human lung cancer H322 cells were calculated, as shown in Table 5 below. 002_6_h4, which had the highest inhibitory function among the humanized antibodies, was selected as the lead antibody for further development. This antibody showed a favorable IC50 value compared to benchmark antibodies.

[0091] [Table 5]

[0092] The humanized antibody 002_6_h4 selected as the lead antibody above was compared with existing antibodies (commercially available anti-CD73 antibody (biolegend), benchmark antibody) and the base chimeric antibody 002_6_c, and its binding to various cells was confirmed by flow cytometry.

[0093] Binding analysis to target-expressing cells was performed by FACS analysis using CD73 native-expressing cells (human breast cancer cell line MDA-MB-231), CD73-expressing human breast cancer cell line MDA-MB-231 cells, CD73-expressing HEK293T wild-type cells (derived from human embryonic kidney cells) that do not express CD73, and HEK293T cells overexpressing CD73. CD73-overexpressing cells were generated using a lentiviral vector system (Origene). HEK293T cells were infected with lentiviral particles carrying the human CD73 gene (NM_002526) and a puromycin resistance gene, and then cultured in the presence of puromycin. HEK293T cells, which had integrated the target gene into their genome and become CD73-positive, were collected and used for binding analysis. After reacting the test antibody or isotype control antibody with the cells, the cells were stained with a fluorescently labeled secondary antibody and subjected to flow cytometry analysis, and the antibody binding was evaluated based on the obtained fluorescence intensity.

[0094] The results are shown in Figure 8. As a result, it was found that both 002_6_c and 002_6_h4 exhibited binding properties to various cells similar to those of existing antibodies (commercially available anti-CD73 antibodies (biolegend), benchmark antibodies), confirming their binding to the CD73 antigen.

[0095] Next, humanized antibody 002_6_h4 was compared with existing antibodies (commercially available anti-CD73 antibody (biolegend), benchmark antibody) and the base chimeric antibody 002_6_c. Antibody function screening was performed using human breast cancer MDA-MB-231 cells to measure their AMP degradation inhibitory function using the same method as in (1-3). The results are shown in Figure 9. Based on these results, the IC50 values ​​of the humanized antibodies for AMP degradation inhibitory function using human breast cancer MDA-MB-231 cells were calculated, and the results are shown in Table 6 below. Together with the results of the test using human lung cancer H322 cells described above, this indicates that humanized antibody 002_6_h4, like chimeric antibody 002_6_c, has AMP degradation inhibitory function in multiple cancer types.

[0096] [Table 6]

[0097] Example 7: Inhibitory function against recombinant antigens This example was carried out with the aim of confirming the function of the anti-CD73 antibody obtained in Example 6 in inhibiting the enzymatic reaction of recombinant CD73 antigen.

[0098] The experiment was performed by immobilizing CD73 antigen (terminal His-Tag, self-prepared) on a 96-well ELISA plate via an anti-His-Tag antibody, adding humanized anti-CD73 antibody 002_6_h4 and the substrate AMP (final concentration 100 μM), incubating at 37°C for 1 hour, and measuring the decomposition of AMP to adenosine by CD73, as in Example 1 (1-3).

[0099] The supernatant was collected, and ATP (final concentration 100 μM) and Cell TiterGlo (Promega, G9243) were added and mixed. Chemiluminescence was then measured using a microplate reader. Based on the principle that AMP inhibits the luminescence produced by the reaction of ATP and Cell TiterGlo, the inhibitory effect of the antibody on enzyme activity was evaluated based on the amount of remaining AMP. The same experiment was performed using a benchmark antibody as a positive control.

[0100] The inhibitory function of 002_6_h4 on the AMP degradation of recombinant CD73 antigen is shown in Figure 10. The results suggest that 002_6_h4 exhibits inhibitory function on the AMP degradation of recombinant CD73 antigen, directly inhibiting the enzyme.

[0101] Example 8: Evaluation of effects on T cell division This example was carried out with the aim of confirming the proliferation-promoting activity of the anti-CD73 antibody obtained in Example 6 on human peripheral blood T cells.

[0102] CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) derived from healthy volunteers using the CD4+ T Cell Isolation Kit, human (Miltenyi Biotech, 130-096-533), and fluorescently labeled using the CellTrace CFSE cell proliferation kit (ThermoFisher Scientific, #C34554).

[0103] Dynabeads TM CD4+ T cells were activated using Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Veritas, DB11131), seeded into 96-well plates (1x10^5 cells / well) and cultured for 3-7 days in the presence of the humanized anti-CD73 antibody 002_6_h4 and the substrate AMP (100μM). The cells were subjected to flow cytometry analysis to measure fluorescence intensity, and the percentage of divided cells was analyzed based on the fluorescence intensity.

[0104] The effect of 002_6_h4 on the division (proliferation) of human peripheral blood T cells is shown in Figure 11. The percentage of divided T cells increased in an antibody concentration-dependent manner, demonstrating the effect of anti-CD73 antibodies in promoting T cell division.

[0105] Example 9: Evaluation of antibody function (concentration-dependence evaluation) This example was carried out with the aim of evaluating the functions of antibodies other than the 002_m antibody obtained in Example 1 (i.e., 003_m antibody, 004_m antibody, 005_m antibody, 006_m antibody, 007_m antibody, 008_m antibody, and 009_m antibody).

