Fusion protein

Abstract

Provided is a novel protein or the like in the form of a trispecific triantibody having an increased anti-tumor effect. The present invention relates to a fusion protein comprising two different chains, i.e., a first chain and a second chain, in which three kinds of combinations of VH / VL binding domains formed in the fusion protein have any kind of binding ability selected from the group consisting of (i) binding ability to 5T4, (ii) binding ability to a CD3 receptor complex of a T cell, and (iii) binding ability to PD-1, PD-L1, or LAG3. The three combined VH / VL binding structural domains have different binding capacities. ).

Description

Technical Field

[0001] The present invention relates to a fusion protein and its use, etc. Specifically, it relates to a fusion protein having the ability to bind to (i) 5T4, (ii) CD3 receptor complex and (iii) PD-1, PD-L1 or LAG3 and its use, etc. Background Art

[0002] CD3-based T-cell derivatives (T-cell Engager (TCE)) are antibodies or antibody-like proteins that simultaneously bind to tumor-associated antigens (TAA) of cancer cells with one arm and to CD3 complexes with the other arm, forming an artificial immune synapse that is independent of TCR and avoids the HLA restriction of T cell-dependent immune responses. TCE is one of the most interesting ways to treat cancers that are resistant to traditional therapies. In the field of hematological malignancies, Blinatumomab is a bispecific T-cell derivative (Bispecific TCE (BiTE)) that simultaneously targets the CD19 antigen of acute lymphoblastic leukemia (ALL) and CD3 on T cells, inducing efficient death of leukemia cells (FDA approved in 2014). On the other hand, an important issue in the treatment of solid tumors with TCE can be manifested by the immunosuppressive tumor microenvironment (TME). Solid tumors mobilize immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs) to form the TME, all of which inhibit the activity of cytotoxic T cells.

[0003] Therefore, in the most effective application of TCE in solid tumors, it may be necessary to use TCE in combination with agents such as immune checkpoint inhibitors, the role of which is to overcome the immunosuppressive TME and turn "immune cold" tumors in a state of immune rejection or immune desert into "immune hot" tumors that have caused inflammation (for example, with reference to non-patent literature 1). Ideally, a single molecule that can have both the functions of TCE and immune checkpoint inhibitors can be designed. Thus, the new construct can overcome the immunosuppressive TME of solid tumors, and can reduce manufacturing costs compared to the respective pharmaceutical manufacturing costs of the two combined agents.

[0004] Carcinoembryonic antigen 5T4, also known as TPBG or WAIF1, is a 72 kDa cell surface glycoprotein containing eight leucine-rich repeats. 5T4 is expressed in the trophoblast of the fetus as early as 9 weeks of gestation. In healthy adults, 5T4 expression is restricted to several specific epithelial cell types and is not detectable in the liver, lung, bronchus, heart, testis, ovary, brain, or muscle.

[0005] On the other hand, 5T4 is reported to be expressed in a variety of cancer types including ovarian cancer, colon cancer, cervical cancer, gastric cancer and lung cancer (for example, see non-patent documents 2 to 6). Furthermore, its expression has also been observed in non-seminomatous germ cell tumors of the bladder, endometrium, esophagus, pancreas, stomach and testis. Choriocarcinoma and placental trophoblastic tumors are also 5T4 positive (for example, see non-patent document 7). Therefore, it has been proposed that 5T4 be used as a target antigen suitable for cancer targeted therapy (for example, see non-patent documents 8 and 9).

[0006] Bispecific antibodies that bind to 5T4 on tumor cells and CD3 expressed on T cells can efficiently induce cancer cell death, thus potentially enabling an interesting strategy to redirect cytotoxic T cells to cancer cells. In clinical trials, there are several bispecific antibodies targeting 5T4 on tumor cells and co-stimulatory molecules on T cells, CD3 or 4-1BB, for the purpose of treating solid tumors. However, to date, there are no anti-5T4 immunotherapies approved for the treatment of cancer.

[0007] In addition, the tribody format is a multispecific antibody comprising a Fab domain as a scaffold, an H chain (V H +CH1) and L chain (V L +C L ) Fab fragments can naturally heterodimerize in vivo, and additional functions such as scFv can be integrated into the scaffold. Among the triantibodies, Tb535H is a bispecific triantibody in which both the Fab domain and the scFv domain target 5T4 (bivalent binding) and the other scFv targets CD3 (monovalent binding), and it is known that it can exert a certain anti-tumor effect (for example, refer to Patent Document 1).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: WO 2016 / 097408 A1

[0011] Patent Document 2: WO 2019 / 180201 A2

[0012] Patent Document 3: WO 99 / 37791

[0013] Non-patent literature

[0014] Non-patent literature 1: Hegde PS, Chen DS. Top 10 challenges in cancer immunotherapy. Immunity. (2020) 52: 17-35. doi: 10.1016 / j.immuni.2019.12.011

[0015] Non-patent literature 2: Int J Gynecol Cancer. 1995; 5: 269-274

[0016] Non-patent literature 3: Starzynska T, et al. Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma. Br J Cancer. 1994; 69: 899-902

[0017] Non-patent document 4: Jones H, et al., Investigation of expression of 5T4 antigen in cervical cancer. Br J Cancer. 1990; 6: 69-100

[0018] Non-patent document 5: Starzynska T, et al. The expression of 5T4 antigen incolorectal and gastric carcinoma. Br J Cancer. 1992; 66: 867-869

[0019] Non-patent literature 6: Forsberg G, et al. Therapy of human non-small-cell lung carcinoma using antibody targeting of modified superantigen. Br J Cancer. 2001; 85: 129-136

[0020] Non-patent literature 7: Br J Cancer. 1990 Jan; 61(1): 89-95. doi: 10.1038 / bjc.1990.20.

[0021] Non-patent literature 8: Woods AM, et al. Characterization of the murine5T4oncofoetal antigen: a target for immunotherapy in cancer. Biochem J. 2002; 366: 353-365

[0022] Non-patent literature 9: Hole N, et al. Isolation and characterization of 5T4, Atumor-associated antigen. Int J Cancer. 1990; 45: 179-184

[0023] Non-patent literature 10: Sasso, et al. MABS 2018, VOL. 10, NO. 7, 1060-1072

[0024] Non-patent literature 11: Passariello et al. IJMS 2022, 23, 3466 Summary of the invention

[0025] Problem that the invention aims to solve

[0026] Under such circumstances, the development of new proteins in the form of multispecific triabodies with further enhanced anti-tumor effects is desired.

[0027] Solutions for solving problems

[0028] The present invention has been made in consideration of the above circumstances, and provides the following fusion proteins and uses thereof (pharmaceutical compositions, etc.), etc.

[0029] [1] A fusion protein comprising two different chains, a first chain and a second chain,

[0030] The first chain contains two antibody variable regions VH1 and VH2, one antibody variable region VL2, and one antibody constant region CH1 or CL.

[0031] The second chain contains two antibody variable regions VL1 and VL3, one antibody variable region VH3, and one antibody constant region CL or CH1.

[0032] The first chain has a heterodimer interaction with the second chain (preferably, the heterodimer interaction is between the CH1 domain of one chain and the CL domain of the other chain),

[0033] The VH1 / VL1 binding domain, VH2 / VL2 binding domain, and VH3 / VL3 binding domain as the three combined VH / VL binding domains formed in the fusion protein have a structure selected from:

[0034] (i) Binding ability to 5T4,

[0035] (ii) binding capacity to the CD3 receptor complex, and

[0036] (iii) Binding ability to PD-1, PD-L1 or LAG3

[0037] any binding ability among (wherein the above three combinations of VH / VL binding domains have different binding abilities from each other.).

[0038] [2] The fusion protein according to [1] above, wherein the fusion protein is a heterodimeric protein comprising the first chain and the second chain.

[0039] [3] The fusion protein according to [1] or [2] above, wherein the two different chains comprise:

[0040] a) Chain 1:

[0041] VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0042] VH(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0043] Chain 2:

[0044] VL(5T4)-CL-L1-VH(X)-L2-VL(X) or

[0045] VL(5T4)-CL-L1-VL(X)-L2-VH(X)

[0046] combination of;

[0047] b) Chain 1:

[0048] VH(5T4)-CH1-L1-VH(X)-L2-VL(X) or

[0049] VH(5T4)-CH1-L1-VL(X)-L2-VH(X), and

[0050] Chain 2:

[0051] VL(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0052] VL(5T4)-CL-L1-VL(CD3)-L2-VH(CD3)

[0053] combination of;

[0054] c) Chain 1:

[0055] VL(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0056] VL(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0057] Chain 2:

[0058] VH(5T4)-CL-L1-VH(X)-L2-VL(X) or

[0059] VH(5T4)-CL-L1-VL(X)-L2-VH(X)

[0060] combination of;

[0061] d) Chain 1:

[0062] VL(5T4)-CH1-L1-VH(X)-L2-VL(X) or

[0063] VL(5T4)-CH1-L1-VL(X)-L2-VH(X), and

[0064] Chain 2:

[0065] VH(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0066] VH(5T4)-CL-L1-VL(CD3)-L2-VH(CD3)

[0067] combination of;

[0068] e) Chain 1:

[0069] VH(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0070] VH(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0071] Chain 2:

[0072] VL(CD3)-CL-L1-VH(X)-L2-VL(X) or

[0073] VL(CD3)-CL-L1-VL(X)-L2-VH(X)

[0074] combination of;

[0075] f) Chain 1:

[0076] VH(CD3)-CH1-L1-VH(X)-L2-VL(X) or

[0077] VH(CD3)-CH1-L1-VL(X)-L2-VH(X), and

[0078] Chain 2:

[0079] VL(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0080] VL(CD3)-CL-L1-VL(5T4)-L2-VH(5T4)

[0081] combination of;

[0082] g) Chain 1:

[0083] VL(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0084] VL(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0085] Chain 2:

[0086] VH(CD3)-CL-L1-VH(X)-L2-VL(X) or

[0087] VH(CD3)-CL-L1-VL(X)-L2-VH(X)

[0088] combination of;

[0089] h) First chain:

[0090] VL(CD3)-CH1-L1-VH(X)-L2-VL(X) or

[0091] VL(CD3)-CH1-L1-VL(X)-L2-VH(X), and

[0092] Chain 2:

[0093] VH(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0094] VH(CD3)-CL-L1-VL(5T4)-L2-VH(5T4)

[0095] combination of;

[0096] i) Chain 1:

[0097] VH(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0098] VH(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0099] Chain 2:

[0100] VL(X)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0101] VL(X)-CL-L1-VL(CD3)-L2-VH(CD3)

[0102] combination of;

[0103] j) Chain 1:

[0104] VH(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0105] VH(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0106] Chain 2:

[0107] VL(X)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0108] VL(X)-CL-L1-VL(5T4)-L2-VH(5T4)

[0109] combination of;

[0110] k) Chain 1:

[0111] VL(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0112] VL(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0113] Chain 2:

[0114] VH(X)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0115] VH(X)-CL-L1-VL(CD3)-L2-VH(CD3)

[0116] combination of;

[0117] or,

[0118] l) Chain 1:

[0119] VL(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0120] VL(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0121] Chain 2:

[0122] VH(X)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0123] VH(X)-CL-L1-VL(5T4)-L2-VH(5T4)

[0124] A combination of

[0125] [Here, in the above a) to l),

[0126] VH and VL are antibody variable regions.

