Multispecific antigen-binding molecule targeting CD39

Abstract

The present invention makes it possible to obtain a novel CD39 binding domain usable for producing a multispecific antigen-binding molecule targeting CD39 on cancer cells, or an article or a method using the CD39 binding domain. Provided is: a CD39 binding domain that includes heavy chains CDR1-3 and light chains CDR1-3 comprising a specific amino acid sequence; or a multispecific molecule comprising the CD39 binding domain. The CD39 binding domain or the multispecific molecule may have binding ability to CD39 on the cancer cell surface.

Description

Multispecific antigen-binding molecules targeting CD39

[0001] The technical field of the present invention relates to CD39 binding domains or multispecific antigen-binding molecules that target CD39.

[0002] CD39 is a double-transmembrane glycoprotein that has been reported to be expressed in cancer cells such as lung cancer, gastric cancer, ovarian cancer, and melanoma (e.g., Non-Patent Documents 1 and 2). Patent Documents 1 and 2 describe antibodies that bind to CD39 and antibodies that have the ability to inhibit the enzymatic activity of CD39.

[0003] In the field of cancer treatment, research and development of molecules that can bind to multiple antigens (multispecific antigen-binding molecules), such as bispecific antibodies, has been ongoing (e.g., Non-Patent Documents 3 to 12). For example, a known technique involves administering to a patient a bispecific antibody that binds to both a cancer cell surface antigen (e.g., CD19) and a T cell surface antigen (e.g., CD3), thereby attracting T cells to the vicinity of cancer cells and allowing the T cells to effectively kill the cancer cells.

[0004] According to Non-Patent Document 3, more than 140 different molecules are undergoing clinical trials, and Figure 1 and Table 1 of Non-Patent Document 3 list numerous formats of multispecific antibodies undergoing clinical trials. Multispecific antigen-binding molecules approved for cancer treatment in the United States include blinatumomab, teclistamab, and tebentafusp.

[0005] Blinatumomab is a bispecific antibody that targets CD19 on malignant B cells, with the CD19-binding domain and CD3-binding domain linked via a linker. Teclistamab is a bispecific antibody that targets BCMA on myeloma cells, with the BCMA-binding domain and CD3-binding domain linked via an Fc region. Tebentafsp is a bispecific fusion protein that targets gp100 on melanoma, with the gp100-binding domain and CD3-binding domain linked via a linker.

[0006] International Publication No. 2018 / 167267 U.S. Patent Application Publication No. 2019 / 0062448

[0007] Xia et al., Mol Cancer. 2023 Mar 2;22(1):44.Cai et al., Oncol Lett. 2016 Nov; 12(5): 4080-4086.Elshiaty et al., Int J Mol Sci. 2021 May 26;22(11):5632.Li et al., Cell Mol Immunol. 2020 May; 17(5): 451-461.Wei et al., Front Immunol. 2022; 13: 1035276.Nie et al., Antib Ther. 2020 Jan; 3(1): 18-62.Blanco et al., Clin Cancer Res. 2021 Oct 15; 27(20): 5457-5464.Kang et al.,Front Immunol. 2022 Oct 20;13:1020003.Brinkmann et al., MAbs. 2017 Feb / Mar;9(2):182-212.Salvaris et al., J Pers Med . 2021 Apr 29;11(5):355.Knudson et al., Oncoimmunology. 2018; 7(5): e1426519.Chen et al., Mol Ther Oncolytics. 2021 Apr 2;21:144-157.

[0008] As mentioned above, CD39 is expressed on cancer cells, and to effectively kill cancer cells, it is necessary to prepare an optimal CD39-binding domain when designing a multispecific antigen-binding molecule that targets CD39 on cancer cells.

[0009] As described in the Examples below, the present inventors generated novel specific CD39-binding domains and prepared multispecific antigen-binding molecules containing the CD39-binding domains. As a result, multispecific antigen-binding molecules were obtained that exhibited excellent one or more properties, such as affinity for CD39 on the cancer cell membrane, or antitumor or cytokine production effects associated with engagement between cancer cells and immune cells.

[0010] Objects of the present invention include providing a novel CD39-binding domain that can be used to prepare multispecific antigen-binding molecules that target CD39 on cancer cells, or a product or method utilizing the CD39-binding domain.

[0011] According to one aspect of the present invention, there is provided a CD39-binding domain 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 CD39-binding domain comprising 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 CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18, or (d) a CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24. The use of this multispecific antigen-binding molecule can produce excellent anti-tumor effects.

[0012] According to one aspect of the present invention, there is provided a polynucleotide or vector encoding a multispecific antigen-binding molecule comprising a CD39-binding domain comprising heavy chain CDR1-3 and light chain CDR1-3 of any of (a) to (d) above. According to one aspect of the present invention, there is provided a cell comprising the above polynucleotide or vector. According to one aspect of the present invention, there is provided a method for producing a multispecific antigen-binding molecule, the method comprising the step of growing the above cells. According to one aspect of the present invention, there is provided a composition comprising the above multispecific antigen-binding molecule. According to one aspect of the present invention, there is provided a pharmaceutical composition for treating malignant tumors, the pharmaceutical composition comprising the above multispecific antigen-binding molecule. According to one aspect of the present invention, there is provided a composition for promoting cytokine production by immune cells, the pharmaceutical composition comprising the above multispecific antigen-binding molecule. According to one aspect of the present invention, there is provided a kit comprising the above multispecific antigen-binding molecule.

[0013] This figure shows the results of epitope binning of anti-human CD39 antibodies. Epitope binning was performed using Biacore 8K. The antibodies (1 in the vertical column in Figure 1) were applied to a Ni-NTA sensor chip that captured recombinant human CD39 antigen. st Ab), followed by the antibodies listed next to Figure 1 (2 nd Ab) and 2 ndThe binding of Abs was evaluated based on the presence or absence of a response. Combinations that did not produce a response are shown in dark gray, while combinations that produced a response are shown in white. Combinations in which the response varied depending on the flow order are shown in light gray. Clones showing similar competition were grouped into four bins. This figure shows the results of measuring the affinity of anti-human CD39 antibodies for recombinant antigens by surface plasmon resonance. Each anti-human CD39 antibody was captured onto a CM5 sensor chip using the Human Antibody Capture Kit (Cytiava), and recombinant human CD39 prepared in five 2-fold dilutions starting from 50 nM or 12.5 nM was run as the analyte. The binding rate (ka) and dissociation rate (kd) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. This figure shows the results of measuring the binding affinity of anti-human CD39 antibodies to cell membrane antigens. The binding of each anti-CD39 antibody to HEK293T cells stably expressing human CD39 was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 2 shows a schematic diagram of the bispecific molecule designed in Example 2.1. The bispecific molecule was designed to have a first antigen-binding domain (CD39-binding domain) targeting human CD39 and a second antigen-binding domain (4-1BB-binding domain) targeting human 4-1BB, an immune cell surface antigen. The first antigen-binding domain was an scFv-formatted anti-human CD39 antibody. The second antigen-binding domain was a trimer of the extracellular domain of human 4-1BBL. A hole mutation (Hole-mutated Fc region: SEQ ID NO: 38) from Knob-into-Hole (US7183076B2, Knob: Seq No. 2, Hole: Seq No. 3) was introduced into the Fc region of the polypeptide chain containing the first antigen-binding domain, and a knob mutation (Knob-mutated Fc region: SEQ ID NO: 45) was introduced into the Fc region of the polypeptide chain containing the second antigen-binding domain. Figure 1 shows the results of measuring the affinity of the bispecific molecule for human CD39 and 4-1BB by surface plasmon resonance.Each bispecific molecule was captured onto a CM5 sensor chip using the Human Antibody Capture Kit. Analyte samples were prepared using recombinant human CD39 (50 nM or 12.5 nM, 2-fold dilutions in five steps) or recombinant human 4-1BB (2 uM, 4-fold dilutions in four steps). The association rate (ka) and dissociation rate (kd) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. Figure 1 shows the binding of bispecific molecules to a human cancer cell line (OAW42). The binding of each bispecific molecule to OAW42 cells was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 2 shows the binding of bispecific molecules to a human cancer cell line (SKMEL28). The binding of each bispecific molecule to SKMEL28 cells was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 1 shows the results of measuring the inhibitory ability of bispecific molecules on human CD39 enzyme activity. OAW42 cells were plated in a 96-well plate at 6.25 x 10 3 Cells were seeded at 100% ATP / well, and serially diluted bispecific molecules were added. ATP was then added, and the amount of ATP remaining in the supernatant after 30 minutes was quantified using CellTiter-Glo® 2.0 (Promega, G9242). The ATP remaining value in wells without cells seeded was set to 100%, and the ATP remaining value in wells seeded with cells but without antibody was set to 0%. The inhibition rate of ATP degradation for each bispecific molecule was calculated from the ratio of the ATP remaining value of the measurement sample. Figure 1 shows the results of measuring cytokine production in a co-culture system of cancer cells and PBMCs. SKMEL28 cells irradiated with 50 Gy of X-rays were plated at 2 x 10 cells in a 96-well plate as target cells. 4Cells were seeded at 1000 cells / well, and PBMCs were added as effector cells at an effector cell / target cell ratio (E / T) of 10. Alternatively, wells containing only effector cells were prepared. Furthermore, 10 μg / mL of each bispecific molecule and 1 nM of an anti-human CD3 antibody (OKT3) were added and incubated at 37°C for 2 days. After 2 days, the culture supernatants were collected and cytokine (IL-2, TNF-α, and IFN-γ) concentrations were measured by Luminex. Figure 1 shows the results of surface plasmon resonance assays of the affinity of humanized anti-human CD39 antibodies for recombinant antigens. Each anti-human CD39 antibody was captured onto a CM5 sensor chip using the Human Antibody Capture Kit, and recombinant human CD39 prepared in five 2-fold dilutions starting from 40 nM or 10 nM was loaded as the analyte. The association rate (ka) and dissociation rate (kd) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. Figure 1 shows the results of binding analysis of humanized anti-human CD39 antibodies to cell membrane antigens. The binding of each anti-CD39 antibody to HEK293T cells stably expressing human CD39 was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 2 shows the results of surface plasmon resonance analysis of the affinity of bispecific molecules for human CD39 and 4-1BB. Each bispecific molecule was captured onto a CM5 sensor chip using the Human Antibody Capture Kit. Recombinant human CD39 was diluted 2-fold from 50 nM or 12.5 nM in five steps, and recombinant human 4-1BB was diluted 4-fold from 2 uM in four steps. The binding rate (ka) and dissociation rate (kD) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. Figure 1 shows the results of measuring the binding of bispecific molecules to a human cancer cell line (OAW42). Binding of each bispecific molecule to OAW42 cells was measured by flow cytometry, and mean fluorescence intensity was plotted against antibody concentration. Figure 2 shows the results of measuring the binding of bispecific molecules to a human cancer cell line (SKMEL28).The binding of each bispecific molecule to SKMEL28 cells was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 1 shows the results of measuring IL-2 production in a co-culture system of cancer cells and PBMCs. SKMEL28 cells were cultured as target cells at 2x10 in a 96-well plate. 4 Cells were seeded at 1000 cells / well, and PBMCs were added as effector cells to achieve an effector cell / target cell ratio (E / T) of 1.6. Alternatively, wells containing only effector cells were prepared. Furthermore, 1 μg / mL of each bispecific molecule and 1 nM of anti-human CD3 antibody (OKT3) were added, and the cells were incubated at 37°C for 4 or 6 days. After incubation, the culture supernatant was collected, and the IL-2 concentration was measured using Luminex. This figure shows the results of measuring TNF-α production in a co-culture system of cancer cells and PBMCs. For conditions, see the legend for Figure 15. This figure shows the results of measuring IFN-γ production in a co-culture system of cancer cells and PBMCs. For conditions, see the legend for Figure 15. This figure summarizes the experimental conditions and p values ​​for measuring cytokine production in Figures 15, 16, and 17. This figure shows the results of measuring the inhibitory effect of bispecific molecules on cancer cell proliferation. SKMEL28 cells were cultured at 4 x 10 319. The cells were seeded at 1000 cells / well, and PBMCs were added as effector cells to give an effector cell / target cell ratio (E / T) of 2. Furthermore, 1 μg / mL of each bispecific molecule and 1 nM of an anti-human CD3 antibody (OKT3) were added, followed by incubation at 37°C for 7 days. After 7 days, the culture supernatant was removed, and the number of cells remaining on the plate bottom was quantified using CellTiter-Glo® 2.0 (Promega, G9242) using the amount of ATP as an index. Figure 19 summarizes the P values ​​for the growth inhibitory effect measurement results shown in Figure 19. Figure 19 shows the amino acid sequences of the CDRs of the molecules used in the Examples. Figure 19 shows the amino acid sequences of the variable regions of the molecules used in the Examples. Figure 19 shows the amino acid sequences of the Fc region of the molecules used in the Examples. Figure 19 shows the amino acid sequences of each region of the molecules used in the Examples. Figure 19 shows the amino acid sequences of each region of the anti-CD3 antibody used in the Examples. Figure 19 shows the results of measuring the affinity of bispecific molecules for recombinant antigens by surface plasmon resonance. The bispecific molecules were captured onto a CM5 sensor chip using a Human Antibody Capture Kit (Cytiava), and recombinant human CD39, prepared in five 2-fold dilutions starting from 50 nM or 12.5 nM, was run as the analyte. The association rate (ka) and dissociation rate (kd) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the kd / ka ratio. Figure 1 shows the binding of bispecific molecules to a human cancer cell line (SKMEL28). The binding of each bispecific molecule to SKMEL28 cells was measured by flow cytometry, and the mean fluorescence intensity was plotted against the antibody concentration. Figure 2 shows the inhibitory effect of bispecific molecules on cancer cell proliferation. SKMEL28 cells were plated at 4 x 10 3The cells were seeded at 2 x 10 cells / well, and PBMCs were added as effector cells to give an effector cell / target cell ratio (E / T) of 2. Furthermore, 1 μg / mL of each bispecific molecule and 1 nM of anti-human CD3 antibody (OKT3) were added, and the cells were incubated at 37°C for 7 days. After 7 days, the culture supernatant was removed, and the number of cells remaining on the plate bottom was quantified using CellTiter-Glo® 2.0 (Promega, G9242) using ATP content as an index. This figure shows the results of measuring the amount of IL-2, TNF-α, or IFN-γ produced in a co-culture system of cancer cells and PBMCs. SKMEL28 cells were seeded as target cells at 2 x 10 cells / well in a 96-well plate. 4 Cells were seeded at 1000 cells / well and PBMCs were added as effector cells to achieve an effector cell / target cell ratio (E / T) of 1.6. Alternatively, effector cells alone were added to some wells. Furthermore, 1 μg / mL of each bispecific molecule and 1 nM of an anti-human CD3 antibody (OKT3) were added and incubated at 37°C for 4 or 6 days. After incubation, the culture supernatant was collected and the concentrations of IL-2, TNF-α, and IFN-γ were measured using Luminex. This figure shows the results of measuring the production of Granzyme B or Perforin in co-cultures of cancer cells and PBMCs. For conditions, see the legend for Figure 29. Concentration measurements were performed using MILLIPLEX MAP 7-Plex Human CD8+ / Cytokine Magnetic Bead Panel (Millipore, #HCD8MAG15K17PMX). The total numbers of CD4-positive T cells and CD8-positive T cells were calculated by flow cytometry analysis, and each was classified according to the expression patterns of CD39 and PD1.