[0106] The antibody function evaluation was carried out by the same method as the antibody function screening described in Example 1 (1-3). That is, human lung cancer cell line H322 cells or human breast cancer cell line MDA-MB-231 cells (ATCC® HTB-26 TM In this example, the AMP degradation reaction was measured in the same manner as in Example 1, and the antibody concentration dependency of the AMP degradation inhibitory function was analyzed to calculate the IC50 value (50% inhibitory concentration: here, this refers to the antibody concentration that can inhibit 50% of the enzymatic function of CD73) to quantitatively evaluate the antibody function.

[0107] The inhibitory functions of the 003_m antibody, 004_m antibody, 005_m antibody, 006_m antibody, 007_m antibody, 008_m antibody, and 009_m antibody prepared in Example 1 on AMP degradation in H322 cells are shown in Figure 12, and their inhibitory functions on AMP degradation in MDA-MB-231 cells are shown in Figure 13. Although some of these antibodies are mouse antibodies, they were found to exhibit IC50 values ​​lower than or close to those of the benchmark antibodies (see Table 7).

[0108] [Table 7]

[0109] Furthermore, clones judged to be highly functional based on the tests shown in Figures 12 and 13 above (004_m, 006_m, 007_m, 008_m) were selected. The AMP degradation inhibitory function (MDA-MB-231 cells) of these four antibodies is shown below. The IC50 values ​​of these clones were favorable compared to the benchmark antibody (under clinical development) and were almost equivalent to that of 002_6_c (see Table 8).

[0110] [Table 8]

[0111] Example 10: Antibody gene sequence analysis This example was carried out with the aim of determining the amino acid sequences of the 003_m to 009_m antibodies obtained in Example 1.

[0112] For the clones of antibodies 003_m to 009_m, which were found to have the ability to inhibit AMP degradation in functional analysis, single colonies were recovered from an inoculated plate of Escherichia coli transfected with the ligation product prepared in Example 1 (1-1), and plasmid DNA was obtained.

[0113] The resulting plasmid DNA was analyzed by Sanger sequencing to determine the gene sequences defining the heavy chain variable region and the light chain variable region, and the amino acid sequence was determined based on the sequences.

[0114] The heavy chain variable region amino acid sequences (Figure 14-1) and light chain variable region amino acid sequences (Figure 14-2) of the 003_m antibody, 004_m antibody, 005_m antibody, 006_m antibody, 007_m antibody, 008_m antibody, and 009_m antibody are shown. SEQ ID NOs were assigned to each amino acid sequence as shown in Table 9 below.

[0115] [Table 9]

[0116] Example 11: Analysis of binding to target-expressing cells This example was carried out with the aim of evaluating the binding ability of each of the antibodies 003_m to 009_m obtained in Example 1.

[0117] Binding analysis to target-expressing cells was performed by FACS analysis using CD73 native-expressing cells (human lung cancer cell line H322 or human breast cancer cell line MDA-MB-231) or CD73-overexpressing cells. CD73-overexpressing cells were generated using a lentiviral vector system (Origene). HEK293T cells were infected with lentiviral particles carrying the human CD73 gene (NM_002526) and a puromycin resistance gene. The cells were then cultured in the presence of puromycin. HEK293T cells, which had integrated the target gene into their genome and become CD73-positive, were collected and used for binding analysis. HEK293T cells that do not express CD73 were used as a negative control.

[0118] After reacting the test antibody or isotype control antibody with the cells, the cells were stained with a fluorescently labeled secondary antibody and subjected to flow cytometry analysis, and the antibody binding was evaluated based on the obtained fluorescence intensity.

[0119] The binding of the 003_m to 009_m antibodies to target-expressing cells (H322 cells) is shown in Figure 15, the binding of the 003_m to 009_m antibodies to target-expressing cells (MDA-MB-231 cells) is shown in Figure 16, and the binding of the 003_m to 009_m antibodies to target-expressing cells (CD73-overexpressing HEK-293T cells) is shown in Figure 17. In Figures 15 to 17, the solid lines and gray fills indicate the fluorescence intensity histograms of the test antibodies and isotype control antibodies, respectively.

[0120] Example 12: Evaluation of effects on T cell division This example was carried out with the aim of confirming the proliferation-promoting activity of the anti-CD73 antibody obtained in Example 9 on human peripheral blood T cells.

[0121] The same method as in Example 8 was used, and the percentage of dividing cells was compared when the antibody concentration was set to 1 nM. The results are shown in Figure 18. The dotted line indicates the percentage of dividing cells in the case of the isotype control antibody.

[0122] Specifically, CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs) derived from healthy volunteers using a CD4+ T Cell Isolation Kit, human (Miltenyi Biotech, 130-096-533), and the cells were fluorescently labeled using a CellTrace CFSE cell proliferation kit (ThermoFisher Scientific, #C34554).