[0127] VH(5T4) and VL(5T4) are the variable regions of the antibody against 5T4.

[0128] VH(CD3) and VL(CD3) are the variable regions of antibodies targeting the CD3 receptor complex.

[0129] VH(X) and VL(X) are variable regions of antibodies targeting PD-1, PD-L1 or LAG3.

[0130] CH1 and CL are antibody constant regions.

[0131] L1 and L2 are connectors. ].

[0132] [4] The fusion protein according to [1] or [2] above, wherein the amino acid sequences of the complementarity determining regions (CDR) 1, CDR2 and CDR3 in VH1, VH2 and VH3 are respectively composed of the following sequences:

[0133] The amino acid sequences shown in SEQ ID NOs: 3, 4 and 5,

[0134] The amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11,

[0135] The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or 27,

[0136] The amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39,

[0137] The amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 48 or 49 and SEQ ID NO: 50, or

[0138] The amino acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59 or 49 and SEQ ID NO: 60, and

[0139] The amino acid sequences of CDR1, CDR2 and CDR3 in VL1, VL2 and VL3 are respectively composed of the following sequences:

[0140] The amino acid sequences shown in SEQ ID NOs: 19, 20 and 21,

[0141] The amino acid sequences shown in SEQ ID NOs: 14, 15 and 16,

[0142] The amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, 32 or 33,

[0143] The amino acid sequences shown in SEQ ID NOs: 41, 42 and 43,

[0144] The amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, or

[0145] The amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64.

[0146] [5] The fusion protein according to [1] or [2] above, wherein the amino acid sequences of VH1, VH2 and VH3 are different from each other and consist of, contain or have at least 90% identity with the amino acid sequence of SEQ ID NO: 88, 90, 8, 92, 23, 78, 46, 81, 57, 109, 82, 83, 84 or 36, and

[0147] The amino acid sequences of VL1, VL2 and VL3 are different from each other, and are composed of the amino acid sequence shown in sequence numbers 89, 91, 13, 93, 28, 79, 80, 51, 61, 85, 86, 87 or 40, or contain the amino acid sequence, or have at least 90% identity with the amino acid sequence.

[0148] [6] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining regions (CDR) 1, CDR2 and CDR3 of VH(5T4) are respectively composed of the amino acid sequences represented by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and

[0149] The amino acid sequences of CDR1, CDR2 and CDR3 of VL(5T4) are composed of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0150] [7] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining regions (CDR) 1, CDR2 and CDR3 of VH (CD3) are respectively composed of the amino acid sequences represented by SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and

[0151] The amino acid sequences of CDR1, CDR2 and CDR3 of VL (CD3) are composed of the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0152] [8] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining region (CDR) 1, CDR2 and CDR3 of VH(X) (wherein X is PD-1) are respectively composed of the amino acid sequences represented by SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or 27, and

[0153] The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) (wherein X is PD-1) are respectively composed of the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, 32 or 33.

[0154] [9] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining region (CDR) 1, CDR2 and CDR3 of VH(X) (wherein X is PD-L1) are respectively composed of the amino acid sequences represented by SEQ ID NO: 47, SEQ ID NO: 48 or 49 and SEQ ID NO: 50, in that order; and

[0155] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(X) (wherein X is PD-L1) are composed of the amino acid sequences represented by SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, respectively.

[0156]

[10] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining region (CDR) 1, CDR2 and CDR3 of VH(X) (wherein X is PD-L1) are respectively composed of the amino acid sequences represented by SEQ ID NO: 58, SEQ ID NO: 59 or 49 and SEQ ID NO: 60, and

[0157] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(X) (wherein X is PD-L1) are composed of the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64, respectively.

[0158]

[11] The fusion protein according to [3] above, wherein the amino acid sequences of the complementarity determining region (CDR) 1, CDR2 and CDR3 of VH(X) (wherein X is LAG3) are respectively composed of the amino acid sequences represented by SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and

[0159] The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) (wherein X is LAG3) are composed of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0160]

[12] The fusion protein according to [3] above, wherein the amino acid sequence of VH(5T4) consists of, or contains, or has at least 90% identity with the amino acid sequence of SEQ ID NO: 88 or 90, and

[0161] The amino acid sequence of VL(5T4) consists of the amino acid sequence shown in SEQ ID NO: 89 or 91, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0162]

[13] The fusion protein according to [3] above, wherein the amino acid sequence of VH (CD3) consists of the amino acid sequence of SEQ ID NO: 8 or 92, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0163] The amino acid sequence of VL (CD3) consists of the amino acid sequence shown in SEQ ID NO: 13 or 93, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0164]

[14] The fusion protein according to [3] above, wherein the amino acid sequence of VH(X) (wherein X is PD-1) consists of the amino acid sequence of SEQ ID NO: 23 or 78, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0165] The amino acid sequence of VL(X) (wherein X is PD-1) consists of the amino acid sequence shown in SEQ ID NO: 28, 79 or 80, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0166]

[15] The fusion protein according to [3] above, wherein the amino acid sequence of VH(X) (wherein X is PD-L1) consists of the amino acid sequence of SEQ ID NO: 46 or 81, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0167] The amino acid sequence of VL(X) (wherein X is PD-L1) consists of the amino acid sequence shown in SEQ ID NO: 51, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0168]

[16] The fusion protein according to [3] above, wherein the amino acid sequence of VH(X) (wherein X is PD-L1) consists of, or contains, or has at least 90% identity with the amino acid sequence of SEQ ID NO: 57, 109, 82, 83 or 84, and

[0169] The amino acid sequence of VL(X) (wherein X is PD-L1) consists of the amino acid sequence shown in SEQ ID NO: 61, 85, 86 or 87, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0170]

[17] The fusion protein according to [3] above, wherein the amino acid sequence of VH(X) (wherein X is LAG3) consists of the amino acid sequence of SEQ ID NO: 36, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0171] The amino acid sequence of VL(X) (wherein X is LAG3) consists of the amino acid sequence shown in SEQ ID NO: 40, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0172]

[18] The fusion protein according to [1] or [2] above, wherein the first chain consists of the amino acid sequence of SEQ ID NO: 1, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0173] The second chain consists of the amino acid sequence shown in SEQ ID NO: 17, 34, 44, 55 or 107, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0174]

[19] The fusion protein according to [3] above, wherein the first chain: VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) consists of the amino acid sequence shown in SEQ ID NO: 1, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence, and

[0175] Chain 2: VL(5T4)CL-L1-VH(X)-L2-VL(X) is composed of the amino acid sequence shown in sequence number 17, 34, 44, 55 or 107, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence.

[0176]

[20] The fusion protein according to any one of [1] to

[19] above, which is a trispecific antibody against (a) 5T4, (b) CD3 receptor complex, and (c) PD-1, PD-L1 or LAG3.

[0177]

[21] The fusion protein according to any one of [1] to

[20] above, which has anti-tumor activity.

[0178]

[22] The fusion protein according to

[21] above, wherein the tumor is a tumor in which 5T4 is expressed in tumor cells or cancer cells.

[0179]

[23] The fusion protein according to

[21] or

[22] above, wherein the tumor is at least one selected from the group consisting of human mesothelioma (e.g., pleural mesothelioma), human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human endometrial cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

[0180]

[24] A polynucleotide encoding the fusion protein according to any one of [1] to

[23] above.

[0181]

[25] A vector comprising the polynucleotide described in

[24] above.

[0182]

[26] A transformant obtained by introducing the vector described in

[25] above into a host cell.

[0183]

[27] A method for producing a fusion protein according to any one of [1] to

[23] , comprising: culturing the transformant according to

[26] , and recovering the fusion protein from the obtained culture.

[0184]

[28] A pharmaceutical composition comprising the fusion protein according to any one of [1] to

[23] above.

[0185]

[29] The pharmaceutical composition according to

[28] above, which is used for the treatment, prevention or diagnosis of tumors.

[0186]

[30] The pharmaceutical composition according to

[29] above, wherein the tumor is a tumor in which 5T4 is expressed in tumor cells or cancer cells.

[0187]

[31] The pharmaceutical composition according to

[29] or

[30] above, wherein the tumor is at least one selected from the group consisting of human mesothelioma (e.g., pleural mesothelioma), human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human endometrial cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

[0188]

[32] A method for treating, preventing or diagnosing a tumor, comprising administering the pharmaceutical composition described in any one of

[28] to

[31] above to a subject.

[0189]

[33] The method according to

[32] above, wherein the tumor is a tumor in which 5T4 is expressed in tumor cells or cancer cells.

[0190]

[34] The method according to

[32] or

[33] above, wherein the tumor is at least one selected from the group consisting of human mesothelioma (e.g., pleural mesothelioma), human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human uterine cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

[0191]

[35] A kit for treating, preventing or diagnosing a tumor, comprising the fusion protein according to any one of [1] to

[23] above.

[0192]

[36] The kit according to

[35] above, wherein the tumor is a tumor in which 5T4 is expressed in tumor cells or cancer cells.