[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, there is provided a novel CD39-binding domain or a multispecific antigen-binding molecule comprising the novel CD39-binding domain, which comprises: (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 CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18, or (d) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24 (hereinafter, these may be abbreviated as any of CDR sets (a) to (d)). Use of such CD39-binding domains enables the production of excellent multispecific antigen-binding molecules, as demonstrated in the Examples below. Superior multispecific antigen-binding molecules include, for example, multispecific antigen-binding molecules that have high affinity for CD39 on the cancer cell membrane, high anti-tumor activity associated with the engagement of cancer cells with immune cells, or high cytokine production activity associated with the engagement of cancer cells with immune cells.

[0016] In one embodiment of the present invention, the CD39 includes a protein also known as ENTPD1 or Ectonucleoside triphosphate diphosphohydrolase 1. CD39 can be expressed on the surface of cancer cells. 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: 61. 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, human- or monkey-derived CD39.

[0017] In one embodiment of the present invention, the anti-tumor effect of the multispecific antigen-binding molecule may be due to, for example, immune cells being attracted to the vicinity of cancer cells by the multispecific antigen-binding molecule that binds to CD39 (a cancer cell surface antigen) and an immune cell surface antigen, and then the cancer cells being killed by the immune cells. Such cancer cell killing through engagement of cancer cells with immune cells has been reported, for example, by Elshiaty et al., Int J Mol Sci. 2021 May 26;22(11):5632., Li et al., Cell Mol Immunol. 2020 May;17(5):451-461., Wei et al., Front Immunol. 2022;13:1035276., Nie et al., Antib Ther. 2020 Jan;3(1):18-62., Blanco et al., Clin Cancer Res. 2021 Oct 15;27(20):5457-5464., Salvaris et al., J Pers Med. 2021 Apr 29;11(5):355., Kang et al., Front Immunol. 2022 Oct 20;13:1020003. or Brinkmann et al., MAbs. 2017 Feb / Mar;9(2):182-212.

[0018] In one embodiment of the present invention, the anti-tumor effect of the multispecific antigen-binding molecule may be due to, for example, trapping anti-inflammatory cytokines by the multispecific antigen-binding molecule that binds to CD39 (a cancer cell surface antigen) and the anti-inflammatory cytokine, thereby reducing anti-inflammatory cytokine signaling in the tumor microenvironment. This signaling includes, for example, signals that induce regulatory T cells. Such anti-tumor effect can be achieved using mechanisms and formats such as those described in Knudson et al., Oncoimmunology. 2018;7(5):e1426519, Chen et al., Mol Ther Oncolytics. 2021 Apr 2;21:144-157, or Elshiaty et al., Int J Mol Sci. 2021 May 26;22(11):5632.

[0019] In one embodiment of the present invention, multispecific antigen-binding molecules may be produced by, for example, the Knobs-into-Holes (KIH) method, quadroma technology, CrossMAb technology, or co-culture method. Methods for producing multispecific antigen-binding molecules, including these production methods, include those described in, for example, Wang et al., Antibodies (Basel). 2019 Aug 2;8(3):43. Brinkmann et al., MAbs. 2017 Feb / Mar;9(2):182-212. or Klein et al., MAbs. 2012 Nov 1;4(6):653-663. The KIH method involves expressing recombinant antibody chains in which a Knob mutation has been introduced into one heavy chain and a Hole mutation has been introduced into the other. By designing the Knob to be inserted into the Hole, heterodimers are preferentially produced. In a bispecific antibody having an IgG-like format, one or two of the two Fabs linked to the Fc region can be replaced with an scFv, a polypeptide containing a ligand for an antigen or an antigen-binding fragment thereof, or a polypeptide containing a receptor for an antigen or an antigen-binding fragment thereof. The KIH method may utilize, for example, the method described in US Pat. No. 7,183,076 B2.

[0020] In one embodiment of the present invention, the multispecific antigen-binding molecule comprises a molecule that has binding affinity to multiple different antigens. Multispecific antigen-binding molecules include, for example, bispecific antigen-binding molecules or trispecific antigen-binding molecules. Multispecific antigen-binding molecules include, for example, multispecific antibodies or multispecific fusion proteins. Multispecific antibodies include, for example, bispecific antibodies or trispecific antibodies. Multispecific fusion proteins include, for example, bispecific fusion proteins or trispecific fusion proteins. "Multispecific" means specificity for two or more different antigens, "bispecific" means specificity for two different antigens, and "trispecific" means specificity for three different antigens.

[0021] In one embodiment of the present invention, the CD39-binding domain preferably comprises the CDR set (a) or (b) above. In this case, a more excellent multispecific antigen-binding molecule can be obtained in terms of high affinity for CD39 on the cancer cell membrane, high antitumor activity associated with engagement of cancer cells with immune cells, or high cytokine production associated with engagement of cancer cells with immune cells.

[0022] In one embodiment of the present invention, the CD39-binding domain is more preferably a CD39-binding domain comprising the CDR set (b) above. In this case, a more excellent multispecific antigen-binding molecule can be obtained in terms of high affinity for CD39 on the cancer cell membrane, high antitumor activity associated with engagement of cancer cells with immune cells, or high cytokine production associated with engagement of cancer cells with immune cells. It is also excellent in terms of suppressing side effects (e.g., neutropenia) caused by CD39 enzyme inhibition.

[0023] In one embodiment of the present invention, the CD39-binding domain preferably has the ability to bind to CD39 on the surface of cancer cells. Methods for assessing the binding ability of a CD39-binding domain include assessing binding to recombinant CD39 (e.g., affinity analysis using surface plasmon resonance or ELISA) and assessing binding to cell surface CD39 (e.g., assessing binding to CD39-overexpressing cells or CD39-expressing cancer cells). The latter method better reflects the in vivo state of CD39 and is therefore more suitable as a target for multispecific antigen-binding molecules.

[0024] In one embodiment of the present invention, the CD39-binding domain comprises a domain comprising a heavy chain variable region and a light chain variable region, in which any one of the CDR sets (a) to (d) is contained within the heavy chain variable region and the light chain variable region.

[0025] In one embodiment of the present invention, the CD39-binding domain may be a humanized CD39-binding domain. A humanized domain may, for example, have an amino acid sequence other than the CDRs derived from a human immunoglobulin. A humanized domain may, for example, have one or more CDRs derived from a non-human species and framework regions derived from a human immunoglobulin, and may comprise a domain that binds to a desired antigen. Domain humanization can be performed, for example, by the procedure described in Example 3.1 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).

[0026] In one embodiment of the present invention, the CD39-binding domain or a polypeptide comprising a CD39-binding domain can be produced by, but is not limited to, the following steps: (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 of the CDR sets (a) to (d) above; (ii) incorporating the antibody variable region sequence into an expression vector (optionally encoding a constant region (e.g., a human constant region)); (iii) transfecting cells with the expression vector; or (iv) recovering and purifying the polypeptide comprising the antibody variable region from the cells. The CD39-binding domain or a polypeptide comprising a CD39-binding domain may also be produced by grafting any of the CDR sets (a) to (d) 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. Polypeptides containing a CD39-binding domain may be produced by immunizing a mammal with CD39 and obtaining anti-CD39 antibody cells. Polypeptides containing a CD39-binding domain may also be produced by screening polypeptides based on their binding strength to CD39 or their ability to inhibit CD39 activity. The CDRs, variable regions, Fc regions, and constant regions are typically regions derived from antibodies. Polypeptides containing a CD39-binding domain may include, for example, antibodies (e.g., full-length antibodies, scFvs, or Fabs).

[0027] In one embodiment of the present invention, the multispecific antigen-binding molecule comprises a multispecific antigen-binding molecule with anti-tumor activity. This anti-tumor activity includes anti-tumor activity resulting from the binding of the multispecific antigen-binding molecule to multiple antigens. This anti-tumor activity includes anti-tumor activity resulting from the binding of the multispecific antigen-binding molecule to CD39 on the surface of cancer cells, immune cell surface antigens, or anti-inflammatory cytokines. This anti-tumor activity includes, for example, cytotoxic activity. Anti-tumor activity does not necessarily include activity resulting from the binding of an Fc region to an Fc receptor. The multispecific antigen-binding molecule comprises a multispecific antigen-binding molecule capable of activating immune cells (e.g., T cells). Activation of immune cells includes promotion of cancer immune responses.

[0028] In one embodiment of the present invention, the multispecific antigen-binding molecule comprises a multispecific antigen-binding molecule that has binding affinity to a first antigen (e.g., CD39) and a second antigen (e.g., an antigen other than CD39). The multispecific antigen-binding molecule comprises a domain that has binding affinity to the first antigen and a domain that has binding affinity to the second antigen. The second antigen includes, for example, an immune cell surface antigen or an anti-inflammatory cytokine.

[0029] In one embodiment of the present invention, the multispecific antigen-binding molecule includes a multispecific antigen-binding molecule comprising a first binding domain and a second binding domain. For example, the multispecific antigen-binding molecule includes a multispecific antigen-binding molecule comprising the CD39-binding domain as the first binding domain and an immune cell surface antigen-binding domain or an anti-inflammatory cytokine-binding domain as the second binding domain. A linker may be present between the first binding domain and the second binding domain. The first binding domain and the second binding domain may be linked via an Fc region. The first binding domain and the second binding domain may be linked via a disulfide bond. The second binding domain may have the ability to activate immune cells (e.g., T cells).

[0030] In one embodiment of the present invention, the immune cells include, for example, lymphocytes, monocytes, NK cells, NKT cells, dendritic cells, or granulocytes. Lymphocytes include, for example, T cells or B cells. T cells include, for example, killer T cells, helper T cells, regulatory T cells, or γδ T cells. Granulocytes include, for example, eosinophils, basophils, or neutrophils. Immune cell surface antigens include antigens expressed on one or more of these immune cells.

[0031] In one embodiment of the present invention, the immune cell surface antigen includes, for example, a costimulatory molecule, an immune checkpoint molecule, or CD3. Costimulatory molecules include, for example, 4-1BB, GITR, CD27, CD40, or NKp30. Immune checkpoint molecules include, for example, TIM-3, LAG-3, BTLA, CTLA-4, or CD73. The immune cell surface antigen-binding domain includes a domain capable of binding to one or more of these immune cell surface antigens. The immune cell surface antigen includes, for example, a molecule that activates immune cells (e.g., T cells).

[0032] In one embodiment of the present invention, the anti-inflammatory cytokine includes, for example, a molecule that induces regulatory T cells (Tregs). Anti-inflammatory cytokines include, for example, TGFβ, IL-4, IL-10, or IL-11. Tregs can be differentiated from naive T cells in peripheral blood by antigen stimulation in the presence of an anti-inflammatory cytokine (e.g., TGFβ).

[0033] In one embodiment of the present invention, the multispecific antigen-binding molecule includes, for example, a multispecific antigen-binding molecule comprising the CD39-binding domain as a first binding domain and a 4-1BB, GITR, CD27, CD40, NKp30, TIM-3, LAG-3, BTLA, CTLA-4, CD73, CD3, or TGFβ-binding domain as a second binding domain, resulting in a multispecific antigen-binding molecule with particularly excellent anti-tumor activity.

[0034] In one embodiment of the present invention, the 4-1BB includes a protein also known as CD137. 4-1BB is a costimulatory molecule belonging to the TNF receptor family and has been reported to be involved in the activation and survival of T cells and NK cells. Details of the amino acid sequence of 4-1BB can be found on websites such as NCBI or UniProt. The primary accession number for 4-1BB listed in UniProt is, for example, Q07011. The amino acid sequence of human 4-1BB is, for example, SEQ ID NO: 62. 4-1BB includes, for example, 4-1BB derived from human or monkey.

[0035] In one embodiment of the present invention, the 4-1BB-binding domain comprises, for example, a polypeptide having binding affinity to 4-1BB. The 4-1BB-binding domain may comprise, for example, an amino acid sequence in a ligand for 4-1BB or an anti-4-1BB antibody. The 4-1BB-binding domain may specifically comprise, for example, a region or domain in a ligand for 4-1BB or an anti-4-1BB antibody having binding affinity to 4-1BB, or a region determining binding affinity to 4-1BB. The region in a ligand for 4-1BB having binding affinity to 4-1BB may be, for example, an extracellular domain. The region in a ligand for 4-1BB having binding affinity to 4-1BB may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 39. The ligand for 4-1BB may comprise, for example, 4-1BBL. The 4-1BB-binding domain may comprise, for example, an amino acid sequence derived from a ligand for 4-1BB, such as (i) the amino acid sequence set forth in SEQ ID NO: 39, (ii) an amino acid sequence showing 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 39, or (iii) an amino acid sequence having deletions, substitutions, insertions, or additions of up to several amino acids relative to the amino acid sequence set forth in SEQ ID NO: 39. The amino acid sequence of a ligand for 4-1BB may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 40. The 4-1BB-binding domain may comprise, for example, an amino acid sequence derived from a ligand for 4-1BB, such as (i) the amino acid sequence set forth in SEQ ID NO: 40, (ii) an amino acid sequence showing 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 40, or (iii) an amino acid sequence having deletions, substitutions, insertions, or additions of one to several amino acids relative to the amino acid sequence set forth in SEQ ID NO: 40. The 4-1BB-binding domain may comprise, for example, a trimer of a polypeptide comprising an amino acid sequence derived from a ligand for 4-1BB. The ligand for 4-1BB may comprise, for example, 4-1BBL. The region in the anti-4-1BB antibody that determines the binding ability to 4-1BB may include, for example, the amino acid sequence of heavy chain CDR1 to 3 and light chain CDR1 to 3. The region in the anti-4-1BB antibody that has binding ability to 4-1BB may include, for example, the amino acid sequence of the variable region (including, for example, the heavy chain variable region or the light chain variable region).The 4-1BB-binding domain may comprise, for example, the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 as amino acid sequences derived from an anti-4-1BB antibody. The 4-1BB-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as amino acid sequences derived from an anti-4-1BB antibody. The 4-1BB-binding domain may comprise, for example, the amino acid sequence derived from an anti-4-1BB antibody, (i) the amino acid sequence of a variable region of an anti-4-1BB antibody, (ii) an amino acid sequence showing 90% or more homology to said amino acid sequence, or (iii) an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions to said amino acid sequence. The 4-1BB-binding domain comprises a 4-1BB-binding domain capable of activating immune cells (e.g., T cells). The above-mentioned anti-4-1BB antibody may be an agonist antibody. The anti-4-1BB antibody may be, for example, an anti-4-1BB antibody described in the literature or a commercially available anti-4-1BB antibody (e.g., Bartkowiak et al., Front Oncol. 2015 Jun 8;5:117 (e.g., α4-1BB (PF-05082566) or Urelumab), WO / 2000 / 029445 (e.g., Hz4B4-1), or Shen et al., Am J Cancer Res. 2023 Jul 15;13(7):3246-3256. eCollection 2023, etc.).

[0036] In one embodiment of the present invention, the GITR includes a protein also known as CD357. Details of GITR, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number of GITR listed in UniProt is, for example, Q9Y5U5. GITR includes, for example, GITR derived from human or monkey.

[0037] In one embodiment of the present invention, the GITR-binding domain comprises, for example, a polypeptide having binding ability to GITR. The GITR-binding domain may comprise, for example, an amino acid sequence in a ligand for GITR or an anti-GITR antibody. The GITR-binding domain may specifically comprise, for example, a region or domain having GITR-binding ability, or a region determining GITR-binding ability, in a ligand for GITR or an anti-GITR antibody. The region having GITR-binding ability in a ligand for GITR may be, for example, an extracellular domain. The ligand for GITR may comprise, for example, GITRL (Q9UNG2, UniProt Primary accession number). The GITR-binding domain may comprise, for example, an amino acid sequence derived from a ligand for GITR, such as (i) the amino acid sequence of a ligand for GITR or its extracellular domain, (ii) an amino acid sequence having 90% or more homology to the amino acid sequence, or (iii) an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions to the amino acid sequence. The region determining GITR-binding ability in an anti-GITR antibody may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3. The region having GITR-binding ability in an anti-GITR antibody may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The GITR-binding domain may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3 as an amino acid sequence derived from an anti-GITR antibody. The GITR-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as an amino acid sequence derived from an anti-GITR antibody. The GITR-binding domain may comprise, for example, the amino acid sequence derived from an anti-GITR antibody: (i) the amino acid sequence of the variable region of an anti-GITR antibody; (ii) an amino acid sequence having 90% or more homology to the amino acid sequence; or (iii) an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence. The GITR binding domain comprises a GITR binding domain capable of activating immune cells (eg, T cells).The anti-GITR antibody may be an agonist antibody. For example, the anti-GITR antibody may be an anti-GITR antibody described in the literature or commercially available (e.g., Knee et al., Eur J Cancer. 2016 Nov;67:1-10. or Schaer et al., Curr Opin Investig Drugs.).