[0123] Dynabeads TM CD4+ T cells were activated using Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Veritas, DB11131), then seeded into 96-well plates (1x10^5 cells / well) and cultured for 3-7 days in the presence of anti-CD73 antibodies (006_m, 007_m, 008_m) and the substrate AMP (100μM). The cells were subjected to flow cytometry analysis to measure fluorescence intensity, and the percentage of divided cells was analyzed based on the fluorescence intensity.

[0124] The effect of 1 nM anti-CD73 antibody (006_m antibody, 007_m antibody, or 008_m antibody) on the division (proliferation) of human peripheral blood T cells is shown in Figure 18. In the figure, "w / o AMP" and "with AMP" (100 μM AMP) were included to examine the effect of AMP without antibody, since AMP has an inhibitory effect on T cell division. On the other hand, in groups treated with 1 nM, 0.1 nM, or 0.01 nM of humanized anti-CD73 antibody (006_m antibody, 007_m antibody, 008_m antibody), the percentage of divided T cells increased in an antibody concentration-dependent manner (data not shown), demonstrating the effect of anti-CD73 antibody in promoting T cell division.

[0125] All of the 003_m to 009_m antibodies promoted cell division more than the isotype control antibody, and were almost equivalent to the benchmark antibodies, 002_6_c and 002_6_h4.

[0126] Example 13: Preparation of humanized antibodies (2) This example was carried out with the aim of producing humanized antibodies based on the 004_m antibody, 006_m antibody, 007_m antibody, and 008_m antibody selected in Example 9 from each of the antibodies 003_m to 009_m obtained in Example 1.

[0127] Humanized antibodies were produced using the publicly available software Tabhu (http: / / circe.med.uniroma1.it / tabhu / ), as in Example 6. Using this software, sequences with similar sequence and conformational characteristics to the 004_m, 007_m, and 008_m antibodies were extracted from a human antibody database, and five sequences for the 004_m, four sequences for the 007_m, and four sequences for the 008_m antibodies were selected as candidate sequences. Sequences other than the CDR regions of these antibodies were then grafted with the sequences of the CDR regions of the 004_m, 007_m, and 008_m antibodies, respectively.

[0128] For the 006_m antibody, six candidate humanized antibody sequences were created using commercially available biological software.

[0129] Next, we backmutated some amino acid residues from the grafted 004m, 007m, and 008m antibodies to their mouse sequences. The amino acid residues targeted for backmutation were selected based on the structural differences between humanized and mouse antibodies (high TubHu scores), with a maximum of 10 amino acid residues per sequence. Details were obtained according to the method described in Bioinformatics. 2015 Feb 1;31(3):434-5.

[0130] The amino acid sequences of the variable regions of the humanized antibodies (004_h3 antibody, 006_hKB antibody, 007_h1 antibody, 008_h4 antibody) obtained based on the 004_m antibody, 006_m antibody, 007_m antibody, and 008_m antibody are shown in Table 10. SEQ ID NO: 81 to SEQ ID NO: 88 were assigned to each amino acid sequence, as shown in Table 10.

[0131] [Table 10]

[0132] Each of the obtained humanized antibodies (004_h3 antibody, 006_hKB antibody, 007_h1 antibody, 008_h4 antibody) was compared with existing antibodies (commercially available anti-CD73 antibody (biolegend), benchmark antibody) and the base chimeric antibody 002_6_c, and their binding to various cells was confirmed by flow cytometry.

[0133] Binding analysis to human target-expressing cells was performed by FACS analysis using CD73 native-expressing cells (human lung cancer cell line H322). Binding analysis to target-expressing cells was performed using CD73 native-expressing cells (human breast cancer cell line MDA-MB-231), human breast cancer cell line MDA-MB-231 cells expressing the CD73 antigen, HEK293T wild-type cells (derived from human embryonic kidney cells) that do not express the CD73 antigen, and HEK293T cells overexpressing the CD73 antigen. CD73-overexpressing cells were generated using a lentiviral vector system (Origene). Lentiviral particles carrying the human CD73 gene (NM_002526), ​​the rhesus macaque CD73 gene (XM_001086989.2) (the amino acid sequence is identical to that of cynomolgus macaque CD73), and a puromycin resistance gene were produced and used to infect HEK293T cells. The cells were then cultured in the presence of puromycin, and the target gene was integrated into the genome, resulting in CD73-positive HEK293T cells. These cells were then harvested and used for binding analysis. After reacting with the test antibody or isotype control antibody, the cells were stained with a fluorescently labeled secondary antibody and subjected to flow cytometry analysis. Antibody binding was assessed based on the fluorescence intensity obtained.

[0134] The results are shown in Figures 19 to 21. Figures 19 and 20 show the binding of the antibodies to cells expressing the human CD73 gene, and Figure 21 shows the binding of the antibodies to cells expressing the rhesus monkey CD73 gene. Of the humanized antibodies tested, the 004_h3, 006_hKB, and 008_h4 antibodies were confirmed to exhibit binding to the human CD73 antigen similar to that of the comparative antibody 002_6_h4, confirming their binding to the CD73 antigen. Furthermore, the 006_hKB antibody was confirmed to exhibit binding to the rhesus monkey CD73 antigen (cynomolgus monkey CD73 antigen) similar to that of the 002_6_h4 antibody, indicating that rhesus monkeys or cynomolgus monkeys can be used in future animal experiments to confirm the effects of the antibodies of the present invention.

