Anti-CD39 antibody

Abstract

Obtained is an excellent anti-CD39 antibody. Provided is an antibody or an antigen-binding fragment that binds to CD39, and contains heavy-chain CDRs 1-3 and light-chain CDRs 1-3 containing a specific amino acid sequence. This antibody may be a neutralizing antibody against CD39. This antibody may have the ability to inhibit malignant tumor proliferation.

Description

Anti-CD39 antibody

[0001] The technical field of the present invention relates to anti-CD39 antibodies.

[0002] CD39 is a two-transmembrane glycoprotein that has been reported to be involved in the immunosuppression of immune cells through its ATPase activity or ADPase activity (Non-Patent Document 1).

[0003] Patent Documents 1 and 2 describe antibodies that bind to CD39 and antibodies that have the ability to inhibit the enzymatic activity of CD39.

[0004] International Publication No. 2018 / 167267 U.S. Patent Application Publication No. 2019 / 0062448

[0005] Allard et al., Immunol Rev. 2017 Mar;276(1):121-144.

[0006] However, the antibodies of Patent Documents 1 and 2 do not have a sufficiently strong effect of inhibiting the proliferation of cancer cells, and there is room for improvement.

[0007] An object of the present invention is to provide an excellent anti-CD39 antibody, among other things.

[0008] As described in the Examples below, the present inventors immunized mice with a human CD39 recombinant antigen and produced a specific anti-CD39 antibody through screening. The effect of the obtained antibody on cancer cells was then examined, and surprisingly, it was found to have an excellent effect of inhibiting cancer cell proliferation. In particular, this antibody showed an unexpectedly significantly superior effect of inhibiting cancer cell proliferation compared to the anti-CD39 antibodies produced based on the above-mentioned Patent Documents 1 and 2.

[0009] According to one aspect of the present invention, there is provided an antibody or antigen-binding fragment that binds to CD39, comprising: (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (b) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; (c) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (d) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 21, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24; (e) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30;(f) an antibody or antigen-binding fragment comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, or (g) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42. The use of this antibody or antigen-binding fragment can produce an excellent inhibitory effect on cancer cell proliferation.

[0010] According to one aspect of the present invention, there is provided a polynucleotide or vector encoding an antibody or antigen-binding fragment comprising heavy chain CDR1 to 3 and light chain CDR1 to 3 of any one of (a) to (g) above. The above antibody can be produced using this polynucleotide or vector. According to one aspect of the present invention, there is provided a cell comprising the above polynucleotide or vector. The above antibody can be produced using this cell. According to one aspect of the present invention, there is provided a method for producing an antibody or antigen-binding fragment, comprising the step of growing the above cell. The above antibody can be produced using this production method.

[0011] According to one aspect of the present invention, there is provided a composition comprising an antibody or antigen-binding fragment that binds to CD39, the antibody or antigen-binding fragment comprising heavy chain CDR1-3 and light chain CDR1-3 of any one of (a) to (g) above. Use of this composition can produce excellent effects in inhibiting cancer cell growth or promoting the cytotoxic activity of immune cells. According to another aspect of the present invention, there is provided a pharmaceutical composition for treating malignant tumors, the composition comprising an antibody or antigen-binding fragment that binds to CD39, the antibody or antigen-binding fragment comprising heavy chain CDR1-3 and light chain CDR1-3 of any one of (a) to (g) above. Use of this composition can produce excellent effects in inhibiting cancer cell growth or promoting the cytotoxic activity of immune cells. According to another aspect of the present invention, there is provided a composition for inhibiting CD39 activity, the composition comprising an antibody or antigen-binding fragment that binds to CD39, the antibody or antigen-binding fragment comprising heavy chain CDR1-3 and light chain CDR1-3 of any one of (a) to (g) above. Use of this composition for inhibiting CD39 activity can produce excellent effects in inhibiting CD39 activity. According to one aspect of the present invention, there is provided a composition for promoting TNFα production by immune cells, comprising an antibody or antigen-binding fragment that binds to CD39, and which comprises heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above. Use of this composition for promoting TNFα production can produce an excellent effect of promoting TNFα production. According to one aspect of the present invention, there is provided a composition for promoting the cytotoxic activity of immune cells, comprising an antibody or antigen-binding fragment that binds to CD39, and which comprises heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above. Use of this promoting composition can produce an excellent effect of promoting cytotoxic activity.

[0012] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating malignant tumors, comprising an antibody or antigen-binding fragment that binds to CD39, comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above, for use in combination therapy with a chemotherapeutic agent and an antibody or antigen-binding fragment that binds to CD39, comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above. Use of this pharmaceutical composition enables excellent treatment of malignant tumors. According to one aspect of the present invention, there is provided a pharmaceutical composition for treating malignant tumors, comprising a chemotherapeutic agent and used in combination therapy with an antibody or antigen-binding fragment that binds to CD39, comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above. Use of this pharmaceutical composition enables excellent treatment of malignant tumors. According to one aspect of the present invention, there is provided a kit comprising the antibody or antigen-binding fragment. Use of this kit can produce excellent cancer cell proliferation inhibitory effects. According to one aspect of the present invention, there is provided a pharmaceutical composition for treating malignant tumors, comprising an antibody or antigen-binding fragment that binds to CD39, comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above, for use in combination therapy with a chemotherapeutic agent and an antibody or antigen-binding fragment that binds to CD39, comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (g) above. Use of this pharmaceutical composition enables excellent treatment of malignant tumors. According to one aspect of the present invention, there is provided a kit comprising the antibody or antigen-binding fragment. Use of this kit can produce excellent cancer cell proliferation inhibitory effects. According to one aspect of the present invention, there is provided a kit for treating malignant tumors, comprising the antibody or antigen-binding fragment. hi A composition for selective cell removal is provided. This composition can be used to selectively remove CD39 hi Cells can be selectively removed.

[0013] Figure 1 shows the results of CD39 inhibitory activity measurements. Figure 2 shows the results of CD39 inhibitory activity measurements. Figure 3 shows the results of CD39 inhibitory activity measurements. Figure 4A shows the results of CD39 inhibitory activity measurements. Figure 4B shows the results of CD39 inhibitory activity measurements. Figure 5 shows the results of CD39 inhibitory activity measurements. Figure 6 shows the results of SPR analysis. Figure 7 shows the results of SPR analysis. Figure 8 shows the results of FCM analysis (human CD39). Figure 9 shows the results of FCM analysis (human CD39). Figure 10 shows the results of FCM analysis (human CD39). Figure 11A shows the results of FCM analysis (human CD39). Figure 11B shows the results of FCM analysis (human CD39). Figure 12 shows the results of FCM analysis (monkey CD39). Figure 13 shows the results of FCM analysis (monkey CD39). Figure 14 shows the results of FCM analysis (monkey CD39). Figure 15 shows the results of FCM analysis (monkey CD39). Figure 16 shows the results of FCM analysis (monkey CD39). Figure 17 shows the results of measuring CD39 inhibitory activity. Figure 18 shows the results of FCM analysis (Treg CD39). Figure 19 shows the results of measuring CD4-positive T cell proliferation. Figure 20 shows the results of measuring CD8-positive T cell proliferation. Figure 21 shows the results of measuring CD4-positive T cell proliferation. Figure 22 shows the results of measuring CD8-positive T cell proliferation. Figure 23 shows the results of measuring CD3-positive T cell proliferation. Figure 24 shows the results of measuring CD4-positive T cell proliferation. Figure 25 shows the results of measuring CD8-positive T cell proliferation. Figure 26 shows the results of measuring TNFα concentration. Figure 27 shows the results of measuring antibody concentration after blood exposure. Figure 28 shows the results of measuring antibody antitumor activity. Figure 29 shows the results of measuring antibody antitumor activity. Figure 30 shows the results of measuring antibody antitumor activity. Figure 31 shows the results of measuring the antitumor activity of antibodies. Figure 32 shows the results of measuring the antitumor activity of antibodies. Figure 33 shows the results of measuring the antitumor activity of antibodies (in vivo, single agent). Figure 34 shows the results of measuring the antitumor activity of antibodies (in vivo, combined use).Figure 35 shows the results of measuring heart weight. Figure 36 shows the amino acid sequence of the CDR of the antibody of the example. Figure 37 shows the amino acid sequence of the variable region of the antibody of the example. Figure 38 shows the amino acid sequence of the variable region of the antibody of the example. Figure 39 shows the amino acid sequence of the variable region of the antibody of the example. Figure 40 shows the amino acid sequence of the variable region of the antibody of the example. Figure 41 shows the amino acid sequence of the constant region of the antibody of the example. Figure 42 shows the results of SPR analysis. Figure 43 shows the results of measuring CD39 inhibitory activity. Figure 44 shows CD4. + Figure 45 shows the results of measuring the number of CD4 T cells. + Figure 46 shows the results of measuring the percentage of T cells that have undergone six divisions. + Figure 47 shows the results of measuring the number of CD8 T cells. + Figure 48 shows the results of measuring the proportion of T cells that have divided six times. Figure 48 shows the results of glycosylation analysis. Figure 49 shows the results of binding analysis to activated Treg. Figure 50 shows the results of binding analysis to activated Treg. Figure 51 shows the results of ADCC activity measurement (reporter assay). Figure 52 shows the results of ADCC activity measurement (cancer cell line). Figure 53 shows the results of SPR analysis. Figures 54A-C are diagrams summarizing ADCC activity, affinity to FcγRIIIa, and fucosylation rate. Figure 55 shows the results of cell type analysis. Figure 56 shows the results of Treg elimination evaluation. Figure 57 shows the results of Treg elimination evaluation. Figure 58 shows CD8 + T CM and CD8 + T EMFigure 59 shows the results of a cytotoxic T cell induction test. Figure 60 shows the results of a cancer cell growth inhibition test using Treg-depleted PBMC. Figure 61 shows the results of SPR analysis. Figure 62 shows the results of SPR analysis. Figure 63 shows the results of CD39 inhibitory activity measurement. Figure 64 shows the results of glycan analysis. Figure 65 shows the results of ADCC activity measurement (Treg target). Figure 66 shows the correlation between ADCC activity and FcγRIIIa binding activity. Figure 67 shows the correlation between ADCC activity and fucosylation rate. Figure 68 shows the results of ADCC activity measurement (OAW-42 target). Figure 69 shows the results of ADCC activity measurement (OAW-42 target). Figure 70 shows the results of CD4 + Figure 71 shows the results of cell type analysis of T cells. + Figure 72 shows the results of cell type analysis of T cells. + FIG. 1 shows the results of cell type analysis of T cells.

[0014] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0015] According to one embodiment of the present invention, a novel anti-CD39 antibody is provided, which comprises, for example: (a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, (b) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12, (c) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (d) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 20, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 21, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 24; (e) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30;(f) an antibody comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, or (g) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42 (hereinafter, these may be abbreviated as any of CDR sets (a) to (g)). Use of such an antibody can produce excellent cancer cell proliferation inhibitory effects, as demonstrated in the Examples below. The excellent cancer cell proliferation inhibitory effect includes, for example, a high proliferation inhibitory effect, a proliferation inhibitory effect occurring in a short period of time, a high TNFα production promoting effect, or a high immunosuppression relieving effect, as compared with conventional anti-CD39 antibodies. The excellent cancer cell proliferation inhibitory effect includes, for example, an inhibitory effect on the decrease in heart weight when co-administered with doxorubicin, a Treg function reducing effect, a Teff division promoting effect, a Treg lysis effect, a Treg division suppressing effect, a Treg frequency reducing effect, or a CD39 hi The anti-CD39 antibody may have a selective cell removal effect. The cancer cell proliferation inhibitory effect of the anti-CD39 antibody may be an effect that occurs by promoting cytotoxic activity caused by immune cells (e.g., T cells or PBMCs). Promotion of cytotoxic activity may occur when the anti-CD39 antibody inhibits CD39 activity, thereby inhibiting the production of adenosine. Promotion of cytotoxic activity leads to an active reduction in cancer cells, and is therefore very beneficial for cancer treatment. Furthermore, the above-mentioned antibody may be used as an antibody drug with enhanced cancer treatment effects due to low fucose content, or as a CD39 antibody. hi It is an excellent antibody for preparing antibody drugs with excellent selective cell removal activity.

[0016] In one embodiment of the present invention, CD39 includes a protein designated ENTPD1 or Ectonucleoside triphosphate diphosphohydrolase 1. Details of CD39, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for CD39 listed in UniProt is, for example, P49961. The amino acid sequence of human CD39 is, for example, SEQ ID NO: 98. The origin of CD39 is not limited as long as it has CD39 activity. CD39 activity includes, for example, ATP hydrolysis activity, ADP hydrolysis activity, or the activity of converting ATP or ADP to AMP. CD39 activity may be assessed by incubating CD39 in an environment where it can come into contact with ATP and measuring the amount of ATP reduction. The amount of ATP reduction may be assessed, for example, by measuring chemiluminescence using CellTiter-Glo (Promega). CD39 includes, for example, CD39 derived from humans, monkeys, mice, rats, dogs, or cats.