[0193]

[37] A kit according to the above

[35] or

[36] , wherein the tumor is at least one selected from the group consisting of human mesothelioma (e.g., pleural mesothelioma), human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human endometrial cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

[0194] Effects of the Invention

[0195] According to the present invention, a fusion protein in the form of a multispecific tri-antibody having a significantly increased anti-tumor effect compared to the conventional fusion protein Tb535H as a bispecific tri-antibody can be provided. The fusion protein of the present invention is a trispecific tri-antibody that has a binding ability not only to 5T4 and CD3 receptor complexes, but also to PD-1, PD-L1 or LAG3 (i.e., also has immune checkpoint inhibitory activity), and can exert an excellent anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1It is a schematic diagram of the structure of the tri-antibody. A. Scheme of the construction of the 53X tri-antibody molecule. SP: human interleukin (IL)-2 signal peptide; VH_5T4, VL_5T4: amino acid sequences encoding the immunoglobulin heavy chain variable region and light chain variable region of Tb535H specific to human 5T4, respectively; CH1, CL: amino acid sequences encoding the human immunoglobulin heavy chain constant region 1 and kappa light chain constant region, respectively; VH_CD3, VL_CD3: amino acid sequences encoding the heavy chain variable region and light chain variable region of humanized OKT3 derived from constructing a human CD3-specific scFv, respectively; L1, L2: amino acid sequences with flexible linkers (GPGGGSPG (sequence number 6) and GGGGSGGGGSGGGGS [i.e., containing (GGGS)3] (sequence number 106)), respectively. VH_C and VL_C are amino acid sequences encoding the heavy chain variable region and light chain variable region derived from PD-1-1, PD-L1_1, 10.12, LAG3_1 or palivizumab for constructing scFvs specific for human PD-1, PD-L1 or LAG3. The palivizumab-derived scFv was used as an isotype human antibody-derived scFv control. 6xHis is an amino acid sequence encoding a hexahistidine tag. B. Schemes for the three antibody proteins constructed for Tb535H [(5T4)2×CD3], 53D [5T4×CD3×PD-1], 53L1 [5T4×CD3×PD-L1], 53L10 [5T4×CD3×PD-L1], 53G [5T4×CD3×LAG3] and 53P [5T4×CD3×isotype control], respectively.

[0197] Figure 2 Figures showing the binding affinity of Tb535H and the newly constructed 53X tri-antibody to recombinant 5T4 protein in ELISA. A. Binding curve of the tri-antibody (0-500 nM) to recombinant human 5T4 protein based on ELISA test. B. EC of 5T4 binding of each construct 50 (nM) value.

[0198] Figure 3 Figures showing the binding affinity of Tb535H and the newly constructed 53X tri-antibody to recombinant CD3 protein in ELISA. A. Binding curve of the tri-antibody (0-500 nM) to recombinant human CD3δ / ε heterodimer protein based on ELISA test. B. CD3 binding EC of each tri-antibody construct 50 (nM) value.

[0199] Figure 4The following are graphs showing the binding affinity of the three antibodies to the recombinant PD-L1 protein or PD-1 protein in ELISA. A. Binding curves of the 53L1 three antibodies and the 53L10 three antibodies (0-500 nM) to the recombinant human PD-L1 protein based on ELISA test. B. Binding curves of the 53D three antibodies (0-500 nM) to the recombinant human PD-1 protein based on ELISA test.

[0200] Figure 5 Figures showing a T cell activation bioassay in the presence of 5T4-expressing target cells. A. Schematic diagram of TCR / CD3 activation in the presence of 5T4-expressing cells based on the T cell activation bioassay (NFAT) (Promega, catalog number J1621). B. A genetically modified Jurkat T cell line expressing a luciferase reporter gene driven by an NFAT response element (NFAT-RE) ("TCR / CD3 effector cell") was incubated in the presence of CHO-K1-5T4 cells while increasing the concentrations of the three antibodies shown in the figure. After incubation at 37°C for 4 hours, Bio-Glo (trademark) reagent was added and luminescence was measured using a photometer. C. EC of 5T4-mediated TCR / CD3 activation of each construct 50 (nM) value.

[0201] Figure 6 It is a diagram showing the antagonistic effect of 53X tri-antibodies measured by competitive ELISA assay. The ELISA assay was performed based on the determination of the binding of each biotin-labeled recombinant human PD-L1 or MHCII protein (striped pattern column) to PD-1 or LAG-3 immobilized protein, respectively, in the absence or presence of unlabeled competitive anti-PD-L1 (53L1 and 51L10) (A), anti-PD-1 (53D) (B) or anti-LAG-3 (53G) (C) tri-antibodies (black columns) or each parent mAb (gray columns) (used at saturating concentrations (5:1 in the case of anti-PD-L1 or anti-PD-1, 3:1 in the case of anti-LAG-3)). Human IgG4 was used as an isotype control (open column). Binding values ​​are reported as the average of at least 3 measurements obtained in 3 independent experiments. Shown as mean ± standard deviation (SD). P values ​​based on Student's t-test (2 variables) are shown below. **P<0.01, *P<0.05.

[0202] Figure 7It is a diagram showing the cell killing activity of the new three antibodies or Tb535H alone or in combination with the parent mAb for MDA-MB-231 human breast cancer cells co-cultured with hPBMC. MDA-MB-231 human breast cancer cells were co-cultured with hPBMC (effector: target cell ratio 5: 1) and treated with 53L1 and 53L10 (A. striped pattern column), 53D (B. striped pattern column), 53G (C: striped pattern column) for 48 hours. Tb535H (bright gray column), parental immunomodulatory mAb (dark gray column) or their combination (black column) were also tested at the concentrations shown. Untreated co-culture or three antibodies 53P (C: hollow column) were used as negative controls. Cell lysis was determined by detecting LDH release as described in the "Materials and Methods" described later. It is shown in the form of mean ± SD. The P value based on the student's t test (2 variables) is shown below. ***P<0.001, **P<0.01, *P<0.05.

[0203] Figure 8 Graph showing the cell killing activity of the novel tri-antibody or Tb535H alone or in combination with the parent mAb for A549 human lung cancer cells co-cultured with hPBMC. A549 human lung cancer cells were co-cultured with hPBMC (effector: target cell ratio 5:1) and treated with 53L1 and 53L10 (A. striped pattern columns), 53D (B. striped pattern columns), 53G (C: striped pattern columns) for 48 hours. Tb535H (bright gray columns), parental immunomodulatory mAb (dark gray columns) or their combinations (black columns) were also tested at the indicated concentrations. Untreated co-culture or tri-antibody 53P (C: open columns) was used as a negative control. Cell lysis was determined by detecting LDH release as described in the "Materials and Methods" described later. Shown as mean ± SD. P values ​​based on Student's t-test (2 variables) are shown below. ***P<0.001, **P<0.01, *P<0.05.

[0204] Fig. 9It is a figure showing the effect of IFN-γ secretion brought about by administration of new three antibodies or Tb535H alone or in combination with parent mAb based on co-culture of MDA-MB-231 human breast cancer cells and hPBMC. For the culture supernatant of co-culture of MDA-MB-231 human breast cancer cells and hPBMC treated with 53L1 and 53L10 (A. striped pattern column), 53D (B. striped pattern column), 53G (C. striped pattern column) for 48 hours, IFN-γ secretion was determined by ELISA. For Tb535H (bright gray column), parental immunomodulatory mAb (dark gray column) or their combination (black column), the concentration shown in the figure was also tested. Co-culture without treatment or three antibodies 53P (C: hollow column) was used as a negative control. It is shown in the form of mean ± SD. The P value based on Student's t test (2 variables) is as follows. ***P<0.001, **P<0.01, *P<0.05.

[0205] Fig.10 Figures showing the anti-tumor effects of Tb535H and the new tri-antibodies 53L1 and 53L10 in vivo. (A) is the tumor growth curve of A549 subcutaneous tumor in the presence of human PBMCs administered with Tb535H, (B) is the tumor growth curve of A549 subcutaneous tumor in the presence of human PBMCs administered with 53L1, and (C) is the tumor growth curve of A549 subcutaneous tumor in the presence of human PBMCs administered with 53L10. ●: solvent (PBS) administration, ○: 2 μg / mouse administration, △: 20 μg / mouse administration. 5×10 6 A549 human lung cancer cells were subcutaneously transplanted into the right flank of NOD-scid mice together with an equal number of human PBMCs (hPBMCs). hPBMCs were activated by stimulating with Dynabeads Human T-Activator CD3 / CD28 (Veritas) for 4 days before transplantation. The dosage of the test drug shown in the figure was intravenously administered on days 0, 2, 4, 6, 8 and 10 after cell transplantation (a total of 6 doses). Tumor volume is shown as mean ± standard deviation (SD). The significance test was performed by Dunnett's test (relative to the solvent-dosed group). *P < 0.05.

[0206] Fig.11 Figure 1 shows the tumor regression activity of 53L10 tri-antibody in different experimental groups. Figure 2 shows the tumor regression activity of 53L10 tri-antibody in A549 xenograft model. Preparation of A549 cells and hPBMC, subcutaneous transplantation of mixed cells of A549 and hPBMC and Fig.10The same procedure was performed. 53L10 was administered intravenously at a dosage of 2 μg / mouse (○) or 20 μg / mouse (△) on days 0, 2, 4, 6, 8, and 10. The tumor growth of the non-administered group (●) was used as a control group. Tumor volume is shown in the form of mean ± standard deviation (SD). The significance of the difference was tested by Dunnett's test (relative to the non-administered group). *P < 0.05.

[0207] Fig.12 It is a graph showing the anti-tumor effect of 53D and 53G in vivo compared with 53P. (A) Tumor proliferation curve of A549 subcutaneous tumor in the presence of human PBMCs administered with 53P (negative control), (B) Tumor proliferation curve of A549 subcutaneous tumor in the presence of human PBMCs administered with 53D, (C) Tumor proliferation curve of A549 subcutaneous tumor in the presence of human PBMCs administered with 53G. ●: solvent (PBS) administration, ○: 2μg / mouse administration, △: 20μg / mouse administration. Tumor volume is shown in the form of mean ± standard deviation (SD). The difference significance test was performed by Dunnett's test (relative to the solvent administration group). *P<0.05.