[0135] Example 14: Inhibitory function against recombinant antigens This example was carried out with the aim of confirming the function of the humanized anti-CD73 antibody obtained in Example 13 in inhibiting the enzymatic reaction of recombinant CD73 antigen.

[0136] The antibody function evaluation was carried out in the same manner as the antibody function screening described in Example 1 (1-3), using the 004_h3 antibody, 006_hKB antibody, 007_h1 antibody, and 008_h4 antibody as test antibodies, and the 002_6_c antibody, 002_6_h4 antibody, an isotype control antibody, and a benchmark antibody (MEDI9447 antibody) as controls. Specifically, the human breast cancer cell line MDA-MB-231 cells (ATCC® HTB-26 TM ) were seeded into a 96-well plate and incubated overnight at 37°C. The next day, the cells were washed with L-15 medium (Fujifilm Wako Pure Chemical Industries, Ltd., 128-06075), and purified antibodies were added and incubated for 1 hour at 37°C. In this example, as in Example 1 (1-3), AMP (Sigma, A1752), a substrate for CD73, was added to a final concentration of 200 μM. The cells were incubated at 37°C for 4.5 hours to measure the degradation of AMP to adenosine by CD73, and the antibody concentration dependence of the AMP degradation inhibitory function was analyzed. The degradation reaction was performed. After the reaction, the supernatant was collected, and ATP (Sigma, A2383, final concentration 10 μM) and Cell TiterGlo (Promega, G9243) were added and mixed, and chemiluminescence was measured using a microplate reader.

[0137] The AMP degradation inhibitory functions of the 004_h3 antibody, 006_hKB antibody, 007_h1 antibody, and 008_h4 antibody are shown in Figure 22. All of these antibodies were shown to have the function of inhibiting AMP degradation by CD73 present on the cell surface (Figures 22 and 23), and of these antibodies, the 006_hKB antibody in particular was shown to have inhibitory activity equal to or greater than that of the benchmark antibody, as was the case with 002_6_h4.

[0138] Example 15: Evaluation of effects on T cell division This example was carried out with the aim of confirming the proliferation-promoting activity of the anti-CD73 antibody obtained in Example 13 and which showed particularly high efficacy in Example 14 on human peripheral blood T cells.

[0139] Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were incubated with various antibodies (006_hKB antibody, 002_6_h4 antibody, benchmark antibody (MEDI9447 antibody), or isotype control antibody) at a final concentration of 0.0005-1 μg / ml for 30 minutes at room temperature. Subsequently, ATP and Dynabeads (350 μM final concentration) were added. TM Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Veritas, DB11131) was added and the cells were incubated at 37°C and 5% CO2 for 96 hours. After incubation, the cells were stained with T cell marker antibodies (BioLegend, anti-CD3: UCHT1, anti-CD4: RPA-T4, anti-CD8a: HIT8a). Cell counting beads were added to the stained cells and analyzed using a flow cytometer to calculate the number of CD4- and CD8-positive cells per well.

[0140] In addition, the concentrations of IFNγ, TNFα, and IL-2 in the collected culture supernatant were measured by the Luminex method using various antibodies [capture antibodies = IFNγ: M7004 (Thermo), TNFα: MAB602 (R&D Systems), IL-2: MAB610; detection antibodies = IFNγ: M701B (Thermo), IL-2: BAF202 (R&D Systems), TNFα: BAF210 (R&D Systems)].

[0141] The results of evaluating the effect of antibodies on T cell division are shown in Figure 23. Compared to the 002_6_h4 antibody, which was found to exhibit good activity in Example 8, the 006_hKB antibody was shown to have a high T cell division-promoting effect on both CD4-positive T cells and CD8-positive T cells, comparable to that of the 002_6_h4 antibody (Figure 23, upper panel). It was also confirmed that high concentrations of IFNγ, TNFα, and IL-2 were released into the culture supernatant from cells administered with the 006_hKB antibody (Figure 23, lower panel).

[0142] Example 16: Evaluation of antibody uptake into cells This example was carried out with the aim of evaluating the intracellular uptake of the anti-CD73 antibody obtained in Example 6 and the anti-CD73 antibody obtained in Example 13 and which showed particularly high efficacy in Example 14.

[0143] Human breast cancer cells MDA-MB-231 (1×10^5 cells) were seeded onto chamber slides (Thermo Fisher Scientific, 154534PK) and incubated with 10 μg / ml of various antibody solutions (002_6_h4, 006_hKB, MEDI9447) at 37°C for 15 minutes or 19 hours. Before and after incubation, the samples were fixed and permeabilized (Thermo Fisher Scientific, FIX & PERM Fixation and Permeabilization Kit, GAS003) and stained with Anti-human IgG Alexa Fluor 594 (Thermo Fisher Scientific, A-11014). The distribution of the antibody was observed under a fluorescence microscope (Thermo Fisher Scientific, EVOS FLoid Imaging System, 4471136) to determine whether the antibody had been internalized within the cells.