[0017] In one embodiment of the present invention, the anti-CD39 antibody includes an antibody capable of inhibiting CD39 activity. Inhibition of activity includes, for example, inhibition of ATP degradation activity. In one embodiment of the present invention, the anti-CD39 antibody includes an antibody capable of suppressing the proliferation of malignant tumor cells. The suppression of proliferation may be suppression of malignant tumor growth in the presence of immune cells. In one embodiment of the present invention, the anti-CD39 antibody is an antibody having neutralizing activity against CD39 (neutralizing antibody), an antibody having the ability to inhibit CD39 function, an antibody having the ability to promote cytotoxic activity, an antibody having the ability to promote TNFα production by immune cells, an antibody having the ability to activate immune cells, an antibody having the ability to suppress the decrease in heart weight when co-administered with doxorubicin, an antibody having the ability to reduce Treg function, an antibody having the ability to promote Teff division, an antibody having the ability to dissolve Treg, an antibody having the ability to suppress Treg division, an antibody having the ability to reduce Treg frequency, or an antibody capable of inhibiting CD39. hiAntibodies that inhibit CD39 activity include antibodies that have the ability to selectively eliminate cells. Neutralizing antibodies include antibodies that can inhibit CD39 activity. In one embodiment of the present invention, anti-CD39 antibodies include antibodies that induce cytotoxicity against malignant tumor cells but do not induce cytotoxicity against normal cells. In one embodiment of the present invention, immune cells include, for example, T cells or PBMCs. In one embodiment of the present invention, T cells include, for example, Tregs (regulatory T cells). As used herein, antibodies that inhibit CD39 activity include antibodies that have the function of inhibiting CD39 activity or antibodies that have the ability to inhibit CD39 activity. As used herein, antibodies that inhibit the proliferation of malignant tumor cells include antibodies that have the function of inhibiting the proliferation of malignant tumor cells or antibodies that have the ability to inhibit the proliferation of malignant tumor cells.

[0018] In one embodiment of the present invention, the method for producing an anti-CD39 antibody is not particularly limited. For example, the antibody can be produced by (i) using modeling / simulation software (e.g., BIOVIA Discovery Studio 2020 (Dassault Systems)) to design an antibody variable region sequence (e.g., a humanized antibody variable region sequence) comprising any one of the CDR sets (a) to (g) above; (ii) incorporating the antibody variable region sequence into an expression vector encoding a constant region (e.g., a human constant region); (iii) transfecting the expression vector into cells; or (iv) recovering and purifying the antibody from the cells. The anti-CD39 antibody may also be produced by grafting any one of the CDR sets (a) to (g) above into an existing antibody sequence using a grafting technique such as that described in "Safdari et al., Biotechnol Genet Eng Rev. 2013;29:175-86." The anti-CD39 antibody may also be produced by immunizing a mammal with CD39 and recovering and purifying the antibody. Anti-CD39 antibodies may be produced via a step of screening antibodies using as an index the ability to inhibit CD39 activity or the strength of binding to CD39.

[0019] In one embodiment of the present invention, the anti-CD39 antibody is in the form of a monoclonal antibody, which can act against CD39 more efficiently than a polyclonal antibody.

[0020] In one embodiment of the present invention, the anti-CD39 antibody includes an antibody fragment (hereinafter, also referred to as an "antigen-binding fragment") that has CD39-binding activity. The antigen-binding fragment includes an antigen-binding fragment that has any one of the CDR sets (a) to (g) above and has CD39-binding activity.

[0021] In one embodiment of the present invention, the anti-CD39 antibody is D (M) is, for example, 9.0 × 10 -9 , 5.0×10 -9 , 3.0×10 -9 , 1.0×10 -9 , 9.0×10 -10 , 5.0×10 -10 , 3.0×10 -10 , or 1.0 × 10 -10 It may be equal to or less than the above value, or may be within the range of any two of these values. D (M) may be a value measured by SPR. D (M) is the K for CD39 (e.g., human CD39) D (M) may also be used.

[0022] In one embodiment of the present invention, the antibody class of the anti-CD39 antibody is not particularly limited and may be, for example, IgG, IgA, IgM, IgD, or IgE. Furthermore, the antibody subclass is not particularly limited and may be, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0023] In one embodiment of the present invention, the anti-CD39 antibody may be an antibody that binds to a wild-type or mutant form of CD39. Mutant forms of CD39 include those resulting from differences in DNA sequence between individuals, such as SNPs.

[0024] In one embodiment of the present invention, CDRs (complementarity-determining regions) are regions that form the antigen-binding site. Typically, CDRs are located on the Fv (variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)) of an antibody. Typically, CDRs, consisting of CDR1, CDR2, and CDR3, each consisting of approximately 3 to 30 amino acid residues, are present in the heavy chain and the light chain, respectively. It is known that the CDRs of the heavy chain particularly contribute to antibody binding to the antigen. Among the CDRs, CDR3 is known to contribute most to antibody binding to the antigen. The Fv region other than the CDRs is called the framework region, and includes FR1, FR2, FR3, and FR4, which are relatively well conserved among antibodies (e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co.).

[0025] One embodiment of the present invention is an antibody that binds to CD39, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, or a light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, 7, 13, 19, 25, 31, or 37, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, 8, 14, 20, 26, 32, or 38, and the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3. , 9, 15, 21, 27, 33, or 39, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, 10, 16, 22, 28, 34, or 40, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, 11, 17, 23, 29, 35, or 41, and the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, 12, 18, 24, 30, 36, or 42.

[0026] One embodiment of the present invention is an antibody that binds to CD39, comprising a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, or 99, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, or 100. In one embodiment of the present invention, the anti-CD39 antibody comprises: (h) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 43 and 44, respectively; (i) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 45 and 46, respectively; (j) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 47 and 48, respectively; (k) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 49 and 50, respectively; (l) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 51 and 52, respectively; (m) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 53 and 54, respectively; (n) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 55 and 56, respectively; (o) a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 57 and 58; (p) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 59 and 60, respectively; (q) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 61 and 62, respectively; (r) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 63 and 64, respectively; (s) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 65 and 66, respectively; (h) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 67 and 68, respectively; (t) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 69 and 70, respectively; (u) heavy chain variable regions and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 71 and 72, respectively.(v) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 73 and 74, respectively; (w) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 75 and 76, respectively; (x) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 77 and 78, respectively; (y) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 79 and 80, respectively; (z) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 81 and 82, respectively; (a2) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 83 and 84, respectively; (b2) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 85 and 86, respectively; or (c2) heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 99 and 100, respectively. The use of such antibodies can produce an excellent effect of inhibiting the proliferation of cancer cells.

[0027] In one embodiment of the present invention, the anti-CD39 antibody includes an antibody that competes with an anti-CD39 antibody according to an embodiment of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above) (hereinafter, also referred to as a "reference antibody") for binding to CD39. Competition involves an antibody binding to an antigen and thereby inhibiting the binding of another antibody to the same antigen. Competitive antibodies can be identified, for example, by competitive binding assays. Competitive binding assays include, for example, competitive ELISA and competitive FACS analysis (see, for example, Zhou et al., J Gen Virol. 2008 Feb;89(Pt 2):500-508). Binding may also be measured, for example, by surface plasmon resonance. A competitive binding assay may include, for example, the steps of coating an antigen on a microplate, adding a test antibody and incubating the plate to allow binding between the antigen and the test antibody, adding a labeled reference antibody to the well, incubating, and washing, or quantifying the amount of labeled reference antibody bound to the antigen. In this case, for example, the amount of binding of the biotinylated reference antibody may be detected by measuring absorbance at a wavelength of 450 nm using HRP-conjugated streptavidin and 3,3',5,5'-tetramethylbenzidine. The inhibition rate may be evaluated as the percentage decrease in the amount of binding of the labeled reference antibody compared to when the test antibody is not added or when a negative control is added. Inhibition of the reference antibody by a competing antibody includes 20, 30, 40, 50, 60, 70, 80, 90% or more, or 100% inhibition. This inhibition is preferably 40% or more, and particularly preferably 50% or more. The competing antibody may have binding affinity to the epitope bound by the reference antibody.

[0028] In one embodiment of the present invention, the antibody includes an antibody that binds to CD39. Antibodies include molecules or populations thereof that can specifically bind to a specific epitope on an antigen. The antibody may be a polyclonal or monoclonal antibody. Antibodies can exist in various forms, including full-length antibodies (antibodies having Fab and Fc regions), Fv antibodies, Fab antibodies, F(ab')2 antibodies, Fab' antibodies, diabodies, single-chain antibodies (e.g., scFv), dsFv, scFvFc, multispecific antibodies (e.g., bispecific antibodies), antigen-binding peptides or polypeptides, chimeric antibodies, murine antibodies, humanized antibodies, human antibodies, and their equivalents. The antibody may also be a modified or unmodified antibody. Modified antibodies may be bound to various molecules, such as polyethylene glycol. Modified antibodies can be obtained by chemically modifying antibodies using known techniques. The antibody may also be a fusion protein. A fusion protein may be an antibody having a polypeptide or oligopeptide (e.g., a His tag) attached to the N- or C-terminus thereof. The fusion protein may also be an antibody having a mouse or human antibody subsequence fused thereto. The antibody may also be a conjugated antibody (e.g., an antibody-drug conjugate). The drug may be, for example, a cytotoxic drug or an anticancer drug. Such antibody modifications, fusion proteins, and conjugated antibodies are also included in the scope of antibodies. The amino acid sequence, class, or subclass of the antibody may be derived from, for example, a human or non-human mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.). Antibodies include, for example, isolated antibodies, purified antibodies, and recombinant antibodies. Antibodies may be used, for example, in vitro or in vivo.

[0029] In one embodiment of the present invention, monoclonal antibodies include antibodies in which the individual antibodies constituting the population react with substantially the same epitope. Alternatively, they may be antibodies in which the individual antibodies constituting the population are substantially identical (allowing for naturally occurring mutations). The method for producing monoclonal antibodies is not particularly limited, and they may be produced by a method similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug. 7;256(5517):495-497." Alternatively, monoclonal antibodies may be produced by a method similar to the recombinant method described in U.S. Pat. No. 4,816,567. Alternatively, monoclonal antibodies may be isolated from a phage antibody library using a method similar to the technique described in "Clackson et al., Nature. 1991 Aug. 15;352(6336):624-628" or "Marks et al., J. Mol. Biol. 1991 Dec. 5;222(3):581-597." Alternatively, it may be prepared by the method described in "Protein Experiment Handbook, Yodosha (2003): 92-96."

[0030] In one embodiment of the present invention, a chimeric antibody can be constructed by genetic engineering techniques, for example, by linking the variable region and constant region of an antibody between different species. Examples include chimeric antibodies derived from a non-human mammal and a human (e.g., mouse-human chimeric antibodies). Mouse-human chimeric antibodies can be produced, for example, by the method described in Roguska et al., Proc Natl Acad Sci USA, 1994 Feb 1;91(3):969-973. A basic method for producing a mouse-human chimeric antibody involves ligating a mouse leader sequence and variable region sequence present in a cloned cDNA to a sequence encoding a human antibody constant region already present in an expression vector for mammalian cells. Alternatively, the mouse leader sequence and variable region sequence present in a cloned cDNA can be ligated to a sequence encoding a human antibody constant region, and then ligated into a mammalian expression vector. Fragments of the human antibody constant region can be those of any human antibody H chain constant region and L chain constant region, such as Cγ1, Cγ2, Cγ3, or Cγ4 for human H chains, and Cλ or Cκ for L chains.

[0031] In one embodiment of the present invention, a humanized antibody includes, for example, an antibody that binds to a desired antigen and has one or more CDRs derived from a non-human species, a framework region derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin. Antibody humanization can be performed, for example, by the procedures described in Section 1.5 below. Various other techniques known in the art may also be used (e.g., Safdari et al., Biotechnol Genet Eng Rev. 2013;29:175-86). In chimeric or humanized antibodies, the heavy chain constant region may include, but is not limited to, an amino acid sequence of SEQ ID NO: 88-91. The light chain constant region may include, but is not limited to, an amino acid sequence of SEQ ID NO: 87.

[0032] In one embodiment of the present invention, an Fv antibody is an antibody that contains an antigen-recognition site. This region comprises a non-covalently bound dimer of one heavy-chain variable region and one light-chain variable region. In this configuration, the three CDRs of each variable region can interact to form an antigen-binding site on the surface of the VH-VL dimer.

[0033] In one embodiment of the present invention, the Fab antibody is, for example, an antibody fragment obtained by treating an antibody comprising a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are linked via some disulfide bonds. Fab can be obtained, for example, by treating the anti-CD39 antibody according to the above-described embodiment of the present invention comprising a Fab region and an Fc region with the protease papain.

[0034] In one embodiment of the present invention, the F(ab')2 antibody is an antibody that contains two regions corresponding to Fab, among fragments obtainable by treating an antibody containing an Fab region and an Fc region with the protease pepsin. F(ab')2 can be obtained, for example, by treating an anti-CD39 antibody according to an embodiment of the present invention that contains an Fab region and an Fc region with the protease pepsin. Alternatively, F(ab')2 can be prepared, for example, by forming a thioether bond or disulfide bond with the Fab' shown below.

[0035] In one embodiment of the present invention, a Fab' antibody is an antibody obtained by cleaving the disulfide bond in the hinge region of F(ab')2, for example, by treating F(ab')2 with the reducing agent dithiothreitol.