[0208] Fig.13A Figures showing the anti-tumor effects of Tb535H, the combination of Tb535H and pembrolizumab, and the novel tri-antibody 53L10 in vivo. A. Tumor growth curve of A549 subcutaneous tumor in the presence of human PBMCs administered with Tb535H or the novel tri-antibody 53L10. B. Tumor growth curve of A549 subcutaneous tumor in the presence of human PBMCs administered with the combination of Tb535H and pembrolizumab or the novel tri-antibody 53L10.

[0209] Fig. 13B :Refer to the above Fig.13A Instructions in .

[0210] Fig.14 Figure 2 is a graph showing the binding affinity of 53L10 and 53L10-M13 to recombinant 5T4 protein in ELISA (N=2). A. Binding curve of 53L10 and 53L10-M13 (0-500 nM) to recombinant human 5T4 protein based on ELISA assay. B. EC of 5T4 binding of each construct 50 (nM) value.

[0211] Fig.15 Figure 2 is a graph showing the binding affinity of 53L10 and 53L10-M13 to recombinant CD3 protein in ELISA (N=2). A. Binding curve of 53L10 and 53L10-M13 (0-500 nM) to recombinant human CD3δ / ε heterodimer protein based on ELISA assay. B. EC of CD3 binding of each construct50 (nM) value.

[0212] Fig.16 Figure 2 is a graph showing the binding affinity of 53L10 and 53L10-M13 to recombinant PD-L1 protein in ELISA (N=2). A. Binding curve of 53L10 and 53L10-M13 (0-500 nM) to recombinant human PD-L1 protein based on ELISA assay. B. EC of PD-L1 binding of each construct 50 (nM) value.

[0213] Fig.17 Figures showing bioassays of T cell activation in the presence of 5T4 expressing target cells (N=2). A. Genetically modified Jurkat T cell lines expressing a luciferase reporter gene driven by the NFAT response element (NFAT-RE) ("TCR / CD3 effector cells") were incubated in the presence of CHO-K1-5T4 cells with increasing concentrations of the three antibodies shown in the figure. After incubation at 37°C for 6 hours, Bio-Glo (trademark) reagent was added and luminescence was measured using a luminometer. B. 5T4-mediated TCR / CD3 activation of ECs for each construct 50 (nM) value.

[0214] Figure 18A-C Figures showing the T cell activation marker (CD25, CD69) assay of 53L10-M13 and Tb535H in the presence of h5T4, hPD-L1 expressing target cancer cells (A549 human lung cancer cells). A. Flow cytometry was used to confirm the expression of target molecules (h5T4, hPD-L1) on A549 human lung cancer cells. B. The proportion of CD69 positive cells in CD3 positive cells measured by flow cytometry. C. The proportion of CD25 positive cells in CD3 positive cells measured by flow cytometry. D. The EC of T cell activation of each construct evaluated by each T cell activation marker 50 (pM) value and E max (%) value and its 95% confidence interval (CI). nc (Not calculated) means it cannot be calculated.

[0215] Fig.18D :Refer to the above Figure 18A-C Instructions in .

[0216] Fig.19The graphs show the T cell activation marker (CD25, CD69) measurement test of 53L10-M13 and Tb535H in the absence of target cancer cell lines. A. The ratio of CD69 positive cells in CD3 positive cells measured by flow cytometry. B. The ratio of CD25 positive cells in CD3 positive cells measured by flow cytometry.

[0217] Figure 20A-C Figures showing the T cell activation marker (CD25, CD69) assay of 53L10-M13 and Tb535H in the presence of h5T4-expressing target cancer cells (MCF-7 human breast cancer cells). A. Flow cytometry was used to confirm the expression of target molecules (h5T4, hPD-L1) on MCF-7 human breast cancer cells. B. The proportion of CD69-positive cells in CD3-positive cells measured by flow cytometry. C. The proportion of CD25-positive cells in CD3-positive cells measured by flow cytometry. D. The EC of T cell activation of each construct evaluated by each T cell activation marker 50 (pM) value and E max (%) values ​​and their 95% confidence intervals.

[0218] Fig.20D :Refer to the above Figure 20A-C Instructions in .

[0219] Fig.21A The graphs show the concentrations of free cytokines in the culture supernatant in the T cell activation test. A. hTNFα concentration measured by ELISA. B. hIL-2 concentration measured by ELISA. C. hIL-6 concentration measured by ELISA. D. human interferon γ (hIFNγ) concentration measured by ELISA.

[0220] Fig. 21B Refer to the above Fig.21A Instructions in .

[0221] Fig. 21C Refer to the above Fig.21A Instructions in .

[0222] Fig.21D Refer to the above Fig.21A Instructions in .

[0223] Fig. 22 The graphs show the cytotoxicity when hPBMC (without activation treatment) and 53L10-M13 or Tb535H were added to h5T4 and hPD-L1 expressing target cancer cells (A549 human lung cancer cells). A. Cytotoxicity measured by flow cytometry. B. EC of the cell barrier activity of each construct 50 (pM) value and E max(%) values ​​and their 95% confidence intervals.

[0224] Fig.23 The graphs show the cytotoxicity when activated hPBMC and 53L10-M13 or Tb535H were added to h5T4 and hPD-L1 expressing target cancer cells (A549 human lung cancer cells). A. Cytotoxicity measured by flow cytometry. B. EC of the cell barrier activity of each construct 50 (pM) value and E max (%) values ​​and their 95% confidence intervals. DETAILED DESCRIPTION

[0225] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and matters other than the following examples can be appropriately changed and implemented within the scope that does not impair the gist of the present invention.

[0226] It should be noted that this specification includes the entirety of Japanese Patent Application No. 2022-106054 (filed on June 30, 2022), which is the basis for the priority claim of this application. In addition, all publications cited in this specification, such as prior art documents and invention gazettes, patent gazettes, and other patent documents, are incorporated into this specification by reference.

[0227] 1. Summary of the Invention

[0228] As the fusion protein of the present invention, a new trispecific multifunctional triantibody (also referred to as a trispecific antibody; 53X triantibody) is constructed. As the new 53X triantibody, for example, it is composed of 5T4 binding Fab and CD3 binding scFv, but unlike the parent protein Tb535H, the scFv of the remnant can be listed as scFv derived from immune checkpoint molecule-specific antibodies such as PD-1, PD-L1, and LAG-3 (for example, refer to Patent Document 2 and Non-Patent Document 10 described above). Compared with the bispecific T cell derivative targeting 5T4, i.e., the parent protein Tb535H, the new 53X triantibody maintains the same binding properties to 5T4 and CD3 receptor complexes as the Tb535H triantibody, and integrates checkpoint inhibition activity in a single molecule in addition to 5T4-mediated TCR / CD3 activation, further showing an increase in anti-tumor effect. In particular, the new 53X tri-antibody named 53L10, a trispecific T cell derivative targeting the 5T4, CD3, and PD-L1 immune checkpoints, showed the most potent anti-tumor effects both in vitro and in vivo.

[0229] 2. Fusion protein (53X triple antibody)

[0230] The fusion protein of the present invention is a fusion protein comprising two different chains, the first chain and the second chain.

[0231] The first chain contains two antibody variable regions VH1 and VH2, one antibody variable region VL2, and one antibody constant region CH1 or CL.

[0232] The second chain contains two antibody variable regions VL1 and VL3, one antibody variable region VH3, and one antibody constant region CL or CH1.

[0233] Here, "antibody variable region" is a motif of a polypeptide constituting an antibody molecule that can specifically bind or interact with a given antigen (protein, etc.) and an epitope in the antigen. The above-mentioned VH1 to VH3 can all be referred to as a polypeptide consisting of or comprising a heavy chain variable region in an antibody molecule, and the above-mentioned VL1 to VL3 can all be referred to as a polypeptide consisting of or comprising a light chain variable region in an antibody molecule. In addition, VH1 and VL1, VH2 and VL2, VH3 and VL3 form a binding domain with a given antigen by combining with each other. In the present invention, it is referred to as a "combined VH / VL binding domain", and each combination is referred to as a "VH1 / VL1 binding domain", "VH2 / VL2 binding domain" and "VH3 / VL3 binding domain". In addition, the "antibody constant region" is a motif of a polypeptide constituting an antibody molecule, which does not directly participate in antigen binding, but exhibits various effector functions such as antibody dependence, cell-mediated cell killing and complement activation.

[0234] In the fusion protein of the present invention, there is a heterodimer interaction between the first chain and the second chain. The heterodimer interaction can be between the CH1 of one chain and the CL domain of the other chain, or between VH1 and VL1, preferably between the CH1 of one chain and the CL domain of the other chain. Preferably, the fusion protein of the present invention becomes a heterodimer protein of the first chain and the second chain through these interactions. The above-mentioned heterodimer interaction is not limited, and the existing known interactions for heterodimerization in Fab are generally preferred. Fab refers to: a heterodimer between an Fd chain comprising a VH and CH1 antibody domain and an L chain comprising a VL and CL antibody domain, and for the heterodimerization, an interaction (such as a disulfide bond, etc.) is formed between VH1 and VL1 and / or between CH1 and CL. Regarding this interaction, for example, reference can be made to Patent Document 3 described above.

[0235] The fusion protein of the present invention forms the above three combined VH / VL binding domains in the protein, namely, the VH1 / VL1 binding domain, the VH2 / VL2 binding domain and the VH3 / VL3 binding domain. These three binding domains have binding abilities to different substances (antigen proteins). Specifically, it is characterized in that they have any binding abilities selected from the following (i) to (iii).

[0236] (i) Binding ability to 5T4 (tumor-associated antigen (TAA));

[0237] (ii) binding ability to the CD3 receptor complex; and

[0238] (iii) The ability to bind to PD-1 (Programmed death receptor-1), PD-L1 (Programmed cell Death 1-Ligand 1) or LAG3 (Lymphocyte activation gene 3: CD223).

[0239] Combination VH / VL binding domains (or VH and VL themselves) that have binding ability to 5T4 (tumor-associated antigen (TAA)) can be appropriately constructed and obtained, for example, by obtaining or preparing 5T4 as an antigen and preparing a polyclonal or monoclonal antibody against the antigen, and then preparing a desired fragment (VH, VL) derived from the antibody. By the same method, various combination VH / VL binding domains (or VH and VL themselves) that have binding ability to the CD3 receptor complex, binding ability to PD-1, binding ability to PD-L1, and binding ability to LAG3 can also be appropriately constructed and obtained. For detailed and illustrative descriptions of the preparation of such antigens, the preparation of polyclonal or monoclonal antibodies, the preparation of genetically recombinant antibodies (chimeric antibodies, humanized antibodies (humanized antibodies) and human antibodies (fully human antibodies)), and the preparation of antibody fragments, for example, the descriptions in WO 2014 / 054820 and WO 2013 / 077458 can be appropriately referred to and applied.