[0038] In one embodiment of the present invention, the CD27 includes a protein also known as TNFRSF7. Details of the amino acid sequence of CD27 can be found on websites such as NCBI or UniProt. The primary accession number for CD27 listed in UniProt is, for example, P26842. CD27 includes, for example, human- or monkey-derived CD27.

[0039] In one embodiment of the present invention, the CD27-binding domain comprises, for example, a polypeptide having binding affinity to CD27. The CD27-binding domain may comprise, for example, an amino acid sequence found in a CD27 ligand or an anti-CD27 antibody. The CD27-binding domain may specifically comprise, for example, a region or domain having binding affinity to CD27, or a region determining binding affinity to CD27, within a CD27 ligand or anti-CD27 antibody. The region having binding affinity to CD27 within a CD27 ligand may be, for example, the extracellular domain. The CD27 ligand may comprise, for example, CD27L (P32970, UniProt Primary accession number). The CD27-binding domain may comprise, for example, an amino acid sequence derived from a CD27 ligand, such as (i) the amino acid sequence of a CD27 ligand or its extracellular domain, (ii) an amino acid sequence having 90% or more homology to the amino acid sequence, or (iii) an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence. The region in an anti-CD27 antibody that determines CD27-binding ability may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3. The region in an anti-CD27 antibody that has CD27-binding ability may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). A CD27-binding domain may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3 as an amino acid sequence derived from an anti-CD27 antibody. A CD27-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as an amino acid sequence derived from an anti-CD27 antibody. A CD27-binding domain may comprise, for example, the amino acid sequence derived from an anti-CD27 antibody: (i) the amino acid sequence of a variable region of an anti-CD27 antibody; (ii) an amino acid sequence that is 90% or more identical to that amino acid sequence; or (iii) an amino acid sequence that has one to several amino acid deletions, substitutions, insertions, or additions relative to that amino acid sequence. The CD27 binding domain comprises a CD27 binding domain capable of activating immune cells (e.g., T cells).The anti-GITR antibody may be an agonist antibody. The anti-CD27 antibody may be, for example, a literature-described or commercially available anti-CD27 antibody (e.g., Sanmamed et al., Semin Oncol. 2015 Aug;42(4):640-55, or Vogel et al., Eur J Immunol. 2022 Oct;52(10):1620-1629, etc.).

[0040] In one embodiment of the present invention, the CD40 includes a protein also known as TNFRSF5. Details of CD40, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for CD40 listed in UniProt is, for example, P25942. The CD40 includes, for example, human- or monkey-derived CD40.

[0041] In one embodiment of the present invention, the CD40-binding domain comprises, for example, a polypeptide having binding affinity to CD40. The CD40-binding domain may comprise, for example, an amino acid sequence within a CD40 ligand or an anti-CD40 antibody. The CD40-binding domain may specifically comprise, for example, a region or domain within a CD40 ligand or anti-CD40 antibody that has binding affinity to CD40, or a region that determines binding affinity to CD40. The region within a CD40 ligand that has binding affinity to CD40 may be, for example, an extracellular domain. The CD40 ligand may comprise, for example, CD40L (P29965, UniProt Primary accession number). The CD40-binding domain may comprise, for example, an amino acid sequence derived from a CD40 ligand, such as (i) the amino acid sequence of a CD40 ligand or its extracellular domain, (ii) an amino acid sequence that is 90% or more identical to the amino acid sequence, or (iii) an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence. The region in an anti-CD40 antibody that determines CD40-binding ability may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3. The region in an anti-CD40 antibody that has CD40-binding ability may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). A CD40-binding domain may comprise, for example, the amino acid sequence of heavy chain CDR1-3 and light chain CDR1-3 as an amino acid sequence derived from an anti-CD40 antibody. A CD40-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as an amino acid sequence derived from an anti-CD40 antibody. A CD40-binding domain may comprise, for example, the amino acid sequence derived from an anti-CD40 antibody: (i) the amino acid sequence of a variable region of an anti-CD40 antibody; (ii) an amino acid sequence that is 90% or more homologous to that amino acid sequence; or (iii) an amino acid sequence that has one to several amino acid deletions, substitutions, insertions, or additions relative to that amino acid sequence. The CD40 binding domain comprises a CD40 binding domain capable of activating immune cells (e.g., T cells).The anti-GITR antibody may be an agonist antibody. The anti-CD40 antibody may be, for example, a literature-described or commercially available anti-CD40 antibody (e.g., Lang et al., Int J Mol Sci. 2022 Oct 25;23(21):12869, or Hassan et al., Immunopharmacol Immunotoxicol. 2014 Apr;36(2):96-104, etc.).

[0042] In one embodiment of the present invention, the NKp30 includes a protein also known as CD337. Details of the amino acid sequence of NKp30 can be found on websites such as NCBI or UniProt. The primary accession number of NKp30 listed in UniProt is, for example, O14931. NKp30 includes, for example, NKp30 derived from human or monkey.

[0043] In one embodiment of the present invention, the NKp30-binding domain comprises, for example, a polypeptide having binding affinity to NKp30. The NKp30-binding domain may comprise, for example, an amino acid sequence in a ligand for NKp30 or an anti-NKp30 antibody. The NKp30-binding domain may specifically comprise, for example, a region or domain in a ligand for NKp30 or an anti-NKp30 antibody that has binding affinity to NKp30, or a region that determines binding to NKp30. The region in a ligand for NKp30 that has binding affinity to NKp30 may be, for example, an extracellular domain. The ligand for NKp30 may comprise, for example, B7-H6 (Q68D85, UniProt Primary accession number). The NKp30-binding domain may comprise, for example, an amino acid sequence derived from a ligand for NKp30, such as (i) the amino acid sequence of a ligand for NKp30 or its extracellular domain, (ii) an amino acid sequence that is 90% or more homologous to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The region in an anti-NKp30 antibody that determines the binding ability to NKp30 may comprise, for example, the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3. The region in an anti-NKp30 antibody that has binding ability to NKp30 may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region). The NKp30-binding domain may comprise, for example, the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 as amino acid sequences derived from an anti-NKp30 antibody. The NKp30-binding domain may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region; the same applies below) derived from an anti-NKp30 antibody. The NKp30-binding domain may comprise, for example, the amino acid sequence derived from an anti-NKp30 antibody, (i) the amino acid sequence of a variable region of an anti-NKp30 antibody, (ii) an amino acid sequence that is 90% or more homologous to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence.The NKp30-binding domain comprises an NKp30-binding domain capable of activating immune cells (e.g., T cells). The anti-GITR antibody may be an agonist antibody. The anti-NKp30 antibody may be, for example, a literature-described or commercially available anti-NKp30 antibody (e.g., Zou et al., PLoS One. 2015 Aug. 4;10(8):e0134568, or Queiroz et al., BMC Infect Dis. 2019 May 17;19(1):433, etc.).

[0044] In one embodiment of the present invention, the TIM-3 includes a protein also known as CD366. Details of the amino acid sequence of TIM-3 can be found on websites such as NCBI or UniProt. The primary accession number for TIM-3 listed in UniProt is, for example, Q8TDQ0. TIM-3 includes, for example, TIM-3 derived from human or monkey.

[0045] In one embodiment of the present invention, the TIM-3-binding domain comprises, for example, a polypeptide that has binding affinity to TIM-3. The TIM-3-binding domain may comprise, for example, an amino acid sequence found in an anti-TIM-3 antibody. The TIM-3-binding domain may specifically comprise, for example, a region or domain within the anti-TIM-3 antibody that has binding affinity to TIM-3, or a region that determines binding to TIM-3. The region within the anti-TIM-3 antibody that determines binding to TIM-3 may comprise, for example, the amino acid sequence of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3. The region within the anti-TIM-3 antibody that has binding affinity to TIM-3 may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The TIM-3-binding domain may comprise, for example, the amino acid sequence of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 derived from the anti-TIM-3 antibody. The TIM-3-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) derived from an anti-TIM-3 antibody. For example, the TIM-3-binding domain may comprise, as an amino acid sequence derived from an anti-TIM-3 antibody, (i) the amino acid sequence of a variable region of an anti-TIM-3 antibody, (ii) an amino acid sequence that is 90% or more identical to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The TIM-3-binding domain comprises a TIM-3-binding domain capable of activating immune cells (e.g., T cells). The above-mentioned anti-TIM-3 antibody may be an antagonist antibody. The above-mentioned anti-TIM-3 antibody can be, for example, an anti-TIM-3 antibody described in the literature or a commercially available anti-TIM-3 antibody (e.g., Bewersdorf et al., Genes Dev. 2022 Mar 1;36(5-6):259-277. or Bailly, Acta Trop. 2023 Feb;238:106792.).

[0046] In one embodiment of the present invention, the LAG-3 includes a protein also known as CD223. Details of the amino acid sequence of LAG-3 can be found on websites such as NCBI or UniProt. The primary accession number for LAG-3 listed in UniProt is, for example, P18627. LAG-3 includes, for example, LAG-3 derived from human or monkey.

[0047] In one embodiment of the present invention, the LAG-3-binding domain comprises, for example, a polypeptide having binding affinity to LAG-3. The LAG-3-binding domain may comprise, for example, an amino acid sequence found in an anti-LAG-3 antibody. The LAG-3-binding domain may specifically comprise, for example, a region or domain within an anti-LAG-3 antibody that has binding affinity to LAG-3, or a region determining binding affinity to LAG-3. The region determining binding affinity to LAG-3 within an anti-LAG-3 antibody may comprise, for example, the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3. The region within an anti-LAG-3 antibody that has binding affinity to LAG-3 may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The LAG-3-binding domain may comprise, for example, the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 as amino acid sequences derived from an anti-LAG-3 antibody. The LAG-3-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) derived from an anti-LAG-3 antibody. The LAG-3-binding domain may comprise, for example, the amino acid sequence of a variable region of an anti-LAG-3 antibody, (i) an amino acid sequence that is 90% or more identical to that amino acid sequence, or (iii) an amino acid sequence that has one to several amino acid deletions, substitutions, insertions, or additions relative to that amino acid sequence. The LAG-3-binding domain comprises a LAG-3-binding domain capable of activating immune cells (e.g., T cells). The anti-LAG-3 antibody may be an antagonist antibody. The above-mentioned anti-LAG-3 antibody can be, for example, an anti-LAG-3 antibody described in the literature or a commercially available anti-LAG-3 antibody (e.g., Sauer et al., Int J Mol Sci. 2022 Sep 1;23(17):9958, or Kozlowski et al., Biomedicines. 2022 Nov 5;10(11):2826, etc.).

[0048] In one embodiment of the present invention, the BTLA includes a protein also known as CD272. Details of BTLA, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for BTLA listed in UniProt is, for example, Q7Z6A9. BTLA includes, for example, BTLA derived from human or monkey.

[0049] In one embodiment of the present invention, the BTLA-binding domain comprises, for example, a polypeptide that has binding affinity to BTLA. The BTLA-binding domain may comprise, for example, an amino acid sequence found in an anti-BTLA antibody. The BTLA-binding domain may specifically comprise, for example, a region or domain within an anti-BTLA antibody that has binding affinity to BTLA, or a region that determines binding affinity to BTLA. The region within an anti-BTLA antibody that determines binding affinity to BTLA may comprise, for example, the amino acid sequence of heavy chain CDRs 1-3 and light chain CDRs 1-3. The region within an anti-BTLA antibody that has binding affinity to BTLA may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The BTLA-binding domain may comprise, for example, the amino acid sequence of heavy chain CDRs 1-3 and light chain CDRs 1-3 as amino acid sequences derived from an anti-BTLA antibody. The BTLA-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as amino acid sequences derived from an anti-BTLA antibody. The BTLA-binding domain may comprise, for example, an amino acid sequence derived from an anti-BTLA antibody, such as (i) the amino acid sequence of the variable region of an anti-BTLA antibody, (ii) an amino acid sequence that is 90% or more identical to that amino acid sequence, or (iii) an amino acid sequence that has one to several amino acid deletions, substitutions, insertions, or additions relative to that amino acid sequence. The BTLA-binding domain comprises a BTLA-binding domain that has the ability to activate immune cells (e.g., T cells). The above-mentioned anti-BTLA antibody may be an antagonist antibody. The above-mentioned anti-BTLA antibody may be, for example, an anti-BTLA antibody described in the literature or commercially available (e.g., Sordo-Bahamonde et al., Mol Cancer. 2023 Aug 30;22(1):142, or Chen et al., J Immunother Cancer. 2019 Nov 21;7(1):313, etc.).

[0050] In one embodiment of the present invention, the CTLA-4 includes a protein also known as CD152. Details of the amino acid sequence of CTLA-4 can be found on websites such as NCBI or UniProt. The primary accession number for CTLA-4 listed in UniProt is, for example, P16410. CTLA-4 includes, for example, human or monkey-derived CTLA-4.

[0051] In one embodiment of the present invention, the CTLA-4-binding domain comprises, for example, a polypeptide having binding affinity to CTLA-4. The CTLA-4-binding domain may comprise, for example, an amino acid sequence within an anti-CTLA-4 antibody. The CTLA-4-binding domain may specifically comprise, for example, a region or domain within an anti-CTLA-4 antibody having binding affinity to CTLA-4, or a region determining binding affinity to CTLA-4. The region determining binding affinity to CTLA-4 within an anti-CTLA-4 antibody may comprise, for example, the amino acid sequence of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3. The region having binding affinity to CTLA-4 within an anti-CTLA-4 antibody may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region). The CTLA-4-binding domain may comprise, for example, the amino acid sequence of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 as amino acid sequences derived from an anti-CTLA-4 antibody. The CTLA-4-binding domain may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region; the same applies below) derived from an anti-CTLA-4 antibody. The CTLA-4-binding domain may comprise, for example, the amino acid sequence of a variable region of an anti-CTLA-4 antibody, (i) an amino acid sequence that is 90% or more homologous to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The CTLA-4-binding domain comprises a CTLA-4-binding domain capable of activating immune cells (e.g., T cells). The above-mentioned anti-CTLA-4 antibody may be an antagonist antibody. The anti-CTLA-4 antibody may be, for example, a literature-described or commercially available anti-CTLA-4 antibody (e.g., Seidel et al., Front Oncol. 2018 Mar 28;8:86, or Blank et al., Int Immunol. 2015 Jan;27(1):3-10, etc.).

[0052] In one embodiment of the present invention, the CD73 includes a protein also known as Ecto-5'-nucleotidase. Details of CD73, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for CD73 listed in UniProt is, for example, P21589. CD73 includes, for example, human- or monkey-derived CD73.