[0036] In one embodiment of the present invention, the scFv antibody includes an antibody in a form in which the VH and VL are linked via a suitable peptide linker. For example, the scFv antibody can be produced by obtaining cDNA encoding the VH and VL of the anti-CD39 antibody according to the above-mentioned embodiment of the present invention, constructing a polynucleotide encoding VH-peptide linker-VL, inserting the polynucleotide into a vector, and using an expression cell.

[0037] In one embodiment of the present invention, a diabody is an antibody in the form of dimerized scFv and has bivalent antigen-binding activity. The bivalent antigen-binding activities can be the same, or one of the two can have a different antigen-binding activity. For example, a diabody can be produced by constructing a polynucleotide encoding an scFv so that the amino acid sequence of the peptide linker is 8 residues or less, incorporating the resulting polynucleotide into a vector, and using an expression cell.

[0038] In one embodiment of the present invention, a dsFv is an antibody in which polypeptides having cysteine ​​residues introduced into VH and VL are linked via a disulfide bond between the cysteine ​​residues. The position at which the cysteine ​​residue is introduced can be selected based on the predicted three-dimensional structure of the antibody according to the method described by Reiter et al. (Reiter et al., Protein Eng. 1994 May;7(5):697-704.).

[0039] In one embodiment of the present invention, scFvFc includes an antibody in a form in which an scFv and an Fc region are linked via a suitable linker. For example, a polynucleotide encoding the scFv, linker, and Fc region can be inserted into a vector and produced using an expression cell. Alternatively, it can be produced, for example, by the procedure described in Section 1.7 below.

[0040] In one embodiment of the present invention, the "antigen-binding peptide or polypeptide" is an antibody comprising an antibody VH, VL, or their CDR1, 2, or 3. A peptide comprising multiple CDRs can be linked directly or via a suitable peptide linker.

[0041] The method for producing the above-mentioned Fv antibodies, Fab antibodies, F(ab')2 antibodies, Fab' antibodies, scFv antibodies, diabodies, dsFv antibodies, scFvFc, and antigen-binding peptides or polypeptides (hereinafter sometimes referred to as "Fv antibodies, etc.") is not particularly limited. For example, DNA encoding the Fv antibody or other domains of the anti-CD39 antibodies according to the above-mentioned embodiments of the present invention can be inserted into an expression vector and produced using expression cells. Alternatively, they may be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBOC method (t-butyloxycarbonyl method). The antigen-binding fragment according to the above-mentioned embodiments of the present invention may be one or more of the above-mentioned Fv antibodies, etc.

[0042] In one embodiment of the present invention, the anti-CD39 antibody includes a glycomodified antibody. Glycomodified antibodies include, for example, antibodies in which the sugar chain structure attached to the Fc region of the antibody has been modified. Glycomodified antibodies include, for example, low-fucose antibodies. Low-fucose antibodies include, for example, antibodies in which the sugar chain attached to the Fc region has been reduced in fucose. In the glycomodified antibody, the Fc region may be, for example, IgG1. In one embodiment of the present invention, the anti-CD39 antibody includes an antibody comprising an Fc region having an N-glycan, wherein the N-glycan does not contain core fucose.

[0043] One embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody, wherein the anti-CD39 antibody has N-glycans, and 40% or more of the N-glycans in the composition do not contain core fucose. Use of such a pharmaceutical composition can produce excellent ADCC activity, as demonstrated in the Examples below. This pharmaceutical composition can be used for the treatment of malignant tumors. Administration of this pharmaceutical composition to a patient can provide a method for treating malignant tumors. The anti-CD39 antibody includes the anti-CD39 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above). The anti-CD39 antibody may have an N-glycan in its Fc region. The N-glycan includes an N-glycan or an N-linked glycan. The N-glycan includes a glycan bound to the amide moiety of asparagine in the Fc region. The asparagine includes asparagine or Asn297 in the CH2 domain. The core fucose includes fucose attached via an α1-6 linkage to a GlcNAc residue at the reducing end of the N-glycan. Glycans may be analyzed, for example, by LC / MS, or by outsourcing the analysis to a contract company (e.g., Sumitomo Bakelite Co., Ltd.). The fucosylation percentage (%) may be calculated, for example, by (peak area of ​​core fucose-containing glycans) / (peak area of ​​all N-glycans) x 100 in an LC chromatogram. The percentage of glycans free of core fucose may be, for example, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or may be any of these values ​​or greater, or may be within a range of any two of these values. To achieve a better cancer cell proliferation inhibitory effect, this percentage is preferably 45% or greater, more preferably 60% or greater, even more preferably 65% ​​or greater, and most preferably 67% or greater. This percentage is also useful for achieving better CD39 activity. hi CD4 + FoxP3 + In order to eliminate Tregs and effector Tregs, the percentage is preferably 60% or more, more preferably 65% ​​or more, and even more preferably 67% or more. This percentage is determined by the CD39 expression level at the time of induction of antigen-specific cytotoxic T cells. + / - PD1 + Tim-3 +To reduce the fraction ratio and minimize exhaustion, a fraction ratio of 60% or more is preferred, more preferably 65% ​​or more, and even more preferably 67% or more. In another aspect, one embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody, wherein the anti-CD39 antibody has N-glycosylation, and 40% or more of the antibody in the composition have N-glycosylation without core fucose. In another aspect, one embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody, wherein the anti-CD39 antibody has N-glycosylation, and less than 60% of the N-glycosylation in the composition contains core fucose. Use of such a pharmaceutical composition can produce excellent ADCC activity, as demonstrated in the Examples described below. The fraction containing core fucose may be, for example, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 0%, or may be less than any of these values ​​or within a range of any two of these values. This ratio is preferably less than 55%, more preferably less than 40%, even more preferably less than 35%, and most preferably less than 33%, in order to produce a better inhibitory effect on cancer cell proliferation. hi CD4 + FoxP3 + To eliminate Tregs and effector Tregs, the percentage is preferably less than 40%, more preferably less than 35%, and even more preferably less than 33%. This percentage is determined by the CD39 expression level at the time of induction of antigen-specific cytotoxic T cells. + / - PD1 + Tim-3 + In order to reduce the fraction ratio and the degree of exhaustion, it is preferably less than 40%, more preferably less than 35%, and even more preferably less than 33%.

[0044] In one embodiment of the present invention, the heavy chain constant region of the anti-CD39 antibody may have, for example, the amino acid sequence of SEQ ID NO: 88 to 91, or 93. In one embodiment of the present invention, the light chain constant region of the anti-CD39 antibody may have, for example, the amino acid sequence of SEQ ID NO: 87 or 92. The heavy chain constant region and the light chain constant region are not particularly limited, but in the Examples described below, 6-3m1 and 23-7m1 are SEQ ID NOs: 93 and 92, P4_h1lala is SEQ ID NOs: 89 and 87, 6-3h4_IB, 6-3h4_IG, 6-3h4_IF, 6-3h4_CB, 6-3h4_HB, 6-3h4_KB, B23-7h4_IB, B23-7h4_IG, B23-7h4_IF, B23-7h4_C, B, B23-7h4_HB, and B23-7h4_KB have heavy chain constant regions and light chain constant regions shown in the amino acid sequences of SEQ ID NOs: 90 and 87, and P4_sc, T86_sc, I67_sc, Tre291_sc, mAnE67_sc, HuP4_11_sc, HuP4_41_sc, HuP4_51_sc, and HuP4_IB_sc have a heavy chain constant region shown in the amino acid sequence of SEQ ID NO: 91.

[0045] One embodiment of the present invention is a polynucleotide or vector encoding the anti-CD39 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above). A transformant can be produced by introducing this polynucleotide or vector into a cell. The transformant may be a human or non-human mammalian cell (e.g., rat, mouse, guinea pig, rabbit, bovine, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey COS-7 cells, and human embryonic kidney cells (e.g., HEK293 cells). Alternatively, the transformant may be Escherichia bacteria, yeast, or the like. The polynucleotide or vector may be constructed to be capable of expressing an anti-CD39 antibody. The polynucleotide or vector may contain components necessary for protein expression, such as a promoter, an enhancer, an origin of replication, or an antibiotic resistance gene.

[0046] Examples of the vector that can be used include E. coli-derived plasmids (e.g., pET-Blue), Bacillus subtilis-derived plasmids (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5 / His-TOPO), bacteriophages such as λ phage, and virus-derived vectors. The vector may be an expression vector or may be circular.

[0047] Methods for introducing the above-mentioned polynucleotides or vectors into cells include, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, and microinjection (New Genetic Engineering Handbook, 4th revised edition, Yodosha (2003): 152-179). Antibodies can be produced using cells, for example, by the method described in Protein Experiment Handbook, Yodosha (2003): 128-142.

[0048] One embodiment of the present invention is a method for producing an anti-CD39 antibody, comprising the step of growing cells containing the polynucleotide or vector according to any one of the above embodiments of the present invention. The growing step comprises a culturing step. This production method may also comprise the step of recovering the anti-CD39 antibody. This production method may also comprise the step of preparing a cell culture medium. This production method may also comprise the step of purifying the anti-CD39 antibody.

[0049] In one embodiment of the present invention, antibodies can be purified using, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography (Protein Experiment Handbook, Yodosha (2003): 27-52).

[0050] One embodiment of the present invention is a composition comprising an anti-CD39 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above). Use of this composition enables efficient detection of CD39. Furthermore, it can produce an excellent effect of inhibiting cancer cell proliferation. The components contained in this composition are not particularly limited, and it may contain, for example, a buffer solution. One or more of the various embodiments of the inhibitors and pharmaceutical compositions described below (e.g., the inclusion of a carrier) may be applied to this composition. This composition can be used for inhibiting CD39 activity, detecting CD39, inhibiting malignant tumor cell proliferation, activating immune cells, promoting TNFα production by immune cells, suppressing Treg function, promoting Teff division, suppressing Treg division, reducing Treg frequency, or inhibiting CD39. hi One embodiment of the present invention includes a method for inhibiting CD39 activity, a method for detecting CD39, a method for suppressing the proliferation of malignant tumor cells, a method for activating immune cells, a method for promoting TNFα production by immune cells, a method for suppressing Treg function, a method for promoting Teff division, a method for suppressing Treg division, a method for reducing Treg frequency, or a method for inhibiting CD39 activity, each of which comprises a step of contacting CD39 (or a CD39-expressing cell) with the anti-CD39 antibody according to the embodiment of the present invention (an antibody having any of the CDR sets (a) to (g) above). hi This method is a method for selectively removing cells. The occurrence of immune cell activation may be evaluated, for example, by an increase in the proliferation rate of immune cells, an increase in the cytotoxicity of immune cells against cancer cells, or an increase in cytokine secretion from immune cells. CD39-expressing cells include, for example, immune cells or malignant tumor cells that express CD39.

[0051] One embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above). This pharmaceutical composition may comprise one or more pharmacologically acceptable carriers. The pharmaceutical composition includes, for example, a pharmaceutical composition for treating malignant tumors. The malignant tumor includes a CD39-positive malignant tumor. One embodiment of the present invention is a method for treating a disease, comprising the step of administering to a patient an anti-CD39 antibody according to the above-described embodiments of the present invention (or a pharmaceutical composition comprising an anti-CD39 antibody). The treatment includes, for example, treatment of malignant tumors and cancer immunotherapy. One embodiment of the present invention is use of an anti-CD39 antibody according to the above-described embodiments of the present invention for producing a pharmaceutical composition.

[0052] One embodiment of the present invention is a kit comprising an anti-CD39 antibody according to the above-described embodiments of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above). Use of this kit can produce an excellent effect of suppressing the proliferation of cancer cells. This kit can be used for treating malignant tumors, inhibiting CD39 activity, detecting CD39, suppressing the proliferation of malignant tumor cells, activating immune cells, promoting TNFα production by immune cells, suppressing Treg function, promoting Teff division, suppressing Treg division, reducing Treg frequency, or inhibiting CD39. hi Kits for selective cell removal are included. The kits may include, for example, instructions, buffers, containers, or packaging.

[0053] In one embodiment of the present invention, treatment includes exerting a symptom-ameliorating, suppressive, or preventive effect on a patient's disease or one or more symptoms associated with the disease. In one embodiment of the present invention, the pharmaceutical composition may be prepared by any method known in the technical field of pharmaceuticals, for example, by mixing an active ingredient with one or more pharmacologically acceptable carriers. Furthermore, the pharmaceutical composition may be used in any form, as long as it is used for treatment. It may contain the active ingredient alone or a mixture of the active ingredient with any other component. The form of the carrier is also not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The content of the carrier may be, for example, a pharmaceutically effective amount. An effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient. For example, a buffer solution is effective for stabilizing the active ingredient in a vial. The dosage, administration interval, administration method, and administration route are not particularly limited and can be selected appropriately depending on the patient's age, weight, symptoms, target organ, etc. Furthermore, the pharmaceutical composition preferably contains a therapeutically effective amount, or an amount effective to exert the desired effect, of the active ingredient.

[0054] In one embodiment of the present invention, a subject (including a patient) includes a human or non-human mammal (e.g., one or more of a mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee), and may be a patient diagnosed with or in need of treatment for a malignant tumor.