[0144] The intracellular uptake of 002_6_h4 and 006_hKB is shown in Figure 24. For both 002_6_h4 and 006_hKB, the antibody was distributed on the cell surface after 15 minutes of incubation (top panel of Figure 24), whereas after 19 hours of incubation (bottom panel of Figure 24), the antibody accumulated inside the cells, indicating that the antibody had been internalized. This result suggests that the uptake of anti-CD73 antibodies may induce the disappearance of antigens from the cell surface.

[0145] The internalization of such antibodies into T cells was quantified over time. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with Dynabeads. TM After 6 days of stimulation with Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Veritas, DB11131), the cells were harvested and 10 μg / ml of various antibodies (002_6_h4 antibody, 006_hKB antibody, benchmark antibody (MEDI9447 antibody), isotype control antibody) were added and incubated on ice for 30 minutes.

[0146] After washing, the cells were suspended in FACS buffer (PBS, 0.5% BSA, 2 mM EDTA) and incubated at 37°C for up to 240 minutes. Cell samples were taken at 30, 120, and 240 minutes. Each sample was stained with a fluorescently labeled secondary antibody (APC anti-human IgG Fc) (BioLegend, 409306) and subjected to flow cytometry analysis. The percentage of antibody internalization was calculated using the following formula: % Control = (Anti-CD73 antibody (X min) MFI - Isotype control (X min) MFI) / (Anti-CD73 antibody (0 min) MFI - Isotype control (0 min) MFI) x 100

[0147] The results are shown in Figure 25. 002_6_h4 and 006_hKB had a higher internalization rate than the benchmark antibody at each measurement time. This result also suggests that the uptake of anti-CD73 antibodies may induce the loss of antigen from the cell surface.

[0148] Internalization of cell surface CD73 in cancer cells treated with the antibodies of the present invention was quantified over time. Human breast cancer cells MDA-MB-231 and human melanoma cells HT-144 were suspended in fetal bovine serum-free medium (McCoy's 5A (Modified) (Gibco, 16600082)) for MDA-MB-231 and HT-144, respectively. After suspension, 10 μg / ml of various antibodies (002_6_h4 antibody, 006_hKB antibody, benchmark antibody (MEDI9447 antibody), and isotype control antibody) were added and incubated at 37°C for 1, 3, 6, 22, and 48 hours.

[0149] Each antibody was added again at 10 μg / mL and allowed to react. After washing, the cells were stained with a fluorescently labeled secondary antibody and subjected to flow cytometry analysis. The internalization rate of the antigen was evaluated based on the fluorescence intensity obtained. The internalization rate of the antigen was calculated using the following formula. %CD73 Ag = (anti-CD73 antibody (X min) MFI - isotype control (X min) MFI) / (anti-CD73 antibody (0 min) MFI - isotype control (0 min) MFI) x 100

[0150] The results are shown in Figure 26. 002_6_h4, 006_hKB, and the benchmark antibody all reduced the amount of CD73 expression on the cancer cell surface over time when incubated with cancer cells. This suggests that the CD73-bound anti-CD73 antibody was internalized along with the antigen, thereby reducing the amount of CD73 expression on the cell surface.

[0151] Example 17: Measurement of KD value This example was conducted with the aim of evaluating the dissociation constant (KD value), which is an indicator of binding affinity for the CD73 antigen, of the anti-CD73 antibody obtained in Example 6 and the anti-CD73 antibody obtained in Example 13 and which showed particularly high efficacy in Example 14.

[0152] The KD values ​​of antibody binding to the target antigen were measured using a Biacore 8K (GE Healthcare). The 002_6_h4 and 006_hKB antibodies were immobilized on a sensor chip (CM5) using the Human Antibody Capture Kit Type 2 (Cytiva, 26-2346-00). Human CD73 antigen was immobilized at 5 × 10 -9 Four serially diluted solutions were prepared from M and subjected to multi-cycle kinetic analysis. The KD values ​​in the 1:1 binding model were as shown in Table 11 below.

[0153] [Table 11]

[0154] Example 18: Human-immunized mouse study This example was conducted to confirm the antitumor effects of the anti-CD73 antibody obtained in Example 6 and the anti-CD73 antibody obtained in Example 13, which showed particularly high efficacy in Example 14, in mice transplanted with human CD34-positive cells.

[0155] Human CD34-positive cell-transplanted NSG mice (hu-NSG, Jackson Laboratories) were generated from multiple healthy donors. The human breast cancer cell line MDA-MB-231 was transplanted into the mammary fat pad of these mice to create a human breast cancer cell-bearing mouse model. The transplanted human breast cancer cells proliferated to 100 mm 3 At this stage, 002_6_h4 antibody or isotype control antibody was intraperitoneally administered at a dose of 20 mg / kg once every four days for a total of seven times (referred to as "Q4Dx7"). The day of administration was first designated as day 0, and the mice were observed until day 34.

[0156] After the observation period, tumors were excised and prepared into cell suspensions using a Gentle MACS (Miltenyi). Cells were stained with human (hu)CD45 AF700 clone HI30 (BioLegend), huCD3 APCCy7 clone HITa (BioLegend), huCD4 PECy7 clone SK3 (BioLegend), and huCD8 FITC clone SK1 (BioLegend), and the number of tumor-infiltrating CD45+, CD3+, CD8+ T cells and CD45+, CD3+, CD4+ T cells was measured by flow cytometry.