[0053] In one embodiment of the present invention, the CD73-binding domain comprises, for example, a polypeptide having binding affinity to CD73. The CD73-binding domain may comprise, for example, an amino acid sequence found in an anti-CD73 antibody. The CD73-binding domain may specifically comprise, for example, a region or domain within an anti-CD73 antibody that has binding affinity to CD73, or a region determining binding affinity to CD73. The region determining binding affinity to CD73 within an anti-CD73 antibody may comprise, for example, the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3. The region within an anti-CD73 antibody that has binding affinity to CD73 may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The CD73-binding domain may comprise, for example, the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 as amino acid sequences derived from an anti-CD73 antibody. The CD73-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) derived from an anti-CD73 antibody. The CD73-binding domain may comprise, for example, the amino acid sequence of a variable region of an anti-CD73 antibody, (i) an amino acid sequence that is 90% or more identical to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The CD73-binding domain comprises a CD73-binding domain that has the ability to activate immune cells (e.g., T cells). The above-mentioned anti-CD73 antibody may be an antagonistic antibody or a neutralizing antibody. The anti-CD73 antibody may be, for example, an anti-CD73 antibody described in the literature or a commercially available anti-CD73 antibody (e.g., Kurago et al., Front Immunol. 2023 Jun 26;14:1212209, or Minor et al., Am J Physiol Cell Physiol. 2019 Dec 1;317(6):C1079-C1092, etc.).

[0054] In one embodiment of the present invention, the CD3 comprises a protein complex that noncovalently associates with the T cell receptor (TCR) and functions as a co-receptor for T cells. CD3 comprises gamma, delta, epsilon, and zeta subunits. Details of the amino acid sequence of CD3 can be found on websites such as NCBI or UniProt. The primary accession number for CD3 listed in UniProt is, for example, P07766. The amino acid sequence of human CD3 epsilon (CD3E) comprises, for example, the amino acid sequence set forth in SEQ ID NO: 63. CD3 includes, for example, CD3 derived from a human or monkey.

[0055] In one embodiment of the present invention, the CD3-binding domain comprises, for example, a polypeptide that binds to CD3. The CD3-binding domain may comprise, for example, an amino acid sequence found in an anti-CD3 antibody. The CD3-binding domain may specifically comprise, for example, a region or domain within an anti-CD3 antibody that binds to CD3, or a region that determines binding to CD3. The region within an anti-CD3 antibody that determines binding to CD3 may comprise, for example, the amino acid sequences of heavy chain CDRs 1-3 and light chain CDRs 1-3. The region within an anti-CD3 antibody that binds to CD3 may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region). The CD3-binding domain may comprise, for example, the amino acid sequences of heavy chain CDRs 1-3 and light chain CDRs 1-3 as amino acid sequences derived from an anti-CD3 antibody. The CD3-binding domain may comprise, for example, the amino acid sequence of a variable region (e.g., including a heavy chain variable region or a light chain variable region; the same applies below) as amino acid sequences derived from an anti-CD3 antibody. The CD3-binding domain may comprise, for example, an amino acid sequence derived from an anti-CD3 antibody: (i) the amino acid sequence of the variable region of an anti-CD3 antibody, (ii) an amino acid sequence that is 90% or more identical to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The CD3-binding domain comprises a CD3-binding domain that has the ability to activate immune cells (e.g., T cells). The above-mentioned anti-CD3 antibody may be an agonist antibody. The anti-CD3 antibody may be, for example, a literature-described or commercially available anti-CD3 antibody (e.g., Goebeler et al., Leuk Lymphoma. 2016 May;57(5):1021-32; Sun et al., Sci Transl Med. 2015 May 13;7(287):287ra70; Pessano et al., EMBO J. 1985 Feb;4(2):337-44; JP2018-501297; WO2007042261). The anti-CD3 antibody may be, for example, OKT3, SP34, or an antibody comprising heavy chain CDR1-3 or light chain CDR1-3 thereof.The above-mentioned anti-CD3 antibody may, for example, have heavy chain CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 46 to 48, respectively, and light chain CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively. The above-mentioned anti-CD3 antibody may, for example, have a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 53. The above-mentioned anti-CD3 antibody may, for example, have heavy chain CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 70 to 72, respectively, and light chain CDR1 to CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 73 to 75, respectively. The above-mentioned anti-CD3 antibody may, for example, have a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 76, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 77.

[0056] In one embodiment of the present invention, the TGFβ includes a protein also known as transforming growth factor β. TGFβ includes the isoforms TGFβ-1, TGFβ-2, and TGFβ-3. Details of the amino acid sequence of TGFβ can be found on websites such as NCBI or UniProt. The primary accession numbers for TGFβ listed in UniProt are, for example, P01137, P61812, or P10600 for each isoform. The amino acid sequence of human TGFβ includes, for example, the amino acid sequences shown in SEQ ID NOs: 64 to 66 for each isoform. TGFβ includes, for example, TGFβ derived from human or monkey.

[0057] In one embodiment of the present invention, the TGFβ-binding domain comprises, for example, a polypeptide having binding ability to TGFβ. The TGFβ-binding domain may comprise, for example, an amino acid sequence in a TGFβ receptor or an anti-TGFβ antibody. The TGFβ-binding domain may particularly comprise, for example, a region or domain having binding ability to TGFβ, or a region determining binding ability to TGFβ, in a TGFβ receptor or an anti-TGFβ antibody. The region having binding ability to TGFβ in a TGFβ receptor may be, for example, an extracellular domain (e.g., the amino acid sequence of positions 23-166). The region having binding ability to TGFβ in a TGFβ receptor may comprise, for example, the amino acid sequence shown in SEQ ID NO: 54. The TGFβ receptor may comprise, for example, TGFβR2 (P37173, UniProt Primary accession number). The TGFβ-binding domain may comprise, for example, an amino acid sequence derived from a TGFβ receptor: (i) the amino acid sequence shown in SEQ ID NO: 54, (ii) an amino acid sequence showing 90% or more homology to the amino acid sequence shown in SEQ ID NO: 54, or (iii) an amino acid sequence having deletions, substitutions, insertions, or additions of up to several amino acids relative to the amino acid sequence shown in SEQ ID NO: 54. The amino acid sequence of a TGFβ receptor may comprise, for example, the amino acid sequence shown in SEQ ID NO: 55. The TGFβ-binding domain may comprise, for example, an amino acid sequence derived from a TGFβ receptor: (i) the amino acid sequence shown in SEQ ID NO: 55, (ii) an amino acid sequence showing 90% or more homology to the amino acid sequence shown in SEQ ID NO: 55, or (iii) an amino acid sequence having deletions, substitutions, insertions, or additions of one to several amino acids relative to the amino acid sequence shown in SEQ ID NO: 55. The region determining the binding ability to TGFβ in an anti-TGFβ antibody may comprise, for example, the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3. The region of an anti-TGFβ antibody that has TGFβ-binding ability may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region). The TGFβ-binding domain may comprise, for example, the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 derived from an anti-TGFβ antibody.The TGFβ-binding domain may comprise, for example, the amino acid sequence of a variable region (including, for example, a heavy chain variable region or a light chain variable region; the same applies below) as an amino acid sequence derived from an anti-TGFβ antibody. The TGFβ-binding domain may comprise, for example, as an amino acid sequence derived from an anti-TGFβ antibody, (i) the amino acid sequence of a variable region of an anti-TGFβ antibody, (ii) an amino acid sequence that is 90% or more homologous to that amino acid sequence, or (iii) an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence. The TGFβ-binding domain comprises a TGFβ-binding domain that has the ability to activate immune cells (e.g., T cells). The above-mentioned anti-TGFβ antibody may be an agonist antibody. The above-mentioned anti-TGFβ antibody can be, for example, an anti-TGFβ antibody described in the literature or a commercially available anti-TGFβ antibody (e.g., Yi et al., J Hematol Oncol. 2022 Oct 8;15(1):142. or Niu et al., J Hematol Oncol. 2023 Aug 12;16(1):94., etc.).

[0058] In one embodiment of the present invention, the multispecific antigen-binding molecule may be a humanized multispecific antigen-binding molecule. A humanized multispecific antigen-binding molecule may, for example, have amino acid sequences other than the CDRs derived from human immunoglobulin. A humanized multispecific antigen-binding molecule may, for example, have one or more CDRs derived from a non-human species, framework regions derived from human immunoglobulin, and optionally constant regions derived from human immunoglobulin, and may contain a domain that binds to a desired antigen. Multispecific antigen-binding molecules can be humanized, for example, by the procedure described in Example 3.1 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).

[0059] In one embodiment of the present invention, the multispecific antigen-binding molecule may comprise, for example, an Fc region. The Fc region may be, for example, an IgG, IgA, IgM, IgE, or IgD type Fc region. IgG includes, for example, IgG1, IgG2, IgG3, or IgG4. In one embodiment of the present invention, the Fc region may have, for example, a knob mutation and a hole mutation in separate chains. In one embodiment of the present invention, the Fc region may have, for example, an L234A mutation and an L235A mutation.

[0060] In one embodiment of the present invention, the multispecific antigen-binding molecule may be, for example, a T cell engager. The multispecific antigen-binding molecule may exert an anti-tumor effect, for example, by redirecting T cells.

[0061] In one embodiment of the present invention, the multispecific antigen-binding molecule may have a first arm and a second arm. The first arm may, for example, have the first binding domain. The second arm may, for example, have the second binding domain.

[0062] In one embodiment of the present invention, the multispecific antigen-binding molecule may comprise a first binding domain and a second binding domain, and may further comprise a third binding domain or a fourth binding domain. The first binding domain may be located at the N-terminus of the Fc region of the multispecific antigen-binding molecule. The second binding domain may be located at the N-terminus or C-terminus of the Fc region of the multispecific antigen-binding molecule. The third binding domain and the fourth binding domain may be located at the C-terminus of the Fc region of the multispecific antigen-binding molecule.

[0063] In one embodiment of the present invention, the first binding domain and the second binding domain, or the polypeptide comprising the first binding domain and the polypeptide comprising the second binding domain, may be linked via a linker. In one embodiment of the present invention, the linker is a compound for bridging two or more entities and includes a compound that is not directly involved in binding to an antigen. The linker includes, for example, a peptide linker. The peptide linker may be a GS linker or a G linker. The GS linker includes, for example, a linker composed of glycine (G) and serine (S) residues. The GS linker may include, for example, a GGGGS motif (SEQ ID NO: 67). The GS linker may be, for example, a linker represented by (GGGGS)n (n represents a number, and may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8, or may be equal to or greater than any of these values, or may be within a range between any two of these values). The G linker may be, for example, a linker represented by Gn (n represents a number, e.g., 3, 4, 5, 6, 7, or 8, or may be any one of these values ​​or more, or may be within a range between any two of these values). The number of amino acid residues contained in the peptide linker may be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40, or may be any one of these values ​​or more, or may be within a range between any two of these values. Peptides, constant regions, and Fc regions can also be used as linkers. For example, the multispecific antigen-binding molecule can have a first arm comprising, from the N-terminus to the C-terminus, a first binding domain-peptide linker, and a second arm comprising, from the N-terminus to the C-terminus, a second binding domain-peptide linker-constant region, linked via an Fc region. A peptide linker may be further interposed between any of these.

[0064] In one embodiment of the present invention, the multispecific antigen-binding molecule is, for example, 5.0 x 10 -7 The following K D (M) may bind to human CD39 on the cell membrane. This value is, for example, 5.0 × 10 -7 , 1.0×10 -7 , 5.0×10-8 , 1.0×10 -8 , 5.0×10 -9 , 1.0×10 -9 , 5.0×10 -10 , or 1.0 × 10 -10 , or may be equal to or less than any of these values, or may be within a range of any two of these values. D (M) may be a value calculated from the results obtained by flow cytometry. D (M) may be a value measured by surface plasmon resonance (SPR). In one embodiment of the present invention, the multispecific antigen-binding molecule is, for example, 1.0 × 10 -8 The following K D (M) may bind to human CD39 isolated at 1.0×10 -8 , 5.0×10 -9 , 1.0×10 -9 , 5.0×10 -10 , or 1.0 × 10 -10 , or may be equal to or less than any of these values, or may be within a range between any two of these values.

[0065] In one embodiment of the present invention, the multispecific antigen-binding molecule is, for example, 5.0 x 10 -6 The following K D The antibody may bind to an antigen other than human CD39 isolated in (M) (e.g., an immune cell surface antigen or an anti-inflammatory cytokine). This value is, for example, 5.0 × 10 -6 , 1.0×10 -6 , 5.0×10 -7 , 1.0×10 -7 , 5.0×10 -8 , 1.0×10 -8 , 5.0×10 -9 , or 1.0 × 10 -9 , or may be equal to or less than any of these values, or may be within a range between any two of these values.

[0066] In one embodiment of the present invention, the multispecific antigen-binding molecule comprises, for example, a form of a multispecific monoclonal antibody.

[0067] In one embodiment of the present invention, the multispecific antigen-binding molecule may be a multispecific antigen-binding molecule that has binding affinity to wild-type or mutant forms of CD39. Mutant forms of CD39 include those resulting from differences in DNA sequences between individuals, such as SNPs.

[0068] In one embodiment of the present invention, the multispecific antigen-binding molecule may be in an IgG format, an IgG-like format (e.g., a format with an Fc region), or a fragment-based format (an Fc region-free format) (see, for example, Elshiaty et al., Int J Mol Sci. 2021 May 26;22(11):5632; Nie et al., Antib Ther. 2020 Jan;3(1):18-62; Kang et al., Front Immunol. 2022 Oct 20;13:1020003; or Brinkmann et al., MAbs. 2017 Feb / Mar;9(2):182-212). The IgG format includes, for example, an IgG-like molecule having two arms and an Fc region, in which the first arm contains a Fab region that recognizes a first antigen and the second arm contains a Fab region that recognizes a second antigen. Note that the first antigen and the second antigen are different types of antigens.The IgG-like format includes, for example, (i) an IgG-like molecule having two arms and an Fc region, in which the first arm contains a polypeptide (e.g., Fab or scFv) comprising a variable region (e.g., heavy chain variable region or light chain variable region) that recognizes a first antigen, and the second arm contains a polypeptide (e.g., Fab or scFv) comprising a variable region (e.g., heavy chain variable region or light chain variable region) that recognizes a second antigen, and when one arm is Fab, the other arm is a format other than Fab; (ii) an IgG-like molecule having an Fc region, in which the Fc region is bound to a bispecific diabody, scDb (single-chain diabody), or tandem diabody. (iii) a format in which an IgG-like molecule having two arms and an Fc region comprises a polypeptide (e.g., Fab or scFv) comprising a variable region (e.g., a heavy chain variable region or a light chain variable region) that recognizes a first antigen, and the second arm comprises a polypeptide comprising a ligand or receptor for a second antigen or an antigen-binding fragment thereof; or (iv) a format in which one or more antigen-binding domains are further linked to the IgG-like formats of (i) to (iii) above. Fragment-based formats include, for example, (i) a format that does not have an Fc region and includes a polypeptide comprising a variable region (e.g., a heavy chain variable region or a light chain variable region) that recognizes a first antigen and a polypeptide comprising a variable region (e.g., a heavy chain variable region or a light chain variable region) that recognizes a second antigen, where these polypeptides are linked by a linker or are conjugated together; or (ii) a format that does not have an Fc region and includes a polypeptide comprising a variable region (e.g., a heavy chain variable region or a light chain variable region) that recognizes a first antigen and a polypeptide comprising a ligand or receptor for a second antigen or an antigen-binding fragment thereof, where these polypeptides are linked by a linker or are conjugated together.Multispecific antigen-binding molecules may be in the format of scFv-Lig-Fc, Fab-Lig-Fc, scFv-Fab-Fc, scFv-scFv-Fc, Fab-Fab-Fc, diabody-Lig-Fc, diabody-Fab-Fc, or diabody-diabody-Fc (indicating an intervening sequence between the first arm, second arm, and arms). Here, Lig is a polypeptide comprising a ligand or receptor for an antigen or an antigen-binding fragment thereof. Multispecific antigen-binding molecules may also be in the format of, for example, a bispecific diabody, a single-chain diabody (scDb), or a tandem scFv. A multispecific antigen-binding molecule may be in a format that comprises, in order from N-terminus to C-terminus, a first binding domain, an optional linker, and a first chain constituting an Fc region; and, in order from N-terminus to C-terminus, a second binding domain, an optional linker, a constant region, and a second chain constituting an Fc region, with the first and second chains linked by a disulfide bond. The constant region may be, for example, a complex (e.g., a first constant region (e.g., a heavy chain constant region (e.g., CH1)) with a second constant region (e.g., a light chain constant region (e.g., Cκ)) linked by a disulfide bond. The second binding domain may, for example, form a trimer (first to third chains), in which the first and second chains are linked by a linker, and one of the first and second chains is linked to the first constant region via a linker. The third chain may, for example, be linked to the second constant region via a linker. Multispecific antigen-binding molecules can be produced, for example, using the method described in Wang et al., Antibodies (Basel). 2019 Aug 2;8(3):43.