[0055] The effect of inhibiting malignant tumor cell proliferation may be evaluated, for example, by observing changes in malignant tumor cell proliferation over time after antibody administration. A state in which cell proliferation is inhibited includes a state in which the proliferation rate of the test cells is significantly reduced compared to before antibody treatment. The proliferation rate may be measured, for example, using absorbance as an indicator, or may be determined from image data. The therapeutic effect of malignant tumors may be evaluated, for example, by observing the reduction in tumor weight after antibody administration. In this case, a significant decrease in tumor weight compared to before antibody administration or after administration of a negative control may be considered to be therapeutic. The therapeutic effect of malignant tumors may be measured, for example, using the amount of malignant tumor marker in the patient or a patient-derived sample as an indicator. In this case, a significant decrease in marker amount compared to before antibody administration or after administration of a negative control may be considered to be therapeutic. The tumor weight or marker amount after antibody administration may be reduced to 0.9, 0.7, 0.5, 0.3, or 0.1 times or less compared to before administration or after administration of a negative control. The therapeutic effect may also be evaluated by diagnostic imaging.

[0056] In one embodiment of the present invention, malignant tumors include, for example, tumors that develop as a result of mutations in normal cells. Malignant tumors can arise from any organ or tissue throughout the body. Examples of malignant tumors include one or more selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, ureter cancer, renal pelvis cancer, ureter cancer, penile cancer, testicular cancer, brain tumor, central nervous system cancer, peripheral nervous system cancer, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, myeloma, and hematological malignancies. Malignant tumors include CD39-positive malignant tumors.

[0057] One embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody used in combination therapy with an anti-CD39 antibody and a chemotherapeutic agent. This pharmaceutical composition can be used to treat malignant tumors. It can also suppress a decrease in heart weight caused by the chemotherapeutic agent. One embodiment of the present invention is a pharmaceutical composition comprising an anti-CD39 antibody for administration to a patient who has been administered a chemotherapeutic agent. One embodiment of the present invention is a pharmaceutical composition comprising a chemotherapeutic agent and an anti-CD39 antibody. One embodiment of the present invention is a pharmaceutical composition comprising a chemotherapeutic agent for use in combination therapy with an anti-CD39 antibody and a chemotherapeutic agent. One embodiment of the present invention is a pharmaceutical composition comprising a chemotherapeutic agent for administration to a patient who has been administered an anti-CD39 antibody. One embodiment of the present invention is a method for treating malignant tumors, comprising the steps of administering an anti-CD39 antibody to a patient and administering a chemotherapeutic agent to a patient. In this administration method, the anti-CD39 antibody and the chemotherapeutic agent may be administered simultaneously or sequentially (in any order). In one embodiment of the present invention, "combination" includes administration of an anti-CD39 antibody and a chemotherapeutic agent simultaneously or separately (in any order), or administration of a combination of these. Another embodiment of the present invention is use of an anti-CD39 antibody for the manufacture of a pharmaceutical composition for use in combination therapy with an anti-CD39 antibody and a chemotherapeutic agent. Another embodiment of the present invention is use of a chemotherapeutic agent for the manufacture of a pharmaceutical composition for use in combination therapy with an anti-CD39 antibody and a chemotherapeutic agent.

[0058] In one embodiment of the present invention, the chemotherapeutic agent is not particularly limited and includes, for example, an anticancer agent. Anticancer agents include, for example, microtubule inhibitors, DNA synthesis inhibitors, growth factor inhibitors, tyrosine kinase inhibitors, or cytotoxic substances. Microtubule inhibitors include, for example, vinca alkaloids or taxanes. Vinca alkaloids include, for example, vincristine, vinblastine, vindesine, vinorelbine, or eribulin. Taxanes include, for example, paclitaxel or docetaxel. DNA synthesis inhibitors include, for example, antimetabolites, topoisomerase inhibitors, platinum compounds, antitumor antibiotics, or alkylating agents. Antimetabolites include, for example, pemetrexed, 5-fluorouracil, S-1, gemcitabine, or capecitabine. Topoisomerase inhibitors include, for example, irinotecan, nogitecan, etoposide, or zobuzoxacin. Platinum agents include, for example, cisplatin, oxaliplatin, nedaplatin, or carboplatin. Antitumor antibiotics include, for example, anthracyclines (e.g., doxorubicin, liposomal doxorubicin, daunorubicin, epirubicin, idarubicin, aclarubicin, amrubicin, mitoxatrone, or pirarubicin), mitomycin C, actinomycin D, bleomycin, beplomycin, or zinostatin stimur. Alkylating agents include, for example, cyclophosphamide, dacarbazine, or ifosfamide. Growth factor inhibitors include, for example, inhibitors of EGF, VEGF, FGF, or IGF. Growth factor inhibitors include, for example, bevacizumab, cetuximab, or panitumumab. Tyrosine kinase inhibitors include, for example, gefitinib or erlotinib. Cytotoxic agents include, for example, saporin, emtansine, deruxtecan, or vedotin. Chemotherapeutic forms include, for example, low molecular weight compounds or high molecular weight compounds. Chemotherapeutic agents include salts of any one or more compounds described herein. In one embodiment of the present invention, a form of the compound or a salt thereof includes a solvate thereof.

[0059] In one embodiment of the present invention, the salt is not particularly limited and includes, for example, inorganic salts or organic salts (see, for example, "Bharate et al., Drug Discov Today. 2021 Feb;26(2):384-398" or "Berge et al., J Pharm Sci. 1977 Jan;66(1):1-19"). Salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, and the like. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. Salts include pharmaceutically acceptable salts. In one embodiment of the present invention, pharmaceutically acceptable salts include forms that have reasonable benefits for pharmaceutical use. In one embodiment of the present invention, salts of doxorubicin include, for example, doxorubicin hydrochloride. In one embodiment of the present invention, a solvate includes a form of a compound formed by a solute and a solvent (see, for example, Healy et al., Adv Drug Deliv Rev. 2017 Aug 1;117:25-46.) The solvate includes, but is not limited to, a hydrate (e.g., a monohydrate, a dihydrate, a trihydrate, etc.) or an organic solvent (e.g., a solvate with an alcohol (e.g., methanol, ethanol, propanol, etc.), acetone, dimethylformamide, or ethyl acetate, etc.).Solvents include those that are capable of substantially maintaining the biological activity of the solute after forming the solvate.

[0060] One embodiment of the present invention is a method for enhancing the ability of a composition to inhibit the proliferation of malignant tumor cells, comprising the step of increasing the proportion of the anti-CD39 antibody according to the above-described embodiment of the present invention (e.g., an antibody having any of the above-described CDR sets (a) to (g)) in the composition. One embodiment of the present invention is a composition containing an anti-CD39 antibody, wherein 90% or more of the anti-CD39 antibody molecules in the composition are the anti-CD39 antibody according to the above-described embodiment of the present invention (e.g., an antibody having any of the above-described CDR sets (a) to (g)). One embodiment of the present invention is an antibody population containing an anti-CD39 antibody, wherein 90% or more of the anti-CD39 antibody molecules in the antibody population are the anti-CD39 antibody according to the above-described embodiment of the present invention (e.g., an antibody having any of the above-described CDR sets (a) to (g)). The above 90% or more may be, for example, 90, 95, 96, 97, 98, 99% or more, or 100%, or may be within a range between any two of these values.

[0061] One embodiment of the present invention is an antibody capable of binding to CD39, which has the amino acid sequences of heavy and light chain CDR1 to CDR3 defined by the IMGT definition (Lefranc et al., Dev Comp Immunol. 2003 Jan;27(1):55-77.), Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), or Chothia definition (Chothia et al., J. Mol. Biol., 1987;196:901-917) in the amino acid sequence 6-3m1, 23-7m1, P4_sc, T86_sc, I67_sc, Tre291_sc, or mAnE67_sc. Use of this antibody can produce excellent inhibitory effects on cancer cell proliferation. In one embodiment of the present invention, the CDRs are defined according to the IMGT or Kabat definitions unless otherwise specified.

[0062] One embodiment of the present invention is an anti-CD39 antibody that has a higher affinity for CD39, a higher inhibitory effect on the proliferation of malignant tumor cells, a faster inhibitory effect on the proliferation of malignant tumor cells, a higher promotion of TNFα production, or a higher immunosuppression relief effect than an anti-CD39 antibody comprising a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NOs: 94 and 95, respectively, or an anti-CD39 antibody comprising a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NOs: 96 and 97, respectively.

[0063] One embodiment of the present invention is a method for measuring cytotoxicity against malignant tumor cells, comprising the steps of contacting malignant tumor cells collected from a subject in vitro with an anti-CD39 antibody according to an embodiment of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above), and measuring whether the ATP metabolism of the malignant tumor cells decreases, whether the cell viability decreases, or whether the secretion of an immunostimulatory substance increases under culture conditions. One embodiment of the present invention is a method for measuring cytotoxicity against malignant tumor cells, comprising the steps of contacting malignant tumor cells collected from a subject with an anti-CD39 antibody according to an embodiment of the present invention (e.g., an antibody having any of the CDR sets (a) to (g) above) in the presence of T cells from the same subject, and measuring the amount of immunostimulatory substance secreted from the T cells under culture conditions. The immunostimulatory substance includes TNFα or IFN-γ.

[0064] One embodiment of the present invention is a method for producing a glycosylated anti-CD39 antibody. This production method may employ, for example, a technique for defucosylation of sugar chains attached to the Fc region. This glycosylation-modified antibody production method may comprise the step of culturing cells expressing the anti-CD39 antibody in the presence of a fucosylation inhibitor. This production method may comprise the steps of transfecting the cells with an expression vector encoding the anti-CD39 antibody, culturing the cells in the presence of a fucosylation inhibitor, or recovering the antibody in the culture supernatant after the culture. In the culture, the number of days from transfection of the vector into the cells to recovery of the antibody may be, for example, 3, 5, 7, 9, 10, 11, 13, 14, 16, or 20 days, or may be any one of more days or a range of any two days.

[0065] One embodiment of the present invention is directed to CD39 antibodies, including anti-CD39 antibodies. hi This composition is for cell removal. hi The cell population can be, for example, an immune cell (e.g., a T cell (e.g., a CD4 + T cells or CD8 + The composition may be a population of CD39 T cells. hi The composition includes a composition for selectively removing cells. hi Cells are CD39 int The composition may be selective for (e.g., preferentially) CD39 cells. hi Because the cells are selectively removed, side effects (e.g., toxicity) are low when administered to a living body. hi The percentage of cells is CD39 int The percentage of cells removed may be higher (e.g., 1.5, 2, 3, or 5 times higher) than the percentage of cells removed. The cell population may be in the form of tissue. The subject may, for example, be a CD39 hi The subject may be a subject in need of cell removal (including selective removal), a subject in need of reduction in side effects caused by administration of an anti-CD39 antibody, or a subject in need of treatment for a malignant tumor. hi Cells and CD39int The cells may be cells expressing CD39 (e.g., CD39-positive cells). hi The cells may be, for example, cells expressing 10,000 or more CD39 molecules per cell. int The cells may be, for example, cells that do not express 10,000 CD39 molecules per cell. hi The present invention is directed to a method for cell removal (including a selective removal method). The method may, for example, comprise contacting a cell population with an anti-CD39 antibody (e.g., comprising administering the anti-CD39 antibody to a subject). In another aspect, the present invention relates to a method for cell removal (including a selective removal method) of a CD39 antibody. hi The present invention relates to the use of an anti-CD39 antibody for the production of a composition for cell removal (including selective removal). hi The cells may be, for example, CD39 hi Immune cells (e.g., CD39 hi T cells). int The cells may be, for example, CD39 int Immune cells (e.g., CD39 int T cells).

[0066] In one embodiment of the present invention, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention comprises a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Furthermore, any amino acid in the amino acid sequence may be L- or D-form. Even in such cases, the antibody according to an embodiment of the present invention can be said to comprise the "specific amino acid sequence." Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.).

[0067] In one embodiment of the present invention, the bond may be either a covalent bond or a non-covalent bond, for example, an ionic bond, a hydrogen bond, a hydrophobic interaction, or a hydrophilic interaction.

[0068] In one embodiment of the present invention, "significantly" may mean, for example, that a statistically significant difference is evaluated using a Student's t-test (one-tailed or two-tailed) and p<0.05 or p<0.01, or that a substantial difference is present.

[0069] As used herein, (a), (b), (c), (d), (e), (f), or (g) is synonymous with one or more selected from the group consisting of (a) to (g).

[0070] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range also includes the two values ​​themselves. In this specification, "A to B" includes A and B.

[0071] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the configurations described in the above embodiments may be combined and adopted.

[0072] The present invention will be further explained below with reference to examples, but is not limited to these.

[0073] 1.1 Isolation and culture of single B cells Mice (BALB / c, female, 7 weeks old, CLEA Japan) were intraperitoneally administered 10 μg or 40 μg of human CD39 recombinant antigen (SEQ ID NO: 98, autologous preparation) three or four times at two-week intervals. Approximately one week after the final administration, blood was collected from the tail vein, and the increase in CD39-specific antibody titer in the blood was confirmed by antigen-fixed ELISA, after which the mouse spleens were collected.