[0157] The number of tumor-infiltrating T cells in mice was measured on day 34 after administration of the 002_6_h4 antibody or isotype control antibody. The results are shown in Figure 27. The number of tumor-infiltrating T cells tended to increase with administration of the 002_6_h4 antibody compared with administration of the isotype control antibody, suggesting that the anti-CD73 antibody has an anti-tumor effect.

[0158] In this example, it was further confirmed whether the developed antibody enhances the cancer cell cytotoxicity of T cells, and if so, to what extent, by comparing it with a benchmark antibody.

[0159] Human lung cancer cells NCI-H292 or HCC827 transfected with RFP protein were seeded onto a 96-well plate and cultured overnight. The next day, the medium was removed, and IL-2 (final concentration 30 IU) and various antibodies suspended in X-VIVO-15 medium (Lonza, 04-418Q) were added and incubated at 37°C for 30 minutes. Dynabeads were then used. TMPBMCs from healthy volunteers were stimulated with Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Veritas, DB11131) for 10 days to allow T cell proliferation. These were added at an E / T ratio of 2 and incubated at 37°C for 30 minutes. ATP was added to NCI-H292 at a final concentration of 500 μM, and ATP to HCC827 at a final concentration of 300 μM. Time-dependent changes in cancer cell proliferation were observed using an IncuCyte ZOOM System (Sartorius) at 37°C and 5% CO2.

[0160] As controls, in addition to an isotype control antibody and a benchmark antibody (MEDI9447), we used an anti-PD-1 antibody (pembrolizumab, Keytruda, MSD) and an anti-PD-L1 antibody (atezolizumab), which work by interfering with the interaction between the PD-1 antigen on cancer cells and the PD-L1 antigen on T cells, thereby inhibiting T cell suppression and activating T cells.

[0161] The results are shown in Figure 28. The developed antibodies (002_6_h4 and 006_hKB were used as examples in this example, but the same applies to other developed antibodies of the present invention) enhanced the effect of T cells in suppressing cancer cell proliferation under conditions in which the action of anti-PD-1 antibodies showed little effect in relieving T cell suppression and failed to stop cancer cell proliferation.

[0162] In this study, we further investigated whether 002_6_h4 could enhance the antitumor effect of a simulated human immune system in mice through animal experiments. We also examined whether an additional effect could be observed when administered in combination with an anti-PD-1 antibody (pembrolizumab), the effectiveness of which has been proven in clinical trials. We established a human breast cancer mouse model by transplanting the human breast cancer cell line MDA-MB-231 into the mammary fat pad of NSG mice (hu-NSG, Jackson Laboratories) (hereafter referred to as "immune humanized mice") transplanted with human CD34-positive cells prepared from multiple healthy donors. In this mouse model, the transplanted human breast cancer cells proliferated to a size of 100 mm. 3 At this stage, the 002_6_h4 antibody or isotype control antibody was administered intraperitoneally at a dose of 20 mg / kg once every four days for a total of seven doses (referred to as "Q4Dx7"), in combination with a checkpoint inhibitor (anti-PD-1 antibody pembrolizumab, Keytruda, MSD) or isotype control antibody at a dose of 10 mg / kg for the first dose and 5 mg / kg thereafter, once every five days for a total of six doses (referred to as "Q5Dx6"). The first day of administration was designated day 0, and tumor size was measured twice weekly until day 34. The antibody administration schedule was as follows: Group 1 = Isotype control antibody only Group 2 = 002_6_h4 antibody and isotype control antibody administration group Group 3 = Checkpoint inhibitor (pembrolizumab) and isotype control antibody treatment group Group 4 = 002_6_h4 antibody and checkpoint inhibitor (pembrolizumab) combination therapy group.

[0163] The mean and standard error of the tumor volume increase rate from day 0 to day 34 for each treatment group in a representative donor are shown in Figure 29. For both donor #5910 and donor #0030, the mean tumor volume increase rate in the combination treatment group (Group 4) was lower than that in the control group (Group 1) and the checkpoint inhibitor treatment group (Group 3).

[0164] Furthermore, in donor #0030, the 002_6_h4 antibody-treated group (Group 2) showed a lower mean tumor volume increase rate compared to the control group (Group 1), suggesting that anti-CD73 antibodies have anti-tumor effects in immunized humanized mice, either alone or in combination with a checkpoint inhibitor (pembrolizumab).

[0165] Example 19: Immunodeficient Mouse Study This example was carried out with the aim of confirming the antitumor effect of the anti-CD73 antibody obtained in Example 6 in immunodeficient mice.

[0166] Human breast cancer cell line MDA-MB-231 was transplanted into the mammary fat pad of NSG mice (JACKSON Laboratories). The transplanted human breast cancer cells grew to 100 mm 3 At this stage, the 002_6_h4 antibody or isotype control antibody was intraperitoneally administered at each dose once every four days for a total of seven times (Q4Dx7) in the groups shown in Table 12. The day administration began was designated day 0, and the observation period continued until day 35, during which tumor size was measured twice a week.