[0069] In one embodiment of the present invention, the multispecific antigen-binding molecule may, if specified, not include a molecule that targets or has binding activity to TIM-3 (T-cell immunoglobulin and mucin domain 3). In one embodiment of the present invention, the multispecific antigen-binding molecule may, if specified, not include a molecule that inhibits the function of TIM-3. In one embodiment of the present invention, the immune cell surface antigen may, if specified, not include TIM-3. The origin of TIM-3 is not limited, and includes, for example, TIM-3 derived from mammals (e.g., humans or monkeys).

[0070] From another aspect, according to one embodiment of the present invention, there is provided a multispecific polypeptide comprising a CD39-binding domain having binding ability to CD39, wherein the CD39-binding domain comprises any one of the CDR sets (a) to (d) above. From another aspect, according to one embodiment of the present invention, there is provided a multispecific complex comprising a CD39-binding domain having binding ability to CD39, wherein the CD39-binding domain comprises any one of the CDR sets (a) to (d) above. Any of the embodiments described above for the multispecific antigen-binding molecule can be applied to this multispecific complex.

[0071] From another perspective, one embodiment of the present invention provides an antigen-binding molecule having multiple antigen-binding domains, one of which is a CD39-binding domain that has binding affinity to CD39, and the CD39-binding domain has any one of the CDR sets (a) to (d) above. Any of the embodiments described above for the multispecific antigen-binding molecule can be applied to this antigen-binding molecule.

[0072] From another perspective, one embodiment of the present invention provides a polypeptide comprising any one of the CDR sets (a) to (d) above. This polypeptide may be, for example, a multispecific antigen-binding molecule. This polypeptide may be, for example, a multispecific antibody or a multispecific fusion protein. This polypeptide may be, for example, an antibody. This polypeptide may be, for example, an scFv or Fab. Any of the embodiments described above for the multispecific antigen-binding molecule may be applied to this polypeptide.

[0073] From another perspective, one embodiment of the present invention provides a polypeptide or complex comprising a first binding domain and a second binding domain. The first binding domain comprises, for example, a domain comprising any of the CDR sets (a) to (d) above. Any of the embodiments described above for the multispecific antigen-binding molecule can be applied to this polypeptide or complex.

[0074] From another perspective, one embodiment of the present invention provides a multispecific antigen-binding molecule comprising a first polypeptide and a second polypeptide. The first polypeptide includes, for example, a polypeptide comprising any one of the CDR sets (a) to (d) above. A linker may be present between the first binding polypeptide and the second binding polypeptide. The first binding polypeptide and the second binding polypeptide may be linked via an Fc region. The first binding polypeptide and the second binding polypeptide may be linked via a disulfide bond. Any of the embodiments described above for the multispecific antigen-binding molecule can be applied to this multispecific antigen-binding molecule.

[0075] From another perspective, one embodiment of the present invention provides a multispecific antigen-binding molecule comprising an anti-CD39 antibody comprising any one of the CDR sets (a) to (d) above. Any of the embodiments described above for the multispecific antigen-binding molecule can be applied to this multispecific antigen-binding molecule.

[0076] From another perspective, one embodiment of the present invention provides an antigen-binding fragment of the multispecific antigen-binding molecule. This antigen-binding fragment may have binding ability to CD39 and binding ability to an immune cell surface antigen or an anti-inflammatory cytokine.

[0077] From another perspective, one embodiment of the present invention provides a domain comprising any one of the CDR sets (a) to (d) above. This domain may be a CD39-binding domain that has the ability to bind to CD39. This CD39-binding domain may be used as a component of or incorporated into a multispecific antigen-binding molecule. Any of the embodiments described above regarding the CD39-binding domain may be applied to this domain.

[0078] From another perspective, one embodiment of the present invention provides a multispecific antigen-binding molecule that competes for binding to CD39 with a multispecific antigen-binding molecule (hereinafter, also referred to as a "reference molecule") comprising a CD39-binding domain according to an embodiment of the present invention (e.g., a CD39-binding domain having any of the CDR sets (a) to (d) above). Competition involves the inhibition of binding of another multispecific antigen-binding molecule to the same antigen by binding to the antigen. Competitive multispecific antigen-binding molecules can be identified, for example, by competitive binding assays. Competitive binding assays include 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, coating an antigen onto a microplate, adding a test polyspecific antigen-binding molecule (hereinafter sometimes referred to as the test molecule) and incubating to allow binding between the antigen and the test molecule, adding a labeled reference molecule to the well, incubating and washing, or quantifying the amount of labeled reference molecule bound to the antigen. In this case, for example, the amount of bound 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 percentage of decrease in the amount of bound labeled reference molecule compared to the case where the test molecule is not added or a negative control is added may be evaluated as the inhibition rate. Inhibition of the competing antibody against the reference molecule 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 multispecific antigen-binding molecule may have binding affinity for the epitope to which the reference molecule binds.

[0079] From another perspective, one embodiment of the present invention provides a polypeptide comprising any one of the CDR sets (a) to (d) above. The polypeptide may be a CD39-binding domain, a multispecific antigen-binding molecule, a variable region, or an antibody.

[0080] From another aspect, according to one embodiment of the present invention, there is provided a composition used for delivering immune cells to the vicinity of malignant tumor cells, the composition comprising a polypeptide comprising any one of the CDR sets (a) to (d) above. From another aspect, according to one embodiment of the present invention, there is provided a method for delivering immune cells to the vicinity of malignant tumor cells, the method comprising contacting malignant tumor cells and immune cells with a polypeptide comprising any one of the CDR sets (a) to (d) above.

[0081] From another aspect, according to one embodiment of the present invention, there is provided a composition used for crosslinking malignant tumor cells and immune cells, comprising a polypeptide comprising any one of the CDR sets (a) to (d) above. From another aspect, according to one embodiment of the present invention, there is provided a method for crosslinking malignant tumor cells and immune cells, comprising contacting a polypeptide comprising any one of the CDR sets (a) to (d) above with the malignant tumor cells and immune cells.

[0082] From another aspect, according to one embodiment of the present invention, there is provided a composition comprising multispecific antigen-binding molecules, wherein 90 or 95% or more of the multispecific antigen-binding molecules in the composition have the same amino acid sequence as the above-mentioned multispecific antigen-binding molecules (e.g., having any of the CDR sets (a) to (d) above). From another aspect, according to one embodiment of the present invention, there is provided a composition comprising multispecific antigen-binding molecules, wherein 90 or 95% or more of the molecules comprising antigen-binding domains in the composition have the same amino acid sequence as the above-mentioned multispecific antigen-binding molecules (e.g., having any of the CDR sets (a) to (d) above).

[0083] From another perspective, one embodiment of the present invention provides a method for enhancing the ability of a composition to suppress the proliferation of malignant tumor cells, comprising the step of increasing the proportion of the above-mentioned CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) above) in the composition.

[0084] From another perspective, one embodiment of the present invention provides a CD39-binding domain, multispecific antigen-binding molecule, or anti-CD39 antibody having the amino acid sequences of heavy and light chain CDRs 1 to 3 or variable regions defined by the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) within the amino acid sequence of P4, T86, I67, Tre291, P4_sc, T86_sc, I67_sc, Tre291_sc, HuP4_IB, or HuT86_IF. In one embodiment of the present invention, the CDRs are defined by the Kabat definition unless otherwise specified.

[0085] From another perspective, one embodiment of the present invention provides a polypeptide comprising a CD39-binding domain and an immune cell surface antigen-binding domain. This polypeptide may be, for example, a multispecific antigen-binding molecule. Possible embodiments of this multispecific antigen-binding molecule are as described above. For example, the CD39-binding domain may comprise an amino acid sequence within an anti-CD39 antibody (e.g., the amino acid sequence of a CDR or variable region), and the immune cell surface antigen-binding domain may comprise an amino acid sequence within a ligand for an immune cell surface antigen or an amino acid sequence within an antibody for an immune cell surface antigen (e.g., the amino acid sequence of a CDR or variable region).

[0086] From another perspective, one embodiment of the present invention provides a polypeptide comprising a CD39-binding domain and a 4-1BB-binding domain. This polypeptide may be, for example, a multispecific antigen-binding molecule. Possible embodiments of this multispecific antigen-binding molecule are as described above. For example, the CD39-binding domain may comprise an amino acid sequence within an anti-CD39 antibody (e.g., the amino acid sequence of a CDR or variable region), and the 4-1BB-binding domain may comprise an amino acid sequence within a ligand for 4-1BB or an amino acid sequence within an anti-4-1BB antibody (e.g., the amino acid sequence of a CDR or variable region).

[0087] From another perspective, one embodiment of the present invention provides a polypeptide comprising a CD39-binding domain and a CD3-binding domain. This polypeptide may be, for example, a multispecific antigen-binding molecule. Possible embodiments of this multispecific antigen-binding molecule are as described above. For example, the CD39-binding domain may comprise an amino acid sequence within an anti-CD39 antibody (e.g., the amino acid sequence of a CDR or variable region), and the CD3-binding domain may comprise an amino acid sequence within a ligand for CD3 or an amino acid sequence within an anti-CD3 antibody (e.g., the amino acid sequence of a CDR or variable region).

[0088] 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.).

[0089] In one embodiment of the present invention, a bispecific antibody includes an antibody that has binding affinity to two different types of antigens. The bispecific antibody may have a CD39-binding domain as a first binding domain. The bispecific antibody may have an immune cell surface antigen-binding domain or an anti-inflammatory cytokine-binding domain as a second binding domain. The bispecific antibody may have the first binding domain and the second binding domain present in a single molecule or in a complexed form. In this case, an intervening sequence (e.g., a linker, an Fc region, or a constant region) may be present between the first binding domain and the second binding domain. The bispecific antibody includes an antibody in which the two binding domains have antibody-derived sequences (e.g., CDRs, variable regions). The bispecific antibody may form any of the formats of the multispecific antigen-binding molecules described above.

[0090] In one embodiment of the present invention, the bispecific antigen-binding fusion protein includes a fusion protein capable of binding to two different antigens. The bispecific antigen-binding fusion protein may have a CD39-binding domain as the first binding domain. The bispecific antigen-binding fusion protein may have an immune cell surface antigen-binding domain or an anti-inflammatory cytokine-binding domain as the second binding domain. The bispecific antigen-binding fusion protein may have the first binding domain and the second binding domain present in a single molecule or in a complexed form. In this case, an intervening sequence (e.g., a linker, Fc region, or constant region) may be present between the first binding domain and the second binding domain. The bispecific antigen-binding fusion protein includes a fusion protein in which at least one domain (e.g., the second binding domain) does not have an antibody-derived sequence (e.g., a CDR or variable region). The bispecific antigen-binding fusion protein may have at least one domain (e.g., the second binding domain) comprising a ligand or receptor for an antigen, or an antigen-binding fragment thereof. The bispecific antigen-binding fusion protein may form any of the formats of the multispecific antigen-binding molecules described above.

[0091] In one embodiment of the present invention, an antibody includes a molecule or population thereof capable of specifically binding to a specific epitope on an antigen. The antibody may be a monoclonal antibody. Antibodies can exist in various forms, including one or more forms selected from the group consisting of 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. Antibodies also include modified and unmodified antibodies. Modified antibodies may be bound to various molecules, such as polyethylene glycol. Modified antibodies can be obtained by chemically modifying antibodies using known techniques. Antibodies may also be in the form of fusion proteins. Fusion proteins may be antibodies in which a polypeptide or oligopeptide (e.g., a His tag) is bound to the N- or C-terminus of the antibody. The fusion protein may also be in the form of one antibody arm replaced with a polypeptide other than Fab (e.g., a ligand or receptor). The antibody may also be a conjugated antibody (e.g., an antibody-drug complex). 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 antibody form. The amino acid sequence, class, or subclass of the antibody may be derived from, for example, a human or a non-human mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.). The antibody may also be, for example, an isolated antibody, purified antibody, or recombinant antibody. The antibody may also be used, for example, in vitro or in vivo.

[0092] In one embodiment of the present invention, the monoclonal antibody includes antibodies in which the individual antibodies constituting the population react with substantially the same epitope. In the case of bispecific monoclonal antibodies, one antibody may react with two epitopes. Alternatively, the individual antibodies constituting the population may be substantially identical (naturally occurring mutations are allowed). The method for producing monoclonal antibodies is not particularly limited, and they may be produced, for example, 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 phage antibody libraries using techniques similar to those 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, monoclonal antibodies may be produced by the method described in "Protein Experiment Handbook, Yodosha (2003):92-96."

[0093] In one embodiment of the present invention, the chimeric antibody can be constructed by genetic recombination techniques, for example, by linking the variable region and constant region of an antibody from 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 mouse-human chimeric antibodies 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 a mammalian cell expression vector. 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 cell 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.

[0094] In one embodiment of the present invention, the 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. Various other techniques known in the art may also be used (e.g., Safdari et al., Biotechnol Genet Eng Rev. 2013;29:175-86).

[0095] In one embodiment of the present invention, the scFv (single chain Fv) comprises an antibody in a form in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a suitable peptide linker. For example, scFv can be produced by obtaining cDNA encoding VH and VL (e.g., VH and VL comprising any of the CDR sets (a) to (d) above), constructing a polynucleotide encoding VH-peptide linker-VL, inserting the polynucleotide into a vector, and using an expression cell.

[0096] In one embodiment of the present invention, the Fab comprises an antibody in which the heavy chain and light chain, excluding the Fc region, are linked via a disulfide bond. Fab can be produced, for example, using a vector encoding the VH-CH1 region and the light chain.

[0097] In one embodiment of the present invention, the diabody comprises an antibody in the form of dimerized scFvs and possessing 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 such that the amino acid sequence of the peptide linker is 8 to 4 residues (preferably 5 residues) in length, incorporating the resulting polynucleotide into a vector, and using an expression cell. Bispecific diabodies can be produced by combining the VH and VL DNA of two different scFvs and expressing the scFv.

[0098] In one embodiment of the present invention, the scFvFc includes an antibody in a form in which an scFv and an Fc region are linked via a suitable linker. For example, the scFvFc can be produced by inserting a polynucleotide encoding the scFv, linker, and Fc region into a vector and using an expression cell. Alternatively, the scFvFc can be produced, for example, by the procedure described in Section 1.2 below.