[0074] Single-cell suspensions of splenocytes were prepared from the spleen, and IgG-positive B cells were purified using the Memory B Cell Isolation Kit, mouse (Miltenyi Biotec, 130-095-838). The purified B cells were labeled with APC-anti-mouse IgG1 and APC-anti-mouse IgG2a antibodies provided with the kit. They were then stained with PE-human CD39 (self-prepared), PE / Cy7-anti-mouse / human CD45R / B220 (BioLegend, 103222), and BV421-anti-mouse IgM (BioLegend, 406518). From the stained cells, IgM-negative, IgG-positive, B220-positive, and human CD39-binding cell populations were sorted individually into 96-well plates containing 150 μl of medium using a FACS Aria III (BD). The medium used was 40% FBS (Equitech-Bio, SFBM30-0500), RPMI-1640 (Wako Pure Chemical Industries, Ltd., 189-02025), and 1% penicillin-streptomycin (Nacalai Tesque, 09367-34). After incubation at 37°C and 5% CO2 for 1 hour, 3–5 × 10 cells were suspended in RPMI-1640 containing cytokines. 4100 μl of feeder cells from the 100 cells were added to a 96-well plate and cultured at 37°C, 5% CO2 for 12-14 days. The feeder cells were cryopreserved mouse CD40L-expressing 3T3 cells irradiated with 50 Gy of X-rays and then resuspended. The final concentrations of cytokines were 10 ng / mL Mouse BAFF (ACRO Biosystems, BAF-M521y-50ug), 10 ng / mL Mouse IL-1β (BioLegend, 575104), 10 ng / mL Murine IL-4 (PeproTech, 214-14), 10 ng / mL Murine IL-6 (PeproTech, 214-16), 10 ng / mL Mouse IL-9 (R&D, 409-ML-050), 50 ng / mL Murine IL-21 (R&D, 594-ML-100), and 10 ng / mL Mouse TNF-α (BioLegend, 575204) were added.

[0075] 1.2 Screening for CD39 Function Inhibition by B Cell Culture Supernatant. Two thousand SK-MEL-28 cells (ATCC, HTB-72), a CD39-high-expressing cell line, suspended in 50 μl EMEM (Fujifilm, 055-08975) were seeded into a 96-well V-bottom plate. 150 μl of single B cell culture supernatant was added and incubated at 37°C for 30 minutes. After washing once with DPBS (Nacalai Tesque, 14249-24), 100 μl of 5 μM ATP was added per well and incubated at 20°C for 30 minutes. After centrifugation, 50 μl of the supernatant was removed, 50 μl of CellTiter-Glo (Promega, G9243) was added, and the mixture was vortexed and incubated at room temperature for 10 minutes. ATP levels were then measured by chemiluminescence using a microplate reader. The ability of B cell culture supernatant to inhibit ATP degradation by CD39 was evaluated based on the amount of remaining ATP.

[0076] 1.3 Amplification and isolation of antibody gene sequences. For wells in which ATP degradation was observed to be inhibited in the CD39 function inhibition screening, the genes corresponding to the antibody heavy and light chain variable regions were amplified 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 mixed primer set of 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)). After amplification of the antibody gene, a linker sequence for insertion into an antibody expression vector was added to the end of the gene by PCR, and the gene was then inserted by ligation into a heavy chain or light chain expression vector (containing a CMV promoter, secretion signal, heavy chain variable region or light chain variable region, and heavy chain constant region (SEQ ID NO: 93) or light chain constant region (SEQ ID NO: 92) linked in tandem, respectively). The ligation product was transformed into Escherichia coli, and the antibody expression plasmid was recovered.

[0077] 1.4 Antibody preparation and antibody gene sequence determination. The heavy chain expression plasmid (derived from the H1 or H2 mixed amplification product) and light chain expression plasmid (derived from the k mixed amplification product) obtained by amplification and isolation of the antibody gene sequence were transfected into CHO cells. The antibodies produced in the culture supernatant of the CHO cells were collected, and the CD39 inhibitory activity of the bulk antibodies was confirmed.

[0078] E. coli was transformed with the plasmid encoding the bulk antibody, whose CD39 inhibitory activity had been confirmed. Single colonies for the heavy and light chains were collected from the inoculated plates, and plasmid DNA was isolated. The heavy and light chain expression plasmids derived from the single colonies were co-transfected into CHO cells, and the antibody produced in the culture supernatant was collected from the CHO cells. This antibody was used as a single antibody to prepare the humanized antibody described below in 1.5 and to measure the CD39 enzyme inhibitory activity described below in 1.8.

[0079] The gene sequences of the variable regions of the heavy chain expression plasmid and the light chain expression plasmid derived from a single colony were analyzed by Sanger sequencing, and the amino acid sequences were determined based on the sequences.

[0080] 1.5 Production of Humanized Antibodies Humanized antibodies were produced using the antibodies obtained in the above experiments using the following procedure. The mouse antibody sequence was modified to produce humanized antibodies using biological software Discovery Studio 2020 (Dassault Systems). Multiple humanized antibody variable region sequences were synthesized from a single mouse antibody variable region sequence by changing software parameters. The humanized antibody variable region sequences were then inserted into a plasmid containing a human IgG4 constant region (SEQ ID NO: 90) and a human light chain constant region (SEQ ID NO: 87), creating a humanized antibody expression vector. The resulting expression vector was transfected into CHO cells, and the antibody produced in the culture supernatant from the CHO cells was collected and purified as follows.

[0081] 1.6 Antibody Purification The culture supernatant of the transfectant CHO cells was collected, and the antibodies in the culture supernatant were purified using a MonoSpin ProG or MonoSpin ProA column (GL Science, 7510-11311 or 7510-11310) or a MonoSpin L ProA column (GL Science, 7510-11314) according to the instructions attached thereto.

[0082] 1.7 Antibody cloning by phage display Mice (BALB / c or MRL / lpr, female, 7-8 weeks old, CLEA Japan) were intraperitoneally administered 10-40 μg of human CD39 recombinant antigen (self-prepared) three or four times at weekly or biweekly intervals. Approximately one week after the final administration, blood was collected from the tail vein, and the increase in CD39-specific antibody titer in the blood was confirmed by antigen-fixed ELISA, after which the mouse spleens were collected.

[0083] Total RNA was extracted from the spleen or lymph nodes of immunized mice, converted into cDNA, and the antibody gene sequence (V Ｈ and V κ ) was amplified by PCR. Ｈ and Vκ were linked with a linker to form single chain Fv (hereinafter referred to as scFv), which was then inserted into a pTZ19R-derived phagemid. The scFv-inserted phagemid was transformed into Escherichia coli (DH12S strain) and further infected with helper phage (M13KO7) to generate an scFv phage library (size: approximately 1 × 10 8 types) were produced.

[0084] The scFv antibody phage library was panned using recombinant human CD39. The phagemids were extracted, and the scFv portion was amplified by PCR and then transferred to an scFv secretion expression vector. E. coli cells transfected with the scFv secretion expression vector secrete scFv into the culture supernatant upon IPTG induction, and this was used to screen for scFv clones that inhibit CD39 activity. The nucleotide sequences of the scFvs that showed positive results in the screening were determined. This scFv was linked to a human Fc region (SEQ ID NO: 91, Fc silent) via a linker to form scFvFc, which was then expressed in ExpiCHO cells (Thermo Fisher Scientific) and affinity purified using a Protein A column. The resulting antibodies were used to measure CD39 enzyme inhibitory activity using the same procedure as in 1.8 above. A humanized antibody was also produced using the same procedure as in 1.5 above (however, as before humanization, the heavy chain constant region was designed to be a human Fc region (SEQ ID NO: 91, Fc silent) and to have an scFvFc structure.) The resulting antibody was used to measure CD39 enzyme inhibitory activity using the same procedure as in 1.8 above.

[0085] 1.8 CD39 Enzyme Inhibitory Activity Measurement The CD39 enzyme inhibitory activity of the antibodies obtained in the above experiments was measured as follows. 5,000 to 6,250 cells of the human melanoma cell line SK-MEL-28 (ATCC, HTB-72) or the human ovarian adenocarcinoma cell line OAW-42 (CLS, 300304), both of which highly express CD39, were suspended in 50 μl of EMEM and seeded into a 96-well V-bottom plate. 50 μl of antibody solution (final concentration: 0.001–100 μg / ml) was added and incubated at 37°C for 1 hour. 50 μl of ATP (final concentration: 10 μM) was added and incubated at 37°C for 30 minutes. After centrifugation, 50 μl of the supernatant was removed, 50 μl of CellTiter-Glo was added, and the mixture was vortexed and incubated at room temperature for 10 minutes. Chemiluminescence was measured using a microplate reader. The ability of antibodies to inhibit CD39 ATP degradation was assessed by measuring the amount of ATP consumed by SK-MEL-28 or OAW-42 cells. POM1, a small molecule inhibitor of E-NTPDases, was used as a positive control for human CD39 inhibition in this experiment.

[0086] 1.9 Results 1.9.1 Screening of Mouse Antibodies Through the experiments described above in 1.1 to 1.4, several antibody clones with human CD39 inhibitory activity were isolated. Among the clones obtained, 6-3m1 and 23-7m1 exhibited high inhibitory activity. Figure 1 shows the results of measuring the CD39 inhibitory activity of 6-3m1 and 23-7m1. In all figures, the isotype control is an IgG4 antibody against the scorpion toxic peptide CN2 or a commercially available isotype antibody (unless the antibody name is specified). In all figures, I-394 is an anti-CD39 antibody with the amino acid sequences of the heavy chain variable region and light chain variable region of I-394 (SEQ ID NOs: 94 and 95, respectively) described in WO2018 / 167267. In all figures, T-31414 is an anti-CD39 antibody having the amino acid sequences of the heavy chain variable region and light chain variable region of T-31414 (SEQ ID NOs: 96 and 97, respectively) described in US 2019 / 0062448. The heavy chain constant regions of I-394 and T-31414 whose names end in h1lala have the amino acid sequence of SEQ ID NO: 89, and those whose names end in h4 have the amino acid sequence of SEQ ID NO: 90, and the light chain constant regions of both have the amino acid sequence of SEQ ID NO: 87.

[0087] 1.9.2 Screening by phage display method The experiments described in 1.7 above yielded antibodies P4_sc, T86_sc, I67_sc, Tre291_sc, and mAnE67_sc that have human CD39 inhibitory activity. Ｈ and V κ We then constructed P4_h1lala, a chimeric antibody comprising human IgG1-lala (SEQ ID NO: 89, Fc silent) and a human light chain constant region (SEQ ID NO: 87). Figure 2 shows the results of measuring the CD39 inhibitory activity of P4_h1lala.

[0088] 1.9.3 Evaluation of Humanized Antibodies 6-3m1 was humanized to obtain humanized antibodies 6-3h4_IB, 6-3h4_IG, 6-3h4_IF, 6-3h4_CB, 6-3h4_HB, and 6-3h4_KB. 23-7m1 was humanized to obtain humanized antibodies B23-7h4_IB, B23-7h4_IG, B23-7h4_IF, B23-7h4_CB, B23-7h4_HB, and B23-7h4_KB. These humanized antibodies exhibited excellent CD39 inhibitory activity. Figures 3, 4A, and 4B show the results of CD39 inhibitory activity assays. Furthermore, HuP4_11_sc, HuP4_41_sc, HuP4_51_sc, and HuP4_IB_sc were humanized from P4_sc. These humanized antibodies exhibited good CD39 inhibitory activity. Figure 5 shows the results of measuring CD39 inhibitory activity. The amino acid sequences of the CDRs and variable regions of the antibodies obtained in the above experiments are shown in Figures 36 to 40 (the CDRs of 6-3m1 and 23-7m1 are based on the IMGT definition. The CDRs of P4_sc, T86_sc, I67_sc, Tre291_sc, and mAnE67_sc are based on the Kabat numbering system). The amino acid sequences of the light chain (kappa chain) constant region, IgG1-lala heavy chain constant region, and IgG4 heavy chain constant region used to produce the antibodies are shown in Figure 41.

[0089] 2.1 Affinity measurement (SPR analysis, Surface Plasmon Resonance) The kinetics of antibodies against target antigens was measured using Biacore 8K (Cytiva). Mouse antibodies were immobilized on a sensor chip (CM5) using the Mouse Antibody Capture Kit, type 2 (Cytiva, 29215281), while chimeric and humanized antibodies were immobilized on a sensor chip (CM5) using the Human Antibody Capture Kit, type 2 (Cytiva, 26234600). Human CD39 antigen was immobilized on a sensor chip (CM5) using a concentration of 5 × 10 -9 Four serial dilutions of M were prepared and analyzed using a 1:1 binding model with multi-cycle kinetics under the following conditions: contact time 120 seconds, dissociation time 600 seconds, flow rate 30 μl / min, HBS-EP+ running buffer.

[0090] 2.2 Results The results of SPR analysis are shown in Figures 6 and 7. 6-3m1, 23-7m1, P4_sc, and the humanized antibody showed good affinity for human CD39.

[0091] 3.1 CD39 Binding Analysis (FCM Analysis) (Human CD39) Binding analysis to target antigen-expressing cells was performed by FCM analysis using human CD39-expressing cells. Human CD39-expressing cells were generated using a lentiviral vector system (OriGene, PS100069). Lentiviral particles carrying the human CD39 gene (Gene ID: 953) and a puromycin resistance gene were prepared and used to infect HEK293T or Jurkat cells. The cells were then cultured in the presence of puromycin. HEK293T or Jurkat cells (239T-hCD39 or Jurkat-hCD39) that had become human CD39-positive due to integration of the target gene into their genome were collected and used for FCM analysis. Test antibodies or isotype control antibodies were added at a final concentration of 2 μg / mL or the concentrations specified in the figures at 1 x 10 5 After incubation with 1000 cells at 4°C for 15 minutes, the cells were washed twice with 0.5% BSA, 2 mM EDTA, and D-PBS. After washing, a fluorescently labeled secondary antibody (anti-human IgG Fc, HP6017) was added and stained at 4°C for 15 minutes in the dark, followed by two more washes. After addition of 7AAD (BioLegend, 420404), the fluorescence intensity was measured using a flow cytometer (FACSVerse, BD). Antibody binding was assessed based on the fluorescence intensity.