[0240] The information on the amino acid sequence of human 5T4 (SEQ ID NO: 96) is published, for example, on the Uniprot website (https: / / www.uniprot.org / (hereinafter the same)) as “Primary (citable) accession number: Q13641”.

[0241] The amino acid sequence of the CD3ε subunit of the human CD3 receptor complex (SEQ ID NO: 97) is published on the Uniprot website as “Primary (citable) Accession No.: P07766”, the amino acid sequence of the CD3δ subunit (isoform 1) (SEQ ID NO: 98) is published on the Uniprot website as “Primary (citable) Accession No.: P04234-1”, and the amino acid sequence of the CD3δ subunit (isoform 2) (SEQ ID NO: 99) is published on the Uniprot website as “Primary (citable) Accession No.: P04234-2”.

[0242] The information of the amino acid sequence of human PD-1 (SEQ ID NO: 100) is published on the Uniprot website as “Primary (citable) accession number: Q15116”,

[0243] The information of the amino acid sequence (SEQ ID NO. 101) of human PD-L1 (isoform 1) is published, for example, on the Uniprot website as “Primary (citable) Accession No.: Q9NZQ7-1”, the information of the amino acid sequence (SEQ ID NO. 102) of human PD-L1 (isoform 2) is published, for example, on the Uniprot website as “Primary (citable) Accession No.: Q9NZQ7-2”, and the information of the amino acid sequence (SEQ ID NO. 103) of human PD-L1 (isoform 3) is published, for example, on the Uniprot website as “Primary (citable) Accession No.: Q9NZQ7-3”.

[0244] The amino acid sequence of human LAG3 (isoform 1) (SEQ ID NO: 104) is published on the Uniprot website as "Primary (citable) Accession No.: P18627-1", and the amino acid sequence of human LAG3 (isoform 2) (SEQ ID NO: 105) is published on the Uniprot website as "Primary (citable) Accession No.: P18627-2". These sequence information can be used appropriately in the preparation of antigens, and the production or preparation of antibodies and antibody fragments.

[0245] VH1, VH2 and VH3 in the fusion protein of the present invention have mutually different complementary determining regions (CDR) 1, CDR2 and CDR3, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 that can be used by VH1, VH2 and VH3 are not limited, and preferably, for example, they are respectively composed of the following in sequence:

[0246] The amino acid sequences shown in SEQ ID NOs: 3, 4 and 5,

[0247] The amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11,

[0248] The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 or 27 (it should be noted that the amino acid sequence of SEQ ID NO: 27 can be used instead of the amino acid sequence of SEQ ID NO: 26.),

[0249] The amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39,

[0250] The amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 48 or 49 and SEQ ID NO: 50, or

[0251] The amino acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59 or 49 and SEQ ID NO: 60.

[0252] Similarly, VL1, VL2 and VL3 in the fusion protein of the present invention have mutually different complementary determining regions (CDR) 1, CDR2 and CDR3, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 that can be used by VL1, VL2 and VL3 are not limited, and preferably, for example, they are composed of the following in sequence:

[0253] The amino acid sequences shown in SEQ ID NOs: 19, 20 and 21,

[0254] The amino acid sequences shown in SEQ ID NOs: 14, 15 and 16,

[0255] The amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, 32, or 33 (it should be noted that the amino acid sequences of SEQ ID NO: 32 and 33 can be used instead of the amino acid sequence of SEQ ID NO: 31.),

[0256] The amino acid sequences shown in SEQ ID NOs: 41, 42 and 43,

[0257] The amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, or

[0258] The amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64.

[0259] In addition, the amino acid sequences of VH1, VH2 and VH3 in the fusion protein of the present invention are different from each other, and preferably, for example, those composed of the amino acid sequence shown in SEQ ID NOs: 88, 90, 8, 92, 23, 78, 46, 81, 57, 109, 82, 83, 84 or 36, those containing the amino acid sequence, or those having at least 90% (or, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence. Here, the amino acid of SEQ ID NO. 78 can be preferably used instead of the amino acid of SEQ ID NO. 23, the amino acid of SEQ ID NO. 81 can be preferably used instead of the amino acid of SEQ ID NO. 46, and the amino acids of SEQ ID NOs. 82 to 84 and 109 can be preferably used instead of the amino acid of SEQ ID NO. 57.

[0260] On this basis, the amino acid sequences of VL1, VL2 and VL3 in the fusion protein of the present invention are different from each other, and preferably, for example, those composed of the amino acid sequence shown in sequence numbers 89, 91, 13, 93, 28, 79, 80, 51, 61, 85, 86, 87 or 40, those containing the amino acid sequence or those having at least 90% (or, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) identity with the amino acid sequence. Here, the amino acids of sequence numbers 79 and 80 can be preferably used to replace the amino acid of sequence number 28, and the amino acids of sequence numbers 85 to 87 can be preferably used to replace the amino acid of sequence number 61.

[0261] Furthermore, in the fusion protein of the present invention, the first chain is preferably composed of, contains, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence shown in, for example, SEQ ID NO: 1. On this basis, the second chain is preferably composed of, contains, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence shown in, for example, SEQ ID NO: 17, 34, 44, 55, or 107.

[0262] As another embodiment of the fusion protein of the present invention, for example, the combination of the two different chains, the first chain and the second chain, is preferably a combination shown in the following a) to l).

[0263] a) Chain 1:

[0264] VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0265] VH(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0266] Chain 2:

[0267] VL(5T4)-CL-L1-VH(X)-L2-VL(X) or

[0268] VL(5T4)-CL-L1-VL(X)-L2-VH(X)

[0269] combination.

[0270] b) Chain 1:

[0271] VH(5T4)-CH1-L1-VH(X)-L2-VL(X) or

[0272] VH(5T4)-CH1-L1-VL(X)-L2-VH(X), and

[0273] Chain 2:

[0274] VL(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0275] VL(5T4)-CL-L1-VL(CD3)-L2-VH(CD3)

[0276] combination.

[0277] c) Chain 1:

[0278] VL(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0279] VL(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0280] Chain 2:

[0281] VH(5T4)-CL-L1-VH(X)-L2-VL(X) or

[0282] VH(5T4)-CL-L1-VL(X)-L2-VH(X)

[0283] combination.

[0284] d) Chain 1:

[0285] VL(5T4)-CH1-L1-VH(X)-L2-VL(X) or

[0286] VL(5T4)-CH1-L1-VL(X)-L2-VH(X), and

[0287] Chain 2:

[0288] VH(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0289] VH(5T4)-CL-L1-VL(CD3)-L2-VH(CD3)

[0290] combination.

[0291] e) Chain 1:

[0292] VH(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0293] VH(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0294] Chain 2:

[0295] VL(CD3)-CL-L1-VH(X)-L2-VL(X) or

[0296] VL(CD3)-CL-L1-VL(X)-L2-VH(X)

[0297] combination.

[0298] f) Chain 1:

[0299] VH(CD3)-CH1-L1-VH(X)-L2-VL(X) or

[0300] VH(CD3)-CH1-L1-VL(X)-L2-VH(X), and

[0301] Chain 2:

[0302] VL(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0303] VL(CD3)-CL-L1-VL(5T4)-L2-VH(5T4)

[0304] combination.

[0305] g) Chain 1:

[0306] VL(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0307] VL(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0308] Chain 2:

[0309] VH(CD3)-CL-L1-VH(X)-L2-VL(X) or

[0310] VH(CD3)-CL-L1-VL(X)-L2-VH(X)

[0311] combination.

[0312] h) First chain:

[0313] VL(CD3)-CH1-L1-VH(X)-L2-VL(X) or

[0314] VL(CD3)-CH1-L1-VL(X)-L2-VH(X), and

[0315] Chain 2:

[0316] VH(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0317] VH(CD3)-CL-L1-VL(5T4)-L2-VH(5T4)

[0318] combination.

[0319] i) Chain 1:

[0320] VH(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0321] VH(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0322] Chain 2:

[0323] VL(X)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0324] VL(X)-CL-L1-VL(CD3)-L2-VH(CD3)

[0325] combination.

[0326] j) Chain 1:

[0327] VH(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0328] VH(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0329] Chain 2:

[0330] VL(X)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0331] VL(X)-CL-L1-VL(5T4)-L2-VH(5T4)

[0332] combination.

[0333] k) Chain 1:

[0334] VL(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or

[0335] VL(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and

[0336] Chain 2:

[0337] VH(X)-CL-L1-VH(CD3)-L2-VL(CD3) or

[0338] VH(X)-CL-L1-VL(CD3)-L2-VH(CD3)

[0339] combination.

[0340] l) Chain 1:

[0341] VL(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or

[0342] VL(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and

[0343] Chain 2:

[0344] VH(X)-CL-L1-VH(5T4)-L2-VL(5T4) or

[0345] VH(X)-CL-L1-VL(5T4)-L2-VH(5T4)

[0346] combination.

[0347] Here, in the first chain and / or the second chain shown in a) to l) above,

[0348] VH and VL are antibody variable regions.

[0349] VH(5T4) and VL(5T4) are the variable regions of the antibody against 5T4.

[0350] VH(CD3) and VL(CD3) are the variable regions of antibodies targeting the CD3 receptor complex.

[0351] VH(X) and VL(X) are variable regions of antibodies targeting PD-1, PD-L1 or LAG3.

[0352] CH1 and CL are antibody constant regions.

[0353] L1 and L2 are connectors.