[0167] [Table 12]

[0168] The results of changes in tumor mass volume in the bodies of immunodeficient mice are shown in Figure 30. The 002_6_h4 antibody was shown to have an inhibitory effect on tumor growth compared to the isotype control.

[0169] Example 20: Antitumor effect (knock-in model) This example was carried out with the aim of confirming the anti-tumor effect of the anti-CD73 antibody obtained in Example 6 in knock-in mice expressing human CD73.

[0170] Knock-in mice (B-hCD73 mice) expressing human CD73 instead of mouse CD73 were subcutaneously implanted with 5x10^5 colon cancer cells (MC38-hCD73, Biocytogen). When the implanted MC38-hCD73 cells reached a volume of 50-80 mm3, they were intraperitoneally injected with either the 002_6_h4 antibody or an isotype control antibody at a dose of 10 mg / kg twice weekly for a total of seven doses (referred to as "BIWx7"), and with either the anti-mouse PD-1 antibody clone RMP1-14 (Bio X Cell, BE0146) or a rat IgG2a isotype control antibody (Bio X Cell, BE0089) at a dose of 3 mg / kg once weekly (referred to as "QWx4"). The tumor diameter was measured every 3-4 days, and observations were conducted from the day of administration start date (day 0) until day 18. The antibody administration was as follows: Group 1 = Isotype control antibody only Group 2 = 002_6_h4 antibody and isotype control antibody administration group Group 3 = Checkpoint inhibitor (anti-mouse PD-1 antibody) and isotype control antibody administration group Group 4 = Group administered with the 002_6_h4 antibody in combination with a checkpoint inhibitor (anti-mouse PD-1 antibody).

[0171] The change in mean tumor volume from day 0 to day 18 is shown as the mean and standard error for each treatment group in Figure 31. The mean tumor volume in the combination treatment group (Group 4) was lower than that in the control group (Group 1) and the checkpoint inhibitor treatment group (Group 3), suggesting that anti-CD73 antibodies have an antitumor effect when used in combination with checkpoint inhibitors in mice expressing human CD73. [Industrial Applicability]

[0172] The antibody or human antibody derivative obtained by the present invention suppresses the function of CD73 expressed in cancer cells or immune cells, and relieves exhaustion of immune cells (especially T cells), thereby activating the immune cells and exerting tumor growth inhibitory effects and cancer therapeutic effects.

[0173] The present invention can be widely used as a therapeutic and diagnostic agent for cancer. Because the antibody according to the present invention has the effect of relieving exhaustion of immune cells, it is expected to be particularly effective in combination with other immunotherapies.

Claims

1. (1) Heavy chain complementarity determining region, CDR1 (DX 1 NMD (X 1 is C or S), SEQ ID No.: 1), CDR2 (DINPNNGGTIYNQKFKG, SEQ ID No.: 2), and CDR3 (TNWDYAMDY, SEQ ID No.: 3), and Light chain complementarity determining region, CDR1 (KASQDINSX 2 LS (X 2 is N or D or Q), SEQ ID No.: 4), CDR2 (RANRLID, SEQ ID No.: 5), and CDR3 (X 3 QYDVFPRT(X 3 is L or Q), SEQ ID No.: 6); (2) heavy chain complementarity determining regions, CDR1 (SFGMH, SEQ ID No.: 9), CDR2 (YISSGSRTIYYADTVRG, SEQ ID No.: 10), and CDR3 (DFGSSSPNYFDY, SEQ ID No.: 11); and light chain complementarity-determining regions, CDR1 (RASESVDNYGISFMN, SEQ ID No.: 12), CDR2 (AASNQGS, SEQ ID No.: 13), and CDR3 (QQSKEVPWT, SEQ ID No.: 14); (3) heavy chain complementarity determining regions, CDR1 (GYWMN, SEQ ID No.: 17), CDR2 (RIDPYDSETHYSQKFKD, SEQ ID No.: 18), and CDR3 (SSPITTAPFDY, SEQ ID No.: 19); and light chain complementarity determining regions, CDR1 (RASESVDYYGFSFMN, SEQ ID No.: 20), CDR2 (AASTQGS, SEQ ID No.: 21), and CDR3 (QQSKEVPYT, SEQ ID No.: 22); (4) heavy chain complementarity determining regions, CDR1 (SYGVS, SEQ ID No.: 25), CDR2 (VIWGDGSTNYHSALIS, SEQ ID No.: 26), and CDR3 (TNIFYDYDWYLDV, SEQ ID No.: 27); and light chain complementarity determining regions, CDR1 (RSSQSLVHSNGNTYLH, SEQ ID No.: 28), CDR2 (KVSNRFS, SEQ ID No.: 29), and CDR3 (SHSTHVPWT, SEQ ID No.: 30); (5) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 33), CDR2 (EINPSNARTNYNENFKS, SEQ ID No.: 34), and CDR3 (RGTSGNYFDF, SEQ ID No.: 35); and light chain complementarity determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 36), CDR2 (RANRLVD, SEQ ID No.: 37), and CDR3 (LQYDEFPYT, SEQ ID No.: 38); (6) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 41), CDR2 (KIDPYDSETHYNQKFKD, SEQ ID No.: 42), and CDR3 (IRYGTFDY, SEQ ID No.: 43); and light chain complementarity determining regions, CDR1 (KASQDINNYLS, SEQ ID No.: 44), CDR2 (RANILVD, SEQ ID No.: 45), and CDR3 (LQYDEFPYT, SEQ ID No.: 46); (7) heavy chain complementarity determining regions, CDR1 (SYWMH, SEQ ID No.: 49), CDR2 (EINPSNGRTNYNEKFKN, SEQ ID No.: 50), and CDR3 (RGTSGNYFDY, SEQ ID No.: 51); and light chain complementarity determining regions, CDR1 (KASQDINTYLS, SEQ ID No.: 52), CDR2 (RANRLVD, SEQ ID No.: 53), and CDR3 (LQYDEFPYT, SEQ ID No.: 54); (8) heavy chain complementarity determining regions, CDR1 (SYWMN, SEQ ID No.: 57), CDR2 (RIDPYDSEAHYNQKFKD, SEQ ID No.: 58), and CDR3 (IRYGTFDY, SEQ ID No.: 59); and light chain complementarity determining regions, CDR1 (KASQDINSYLS, SEQ ID No.: 60), CDR2 (RSNSLVD, SEQ ID No.: 61), and CDR3 (LQYDEFPYT, SEQ ID No.: 62); An antibody or humanized antibody derivative comprising a heavy chain or light chain complementarity determining region selected from the group consisting of: an antibody or humanized antibody derivative that binds to the CD73 antigen and activates T cells that are toxic to cancer cells.