[0092] 3.2 Results The results of FCM analysis are shown in Figures 8 to 11B. 6-3m1, 23-7m1, P4_sc, P4_h1lala, and the humanized antibody showed good affinity for human CD39 on the cell surface.

[0093] 4.1 CD39 Binding Analysis (FCM Analysis) (Monkey CD39) Binding analysis to target antigen-expressing cells was performed using cynomolgus monkey CD39-expressing cells. Cross-reactivity was confirmed using this method. Cynomolgus monkey CD39-expressing cells were generated using a lentiviral vector system (OriGene, PS100069). Lentiviral particles carrying the cynomolgus monkey CD39 gene (Gene ID: 102132860) and a puromycin resistance gene were prepared and used to infect HEK293T cells. The cells were then cultured in the presence of puromycin. HEK293T cells (293T-cynoCD39), which had incorporated the target gene into their genome and were positive for human or cynomolgus monkey CD39, were harvested and used for FCM analysis. Cells were incubated with 2 μg / ml of test antibody or isotype control antibody, then stained with a fluorescently labeled secondary antibody. Antibody binding was assessed based on the fluorescence intensity obtained by flow cytometry analysis.

[0094] 4.2 Results The results of FCM analysis are shown in Figures 12 to 16. 6-3m1, 23-7m1, and P4_sc also bound to monkey CD39.

[0095] 5.1 Inhibition and binding of Tregs to CD39. Purchased healthy human PBMCs (Wako Pure Chemical Industries, Ltd., 33000-10M) were cultured at 2x10 5 The cells were seeded at 2x10 cells / well in a 96-well U-bottom plate and cultured for 6 days in the presence of 10 IU / mL IL-2 (NIPRO, 87-890) and 20 ng / mL IL-7 (PeproTech, 200-07). After 6 days, all cells were harvested and Treg populations were isolated using the CD4+CD25+CD127dim / - Regulatory T Cell Isolation Kit II, human (Miltenyi, 130-094-775). The isolated Tregs were cultured at 2x10 4 Seed 6x10 cells / well into a 96-well U-bottom plate. 5Tregs were stimulated for 6 days with Dynabeads Human T-Activator CD3 / CD28 (Gibco, DB11132-D), 10 IU / mL IL-2, and 20 ng / mL IL-7. The medium used was RPMI (Wako Pure Chemical Industries, Ltd., 189-02025) supplemented with 10% Human Serum AB (Gemini Bio, 100-512, H46X00K), 1 mM penicillin-streptomycin (Nacalai Tesque, 09367-34), 1 mM sodium pyruvate (Gibco, 11360070), 10 mM HEPES (Nacalai Tesque, 17557-94), and GlutaMAX (Thermo Fisher Scientific, 35050061). After 6 days, Tregs were harvested and subjected to antibody binding analysis and enzyme inhibitory activity assay.

[0096] The CD39 enzyme inhibitory activity of Tregs was measured using 1x10 4 The experiment was performed using Treg cells and the test antibody at a final concentration of 10 μg / mL. ARL67156, a small molecule inhibitor of E-NTPDases like POM1, was used as a positive control for human CD39 inhibition in this experiment.

[0097] For Treg binding analysis, the collected cells were stained with Treg surface antigens (anti-hCD127: 236A / E7, anti-hCD3: RPA-T8, anti-hCD25: BC96, anti-hCD4: RPA-T4) and the test antibodies at a final concentration of 10 μg / mL, and analyzed using a flow cytometer (FACSVerse, BD).

[0098] 5.2 Results Figure 17 shows the results of measuring CD39 enzyme inhibitory activity. 6-3h4_IF suppressed the function of CD39 on the surface of Treg cells. Figure 18 shows the results of CD39 binding analysis. Binding of 6-3h4_IF to CD39-expressing Treg was confirmed.

[0099] 6.1 T cell proliferation x-vivo TMPeripheral blood mononuclear cells (PBMCs) collected from healthy volunteers were suspended in 15 medium (Lonza, 04-418Q) and seeded (8 x 10) in a 96-well U-bottom plate. 4 After incubation at 37°C and 5% CO2 for 30 minutes, the cells were transferred to the wells using Dynabeads. TM Two microliters of Human T-Activator CD3 / CD28 (VERITAS, DB11132) and 500 μM ATP (Sigma, A2383-1G) were added and cultured for 96 hours at 37°C under 5% CO2. 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 cell suspension and analyzed using a flow cytometer to calculate the number of CD4- and CD8-positive cells per well.

[0100] In addition, the concentration of TNFα in the collected culture supernatant was measured by the Luminex method using antibodies [capture antibody TNFα: MAB602 (R&D Systems), detection antibody TNFα: BAF210 (R&D Systems)].

[0101] 6.2 Results Figures 19 to 25 show the results of measuring T cell proliferation. 6-3m1, 23-7m1, and the humanized antibody promoted the proliferation of CD4-positive and CD8-positive T cells. Figure 26 shows the results of measuring TNFα concentration. Treatment with 6-3m1 and 23-7m1 significantly promoted TNFα production compared to treatment with T-31414h1lala and I-394h4.

[0102] 7.1 Blood exposure SCID mice (CB-17 / Icr-scid / scidJc, female, 6 weeks old) were divided into groups based on body weight and then intraperitoneally administered the antibodies shown in Table 1. Heparinized blood samples were collected 6 and 72 hours after antibody administration. Peripheral blood was centrifuged at 10,000 xg at 4°C for 10 minutes, after which plasma components were collected and the amount of antibody in the plasma was quantified by ELISA.

[0103]

[0104] ELISA was performed as follows. Anti-IgG (H+L chain) (Human) pAb (MBL, 103G) was immobilized on an EIA / RIA plate (Corning, 9018) overnight at 4°C. The plate was then washed three times with 0.05% Tween-20 and PBS. Blocking was performed for 2.5 hours at room temperature with Blocking One (Nacalai, 03953-9) diluted 5-fold with milliQ. After washing the plate three times, diluted plasma was added and incubated for 1 hour at room temperature. After washing the plate three times, anti-IgG (γ chain) (Human) pAb-HRP diluted 1 / 8000 was added and incubated for 30 minutes at room temperature. After the incubation, the plate was washed three times and incubated with the TMB One-Step Substr System (Agilent Technologies, S159985-2) for 10 minutes at room temperature, protected from light. The reaction was stopped by adding an equal volume of 0.5 mol / L sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd., 192-04755) to the TMB, and the absorbance was measured using a Varioskan LUX (Thermo).

[0105] 7.2 Results The antibody concentrations after exposure in the blood are shown in Figure 27. All antibodies showed good concentrations after exposure in the blood.

[0106] 8.1 Antitumor activity Human lung cancer cell line NCI-H292 transfected with red fluorescent protein (RFP) was seeded on a 96-well plate and cultured overnight. The next day, the medium was removed, and X-VIVO-15 TM IL-2 (Miltenyi, 130-097-745) at a final concentration of 30 IU suspended in medium (Lonza, 04-418Q) and anti-human CD39 antibody or isotype control antibody at a final concentration of 0.3-100 μg / mL were added, and the mixture was incubated at 37°C for 30 minutes. TMPBMCs from healthy volunteers stimulated for 10 days with Human T-Activator CD3 / CD28 (VERITAS, DB11131) were added to the cells at an E / T ratio of 2 and incubated at 37°C for 30 minutes. ATP was then added at a final concentration of 500 or 250 μM, and the time course of cancer cell proliferation was observed using an IncuCyte ZOOM System (Sartorius) at 37°C and 5% CO2. This allowed us to evaluate the antitumor activity of the antibody, which is due to its enhancement of PBMC cytotoxicity.

[0107] 8.2 Results Figures 28-32 show the results of measuring the antitumor activity of the antibodies. Treatment with the isotype control increased the proliferation rate of cancer cells (Figure 28). The increased proliferation rate is likely due to immunosuppression caused by the addition of ATP (500 μM) to the experimental system. Treatment with T-31414h1lala reduced the proliferation rate of cancer cells, but only slightly (approximately 0.9) (Figure 28). On the other hand, treatment with 6-3m1, its humanized antibody, or 23-7m1 significantly reduced the proliferation rate of cancer cells (approximately 0.4) (Figures 29 and 30). Furthermore, no significant differences in the proliferation rates of cancer cells were observed among the 6-3m1, its humanized antibody, and 23-7m1 clones.

[0108] Figure 31 shows the results of the antitumor activity of 6-3h4-IF. This assay was performed under conditions with a reduced amount of ATP (250 μM). When treated with the isotype control, proliferation was gradually suppressed up to 100 hours after treatment. The low amount of ATP added to the experimental system likely created an environment that was less susceptible to immunosuppression, leading to gradual suppression of cancer cell proliferation. When treated with I-394h4, there was no difference compared to the isotype control until approximately 50 hours after treatment, and then a slight difference was observed, but the overall difference was small. When treated with 6-3h4-IF, a difference compared to the isotype control began to be observed approximately 15 hours after treatment, and the difference continued to increase thereafter, resulting in a significant overall difference. 6-3h4-IF exerted a rapid inhibitory effect on cancer cell proliferation, even in an environment that was less susceptible to immunosuppression, and the potency of this inhibition was also significant.

[0109] Figure 32 shows the results of measuring the antitumor activity of P4_h1lala. This measurement was performed under conditions with reduced ATP (250 μM) and antibody concentration (10 μg / ml). When treated with the isotype control, proliferation was gradually suppressed up to 80 hours after treatment. The low amount of ATP added to the experimental system created an environment that was less susceptible to immunosuppression, which is thought to have led to gradual suppression of cancer cell proliferation. When treated with I-394h4, there was no difference compared to the isotype control until approximately 80 hours after treatment, and only a slight difference was observed thereafter. When treated with P4_h1lala, a difference compared to the isotype control began to be observed approximately 20 hours after treatment, and the difference continued to widen thereafter, resulting in a significant difference overall. P4_h1lala exerted a rapid inhibitory effect on cancer cell proliferation, even in an environment that was less susceptible to immunosuppression, and the potency of this inhibition was also significant.

[0110] 9.1 Antitumor activity (in vivo, 23-7m1 single agent) Human multiple myeloma-derived cell line MOLP-8 (DMSZ, ACC569) was subcutaneously inoculated (5 x 10 cells) into the right flank of SCID mice (CB-17 / Icr-scid / scidJcl, female, 7 weeks old). 6 On day 11 after transplantation, the mice were divided into groups based on tumor volume, and the antibodies shown in Table 2 were administered twice a week for a total of six doses. On day 34 after transplantation, the mice were euthanized, and the tumors were collected and weighed. Differences in tumor weight were analyzed using Student's t-test. The significance level was set at 5%.

[0111]

[0112] 9.2 Results Figure 33 shows the results of measuring in vivo antitumor activity. Compared to the isotype control antibody-administered group, tumor weight was significantly reduced in the 23-7m1-administered group, demonstrating the in vivo antitumor activity of 23-7m1.

[0113] 10.1 Antitumor activity (in vivo, in combination with doxorubicin) Human multiple myeloma-derived cell line MOLP-8 (DMSZ, ACC569) was subcutaneously implanted (5 x 10 6 The mice were then transplanted into groups based on tumor volume (cells / mouse). 10 days after transplantation, the mice were divided into groups based on tumor volume. The antibody doses listed in Table 3 were administered twice a week for a total of seven doses from the day of grouping. Doxorubicin was administered twice a week for a total of five doses starting seven days after grouping. 24 days after grouping, tumor diameter and body weight were measured. Data analysis of tumor weight (n=5) was performed using GraphPad Prism 9.2.0. One-way ANOVA and Tukey multiple comparisons test were used for statistical analysis. A p**≦0.01 was considered significant.

[0114]

[0115] 10.2 Results Figure 34 shows the results of measuring in vivo antitumor activity (23-7m1 in combination with DOX) (day 24). Compared to the isotype control and vehicle control groups, tumor volume was significantly reduced in the 23-7m1 in combination with doxorubicin group. Figure 35 shows the results of measuring heart weight (day 24). Heart weight decreased on the final day of the test in the doxorubicin-only treatment group, but this tended to improve in the 23-7m1 in combination with doxorubicin group.

[0116] 11.1 Evaluation of Treg Function Teff (effector T cells) (1 × 10) isolated from human PBMCs labeled with CytoTell Green (AAT Bioquest) were used. 4T cells and Tregs isolated from human PBMCs were seeded onto 96-well plates at a ratio of 1:4-32, and Dynabeads™ Human T-Activator CD3 / CD28 (VERITAS) was added. Cultures were then cultured in serum-free X-VIVO 15 medium in the presence of ATP at a final concentration of 500 μM. After 3-5 days, the fluorescence intensity of CytoTell Green was measured using a flow cytometer to assess Teff division. Anti-human CD39 antibody or isotype control antibody was used as the test drug at a final concentration of 0.3-100 μg / mL. The anti-human CD39 antibody reversed the Treg-mediated suppression of Teff division.