[0354] In addition, more specifically, it is preferred that:

[0355] The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH(5T4) are respectively composed of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and

[0356] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(5T4) are respectively composed of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0357] Furthermore, it is more preferred that the amino acid sequence of VH(5T4) consists of the amino acid sequence of SEQ ID NO: 88 or 90, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence, and

[0358] The amino acid sequence of VL(5T4) is composed of the amino acid sequence shown in sequence number 89 or 91, contains the amino acid sequence, or has at least 90% (or, it can be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0359] In addition, more specifically, it is preferred that:

[0360] The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH (CD3) are respectively composed of the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and

[0361] The amino acid sequences of CDR1, CDR2 and CDR3 of VL (CD3) are respectively composed of the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16,

[0362] Furthermore, it is more preferred that the amino acid sequence of VH(CD3) consists of the amino acid sequence of SEQ ID NO: 8 or 92, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence, and

[0363] The amino acid sequence of VL (CD3) is composed of the amino acid sequence shown in SEQ ID NO: 13 or 93, comprises the amino acid sequence, or has at least 90% (or, it may be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0364] In addition, more specifically, it is preferred that:

[0365] The amino acid sequences of the complementarity determining region (CDR) 1, CDR2, and CDR3 of VH(X) (wherein X is PD-1) are respectively composed of the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 or 27, and

[0366] The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) (wherein X is PD-1) are respectively composed of the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, 32 or 33,

[0367] Furthermore, it is more preferred that the amino acid sequence of VH(X) (wherein X is PD-1) consists of the amino acid sequence of SEQ ID NO: 23 or 78, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence, and

[0368] The amino acid sequence of VL(X) (wherein X is PD-1) is composed of the amino acid sequence shown in SEQ ID NO: 28, 79 or 80, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0369] In addition, more specifically, it is preferred that:

[0370] The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH(X) (wherein X is PD-L1 (more specifically, PD-L1.1)) are respectively composed of the amino acid sequences shown in SEQ ID NO: 47, SEQ ID NO: 48 or 49, and SEQ ID NO: 50, and

[0371] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(X) (wherein X is PD-L1 (more specifically, PD-L1.1)) are respectively composed of the amino acid sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54,

[0372] Furthermore, it is more preferred that the amino acid sequence of VH(X) (wherein X is PD-L1 (more specifically, PD-L1.1)) consists of the amino acid sequence of SEQ ID NO: 46 or 81, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence, and

[0373] The amino acid sequence of VL(X) (wherein X is PD-L1 (more specifically, PD-L1.1)) is composed of the amino acid sequence shown in SEQ ID NO: 51, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence.

[0374] In addition, more specifically, it is preferred that:

[0375] The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH(X) (wherein X is PD-L1 (more specifically, 10.12)) are respectively composed of the amino acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59 or 49, and SEQ ID NO: 60, and

[0376] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(X) (wherein X is PD-L1 (more specifically, 10.12)) are respectively composed of the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64,

[0377] Furthermore, it is more preferred that the amino acid sequence of VH(X) (wherein X is PD-L1 (more specifically, 10.12)) consists of the amino acid sequence of SEQ ID NO: 57, 109, 82, 83 or 84, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence, and

[0378] The amino acid sequence of VL(X) (wherein X is PD-L1 (more specifically, 10.12)) is composed of the amino acid sequence shown in SEQ ID NO: 61, 85, 86 or 87, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0379] In addition, more specifically, it is preferred that:

[0380] The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH(X) (wherein X is LAG3) are respectively composed of the amino acid sequences set forth in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, and

[0381] The amino acid sequences of CDR1, CDR2, and CDR3 of VL(X) (wherein X is LAG3) are composed of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.

[0382] Furthermore, it is more preferred that the amino acid sequence of VH(X) (wherein X is LAG3) consists of the amino acid sequence of SEQ ID NO: 36, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence, and

[0383] The amino acid sequence of VL(X) (wherein X is LAG3) is composed of the amino acid sequence shown in SEQ ID NO: 40, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence.

[0384] In addition, more specifically,

[0385] The amino acid sequence of CH1 as the antibody constant region is preferably composed of the amino acid sequence set forth in SEQ ID NO: 94, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence.

[0386] The amino acid sequence of CL as the antibody constant region is preferably composed of the amino acid sequence set forth in SEQ ID NO: 95, comprises the amino acid sequence, or has at least 90% (or may be 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence.

[0387] L1 as a linker is preferably composed of the amino acid sequence shown in SEQ ID NO: 6, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identity with the amino acid sequence.

[0388] L2 as a linker is preferably composed of the amino acid sequence shown in sequence number 12 or 106, contains the amino acid sequence, or has at least 90% (or, it can be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0389] More specifically, the fusion protein of the present invention is more preferably:

[0390] Chain 1: VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) is composed of the amino acid sequence of SEQ ID NO: 1, comprises the amino acid sequence, or has at least 90% (or 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence, and

[0391] Chain 2: VL(5T4)CL-L1-VH(X)-L2-VL(X) is composed of the amino acid sequence shown in sequence number 17, 34, 44, 55 or 107, contains the amino acid sequence, or has at least 90% (or, it can be 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) identity with the amino acid sequence.

[0392] Regarding the fusion proteins of the present invention included in this preferred embodiment, a list of sequence numbers indicating the amino acid sequences of the full lengths of H chain derivatives and various L chain derivatives and their respective domains, regions, and linkers is shown below.

[0393] [Table A]

[0394]

[0395] Specifically, the fusion protein of the present invention as described above is preferably a trispecific antibody (tri-antibody) for (a) 5T4, (b) CD3 receptor complex of T cells and (c) PD-1, PD-L1 or LAG3, whereby the antibody has anti-tumor activity, and compared with the previous Tb535H tri-antibody and the therapy using it in combination with anti-PD-1 antibody and anti-PD-L1 antibody, the anti-tumor effect is significantly increased. Among them, especially in terms of excellent anti-tumor effect, a tri-specific antibody for (a) 5T4, (b) CD3 receptor complex of T cells and (c) PD-L1 is preferred. Therefore, the present invention also provides a fusion protein in the form of a tri-specific antibody that can be used for the treatment, prevention or diagnosis of tumors or cancers.

[0396] Here, as the above-mentioned tumor, there is no limitation, and preferably, those expressing 5T4 in tumor cells or cancer cells are preferred, and specifically, for example, at least one selected from the group consisting of human mesothelioma (e.g., pleural mesothelioma), human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human endometrial cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminomatous germ cell tumor can be preferably cited. The examples of these tumors are also applicable in the following description.

[0397] 3. Polynucleotides, recombinant vectors and transformants

[0398] In the present invention, a polynucleotide (gene, DNA) encoding the above-mentioned fusion protein of the present invention (53X three antibodies) can also be provided. As the polynucleotide, specifically, it is preferred to include a polynucleotide encoding the base sequence of each amino acid sequence shown as an example of the fusion protein of the present invention. In addition, the polynucleotide of the present invention may be composed only of a polynucleotide encoding the fusion protein of the present invention, or it may include the polynucleotide in a part and a known base sequence (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression in other parts and / or an additional sequence (or tag sequence, such as 6×His sequence) that makes protein purification easy, without limitation.

[0399] As a polynucleotide encoding the fusion protein of the present invention, the codons corresponding to each amino acid after translation are not particularly limited, and may include nucleotide DNA that displays codons commonly used in mammals such as humans (preferably codons with high usage frequency) after transcription, and may also include nucleotide DNA that displays codons commonly used in microorganisms such as Escherichia coli and yeast, and plants (preferably codons with high usage frequency).

[0400] The present invention can also provide a recombinant vector comprising the polynucleotide of the present invention and a transformant comprising the recombinant vector.

[0401] In the polynucleotide (gene, DNA) to be integrated into the expression vector used as a recombinant vector, a transcription promoter, SD sequence (when the host is a prokaryotic cell) and Kozak sequence (when the host is a eukaryotic cell) may be connected in advance upstream, and a terminator may be connected downstream, and an enhancer, a splicing signal, a poly A addition signal, a selection marker, etc. may be connected. It should be noted that the various elements required for gene expression such as the above-mentioned transcription promoter may be included in the polynucleotide from the beginning, and when originally included in the expression vector, they may be directly used, and the use of each element is not particularly limited.

[0402] As a method for integrating the polynucleotide into an expression vector, various methods using known gene recombination techniques such as methods using restriction enzymes and methods using topoisomerases can be used. In addition, as an expression vector, there is no limitation as long as it is an expression vector such as plasmid DNA, phage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, etc. that can carry a polynucleotide (gene, DNA) encoding the fusion protein of the present invention, and a vector suitable for the host cell used can be appropriately selected and used.

[0403] Next, the constructed recombinant vector is introduced into a host to obtain a transformant, which is then cultured to express the fusion protein of the present invention. It should be noted that the "transformant" referred to in the present invention refers to a transformant obtained by introducing a foreign gene into a host, including, for example, a transformant obtained by introducing a plasmid DNA into a host (transformation) and a transformant obtained by infecting the host with various viruses and bacteriophages (transduction).

[0404] The host is not limited as long as it can express the fusion protein of the present invention after the introduction of the above-mentioned recombinant vector, and can be appropriately selected. Examples thereof include known hosts such as human, mouse and other animal cells, various plant cells, bacteria, yeast, and plant cells.

[0405] When animal cells are used as hosts, for example, human fibroblasts, human fetal kidney cells, HEK293 cells, 293F cells, CHO cells, monkey COS-7 cells, Vero cells, mouse L cells, rat GH3, human FL cells, etc. In addition, insect cells such as Sf9 cells and Sf21 cells can also be used.

[0406] When bacteria are used as hosts, for example, Escherichia coli, Bacillus subtilis, etc. can be used.

[0407] When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. can be used.

[0408] When plant cells are used as hosts, for example, tobacco BY-2 cells can be used.

[0409] The method for obtaining the transformant is not limited and can be appropriately selected in consideration of the combination of the host and the expression vector. For example, preferably mentioned are electroporation, lipofection, heat shock, PEG, calcium phosphate, DEAE dextran, and methods of infecting the transformant with various viruses such as DNA viruses and RNA viruses.

[0410] In the obtained transformant, the codon type of the polynucleotide contained in the recombinant vector may or may not be the same as the codon type of the host used, and there is no limitation.

[0411] In addition, the present invention also provides a method for producing the fusion protein (53X triantibody) comprising the steps of culturing the above-mentioned transformant and recovering (and further purifying) the fusion protein (53X triantibody) of the present invention from the obtained culture. Furthermore, the fusion protein (53X triantibody) obtained by the production method is also included in the present invention. It should be noted that the methods and conditions of the above-mentioned culture, recovery, purification, etc. can be appropriately selected and adopted based on the technical common sense and known insights of those skilled in the art.