2. The antibody or human antibody derivative of claim 1, wherein the activation of T cells is selected from the group consisting of T cell proliferation, increased cytotoxicity of T cells against cancer cells, and promotion of cytokine secretion by T cells.

3. The antibody or human antibody derivative according to claim 1 or 2, wherein the human antibody derivative is selected from a human antibody variant or a functional fragment thereof selected from a humanized antibody, a chimeric antibody, a multivalent antibody, and a multispecific antibody.

4. The antibody or human antibody derivative according to any one of claims 1 to 3, wherein the functional fragment is F(ab')2.

5. The antibody or humanized antibody derivative according to any one of claims 1 to 4, wherein the amino acid sequence of the heavy chain variable region VH domain of the antibody or humanized antibody derivative is selected from (1) SEQ ID No.: 7, (2) SEQ ID No.: 15, (3) SEQ ID No.: 23, (4) SEQ ID No.: 31, (5) SEQ ID No.: 39, (6) SEQ ID No.: 47, (7) SEQ ID No.: 55, and (8) SEQ ID No.:

63.

6. The antibody or humanized antibody derivative according to any one of claims 1 to 5, wherein the amino acid sequence of the light chain variable region VL domain of the antibody or humanized antibody derivative is selected from (1) SEQ ID No.: 8, (2) SEQ ID No.: 16, (3) SEQ ID No.: 24, (4) SEQ ID No.: 32, (5) SEQ ID No.: 40, (6) SEQ ID No.: 48, (7) SEQ ID No.: 56, and (8) SEQ ID No.:

64.

7. The antibody or humanized antibody derivative according to any one of claims 1 to 6, which induces cytotoxicity against cancer cells but not against normal cells.

8. The antibody or human antibody derivative according to any one of claims 1 to 7, wherein the cancer cells are selected from the group consisting of melanoma, breast cancer, lung cancer, and colon cancer.

9. The antibody or humanized antibody derivative according to any one of claims 1 to 8, which is conjugated with a drug to form an antibody-drug conjugate (ADC).

10. A pharmaceutical composition for cancer treatment, comprising the antibody or human antibody derivative according to any one of claims 1 to 9.

11. 11. The pharmaceutical composition of claim 10, wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, and colon cancer.

12. contacting cancer cells collected from a subject in vitro with the antibody or human antibody derivative according to any one of claims 1 to 9; a step of measuring whether AMP metabolism of cancer cells is reduced under culture conditions, or whether cell viability is reduced, or whether secretion of immune activating substances is enhanced; A method for measuring cytotoxicity to cancer cells, comprising:

13. The method for measuring cytotoxicity against cancer cells described in claim 12, which measures whether the cell viability of cancer cells decreases or whether immune cells derived from peripheral blood lymphocytes are activated in the presence of peripheral blood lymphocytes from the same subject.

14. The method for measuring cytotoxicity against cancer cells according to claim 12 or 13, wherein the method measures the enhancement of cytotoxicity against cancer cells when the antibody or human antibody derivative according to any one of claims 1 to 9 is administered to a subject based on the in vitro AMP metabolism inhibition or cytotoxicity of cancer cells collected from the subject.

15. The method for measuring cytotoxicity against cancer cells according to claim 12 or 13, wherein the method measures the enhancement of cytotoxicity against cancer cells when the antibody or human antibody derivative according to any one of claims 1 to 9 is administered to a subject based on the enhancement of secretion of immune activating substances in vitro.

16. 10. A kit for measuring in vitro the AMP metabolism, cytotoxicity, secretion of immunostimulatory substances, or activation of immune cells of the antibody or human antibody derivative according to any one of claims 1 to 9 against cancer cells collected from a subject, the kit comprising the antibody.

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