[0117] 12.1 Treg Depletion Assessment 12.1.1 Human PBMCs and Tregs isolated from human PBMCs are co-cultured at effector:target cell (E:T) ratios of 40:1, 10:1, 5:1, and 1:2. After 6 hours, TO-PRO-3 iodide (642 / 661; Invitrogen / Molecular Probes) is added, and cell death is measured using a flow cytometer. The test drug is an anti-human CD39 antibody or an isotype control antibody at a final concentration of 0.3-100 μg / mL. The anti-human CD39 antibody lyses Tregs via ADCC.

[0118] 12.1.2 Purchased healthy human PBMC (Wako Pure Chemical Industries, Ltd., 33000-10M) at 2x10 5 The cells were seeded at 100 cells / well in a 96-well U-bottom plate and cultured for 6 days in the presence of 20 IU / mL IL-2 (NIPRO, 87-890) and 40 ng / mL IL-7 (PeproTech, 200-07). After culture, Treg frequency was analyzed using a flow cytometer. The test drug used was an anti-human CD39 antibody or an isotype control antibody at a final concentration of 0.3-100 μg / mL. The anti-human CD39 antibody reduced Treg frequency.

[0119] 13.1 Preparation of Glycoengineered Antibodies 6-3h1_IF_NF1-3 First, the heavy chain constant region sequence of an expression vector incorporating 6-3h4_IF was recombined from human CH (IgG4SP) to human CH (IgG1) to create an expression vector in which the subclass was changed from IgG4 to IgG1. The antibody expressed from this vector was designated 6-3h1_IF (the amino acid sequences of VH and VL are the same as those of 6-3h4_IF; the amino acid sequence of human CH (IgG1) is SEQ ID NO: 88). The glycoengineered antibodies obtained in subsequent experiments were designated 6-3h1_IF_NF1, 6-3h1_IF_NF2, and 6-3h1_IF_NF3. Next, the above expression vectors were transfected into CHO cells using the ExpiCHO Expression System Kit (ThermoFisher Scientific, A29133) to generate transient antibody-expressing CHO cells. The day before transfection, 20 mM D-(-)-arabinose (Nacalai Tesque, 03330-32) was added to the ExpiCHO Expression Medium for 6-3h1_IF_NF1, 20 μM 2F-Peracetyl-Fucose (DMSO suspension) (Merck, 344827) was added to the ExpiCHO Expression Medium for 6-3h1_IF_NF2, and 20 μM D-(-)-arabinose and 20 μM 2F-Peracetyl-Fucose were added to the ExpiCHO Expression Medium for 6-3h1_IF_NF3. After transfection, cell culture was performed according to the instructions included with the ExpiCHO Expression System Kit. For 6-3h1_IF_NF2 and NF3, 20 μM 2F-Peracetyl-Fucose was added to the ExpiCHO Expression Medium on day 6 of culture (day 0, the day of transfection). 20 μM 2F-Peracetyl-Fucose was added to the culture flask with shaking. Culture was continued. For both antibodies, the antibodies produced in the culture supernatant from CHO cells were collected on day 7 of culture and purified.

[0120] 13.2 Affinity Measurement (SPR Analysis) The affinity of 6-3h1_IF_NF1 to 6-3h1_IF_NF3 for human CD39 was measured using the same method as in 2.1 above.

[0121] 13.3 Results The results of SPR analysis are shown in Figure 42. The affinity for human CD39 was almost the same K regardless of the presence or absence of glycosylation or the glycosylation method. D The values ​​were shown.

[0122] 14.1 Measurement of CD39 enzyme inhibitory activity The inhibitory activity of 6-3h1_IF_NF1 to 6-3h1_IF_NF3 against human CD39 was measured using the same method as in 1.8 above.

[0123] 14.2 Results The results of measuring enzyme inhibitory activity are shown in Figure 43. The inhibitory activity against human CD39 was approximately the same regardless of the presence or absence of glycosylation or the glycosylation method.

[0124] 15.1 T cell proliferation test by CD39 function inhibition Frozen PBMC (Wako, 551-37651, Lot: 3010116139) were thawed and Pan T cells were isolated (Pan T Cell Isolation Kit, human, Miltenyi Biotec, 130-096-535). 0.2 μl CytoTell TM X-VIVO stained cells with UltraGreen (AAT Bioquest) TM 15 Suspend in Serum-free Hematopoietic Cell Medium (Lonza, BE02-060F) and 8x10 4Cells were seeded at 25 μl per well into a 96U-bottom plate (non-dividing control cells were treated with mytomaycin C (10 μg / mL) (Nacalai Tesque, 20888-21)). 25 μl of 4x test antibody (40 μg / mL) was added and incubated at 37°C, 5% CO2, for 30 min. Then, 25 μl of 4x ATP (1400 μM) (Sigma, A2383-1G) was added and 25 μl of a 4x CD3 (40 μg / mL) / CD28 (8 μg / mL) mixture was added and incubated at 37°C, 5% CO2, for 96 hr. Surface antigens were stained with various antibodies (PerCP / Cy5.5-CD8, APC-CD3, APC / Cy7-Zombie-NIR, V500-CD4), and Absolute Counting Beads (Invitrogen, C36950) were added. The results were analyzed using FACS Verse. Data were analyzed using GraphPad Prism 9.2.0. Statistical analysis was performed using one-way ANOVA and Dunnett's test. p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001, and p**** ≤ 0.0001 were considered significant.

[0125] 15.2 Results Figure 44 shows the CD4 + The results of measuring the number of T cells are shown below. + The number of T cells was significantly and markedly reduced (see IgG1 ATP. IgG1 is the control antibody). Addition of 6-3h1_IF_NF2 significantly improved this reduction (see NF2 ATP). 6-3h4IF, an IgG4 type, showed a tendency for improvement compared with 6-3h1_IF_NF2, but the improvement was not significant (see 6-3h4IF ATP).

[0126] Figure 45 shows CD4 + The percentage of T cells that have divided six times is shown. + T cell division was significantly reduced. Addition of 6-3h1_IF_NF2 and 6-3h4IF did not produce significant improvement.

[0127] Figure 46 shows CD8 + The results of measuring the number of T cells are shown below.+ The number of T cells was significantly and markedly reduced. This reduction was significantly improved by the addition of 6-3h1_IF_NF2 and IgG4 type 6-3h4IF.

[0128] Figure 47 shows CD8 + The percentage of T cells that have divided six times is shown. + T cell proliferation was significantly reduced. Addition of 6-3h1_IF_NF2 and 6-3h4IF significantly improved this reduction.

[0129] 16.1 Glycosylation analysis of 6-3h1_IF_NF1-3 50 μg of each test antibody was analyzed using the EZGlyco Antibody N-glycan analysis kit. TM The sample was treated according to the package insert of the glycan analyzer (Sumitomo Bakelite, BS-X4410) to prepare 2-aminobenzamide-labeled N-glycans. The prepared samples were analyzed using the glycan LC-MS measurement service (N-glycans) (Sumitomo Bakelite, BS-X4913). The estimated glycan structure was analyzed, and the fucosylation ratio was calculated from the peak area ratio estimated to be core fucose (fucosylation ratio (%) = (peak area of ​​glycans containing core fucose) / (peak area of ​​total N-glycans) x 100). Human serum IgG was used as an experimental control for glycan structure analysis.

[0130] 16.2 Results Figure 48 shows the results of glycosylation analysis (LC chromatogram). The fucosylation rates of 6-3h1_IF, 6-3h1_IF_NF1-NF3 were 85.4%, 49.1%, 30.0%, and 37.5%, respectively. Core fucose addition was suppressed in glycoengineered antibodies, with 6-3h1_IF_NF2 being the most suppressed.

[0131] 17.1 Binding analysis to activated Tregs. Purchased healthy human PBMCs (Wako Pure Chemical Industries, Ltd., 33000-10M) were used in a 2x10 5The cells were seeded at 100 cells / well in a 96-well U-bottom plate and cultured for 6 days in the presence of 10 IU / mL IL-2 (NIPRO, 87-890) and 20 ng / mL IL-7 (PeproTech, 200-07). After 6 days, all cells were harvested and Treg populations were isolated using the CD4+CD25+CD127dim / - Regulatory T Cell Isolation Kit II, human (Miltenyi, 130-094-775). The isolated Tregs were collected at a concentration of 2x10 4 Seed 6x10 cells / well into a 96-well U-bottom plate. 5 Cells were stimulated for 6 days with Dynabeads Human T-Activator CD3 / CD28 (Gibco, DB11132-D), 10 IU / mL IL-2, and 20 ng / mL IL-7. The medium used was RPMI (Wako Pure Chemical Industries, Ltd., 189-02025) supplemented with 10% Human Serum AB (Gemini Bio, 100-512, H46X00K), 1 mM penicillin-streptomycin (Nacalai Tesque, 09367-34), 1 mM sodium pyruvate (Gibco, 11360070), 10 mM HEPES (Nacalai Tesque, 17557-94), and GlutaMAX (Thermo Fisher, 35050061). After 6 days, Tregs were harvested and analyzed for antibody binding using a FACSVerse. Data analysis was performed using GraphPad Prism 9.2.0.

[0132] 17.2 Results Figures 49 and 50 show the results of binding analysis. The affinity (K D [M]) are 3.983 × 10 9 , 1.821×10 9At a concentration of 10 μg / mL, there was no difference in the binding ability to activated Tregs among IgG4-type 6-3h4IF, IgG1-type 6-3h1_IF, and 6-3h1_IF_NF1-3. In comparison, the binding ability of ipilimumab (ipi, YERVOY®, Bristol Myers Squibb) and mogamulizumab (moga, POTELIGEO®, Kyowa Kirin) to activated Tregs was weaker.

[0133] 18.1 ADCC activity measurement (reporter assay) Activated Tregs were used in 3 x 10 4 Cells / well were seeded as target cells, and each test antibody was added at 0.0001 to 10 μg / ml and measured according to the package insert of the ADCC Reporter Bioassay, V Variant (Promega, G7010). The ADCC activity value was expressed as a relative value, with the value obtained when a control IgG1 antibody was added at each concentration being set at 1.

[0134] 18.2 Results The results of the ADCC activity measurements are shown in Figure 51. 6-3h1_IF_NF2 had the highest ADCC activity, followed by NF3 and NF1, all of which exceeded the ADCC activity value of 6-3h1_IF before glycosylation.

[0135] 19.1 ADCC activity measurement (cancer cell line) Human ovarian cancer cell line OVA42 was seeded onto a 96-well plate and cultured overnight. The next day, the medium was removed, and the test antibody and isotype control were added at a final concentration of 10 μg / mL. PBMCs derived from healthy volunteers were then added to the plate to achieve an E / T ratio of 10. After centrifugation at 200 G for 3 minutes, the plate was cultured at 37°C under 5% CO2 for 18 hours. After the culture was completed, the plate was incubated with CytoTox-Glo TMThe number of dead cells was measured using a Cytotoxicity Assay (Promega G9290). ADCC activity was calculated using the RLU values ​​using the following formula: ADCC activity (%) = RLU value when antibody solution at each concentration, effector cells, and target cells were mixed - RLU value when effector cells and target cells were mixed / RLU value when lysis buffer was added to target cells - RLU value when target cells were cultured alone x 100. Data analysis was performed using GraphPad Prism 9.2.0. Statistical analysis was performed using one-way ANOVA and Dunnett's test. p**≦0.01 and p****≦0.0001 were considered to indicate significant differences.

[0136] 19.2 Results The results of the ADCC activity measurement are shown in Figure 52. 6-3h1_IF_NF2 (10 μg / mL) showed significant and pronounced ADCC activity against the human ovarian cancer cell line OAW-42.

[0137] 20.1 Affinity Measurement of 6-3h1_IF_NF1-3 for FcγRIIIa (SPR Analysis) Kinetic measurements of antibody binding to the target antigen were performed using a Biacore 8K (Cytiva). CD16a Protein, Human, Recombinant (ECD, F176V, His Tag) (Sino Biological, 10389-H08H1) labeled with a biotinylation kit (EZ-Link Sulfo-NHS-LC-Biotin) (ThermoFisher Scientific, A39257) was immobilized on a Series S Sensor Chip CAP (Cytiva, 28920234) using a Biotin CAPture Kit, Series S (Cytiva, 28-9201-34). The test antibody was added at a concentration of 2 × 10 -6 Seven serial dilutions of M were prepared and analyzed using a 1:1 binding model with multi-cycle kinetics under the following conditions: contact time 60 seconds, dissociation time 600 seconds, flow rate 30 μl / min, and HBS-EP+ running buffer.

[0138] 20.2 Results The results of SPR analysis are shown in Figure 53. All of the glycoengineered antibodies 6-3h1_IF_NF1 to 3 showed higher affinity for FcγRIIIa (V variant) than the unengineered antibody 6-3h1_IF, with NF2 having the best affinity.

[0139] Figures 54A to 54C show tables and graphs summarizing the results of 16.1 to 16.2, 18.1 to 18.2, and 20.1 to 20.2. A strong correlation was observed between the ADCC activity value and the affinity for FcγRIIIa and the fucosylation rate (R 2 = 0.9598 and 0.9339).