[0412] 4. Pharmaceutical compositions, etc.

[0413] The fusion protein (53X triabody) of the present invention is useful as an active ingredient contained in a pharmaceutical composition.

[0414] As described above, the fusion protein of the present invention may have anti-tumor activity, so the fusion protein of the present invention is preferably used for the treatment, prevention and / or diagnosis of tumors. Therefore, the pharmaceutical composition is useful as a pharmaceutical composition for the treatment and / or prevention of tumors and further for diagnosis. That is, the fusion protein of the present invention is useful as an active ingredient contained in a tumor therapeutic agent and a tumor diagnostic agent. It should be noted that in the present invention, the treatment of the above-mentioned tumor also includes the meaning of tumor growth blocking and growth inhibition. Specifically, for example, a tumor therapeutic agent also includes the form of a tumor growth blocking agent and a growth inhibitor.

[0415] The pharmaceutical composition of the present invention is preferably provided in the form of a pharmaceutical composition comprising the fusion protein of the present invention as an active ingredient and further comprising a pharmaceutically acceptable carrier.

[0416] Regarding the tumor that becomes the application object of the pharmaceutical composition of the present invention, the above-mentioned tumor or cancer illustrative description can be applied in the same manner. The tumor that becomes the application object can be recurrent cancer or metastatic cancer, and the pharmaceutical composition of the present invention (and then the fusion protein of the present invention) can be effectively used as a therapeutic agent, preventive agent and diagnostic agent for recurrent cancer or metastatic cancer.

[0417] "Pharmaceutically acceptable carriers" include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, aromatics, colorants, sweeteners, thickeners, flavoring agents, solubilizers or other additives. By using more than one such carrier, pharmaceutical compositions in the form of injections, liquids, capsules, suspensions, emulsions or syrups can be prepared. These pharmaceutical compositions can be administered orally or parenterally. As other methods for parenteral administration, injections containing more than one active substance and formulated using conventional methods are included. In the case of injections, they can be manufactured by dissolving or suspending in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection.

[0418] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's age, sex, body weight and symptoms, therapeutic effect, administration method, treatment time or the type of fusion protein of the present invention contained in the pharmaceutical composition, etc. Generally, each adult can be administered in the range of 600 μg to 6000 mg per time, but is not limited to this range.

[0419] For example, when the drug is administered by injection, for human patients, the drug can be administered once to several times per day in an amount of 0.1 μg to 100 mg per kg of body weight in one administration. Preferably, the drug can be administered once every 3 days, 1 week, 10 days or 2 weeks or in a single administration (the total number of administrations is 1 time). As the mode of administration, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection, etc., are listed, preferably intravenous injection. In addition, according to the circumstances, the injection can be prepared in the form of a non-aqueous diluent (such as polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), a suspension or an emulsion. Such an injection can be sterilized by filtration sterilization using a filter, a bactericide, etc. The injection can be manufactured in a form prepared at the time of use. That is, a sterile solid composition can be made by freeze drying, etc., and dissolved in sterile distilled water for injection or other solvents before use.

[0420] It should be noted that the present invention also provides the use of the fusion protein of the present invention for manufacturing a drug (medicament) for treating, preventing and / or diagnosing tumors.

[0421] In addition, the present invention also provides the fusion protein of the present invention for use in treating, preventing and / or diagnosing tumors.

[0422] Furthermore, the present invention provides a method for treating, preventing and / or diagnosing a tumor characterized by using the fusion protein of the present invention (i.e., administering the fusion protein of the present invention to a subject (patient)). In addition, the present invention also provides the use of the fusion protein of the present invention for treating, preventing and / or diagnosing a tumor.

[0423] 5. Tumor diagnosis or detection kits, etc.

[0424] The fusion protein of the present invention can be provided as a kit for treating and / or preventing tumors, or as a kit for diagnosing or detecting tumors. The above description can be applied to specific examples of tumors to be diagnosed or detected.

[0425] This diagnosis and detection can be performed, for example, by reacting the fusion protein of the present invention with a sample collected from a living body (hereinafter referred to as a biological sample) and detecting a signal of an antibody or the like after the reaction.

[0426] The signal of the detected antibody, etc. becomes an indicator of the amount of antigen in the biological sample. In the diagnosis and detection of tumors using the fusion protein of the present invention, first, a biological sample collected from a subject as a test object, such as a tissue piece or blood as an inspection object, is combined with the fusion protein (trispecific antibody) of the present invention through an antigen-antibody reaction. Next, the amount of the target antigen in the biological sample is measured based on the measurement result of the amount of bound antibody, thereby performing the measurement. The measurement can be performed according to a known immunological assay, and for example, immunoprecipitation, immunoagglutination, labeled immunoassay, immunoturbidimetry, immunoblotting, flow cytometry, etc. can be used. In the labeled immunoassay, in addition to representing the signal of the antibody with the labeled amount directly detected using the labeled antibody, an antibody with a known concentration or a known antibody titer can also be made into a standard solution and relatively represented. That is, the standard solution and the test object can be measured using a measuring instrument, and the signal of the antibody, etc. in the biological sample can be relatively represented based on the value of the standard solution. Examples of the labeled immunoassay include ELISA, EI, RIA, fluorescent immunoassay (FIA), and chemiluminescence immunoassay, etc. Among them, the ELISA method is particularly preferred because of its simplicity and high sensitivity.

[0427] The test results obtained as above can be used as indicators to evaluate or diagnose the state of the tumor. For example, a test result exceeding a predetermined reference value can be regarded as positive, and a test result below a predetermined reference value can be regarded as negative. In the case of a positive result, it is judged that there is a possibility of suffering from any tumor, and the state of the tumor can be evaluated. Here, the state of the tumor refers to whether the patient suffers from a tumor or the degree of its progression, and examples include whether the patient suffers from a tumor, the degree of progression, the degree of malignancy, whether the patient relapses, etc. Regarding the tumor, examples include whether the patient has metastasized, etc.

[0428] In the above evaluation, one or more of the tumor states may be selected. The evaluation of the presence or absence of a tumor may be performed based on the obtained test results, using a predetermined reference value as a critical value to determine whether the patient has a tumor. The malignancy of a tumor is an indicator of the extent to which the symptoms of a tumor have progressed. A stage evaluation or classification evaluation may be performed based on the test results, such as early-stage cancer or advanced cancer. For example, early-stage cancer or advanced cancer may be evaluated using the test results as an indicator. The metastasis of a tumor may be evaluated using the test results as an indicator by determining whether a new tumor appears in a location away from the primary lesion. The recurrence of a tumor may be evaluated by determining whether the test results exceed a predetermined reference value again after an interval or remission.

[0429] Furthermore, the diagnosis and detection include, for example: reacting the fusion protein of the present invention with a sample collected from an organism (e.g., tumor cells and PBMCs derived from a patient), measuring the signal and cytocidal activity of the antibody after the reaction, etc., thereby serving as an indicator for measuring the effect of the antibody and a drug containing the antibody or determining whether to administer the drug.

[0430] The kit of the present invention may also include a labeling substance or a fixing agent fixed with other antibodies or their labels, etc., in addition to the fusion protein of the present invention. The labeling substance of the antibody refers to a person labeled by an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, etc. The kit of the present invention may also include other reagents for implementing the detection of the present invention, such as when the labeling substance is an enzyme label, an enzyme substrate (chromogenic substrate, etc.), an enzyme substrate dissolving solution, an enzyme reaction stopping solution, or a specimen diluent, etc., in addition. Various buffers, sterile water, various cell culture containers, various reaction containers (Eppendorf tubes, etc.), sealing agents (serum components such as bovine serum albumin (BSA), skim milk, goat serum, etc.), cleaning agents, surfactants, various plates, preservatives such as sodium azide, and experimental operation manuals (instructions), etc., may be included.

[0431] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0432] In this example, when describing various experiments and results related to the fusion protein (trispecific triabody) of the present invention, the item "Results" is described first, and then the item "Materials and Methods" is described.

[0433] Example 1

[0434] 1. Results

[0435] Construction and purification of the new 53X triple antibody

[0436] like Figure 1 A and Figure 1 As shown in B and listed in the following Table 1, a new multifunctional trispecific triabody was constructed.

[0437] [Table 1]

[0438]

[0439] In this example, a novel trispecific multifunctional triabody was generated. The triabody is a multispecific antibody composed of a Fab domain as a scaffold, an H chain (V H +CH1) and L chain (V L +C L)'s Fd fragment can naturally heterodimerize in vivo, and additional functions such as scFv can be integrated into the scaffold. Tb535H is a bispecific tri-antibody with bivalent binding to 5T4 via the Fab domain and the scFv domain and monovalent binding to CD3 via another scFv (Patent Document 1 mentioned above: WO 2016 / 097408 (A1)). The new 53X tri-antibody generated in this example has binding specificity to 5T4, CD3 and PD-L1 (or PD-1 or LAG3) with three different molecules in a single molecule. Specifically, the 5T4-binding scFv arm of the L chain-scFv derivative of Tb535H is replaced with:

[0440] The binding portion derived from PD-1_1 (SEQ ID NO: 22),

[0441] The binding portion derived from PD-L1_1 (SEQ ID NO: 45),

[0442] A binding portion derived from 10.12 (SEQ ID NOs: 56, 75, 76, 77; herein, SEQ ID NOs: 75, 76, 77 are examples of sequences in which any amino acid in SEQ ID NO: 56 is changed for structural stabilization, and a disulfide bond cross-linking sequence is introduced into the scFv) or

[0443] The binding portion derived from LAG3_1 (SEQ ID NO: 35),

[0444] or

[0445] The binding portion derived from Palivizumab (SEQ ID NO: 66) was used as a negative control.

[0446] The novel 53X tri-antibodies of the present invention constructed are also described in Table 1 above, and are respectively referred to as:

[0447] 53D[(Fab)5T4×(scFv)CD3×(scFv)PD-1(PD-1_1)],

[0448] 53L1[(Fab)5T4×(scFv)CD3×(scFv)PD-L1(PD-L1_1)],

[0449] 53L10[(Fab)5T4×(scFv)CD3×(scFv)PD-L1(10.12)],

[0450] 53G[(Fab)5T4×(scFv)CD3×(scFv)LAG3(LAG3_1)] and

[0451] 53P [(Fab)5T4×(scFv)CD3×(scFv)palivizumab] The 53P triabody was constructed to serve as a negative control for the other 53X triabodies.