[0099] In one embodiment of the present invention, the heavy chain variable region may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 25, 27, 29, 31, 33, or 35, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 26, 28, 30, 32, 34, or 36. In one embodiment of the present invention, the heavy chain variable region and the light chain variable region may, for example, comprise an amino acid sequence having 90% or more homology to the amino acid sequences shown in SEQ ID NOs: 25 and 26, respectively, or may comprise an amino acid sequence having a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 25 and 26, respectively, or may comprise an amino acid sequence having 90% or more homology to the amino acid sequences shown in SEQ ID NOs: 27 and 28, respectively, or may comprise an amino acid sequence having a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 27 and 28, respectively, or may comprise an amino acid sequence having 90% or more homology to the amino acid sequences shown in SEQ ID NOs: 29 and 30, respectively, or may comprise an amino acid sequence having a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 29 and 30, respectively. The amino acid sequences shown in SEQ ID NOs: 31 and 32 may each contain an amino acid sequence that is 90% or more identical to the amino acid sequences shown in SEQ ID NOs: 31 and 32, respectively, or may each contain an amino acid sequence that has a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 31 and 32, respectively, or may each contain an amino acid sequence that is 90% or more identical to the amino acid sequences shown in SEQ ID NOs: 33 and 34, respectively, or may each contain an amino acid sequence that has a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 33 and 34, respectively, or may each contain an amino acid sequence that is 90% or more identical to the amino acid sequences shown in SEQ ID NOs: 35 and 36, respectively, or may each contain an amino acid sequence that has a deletion, substitution, insertion or addition of up to several amino acids relative to the amino acid sequences shown in SEQ ID NOs: 35 and 36, respectively.

[0100] In one embodiment of the present invention, the heavy chain variable region may have, for example, FR1 to FR4 comprising the amino acid sequence of FR1 to FR4 in the amino acid sequence shown in SEQ ID NO: 25, 27, 29, 31, 33, or 35, an amino acid sequence that is 90% or more identical to that amino acid sequence, or an amino acid sequence in which a deletion, substitution, insertion, or addition of up to several amino acids is present in that amino acid sequence, and CDR1 to CDR3 comprising the amino acid sequence identical to the amino acid sequence of CDR1 to CDR3 in the amino acid sequence shown in SEQ ID NO: 25, 27, 29, 31, 33, or 35. In one embodiment of the present invention, the light chain variable region may have, for example, FR1 to FR4 having the amino acid sequence of FR1 to FR4 in the amino acid sequence shown in SEQ ID NO: 26, 28, 30, 32, 34, or 36, an amino acid sequence that is 90% or more identical to that amino acid sequence, or an amino acid sequence in which a deletion, substitution, insertion, or addition of up to several amino acids is present in that amino acid sequence, and CDR1 to CDR3 having the same amino acid sequence as the amino acid sequence of CDR1 to CDR3 in the amino acid sequence shown in SEQ ID NO: 26, 28, 30, 32, 34, or 36.

[0101] In one embodiment of the present invention, the Fc region may comprise, for example, the amino acid sequence shown in SEQ ID NO: 37, 38, or 45, an amino acid sequence that is 90% or more homologous to that amino acid sequence, or an amino acid sequence in which a few or fewer amino acids have been deleted, substituted, inserted, or added relative to that amino acid sequence.

[0102] In one embodiment of the present invention, the first constant region may comprise, for example, the amino acid sequence shown in SEQ ID NO: 43, an amino acid sequence having 90% or more homology to that amino acid sequence, or an amino acid sequence having deletions, substitutions, insertions, or additions of up to several amino acids relative to that amino acid sequence. In one embodiment of the present invention, the second constant region may comprise the amino acid sequence shown in SEQ ID NO: 41, an amino acid sequence having 90% or more homology to that amino acid sequence, or an amino acid sequence having deletions, substitutions, insertions, or additions of up to several amino acids relative to that amino acid sequence.

[0103] In one embodiment of the present invention, the homology (%) may be calculated, for example, by calculating the percentage of homologous amino acids between multiple amino acid sequences according to methods known in the art. Before calculating the percentage, the sequences to be compared are aligned, and gaps are introduced into the sequences, if necessary, to maximize the percentage of homologous / identical amino acids. Alignment methods, percentage calculation methods, and related computer programs are well known in the art (e.g., BLAST, GENETYX, etc.). As used herein, homology includes identity, and the percentage of homology can be replaced with the percentage of identity. Homology may also be expressed as (number of homologous residues / number of residues in the original amino acid sequence) x 100. More preferably, the homology may be expressed as a value measured using the default settings of BLASTP (The BLAST Sequence Analysis Tool. The NCBI Handbook, 2nd edition. March 15, 2013., Altschul et al., Nucleic Acids Res0. 1997 Sep 1;25(17):3389-402).

[0104] In one embodiment of the present invention, the above-mentioned "90% or more" may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be equal to or greater than any of these values, or may be within a range between any two of these values. From the viewpoint of further maintaining functional equivalence, this percentage is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more. In one embodiment of the present invention, the above-mentioned "several or fewer" may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, 1, or 0, or may be equal to or less than any of these values, or may be within a range between any two of these values. From the viewpoint of further maintaining functional equivalence, this number is preferably 15 or less, more preferably 10 or less, and particularly preferably 5 or less.

[0105] In one embodiment of the present invention, when substitutions are made to the original sequence, conservative amino acid substitutions (see, e.g., French et al., J. Mol. Evol. (1983) 19, 171-175) are preferred. In one embodiment of the present invention, conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0106] From another perspective, one embodiment of the present invention provides a polynucleotide or vector encoding the CD39-binding domain or multispecific antigen-binding molecule (e.g., having any one of the CDR sets (a) to (d) 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., a monkey). 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.

[0107] 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.

[0108] 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.

[0109] From another perspective, one embodiment of the present invention provides a method for producing a CD39-binding domain or a multispecific antigen-binding molecule, comprising the step of growing cells containing the polynucleotide or vector according to any 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 CD39-binding domain or multispecific antigen-binding molecule. 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 CD39-binding domain or multispecific antigen-binding molecule.

[0110] In one embodiment of the present invention, the CD39-binding domain or multispecific antigen-binding molecule can be purified using, for example, ammonium sulfate precipitation, 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).

[0111] From another perspective, one embodiment of the present invention provides a composition comprising the CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) above). This composition can produce an excellent anti-tumor effect. The components contained in this composition are not particularly limited, and it may contain, for example, a buffer. 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 includes compositions for suppressing the growth of malignant tumor cells, activating immune cells, promoting cytokine production by immune cells, or promoting the release of cytolytic granules. Examples of cytokines include IL-2, TNF-α, or IFN-γ. Examples of cytolytic granules include Granzyme B or Perforin. From another perspective, one embodiment of the present invention provides a method for inhibiting the proliferation of malignant tumor cells, a method for activating immune cells, a method for promoting cytokine production by immune cells, or a method for promoting cytolytic granule release, which comprises the step of contacting malignant tumor cells (e.g., CD39-expressing malignant tumor cells) with the above-described CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) above). The occurrence of immune cell activation may be evaluated, for example, by an increase in the cytotoxicity of immune cells against cancer cells, an increase in cytokine secretion from immune cells, or enhanced expression of PD1 (programmed cell death-1) in CD4- or CD8-positive T cells. Immune cell activation may be, for example, an increase in the cytotoxicity of immune cells against cancer cells, an increase in cytokine secretion from immune cells, enhanced proliferation of CD4- or CD8-positive T cells, or activation accompanied by enhanced expression of PD1 in CD4- or CD8-positive T cells.

[0112] From another aspect, according to one embodiment of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) 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. Malignant tumors include CD39-positive malignant tumors. From another aspect, according to one embodiment of the present invention, there is provided a method for treating a disease, comprising the step of administering to a patient the above-mentioned CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) above). Treatments include, for example, treatment of malignant tumors and cancer immunotherapy. From another aspect, according to one embodiment of the present invention, there is provided use of the above-mentioned CD39-binding domain or multispecific antigen-binding molecule for producing a pharmaceutical composition. Treatment may be carried out by utilizing the anti-tumor activity resulting from the binding of the multispecific antigen-binding molecule to multiple antigens.

[0113] From another perspective, one embodiment of the present invention provides a kit comprising the above-mentioned CD39-binding domain or multispecific antigen-binding molecule (e.g., having any of the CDR sets (a) to (d) above). Use of this kit can produce an excellent anti-tumor effect. This kit includes kits for treating malignant tumors, suppressing the growth of malignant tumor cells, activating immune cells, promoting cytokine production by immune cells, or promoting the release of cytolytic granules. This kit may also include, for example, an instruction manual, a buffer solution, a container, or packaging.

[0114] 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 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. The dosage, administration interval, administration method, and administration route are not particularly limited and can be appropriately selected 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.

[0115] In one embodiment of the present invention, the subject (including the patient) includes a human or non-human mammal (e.g., one or more of a monkey, a marmoset, or a chimpanzee), and may be a patient diagnosed with or in need of treatment for a malignant tumor.

[0116] The effect of inhibiting the proliferation of malignant tumor cells may be evaluated, for example, by observing the time course of malignant tumor cell proliferation after administration of the pharmaceutical composition. 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 treatment with the pharmaceutical composition. The proliferation rate may be measured, for example, using absorbance as an index, 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 administration of the pharmaceutical composition. In this case, a significant decrease in tumor weight compared to before administration of the pharmaceutical composition 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 index. In this case, a significant decrease in marker amount compared to before administration of the pharmaceutical composition or after administration of a negative control may be considered to be therapeutic. The tumor weight or marker amount after administration of the pharmaceutical composition 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.

[0117] In one embodiment of the present invention, malignant tumors include, for example, tumors that develop as a result of mutation of 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.

[0118] In one embodiment of the present invention, the amino acid sequence homology between two different types of antigens may be, for example, 90, 80, 70, 60, 50, or 40%, or may be less than any of these values ​​or within a range of any two of these values.

[0119] In one embodiment of the present invention, when a "binding domain" immediately follows a "protein name," the binding domain includes a domain that recognizes or has binding ability to that protein. For example, a CD39-binding domain includes a domain that recognizes CD39 or a domain that has binding ability to CD39. To more clearly describe the characteristics of the domain, it can also be expressed as a CD39-binding domain that recognizes CD39.

[0120] In one embodiment of the present invention, "multiplex" includes, for example, double, triple, or quadruple. In one embodiment of the present invention, the terms "cell surface antigen," "cell membrane antigen," and "cell-surface antigen" can be used interchangeably and have substantially the same meaning. In one embodiment of the present invention, the terms "multispecific antigen-binding molecule" and "multispecific molecule" can be used interchangeably and have substantially the same meaning. In one embodiment of the present invention, the terms "first (or second) binding domain" and "first (or second) antigen-binding domain" can be used interchangeably and have substantially the same meaning. In one embodiment of the present invention, the antigen includes, for example, a molecule that can have antigenicity. In one embodiment of the present invention, the antigen-binding molecule includes, for example, a molecule that can bind to at least one antigen. For example, a multispecific antigen-binding molecule specific to CD39 can be considered a type of antigen-binding molecule because it is a molecule that can bind to CD39.

[0121] 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 in the 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.).

[0122] 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.

[0123] 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.

[0124] In this specification, (a), (b), (c) or (d) is synonymous with one or more selected from the group consisting of (a) to (d).

[0125] 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.

[0126] 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.

[0127] The present invention will be further explained below with reference to examples, but is not limited to these.

[0128] Example 1.1: Preparation of Recombinant Human CD39. Recombinant human CD39 was transiently expressed in FS293 cells (Thermo Fisher Scientific) and purified. The details are as follows: The extracellular domain (amino acids 38-478) (SEQ ID NO: 60) of human CD39 (Accession No. NM_001776.6 / NP_001767.3) (SEQ ID NO: 61) was inserted into an expression vector, and FS293 cells were transformed by electroporation using MaxCyte-STX (MaxCyte) and cultured for 4-6 days. Recombinant human CD39 in the culture supernatant was batch-purified using Ni-NTA agarose resin (FujiFilm-Wako) and then purified by size exclusion chromatography.

[0129] Example 1.2: Preparation of Mouse Anti-Human CD39 Antibodies Monoclonal antibodies that specifically bind to human CD39 were prepared from the spleens or lymph nodes of immunized mice using phage display. Details are as follows: Mice (BALB / c or MRL / lpr) were immunized with recombinant human CD39 (in house / Example 1.1) or Expi293 cells transiently expressing human CD39 antigen. After confirming an increase in serum antibody titer, the spleens or lymph nodes were collected. Total RNA was extracted from the spleens or lymph nodes of the immunized mice and converted to cDNA, and the antibody gene sequences (VH and Vκ) were amplified by PCR. VH and Vκ were linked with a linker to form single-chain Fv (scFv), which was then inserted into a pTZ19R-derived phagemid. Escherichia coli (DH12S strain) was transformed with the scFv-inserted phagemid and further infected with helper phage (M13KO7) to generate the scFv phage library (size: approximately 1 × 10 8An scFv antibody phage library (types) was constructed by panning using recombinant human CD39. The phagemids after panning were extracted, and the scFv portion was amplified by PCR and transferred to an scFv secretion expression vector. Since E. coli cells transfected with the scFv secretion expression vector secrete scFv into the culture supernatant upon IPTG induction, the culture supernatant was used to screen scFv clones using CD39 antigen binding and CD39 enzyme inhibitory activity as indicators. The nucleotide sequences of four scFv clones that were positive in the screening were determined. The clone IDs of these four scFvs were P4, T86, I67, or Tre291. The amino acid sequences of the CDRs of these antibodies are shown in Figure 21 (CDRs are based on Kabat numbering). The amino acid sequences of the variable regions of all antibodies used in the examples are shown in Figure 22.

[0130] The four scFvs were linked to a modified antibody Fc region (SEQ ID NO: 37, Fc-LALA, which contains L234A and L235A mutations in the Fc region of human IgG1) via a short linker to form scFv-Fc-LALA antibodies. These antibodies were expressed in ExpiCHO cells (Thermo Fisher Scientific) and affinity purified using a Protein A column. The clone IDs of the four resulting anti-human CD39 antibodies were designated P4_sc, T86_sc, I67_sc, and Tre291_sc. These four anti-human CD39 antibodies were derived from P4, T86, I67, or Tre291, respectively, and share the same CDR amino acid sequences.

[0131] Example 1.3: Evaluation of CD39 inhibitory activity CD39 inhibitory activity was evaluated by ATP quantification using wild-type SKMEL28 cells expressing human CD39. The details are as follows: SKMEL28 cells were cultured at 5x10 in E-MEM medium. 3Cells were prepared at 100 μL per well and seeded in 50 μL aliquots onto a 96-well V-bottom plate. 50 μL of serially diluted antibodies were added and incubated in a 37°C incubator for 1 hour. Next, 5 μL of ATP (SIGMA, A2383) prepared at 200 μM in E-MEM medium was added and incubated at room temperature for 30 minutes. The plate was then centrifuged at 700 g for 3 minutes, and 50 μL of the supernatant was transferred to a 96-well white polystyrene plate. 50 μL of CellTiter-Glo® 2.0 (Promega, G9242) for ATP quantification was added to the culture supernatant and incubated at room temperature for 10 minutes. The amount of ATP was measured by luminescence detection using a microplate reader. The ATP remaining value in wells where cells were not seeded was set to 100%, and the ATP remaining value in wells where cells were seeded but no antibody was added was set to 0%. The inhibition rate of ATP degradation by various antibodies was calculated from the ratio of the ATP remaining value of the measurement sample, and the inhibitory activity of the antibodies against ATP degradation of human CD39 was evaluated.

[0132] Example 1.4: Epitope Binning To classify the anti-human CD39 antibodies obtained in Example 1.2 by epitope, epitope binning was performed using Biacore 8K (Cytiva) by intermolecular interaction analysis using surface plasmon resonance (SPR). Details are as follows. Six antibodies were used: P4_sc, T86_sc, I67_sc, Tre291_sc, I-394 (an anti-CD39 antibody having the amino acid sequences of the heavy chain variable region and light chain variable region of I-394 described in WO2018 / 167267 (SEQ ID NOs: 56 and 57, respectively)), and 31414 (an anti-CD39 antibody having the amino acid sequences of the heavy chain variable region and light chain variable region of 31414 described in US2019 / 0062448 (SEQ ID NOs: 58 and 59, respectively)). Recombinant human CD39 (in-house) was captured on a Ni-NTA sensor chip, and the six anti-human CD39 antibodies were added as primary antibodies and allowed to bind until saturation. Subsequently, the six antibodies were added as secondary antibodies, and the presence or absence of binding was assessed.