[0140] 21.1 Effect on T cells. Purchased healthy human PBMC (33000-10, Wako Pure Chemical Industries, Ltd.) was used in a 5x10 5 Cells were seeded at 100 cells / well in 96-well U-bottom plates and cultured for 5 or 6 days in the presence of each test antibody, 100 IU / mL IL-2 (87-890, NIPRO), and 20 ng / mL IL-7 (200-07, PeproTech) to induce Tregs. After washout, the cells were cultured for 11 days in the presence of CMV peptide A02 (TS-0010-1c, MBL), 10 IU / mL IL-2, and 20 ng / mL IL-7 to induce antigen-specific cytotoxic T cells. The culture medium used was RPMI-1640 (189-0202, Wako Pure Chemical Industries) supplemented with 10% Human Serum AB (100-512, H46X00K, Gemini Bio), 1 mM penicillin-streptomycin (09367-34, Nacalai Tesque), 1 mM sodium pyruvate (11360070, Gibco), 10 mM HEPES (17557-9, Nacalai Tesque), 1% GlutaMAX (35050061, Thermo Fisher Scientific), and 0.01 mM 2-mercaptoethanol (21438-82, Nacalai Tesque).

[0141] Various cell types were analyzed using a FACS Verse (BD). Cell counting was performed using CountBright TMAbsolute Counting Beads (C36950, Invitrogen) were used. Data were analyzed using GraphPad Prism 9.2.0. Statistical analysis was performed using one-way ANOVA and Dunnett's test. p<0.05, p<0.01, p<0.001, p<0.0001, and p<0.0001 were considered significant.

[0142] 21.2 Results Figures 55 to 59 show the results of cell type analysis, Treg depletion evaluation, and cytotoxic T cell test. PBMCs from healthy individuals were cultured for 6 days in the presence of 6-3h1_IF_NF2 (10 μg / mL), resulting in the proliferation of CD4 + T cells and CD8 + CD39 in T cells hi The CD39 fraction was selectively removed (Figure 55). Furthermore, by culturing PBMCs from healthy individuals for 5 days in the presence of 6-3h1_IF_NF2 (10 μg / mL), hi CD4 + FoxP3 + Treg and effector Treg (Fr.II, CD4 + CD45RA - FoxP3 hi ) significantly and markedly diminished (Figures 56-57). Furthermore, this was completely abolished by treatment with anti-CD16 (FcγRIIIa) antibody. This indicates that the Treg-eliminating effect of 6-3h1_IF_NF2 is based on CD16 (FcγRIIIa)-mediated ADCC.

[0143] Furthermore, CD8 + T CM (Central Memory, CD8 + CD45RA - CCR7 - ) and CD8 + T EM (Effector Memory, CD8 + CD45RA - CCR7 + ) was elevated, which was completely reversed by anti-CD16 (FcγRIIIa) antibody treatment (Fig. 58).

[0144] Ipilimumab (ipi, YERVOY (registered trademark), Bristol Myers Squibb) and mogamulizumab (moga, POTELIGEO (registered trademark), Kyowa Kirin) were observed to significantly reduce only effector Treg (Fr. II) (Figures 56-57).

[0145] 6-3h1_IF_NF2 (10 μg / mL) significantly induced antigen-specific cytotoxic T cells and CD39 + / - PD1 + Tim-3 + The fraction ratio was lower than that of mogamulizumab, and the degree of exhaustion was low (Figure 59).

[0146] 22.1 Cancer cell proliferation inhibition test using Treg-depleted PBMCs. Purchased healthy human PBMCs (Wako Pure Chemical Industries, 33000-10M) were used at 5x10 5Cells were seeded at 15,000 cells / well in a 96-well U-bottom plate and cultured for 5 days in the presence of 100 IU / mL IL-2 (NIPRO, 87-890), 20 ng / mL IL-7 (PeproTech, 200-07), and 10 μg / mL of each test antibody. Subsequently, A375 (a melanoma cancer cell line) overexpressing CMV antigen and RFP (15,000 cells / well) and PBMCs (45,000 cells / well) cultured for 5 days were mixed and co-cultured at 37°C under 5% CO2 for 5 days. The medium used was RPMI-1640 (189-0202, Wako Pure Chemical Industries) supplemented with 10% Human Serum AB (100-512, H46X00K, Gemini Bio), 1 mM penicillin-streptomycin (09367-34, Nacalai Tesque), 1 mM sodium pyruvate (11360070, Gibco), 10 mM HEPES (17557-9, Nacalai Tesque), 1% GlutaMAX (35050061, Thermo Fisher Scientific), and 0.01 mM 2-mercaptoethanol (21438-82, Nacalai Tesque). RFP fluorescence was measured using a Varioskan LUX multimode microplate reader (ThermoFisher Scientific) to determine the number of A375 cells. Data analysis was performed using GraphPad Prism 9.2.0. Statistical analysis was performed using one-way ANOVA and Dunnett's test. p****≦0.0001 was considered significant.

[0147] 22.2 Results The results are shown in Figure 60. PBMCs treated with 6-3h1_IF_NF2 (10 μg / mL) significantly suppressed the growth of human malignant melanoma A375. The anti-CTLA-4 antibody ipilimumab did not show any inhibitory effect.

[0148] 23.1 Preparation of Glycoengineered Antibodies of B23-7h1_HB and B23-7h1_IF First, the heavy chain constant region sequence of an expression vector incorporating B23-7h4_HB or B23-7h4_IF was recombined from human CH (IgG4SP) to human CH (IgG1) to create an expression vector in which the subclass was changed from IgG4 to IgG1. The antibodies expressed from these vectors were designated B23-7h1_HB or B23-7h1_IF (the amino acid sequences of VH and VL are the same as those of B23-7h4_HB or B23-7h4_IF; the amino acid sequence of human CH (IgG1) is SEQ ID NO: 88). Next, glycoengineered antibodies were prepared under multiple conditions for each clone using the following procedure. The above expression vectors were transfected into CHO cells using the ExpiCHO Expression System Kit (ThermoFisher Scientific, A29133), and each antibody was produced in transient antibody-expressing CHO cells. 2F-Peracetyl-Fucose (DMSO suspension) (Merck, 344827) was added to a final concentration of 20 μM during culture, either the day before transfection and / or 3–10 days after transfection. Cell culture was performed according to the ExpiCHO Expression System Kit package insert, and antibodies produced in the culture supernatant were collected and purified from CHO cells 7–14 days after transfection. Following these procedures, the glycoengineered B23-7h1_HB antibody and glycoengineered B23-7h1_IF antibody listed in the clone name column in Figure 61 were obtained. Antibodies prepared without the addition of 2F-Peracetyl-Fucose are labeled "Parent."

[0149] Furthermore, glycosylated versions of 6-3h1_IF were prepared using the procedure for preparing 6-3h1_IF_NF2 described above in 13.1. The antibody collected on day 7 of culture after transfection was designated 6-3h1_IF_NF2_D7, and the antibody collected on day 13 was designated 6-3h1_IF_NF2_D13.

[0150] 23.2 Affinity for Recombinant CD39 The affinity of glycosylated B23-7h1_HB antibody or glycosylated B23-7h1_IF antibody for human CD39 was measured using the same method as in 2.1 above.

[0151] 23.3 Results Figure 61 shows the results of SPR analysis. The affinity for human CD39 was almost the same K regardless of the presence or absence of glycosylation or the preparation method. D The values ​​were shown.

[0152] 24.1 Affinity for FcγRIIIa (V variant) Using the same method as in 20.1 above, test antibody was diluted to 250 × 10 -9 Five serial dilutions were prepared starting from M, and the affinity of each antibody for FcγRIIIa (V variant) was measured.

[0153] 24.2 Results The results of SPR analysis are shown in Figure 62. There was no change in the affinity of the parent antibody for FcγRIIIa, but the glycosylated antibodies showed changes in their affinity for FcγRIIIa depending on the preparation method, and all showed higher affinity than the parent antibody.

[0154] 25.1 CD39 Enzyme Inhibitory Activity in Human Ovarian Cancer Cell Line OAW-42 Using the same method as in 1.8 above, the inhibitory activity of B23-7h4_IF, B23-7h1_IF, and their glycosylated derivatives against human CD39 was measured.

[0155] 25.2 Results Figure 63 shows the results of enzyme inhibitory activity measurements. The inhibitory activity against human CD39 was almost the same regardless of the antibody subclass, the presence or absence of glycosylation, or the preparation method. The results for the two B23-7h4_IFs were obtained using different plates.

[0156] 26.1 Glycosylation Analysis The fucosylation rate was measured by glycosylation analysis using the same method as in 16.1 above.

[0157] 26.2 Results The results of glycosylation analysis are shown in Figure 64. The fucosylation rates of B23-7h1_IF_1-4 (Parent), B23-7h1_IF_1-5, B23-7h1_IF_1-6, B23-7h1_IF_1-14, and 6-3h1_IF_NF2_D13 were 78.7%, 59.5%, 15.1%, 8.9%, and 13.5%, respectively. Core fucose addition was suppressed in glycoengineered antibodies, with B23-7h1_IF_1-14 being the most suppressed.

[0158] 27.1 ADCC reporter assay (Treg target and OAW-42 target) Using the same method as in 18.1 above, activated Treg and the human ovarian cancer line OAW-42 were used as target cells, and 1.5 x 10 4 or 3 x 10 4 Cells were seeded per well, and the test antibodies were added at various concentrations for measurement.

[0159] 27.2 Results Figure 65 shows the results of ADCC activity measurements (Treg target). In assays targeting activated Treg, the ADCC activity of glycoengineered antibodies varied depending on the preparation method. The results for the two 6-3h1_IF_NF2_D7 antibodies were obtained using different plates. Figure 66 shows the correlation between ADCC activity and FcγRIIIa binding activity, and Figure 67 shows the correlation between ADCC activity and fucosylation rate. The correlation between ADCC activity and affinity for FcγRIIIa (K A The value is K D A strong correlation was observed between the ADCC activity and the fucosylation rate (the reciprocal of the value), and between the ADCC activity and the fucosylation rate.

[0160] The results of ADCC activity measurements (OAW-42 target) are shown in Figures 68 and 69. At the maximum activity value in the assay using OAW-42 as the target cells, B23-7h1_IF_1-14 had an ADCC induction rate that was approximately 2.6 times higher than that of B23-7h1_IF_1-4 (Parent).

[0161] 28.1 CD39 hi Selective removal of T cells Using the same method as in 21.1 above, we confirmed the change in the proportion of various T cells with high CD39 expression by glycoengineered anti-CD39 antibodies.

[0162] 28.2 Results Figures 70 to 72 show the results of cell type analysis. CD4 + T cells and CD8 + CD39 in T cells hi The fraction was significantly reduced, and CD39 int No significant changes were observed in the CD39 fraction of these clones. hi Selective removal ability was demonstrated.

[0163] As described in the above Examples, the present inventors have obtained novel anti-CD39 antibodies, which have been shown to have remarkably excellent properties for use in cancer therapy.

[0164] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment that binds to CD39, (a) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 (b) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 9, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 12 (c) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 15, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO:

18. (d) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 21, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 24 (e) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 25, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 27, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 28, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 30 (f) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 33, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 34, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 35, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 36, or (g) Heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 37, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 38, heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 39, light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and light chain CDR3 containing the amino acid sequence shown in SEQ ID NO:

42. Antibody or antigen-binding fragments containing such fragments.

2. The antibody or antigen-binding fragment according to claim 1, which inhibits CD39 activity.

3. The antibody or antigen-binding fragment according to claim 1, which suppresses the proliferation of malignant tumor cells.

4. The antibody or antigen-binding fragment according to claim 1, which promotes the cytotoxic activity of immune cells.

5. The antibody or antigen-binding fragment according to claim 1, which is a monoclonal antibody.

6. The antibody or antigen-binding fragment according to claim 1, wherein the antibody is a mouse, chimeric, or humanized antibody.

7. K for CD39 D (M) is 9.0 × 10 -9 The antibody or antigen-binding fragment according to claim 1, which is as follows:

8. The antibody or antigen-binding fragment according to claim 1, which is a glycosylated antibody.

9. A polynucleotide or vector encoding an antibody or antigen-binding fragment according to any one of claims 1 to 8.

10. A cell comprising the polynucleotide or vector described in claim 9.

11. A method for producing an antibody or antigen-binding fragment, comprising the step of growing the cells described in claim 10.

12. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

13. A pharmaceutical composition for the treatment of malignant tumors, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

14. A composition for inhibiting CD39 activity, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

15. A composition for promoting TNFα production by immune cells, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

16. A composition for promoting cytotoxic activity, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

17. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment has an N-linked glycan, and 40% or more of the N-linked glycans in the pharmaceutical composition do not contain core fucose.

18. A pharmaceutical composition for the treatment of malignant tumors, comprising the antibody or antigen-binding fragment according to any one of claims 1 to 8, used in combination therapy with a chemotherapeutic agent.

19. A pharmaceutical composition for the treatment of malignant tumors, comprising a chemotherapeutic agent, used in combination therapy with an antibody or antigen-binding fragment according to any one of claims 1 to 8.

20. A kit comprising an antibody or antigen-binding fragment according to any one of claims 1 to 8.

21. CD39, including anti-CD39 antibodies hi A composition for cell-selective removal.