[0133] The results are shown in Figure 1. The antibodies were classified into four epitope bins: (1) P4_sc, I67_sc, and Tre291c_sc, (2) T86_sc, (3) I-394, and (4) 31414. This indicates that the four anti-human CD39 antibodies obtained in Example 1.2 recognize epitopes distinct from those of conventional antibodies. Furthermore, among the four, T86_sc was shown to recognize a unique epitope.

[0134] Example 1.5: Recombinant antigen-binding ability of anti-human CD39 antibodies The affinity of the anti-human CD39 antibodies obtained in Example 1.2 for human CD39 was measured by interaction analysis using surface plasmon resonance (SPR) using a Biacore 8K. Details are as follows: Four anti-human CD39 antibodies prepared were captured on a sensor chip (Cytiva, #29234600) immobilized with an anti-human IgG antibody, and recombinant human CD39 (in-house) was used as the analyte. Data were analyzed using a one-to-one Langmuir binding model to calculate the binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) of each antibody to human CD39 (Figure 2).

[0135] Example 1.6: Analysis of binding of anti-human CD39 antibodies to antigens on cell membranes. The binding of the anti-human CD39 antibodies obtained in Example 1.2 to human CD39 expressed on the cell membrane was analyzed by flow cytometry using HEK293T cells stably expressing human CD39. The details are as follows. Human CD39-expressing HEK293T cells were 2x10 5Cells were plated onto a 96-well V-bottom plate at 100 μl per well and washed once with 200 μl of PBS(-). Next, a 5-fold dilution series of anti-human CD39 antibody was prepared in staining buffer (PBS(-), 1% BSA, 0.09% NaN3), and 100 μl of the antibody was added to the cells. After incubation at 4°C for 30 minutes, the plate was washed once with staining buffer. 100 μl of staining antibody (PE AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch, #109-116-170) diluted 1:100 in staining buffer was added to the cells at a concentration of 100 μl per well. After incubation at 4°C for 20 minutes, the plate was washed twice with staining buffer. 50 μl of fixation buffer (BioLegend, #420801) was added and the plate was incubated at room temperature for 20 minutes for fixation. The stained cells were suspended in staining buffer and their fluorescence intensity (MFI) was measured using a flow cytometer. The MFI values ​​corresponding to each antibody concentration were plotted, and the Kd values ​​were calculated.

[0136] The results are shown in Figure 3. All four anti-human CD39 antibodies (P4_sc, T86_sc, I67_sc, and Tre291_sc) showed good affinity for CD39.

[0137] Example 2.1: Construction of CD39 / 4-1BB Bispecific Molecules Bispecific molecules were constructed based on the anti-human CD39 antibodies obtained in Example 1.2. The bispecific molecules were designed to have a first antigen-binding domain (CD39-binding domain) targeting human CD39 and a second antigen-binding domain (4-1BB-binding domain) targeting human 4-1BB, an immune cell surface antigen (schematically depicted in Figure 4). The construction procedure was detailed as follows: First vectors expressing each of the four anti-human CD39 antibodies (in the form of scFv-Fc) obtained in Example 1.2 were constructed. Second vectors expressing a trimer of the extracellular domain (amino acid sequence from positions 71 to 254 (SEQ ID NO: 39)) of the ligand for human 4-1BB (4-1BBL; Accession No. NM_003811.3 / NP_003802.1) (SEQ ID NO: 40) were constructed. The second vector expressed a polypeptide chain (SEQ ID NO: 42) in which a 4-1BBL monomer and a human IgG Cκ chain (SEQ ID NO: 41) were linked via a short linker, and a polypeptide chain (SEQ ID NO: 44) in which a 4-1BBL dimer linked via a short linker and a human IgG CH chain (SEQ ID NO: 43) were linked via a short linker, and these polypeptide chains formed a trimer through disulfide bonds. To form a heterodimer between the anti-human CD39 antibody and human 4-1BBL, a hole mutation (Hole-into-Hole mutation: SEQ ID NO: 38) was introduced into the Fc region of the anti-human CD39 antibody, and a knob mutation (Knob-into-Hole mutation: SEQ ID NO: 45) was introduced into the Fc region of the human 4-1BBL trimer. The resulting vectors expressing the anti-human CD39 antibody and the human 4-1BBL trimer were co-transfected into ExpiCHO cells (Thermo Fisher Scientific) and expressed. The CD39 / 4-1BB bispecific molecule was purified using an anti-IgG-CH1 column (Thermo Fisher Scientific, 494346201), size exclusion chromatography, and ion exchange chromatography.The clone IDs of the four bispecific molecules obtained were P4_4-1BBL, T86_4-1BBL, I67_4-1BBL, and Tre291_4-1BBL. The CD39-binding domains of these four bispecific molecules were derived from P4_sc, T86_sc, I67_sc, and Tre291_sc, respectively, and the amino acid sequences of their CDRs were the same.

[0138] Example 2.2: Affinity Measurement for Human CD39 and 4-1BB The affinity of the CD39 / 4-1BB bispecific molecules obtained in Example 2.1 for human CD39 and human 4-1BB was measured by the method described in Example 1.5, except that recombinant human CD39 (in-house) and recombinant human 4-1BB (R&D Systems, 9220-4B-100) were used as analytes. Data were analyzed using a one-to-one Langmuir binding model. The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) for each bispecific molecule against anti-human CD39 and human 4-1BBL were calculated.

[0139] The results are shown in Figure 5. All four CD39 / 4-1BB bispecific molecules (P4_4-1BBL, T86_4-1BBL, I67_4-1BBL, and Tre291_4-1BBL) showed good affinity for CD39 and 4-1BB.

[0140] Example 2.3: Measurement of binding to human cancer cell lines. The binding of the CD39 / 4-1BB bispecific molecule obtained in Example 2.1 to human CD39 expressed on the cell membrane was analyzed by flow cytometry using cancer cells expressing human CD39 (OAW42 cells (ECACC: 85073102) or SKMEL28 cells (ATCC: HTB-72)). The details are as follows. OAW42 cells or SKMEL28 cells were cultured at 2 x 10 5Cells were plated onto a 96-well V-bottom plate at 100 μl per well and washed once with 200 μl of PBS(-). Next, a 5-fold dilution series of the CD39 / 4-1BB bispecific molecule was prepared in staining buffer (PBS(-), 1% BSA, 0.09% NaN3), and 100 μl of the dilution was added to the cells. After incubation at 4°C for 1 hour, the plate was washed once with staining buffer. 100 μl / well of staining antibody (PE AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch, #109-116-170) diluted 25-fold in staining buffer was added to the cells. After incubation at 4°C for 30 minutes, the plate was washed twice with staining buffer. 50 μl of fixation buffer (BioLegend, #420801) was added and the plate was incubated at room temperature for 20 minutes for fixation. The stained cells were suspended in staining buffer, and fluorescence intensity was measured using a flow cytometer. The geometric mean fluorescence intensity (gMFI) corresponding to each bispecific molecule concentration was plotted, and Kd values ​​were calculated.

[0141] The results are shown in Figures 6 and 7. All four CD39 / 4-1BB bispecific molecules (P4_4-1BBL, T86_4-1BBL, I67_4-1BBL, and Tre291_4-1BBL) showed good affinity for CD39 on the cancer cell membrane. Among the four, T86_4-1BBL had significantly higher affinity. This was an unexpected result, differing from the trends observed in the affinity measurements in Examples 1.5, 1.6, and 2.2. The CD39-binding domain of T86_4-1BBL is thought to recognize an epitope that readily binds to CD39-expressing cells even when located on only one arm.

[0142] Example 2.4: Measurement of CD39 enzyme inhibitory activity in OAW42 cell line The ability of the CD39 / 4-1BB bispecific molecule obtained in Example 2.1 to inhibit human CD39 enzyme activity was measured by the method described in Example 1.3, except that the OAW42 cell line was used as the human CD39-expressing cells at a concentration of 6.25 x 103 Cells / well were used for evaluation.

[0143] The results are shown in Figure 8. Three CD39 / 4-1BB bispecific molecules (P4_4-1BBL, I67_4-1BBL, and Tre291_4-1BBL) exhibited CD39 enzyme inhibitory activity. Unexpectedly, however, T86_4-1BBL exhibited very weak CD39 enzyme inhibitory activity. This suggests that T86_4-1BBL is superior in terms of suppressing side effects.

[0144] Example 2.5: Measurement of cytokine production in a co-culture system of SKMEL28 cells and PBMCs. The effect of the CD39 / 4-1BB bispecific molecule obtained in Example 2.1 on the antitumor activity in a co-culture system of cancer cells and PBMCs was evaluated. Details are as follows. SKMEL28 cell line (target cells) irradiated with 50 Gy of X-rays were used for the evaluation. Target cells, previously frozen and stored, were thawed on the day of the evaluation, suspended in Assay medium (RPMI-1640 medium containing 10% FCS, 1x NEAA, 1x GlutaMax, and 1% penicillin-streptomycin), and seeded into a 96-well plate. Next, PBMCs from healthy volunteers were suspended in Assay medium and added to the 96-well plate at a volume 10 times the volume of the target cells. Target cells and PBMCs, or PBMCs alone, were seeded into wells with either CD39 / 4-1BB bispecific molecules or isotype 4-1BBL (control) at a final concentration of 10 μg / ml. The CD39 / 4-1BB bispecific molecules used were P4_4-1BBL, T86_4-1BBL, I67_4-1BBL, or Tre291_4-1BBL. Anti-CD3 antibody (OKT3, BioLegend, #317326) was then added to a final concentration of 1 nM, and cells were co-cultured for 2 days at 37°C in 5% CO2. After co-culture, the culture supernatant and non-adherent cells were collected and centrifuged to remove cellular components. IL-2, TNF-α, and IFN-γ in the culture supernatant were measured by Luminex. Standard curves were prepared using known concentrations of recombinant IL-2, TNF-α, and IFN-γ, and the concentrations of each cytokine were calculated.

[0145] The results are shown in Figure 9. Both CD39 / 4-1BB bispecific molecules (P4_4-1BBL and T86_4-1BBL) induced particularly high levels of cytokines (IL-2, TNF-α, and IFN-γ), indicating the induction of large numbers of functional T cells.

[0146] Example 3.1: Preparation of Humanized Antibodies The anti-human CD39 antibodies prepared in Example 1.2 were humanized using the CDR grafting method. Details are as follows. Among the anti-human CD39 antibodies, P4_sc and T86_sc, which were shown to particularly promote cytokine production, were humanized by CDR grafting using Discovery Studio (Dassault Systèmes). From multiple candidates for each antibody, those with affinity and bioactivity equivalent to or greater than those of the original mouse antibody were selected. The humanized antibodies were prepared in the form of scFv-Fc-LALA antibodies using the method described in Example 1.2. The clone IDs of the two humanized antibodies obtained were designated HuP4_IB and HuT86_IF. These two humanized antibodies were derived from P4_sc and T86_sc, respectively, and had the same CDR amino acid sequences.

[0147] Example 3.2: Recombinant antigen-binding ability of humanized anti-human CD39 antibodies The affinity of the humanized anti-human CD39 antibodies obtained in Example 3.1 for human CD39 was measured by the method described in Example 1.5. The data were analyzed using a one-to-one Langmuir binding model, and the binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) for each humanized antibody for human CD39 were calculated (Figure 10).

[0148] Example 3.3: Analysis of binding of humanized anti-human CD39 antibodies to antigens on cell membranes The binding of the humanized anti-human CD39 antibodies obtained in Example 3.1 to human CD39 expressed on cell membranes was measured by the method described in Example 1.6. The geometric mean fluorescence intensity (gMFI) corresponding to each antibody concentration was plotted, and the Kd value was calculated.

[0149] The results are shown in Figure 11. Both of the two humanized anti-human CD39 antibodies (HuP4_IB and HuT86_IF) showed good affinity for CD39.

[0150] Example 4.1: Preparation of CD39 / 4-1BB bispecific molecules using humanized anti-human CD39 antibodies CD39 / 4-1BB bispecific molecules were prepared using the humanized anti-human CD39 antibodies obtained in Example 3.1, following the same procedure as in Example 2.1. In this study, vectors expressing HuP4_IB or HuT86_IF were prepared and used as the first vector. The clone IDs of the two bispecific molecules obtained were designated HuP4_IB_4-1BBL and HuT86_IF_4-1BBL, respectively. The CD39-binding domains of these two bispecific molecules were derived from HuP4_IB or HuT86_IF, respectively, and the amino acid sequences of their CDRs were identical.

[0151] Furthermore, CD39 / 4-1BB bispecific molecules were produced using the same procedure as in Example 2.1, but using 31414 or I-394 as the anti-human CD39 antibody. In this case, vectors expressing 31414 or I-394 were produced and used as the first vector. The clone IDs of the two bispecific molecules obtained were designated 31414_4-1BBL and I-394_4-1BBLL, respectively.

[0152] Example 4.2: Affinity measurement for human CD39 and 4-1BB The affinity of the CD39 / 4-1BB bispecific molecules obtained in Example 4.1 for human CD39 and human 4-1BB was measured by the method described in Example 2.2. Data were analyzed using a 1:1 binding model. The association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) for each bispecific molecule against anti-human CD39 and human 4-1BBL were calculated.

[0153] The results are shown in Figure 12. Both of the two CD39 / 4-1BB bispecific molecules (HuP4_IB_4-1BBL and HuT86_IF_4-1BBL) showed good affinity for CD39 and 4-1BB.

[0154] Example 4.3 Measurement of Binding to Human Cancer Cell Lines The binding of the CD39 / 4-1BB bispecific molecules obtained in Example 4.1 to human CD39 was measured as described in Example 2.3. The geometric mean fluorescence intensity (gMFI) corresponding to each bispecific molecule concentration was plotted, and the Kd value was calculated.

[0155] The results are shown in Figures 13 and 14. Both of the two CD39 / 4-1BB bispecific molecules (HuP4_IB_4-1BBL and HuT86_IF_4-1BBL) showed good affinity for CD39 on cancer cells. In particular, the affinity of HuT86_IF_4-1BBL was remarkably high. This was a different trend from the results of the affinity measurement for isolated CD39 in Example 4.2. The trend was similar to the results in Example 2.3.

[0156] Example 4.4: Measurement of cytokine production in a co-culture system of SKMEL28 cells and PBMCs. The antitumor activity of the CD39 / 4-1BB bispecific molecule obtained in Example 4.1 was evaluated in a co-culture system of cancer cells and PBMCs. Details are as follows. Target cells (SKMEL28) were suspended in assay medium (E-MEM medium containing 10% FCS and 1% penicillin-streptomycin) and seeded into a 96-well plate. Next, PBMCs from healthy volunteers were suspended in assay medium and added to the 96-well plate at a volume 1.6 times that of the target cells. The CD39 / 4-1BB bispecific molecule or control isotype 4-1BBL was added at a final concentration of 1 μg / ml to wells seeded with target cells and PBMCs or PBMCs alone. The CD39 / 4-1BB bispecific molecules used were HuP4_IB_4-1BBL, HuT86_IF_4-1BBL, 31414_4-1BBL, or I-394_4-1BBL. Anti-CD3 antibody (OKT3, BioLegend, #317326) was then added to a final concentration of 1 nM, and co-cultured for 4 or 6 days at 37°C in 5% CO2. After co-culture, the culture supernatant and non-adherent cells were collected and centrifuged to remove cellular components. IL-2, TNF-α, and IFN-γ in the culture supernatant were measured by Luminex. Standard curves were prepared using known concentrations of recombinant IL-2, TNF-α, and IFN-γ, and the concentrations of each cytokine were calculated.

[0157] The results are shown in Figures 15 to 18. Both CD39 / 4-1BB bispecific molecules (P4_4-1BBL and T86_4-1BBL) produced significant amounts of cytokines (IL-2, TNF-α, and IFN-γ). In particular, HuT86_IF_4-1BBL produced significantly higher amounts of cytokines.

[0158] Example 4.5: Measurement of Cytostatic Effect on SKMEL28 Cell Line The CD39 / 4-1BB bispecific molecules obtained in Example 4.1 were evaluated for their antitumor activity in a co-culture system of PBMCs and cancer cells. Details are as follows. The CD39 / 4-1BB bispecific molecules used for evaluation were HuP4_IB_4-1BBL, HuT86_IF_4-1BBL, 31414_4-1BBL, and I-394_4-1BBL, and isotype_4-1BBL was used as a control. On the day before the start of the evaluation, the SKMEL28 cell line was suspended in E-MEM medium containing 10% FCS and 1% penicillin-streptomycin, seeded into a 96-well plate, and cultured at 37°C in 5% CO2. On the first day of evaluation, PBMCs from healthy volunteers were suspended in E-MEM medium containing 10% FCS and 1% penicillin-streptomycin and added to a 96-well plate at a cell number 1.6 times the number of cancer cells seeded the previous day. The bispecific molecule or isotype_4-1BBL used for evaluation was added to the wells containing the cancer cells and PBMCs at a final concentration of 1 μg / ml. Anti-CD3 antibody was then added to a final concentration of 1 nM, and co-cultured at 37°C and 5% CO for 7 days. After co-culture, the culture supernatant and non-adherent cells were collected, and the wells were washed twice with PBS and then 70 μL of PBS was added. 70 μL of CellTiter-Glo® 2.0 (Promega, G9243) was added and shaken at room temperature for 10 minutes. 100 μL of the contents of each well was then aliquoted into a white 96-well plate, and the amount of ATP derived from viable cells was measured using a microplate reader for luminescence detection.

[0159] The results are shown in Figures 19 and 20. Both CD39 / 4-1BB bispecific molecules (P4_4-1BBL and T86_4-1BBL) exhibited antitumor effects in a coculture system of PBMCs and cancer cells. In particular, the antitumor effect of HuT86_IF_4-1BBL was significantly higher.

[0160] Example 5.1: Construction of CD39 / CD3 bispecific molecules. Bispecific molecules were constructed based on the anti-human CD39 antibody obtained in Example 3.1. The bispecific molecules were designed to have a first antigen-binding domain (CD39-binding domain) targeting human CD39 and a second antigen-binding domain (CD3-binding domain) targeting human CD3. The construction procedure is detailed below. The first vector used to express the anti-human CD39 antibody (scFv-Fc form) obtained in Example 3.1 was the one constructed in Example 4.1. A second vector expressing an antibody against human CD3 (OKT3) was constructed. The second vector expressed a polypeptide chain (SEQ ID NO: 69) consisting of an OKT3 Vκ chain (SEQ ID NO: 53) and a human IgG Cκ chain (SEQ ID NO: 41), and another polypeptide chain (SEQ ID NO: 68) consisting of an OKT3 VH chain (SEQ ID NO: 52) and a human IgG CH chain (SEQ ID NO: 43), which formed a dimer through disulfide bonds. To enable heterodimerization of the anti-human CD39 antibody and the anti-human CD3 antibody, a Knob mutation (Knob-into-Hole sequence, US7183076B2, Knob: Seq No. 2, Hole: Seq No. 3) was introduced into the Fc region of the anti-human CD3 antibody (Knob-mutated Fc region: SEQ ID NO: 45). The vectors expressing the anti-human CD39 antibody and the anti-human CD3 antibody were simultaneously transfected into ExpiCHO cells (Thermo Fisher Scientific) for expression. The CD39 / CD3 bispecific molecules were purified using an anti-IgG-CH1 column (Thermo Fisher Scientific, 494346201) and cation exchange chromatography. The clone IDs of the resulting bispecific molecules were OKT3-HuT86_IF_Fsc and OKT3-I-394_Fsc. The CD39-binding domain of OKT3-HuT86_IF-Fsc is derived from T86_sc, and the amino acid sequences of their CDRs are identical.

[0161] Example 5.2: Affinity measurement for human CD39 The affinity of the CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc obtained in Example 5.1 for human CD39 was measured by the method described in Example 1.5. Data were analyzed using a one-to-one Langmuir binding model. The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) of the bispecific molecule for anti-human CD39 were calculated.

[0162] The results are shown in Figure 26. The CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc showed good affinity for CD39.

[0163] Example 5.3: Measurement of binding to human cancer cell lines. The binding of the CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc obtained in Example 5.1 to human CD39 expressed on the cell membrane was measured using the method described in Example 2.3. HuT86_IF_4-1BBL prepared in Example 4.1 was used as a control. The geometric mean fluorescence intensity (gMFI) was plotted against the bispecific molecule concentration, and the Kd value was calculated.

[0164] The results are shown in Figure 27. The CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc exhibited affinity for CD39 on the cancer cell membrane similar to that of HuT86_IF_4-1BBL shown in Example 4.3. This suggests that, like HuT86_IF_4-1BBL, HuT86_IF in OKT3-HuT86_IF_Fsc also recognizes an epitope that easily binds to CD39-expressing cells using only one arm without being affected by the other arm.

[0165] Example 5.4: Measurement of cytotoxic activity against SKMEL28 cell line. The effect of the CD39 / CD3 bispecific molecule obtained in Example 5.1 on the antitumor activity in a co-culture system of PBMCs and cancer cells was measured using the method described in Example 4.5. However, in the CD39 / CD3 bispecific molecule-added group, no anti-CD3 antibody (OKT3, BioLegend, #317326) was added. The number of PBMCs added was equal to that of target cells. The amount of ATP derived from viable cells in wells to which neither the bispecific molecule nor the anti-CD3 antibody was added was defined as 100%, and the viable cell survival rate in each well was calculated from the ATP amount in the measurement sample. The viable cell survival rate was subtracted from 1 to evaluate the cancer cell cytotoxicity of the bispecific molecule and the anti-CD3 antibody.

[0166] The results are shown in Figure 28. Both CD39 / CD3 bispecific molecules (OKT3-HuT86_IF_Fsc and OKT3-I-394_Fsc) exhibited antitumor activity in a coculture system of PBMCs and cancer cells. In particular, the cytotoxic activity of OKT3-I-304_Fsc decreased at the highest concentration, whereas the antitumor activity of OKT3-HuT86_IF_Fsc remained high and concentration-dependently even at the highest concentration.

[0167] Example 5.5: Measurement of cytokine production and cytolytic granule release in a SKMEL28 cell and PBMC coculture system. Cytokine production and cytolytic granule release were measured using the culture supernatant from Example 5.4, as described in Example 4.4. However, in the CD39 / CD3 bispecific molecule-added group, anti-CD3 antibody (OKT3, BioLegend, #317326) was not added. The number of PBMCs added was equal to that of target cells. Granzyme B and perforin were measured using MILLIPLEX MAP 7-Plex Human CD8+ / Cytokine Magnetic Bead Panel (Millipore, #HCD8MAG15K17PMX). Standard curves were prepared using known concentrations of recombinant IL-2, TNF-α, IFN-γ, granzyme B, and perforin, and the concentrations of each cytokine and cytolytic granule were calculated.

[0168] The results are shown in Figures 29 and 30. The amounts of cytokine production and cytolytic granule release by OKT3-HuT86_IF_Fsc were significantly high.

[0169] Example 6.1: Measurement of T cell activation in a co-culture system of SKMEL28 cells and PBMCs. The CD39 / CD3 bispecific molecules obtained in Example 5.1 were evaluated for their effector cell activity in a co-culture system of PBMCs and cancer cells. Details are as follows. OKT3-HuT86_IF_Fsc and OKT3-I-394_Fsc were used as CD39 / CD3 bispecific molecules for evaluation, and OKT3 was used as a control. On the day before the start of the evaluation, SKMEL28 cells were suspended in E-MEM medium containing 10% FCS and 1% penicillin-streptomycin and seeded into 24-well plates at 2.4 x 10^4 cells / well. The cells were then cultured at 37°C in 5% CO2. On the first day of evaluation, PBMCs from healthy volunteers were suspended in E-MEM medium containing 10% FCS and 1% penicillin-streptomycin and added to the wells at an equal cell number to the cancer cells seeded the previous day. The bispecific molecule or OKT3 used for evaluation was added to the wells containing the cancer cells and PBMCs at a final concentration of 1 μg / ml, and co-cultured at 37°C and 5% CO2. On the day before staining, Brefeldin A (BioLegend) was added to a final concentration of 1x and the cells were cultured for 17 hours at 37°C and 5% CO2. On day 6, the cells were harvested into a 96-well V-bottom plate and washed once with 200 μl of PBS(-). To stain for dead cells, 50 μl of Zombie-NIR (BioLegend) diluted 1:1,000 in PBS(-) was added and incubated at room temperature for 15 minutes. After washing once with 200 μl of staining buffer [PBS(-), 1% BSA, 0.09% NaN3], 50 μl of Human TruStain FcX (BioLegend) diluted 2:20 in staining buffer was added and incubated at room temperature for 20 minutes. Afterwards, 50 μl of T cell staining antibodies (BioLegend, anti-CD3: UCHT1, anti-CD4: RPA-T4, anti-CD8a: HIT8a, anti-CD39: A1, anti-PD-1: EH12) diluted appropriately in staining buffer were added and incubated at 4°C for 30 minutes in the dark.Next, the cells were washed once with staining buffer, and 50 μl of fixation buffer was added and left to stand at room temperature for 20 minutes for fixation. They were then washed twice with Intracellular Staining Permeabilization Wash Buffer (BioLegend, #421002). Cytokine staining antibodies (BioLegend, anti-IFN-γ: B27, anti-IL2: MQ1-17H12) were appropriately diluted in Intracellular Staining Permeabilization Wash Buffer and 100 μl of each antibody was added. The cells were then left to stand at 4°C for 40 minutes in the dark. After washing twice with Intracellular Staining Permeabilization Wash Buffer, the cells were suspended in staining buffer, and cell counting beads (Thermo Fisher Scientific, #C36950) were added. The cells were then analyzed using a flow cytometer.

[0170] Figure 31 shows the results of flow cytometry analysis to calculate the total numbers of CD4+ T cells and CD8+ T cells, each classified by the expression patterns of CD39 and PD1. The anti-human CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc prepared in this example significantly increased both CD4+ T cells and CD8+ T cells compared with OKT3-I-394_Fsc and OKT3. Furthermore, this difference was due to an increase in the PD1- and CD39-positive fractions, whose expression is upregulated upon T cell activation. These results demonstrate that the anti-human CD39 / CD3 bispecific molecule OKT3-HuT86_IF_Fsc prepared in this example activates and increases T cells in the presence of CD39-expressing cells. Furthermore, the CD39-positive fraction in T cells can serve as target cells for the anti-human CD39 / CD3 bispecific molecule, but surprisingly has little effect on activated CD39+ T cells.

[0171] 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. (a) a CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (b) a CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12; (c) a CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18; or (d) a CD39-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

24.

2. The multispecific antigen-binding molecule of claim 1, comprising the CD39 binding domain of (a) or (b).

3. A multispecific antigen-binding molecule as described in claim 1 or 2, comprising the CD39 binding domain of (b).

4. A multispecific antigen-binding molecule according to any one of claims 1 to 3, wherein the CD39 binding domain is a domain that recognizes CD39.

5. A multispecific antigen-binding molecule described in any one of claims 1 to 4, which has binding affinity to CD39 on the surface of cancer cells.

6. A multispecific antigen-binding molecule described in any one of claims 1 to 5, having anti-tumor activity.

7. The multispecific antigen-binding molecule of claim 6, wherein the anti-tumor activity is an anti-tumor activity resulting from the binding of the multispecific antigen-binding molecule to multiple types of antigens.

8. A multispecific antigen-binding molecule described in any one of claims 1 to 7, which exerts an anti-tumor effect by binding to CD39 on the surface of cancer cells and to an immune cell surface antigen or an anti-inflammatory cytokine.

9. The multispecific antigen-binding molecule of any one of claims 1 to 8, wherein the CD39 binding domain comprises a heavy chain variable region and a light chain variable region.

10. A multispecific antigen-binding molecule according to any one of claims 1 to 9, which is a bispecific antigen-binding molecule.

11. The multispecific antigen-binding molecule of any one of claims 1 to 10, which is a bispecific antibody or a bispecific antigen-binding fusion protein.

12. A multispecific antigen-binding molecule according to any one of claims 1 to 11, further comprising a domain having binding ability to an antigen other than CD39.

13. A multispecific antigen-binding molecule according to any one of claims 1 to 12, comprising the CD39-binding domain as a first binding domain and an immune cell surface antigen-binding domain or an anti-inflammatory cytokine-binding domain as a second binding domain.

14. The multispecific antigen-binding molecule of claim 13, wherein there is an intervening sequence between the CD39 binding domain and the immune cell surface antigen binding domain or the anti-inflammatory cytokine binding domain.

15. The multispecific antigen-binding molecule of any one of claims 8, 13 and 14, wherein the immune cell is a T cell.

16. A multispecific antigen-binding molecule according to any one of claims 8 and 13 to 15, wherein the immune cell surface antigen is a costimulatory molecule, an immune checkpoint molecule or CD3.

17. The multispecific antigen-binding molecule of any one of claims 8 and 13 to 16, wherein the anti-inflammatory cytokine is a cytokine that induces regulatory T cells.

18. The multispecific antigen-binding molecule of any one of claims 1 to 17, comprising the CD39-binding domain as a first binding domain and a domain having binding affinity to 4-1BB, GITR, CD27, CD40, NKp30, TIM-3, LAG-3, BTLA, CTLA-4, CD73, CD3, or TGFβ as a second binding domain.

19. A multispecific antigen-binding molecule according to any one of claims 1 to 18, comprising the CD39-binding domain as a first binding domain and a domain having binding ability to 4-1BB as a second binding domain.

20. A multispecific antigen-binding molecule according to any one of claims 1 to 18, comprising the CD39-binding domain as a first binding domain and a domain having binding ability to CD3 as a second binding domain.

21. The multispecific antigen-binding molecule of any one of claims 1 to 20, wherein the CD39 binding domain is a humanized CD39 binding domain.

22. A polynucleotide or vector encoding a multispecific antigen-binding molecule according to any one of claims 1 to 21.

23. A cell comprising the polynucleotide or vector of claim 22.

24. A method for producing a multispecific antigen-binding molecule, comprising the step of growing the cell described in claim 23.

25. A composition comprising a multispecific antigen-binding molecule according to any one of claims 1 to 21.

26. A pharmaceutical composition for treating malignant tumors, comprising a multispecific antigen-binding molecule according to any one of claims 1 to 21.

27. The pharmaceutical composition according to claim 26, wherein the treatment is carried out by utilizing the anti-tumor activity resulting from the binding of the multispecific antigen-binding molecule to multiple types of antigens.

28. A composition for promoting cytokine production by immune cells, comprising a multispecific antigen-binding molecule described in any one of claims 1 to 21.

29. The composition of claim 28, wherein the cytokine is IL-2, TNF-α or IFN-γ.

30. A kit comprising a multispecific antigen-binding molecule according to any one of claims 1 to 21.

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