[0452] Binding affinity of 53X trio antibodies to recombinant 5T4 based on enzyme-linked immunosorbent assay (ELISA)

[0453] In order to verify whether the newly developed trispecific 53X triantibodies 53D, 53L1, 53L10, 53G, and 53P retain the ability of Tb535H to bind to the 5T4 antigen, the binding affinity of the purified triantibodies to the 5T4 protein was investigated by ELISA ( Figure 2 ). The recombinant human 5T4 protein (AMSBio) was fixed to a 96-well ELISA plate. The binding test between Tb535H and the purified new tri-antibody was carried out according to the steps described in the "Materials and Methods" described below while increasing the concentration (0 to 500 nM). Figure 2 As shown in A, all three antibodies constructed showed concentration-dependent binding to 5T4, showing Kd (nM) values ​​that were equal to or greater than those of Tb535H ( Figure 2 B). Tb535H has two 5T4 binding arms derived from Fab and scFv, and the constructed new 53X triabodies have a monovalent 5T4 binding arm derived from a single Fab. Even so, the results of all the new 53X triabodies showed that a single arm (Fab) was sufficient to maintain the 5T4 binding of TB535H in the same manner.

[0454] Binding affinity of 53X trio antibodies to recombinant CD3 based on ELISA

[0455] In order to verify whether the newly constructed trispecific 53X triantibodies 53D, 53L1, 53L10, 53G and 53P maintained the ability of Tb535H to bind to the CD3 antigen, the binding affinity of the purified triantibodies to the recombinant CD3 protein was investigated by ELISA ( Figure 3 ). The recombinant human CD3δ / ε heterodimeric protein (AMSBio) was fixed to a 96-well ELISA plate. The binding test between Tb535H and the purified new tri-antibody was carried out according to the steps described in the "Materials and Methods" described below while increasing the concentration (0 to 500 nM). Figure 3 As shown in A, all three antibodies constructed showed concentration-dependent CD3 binding with the same Kd (nM) value ( Figure 3 B). The results showed that the new three antibodies maintained the ability of Tb535H to bind to human CD3 protein.

[0456] ELISA-based novel 53X trio of antibodies containing PD-L1 or PD-1 binding arms to recombinant PD-L1 or PD-1 proteins Combination

[0457] To verify whether the new tri-antibodies containing binding arms against PD-L1 or PD-1 have sufficient binding affinity to their target recombinant proteins, ELISA assays were performed with immobilized PD-1 protein or PD-L1 protein ( Figure 4 ). 53L1 and 53L10, three antibodies with binding arms for PD-L1, showed concentration-dependent binding ability to recombinant PD-L1 protein, with Kd values ​​of 79.85 nM and 29.87 nM, respectively ( Figure 4 A). The three-antibody 53D with a binding arm for PD-1 also showed concentration-dependent binding ability to the recombinant PD-L1 protein ( Figure 4 B).

[0458] T cell activation bioassay in the presence of 5T4-expressing target cells

[0459] Using 5T4-expressing CHO-K1 (CHO-5T4) cells as target cells, we investigated the activation of T cell receptor (TCR) / CD3 on effector cells by the novel tri-antibody and Tb535H. The test was performed using the T cell activation bioassay (NFAT) (Promega, catalog number J1621). Figure 5 A shows a schematic diagram of the test. When the cell surface 5T4 on the target cell and the CD3 on the effector cell are cross-linked by the bispecific T cell derivative, TCR / CD3 transmits intracellular signals, resulting in luminescence mediated by NFAT-RE. The genetically modified Jurkat T cell line ("TCR / CD3 effector cell") expressing a luciferase reporter gene driven by the NFAT response element (NFAT-RE) is shown in the figure. As the concentration increases, in the presence of Tb535H and the new 53X tri-antibody ( Figure 5 B) co-cultured with CHO-5T4 cells (“target cells”). Tb535H induced luciferase activity in a concentration-dependent manner, EC 50 The new tri-antibody has an EC range of 0.03nM to 0.29nM. 50 The values ​​induced concentration-dependent luciferase activity ( Figure 5 C). The new tri-antibodies (except 53D) slightly outperformed Tb535H in 5T4-mediated CD3 activation, but the EC 50 The value converges to the EC of Tb535H 50 Almost 3 times less than the value.

[0460] Antagonistic effect of 53X triple antibody determined by competition ELISA assay

[0461] In order to verify whether the new three antibodies 53D, 53L1 and 53L10, whose binding domains are derived from PD-1.1 and PD-L1.1, respectively, retain the ability of the parent monoclonal antibodies (mAbs) to interfere with the PD-1 / PD-L1 interaction, the following competitive ELISA test was performed: the binding of biotinylated PD-L1 ligand to the immobilized PD-1 receptor was measured in the absence or presence of molar excess (5:1 M / M) of the three antibodies 53D, 53L1, 53L10 or their parent mAbs, i.e., PD-L1_1, 10.12 and PD-1_1, as positive controls in parallel experiments. Human IgG4 was used as an isotype control. Figure 6 A and Figure 6 As shown in Figure B, the binding of biotinylated PD-L1 ligand was greatly reduced in the presence of the above three antibodies relative to the binding signal of biotinylated PD-L1 alone. This indicates that the new three antibodies retain the ability of the parent mAb to block the interaction of PD-L1 and PD-1.

[0462] Similarly, the ability of the triple antibody 53G to block the LAG-3 / MHCII (HLA-DRA) interaction was analyzed by a competition ELISA assay. For this purpose, the LAG-3-His-GST recombinant protein was coated on the plate at a concentration of 50 nM, the triple antibody 53G or its parent mAb, LAG-3_1, was added at a saturating concentration of 2 μM, and the binding of the biotinylated MHCII protein (700 nM) to the LAG3-His-GST recombinant protein was measured. Figure 6 As shown in FIG. 3C , the binding of biotinylated MHCII to LAG3-His-GST recombinant protein was significantly reduced in the presence of the triple antibody 53G or the parent LAG-3_1 (mAb) used as a control, relative to the binding signal when biotinylated MHCII was treated alone.

[0463] In vitro cytotoxic activity of the novel triple antibody in co-culture of human cancer cells and human lymphocytes

[0464] In order to verify whether the new three antibodies targeting PD-L1, PD-1 and LAG-3 efficiently induce the activation of lymphocytes against cancer cells, their effects in the co-culture system of human cancer cells and lymphocytes were investigated. For this purpose, MDA-MB-231 breast cancer cells and human alveolar basal epithelial adenocarcinoma cell line A-549, which highly express PD-L1 and 5T4, were co-cultured with human peripheral blood mononuclear cells (hPBMC) (effector: target ratio 5: 1) and incubated at 37 ° C for 48 hours in the absence or presence of 53D, 53G, 53L1 and 53L10. In parallel, the same concentrations of the parent three antibodies TB535H and the three antibodies (53P) derived from TB535H but lacking immunomodulatory antibody fragments, the above-mentioned parent mAbs, namely PD-L1.1, 10.12, PD-1.1 and LAG-3.1, and the combined use of Tb535H and parent mAbs were also studied. After incubation, cell lysis was measured by lactate dehydrogenase (LDH) assay via LDH released into the cell supernatant. The results showed that, except for 53P used as a negative control, all of the new three antibodies induced tumor cell lysis with greater effectiveness than the parental mAb or its combination with TB535H (ref. Figure 7 and Figure 8 Specifically, all three antibodies increased the LDH release level brought about by the corresponding parent mAb and the combination of the three antibodies with TB535H by at least 2-fold, and 53L1 and 53L10 achieved about 80% to 90% cell lysis at a concentration of 1 nM.

[0465] To elucidate whether the enhancement of tumor cell lysis brought about by the new triple antibodies is associated with increased lymphocyte activation, the concentration of IFN-γ (a functional marker of hPBMC activation) released into the culture supernatant of their co-cultures was measured, and the effects of the new triple antibodies were compared with the effects of combining TB535H and the parental mAbs. Fig. 9 As shown, the IFN-γ concentration in the culture supernatant of co-culture of MDA-MB-231 and hPBMC treated with the new triple antibody (TRB) was significantly higher than the concentration observed when treated with the parental triple antibody TB535H, the parental mAbs, namely PD-L1.1, 10.12, PD-1.1 and LAG-3.1, or a combination thereof.

[0466] Therefore, these results clearly support the strategic validity of inserting immunomodulatory moieties into parental TB535H to improve the antitumor effect.

[0467] In vivo anti-tumor effects of the new 53X triple antibody

[0468] The in vivo antitumor effects of the new tri-antibodies were evaluated using an A549 (human alveolar basal epithelial adenocarcinoma cell line) xenograft mouse model. The expression of 5T4 and PD-L1 on the cell surface in A549 cells was confirmed by flow cytometry analysis (data not shown). Human PBMCs were activated with Dynabeads Human T-activator CD3 / CD28 4 days before transplantation (activated PBMCs). A549 lung cancer cells were subcutaneously transplanted into immunodeficient NOD-SCID mice together with an equal number of activated human PBMCs (day 0). The intravenous administration of the tri-antibodies shown in the figure was performed every other day from day 0 to day 10 (a total of 6 doses) ( Fig.10 ). Compared with the solvent group, the Tb535H group showed significant tumor growth inhibition at a dose of 20 μg / mouse (P < 0.05), but no significant tumor growth inhibition was observed at a dose of 2 μg / mouse ( Fig.10 On the other hand, the anti-tumor activity of 53L1 (20 μg / mouse) was significantly stronger than that of Tb535H at the same dosage ( Fig.10 B). Furthermore, in the 53L10-administered group, compared with the solvent-administered group, significant tumor growth inhibition of A549 tumors was observed in both the 2 μg / mouse and 20 μg / mouse administration doses ( Fig.10 C). The tumor growth inhibition (TGI (%)) of the 53L10 (2 μg / mouse) administration group on day 50 reached 41.5% compared with the solvent administration group (P < 0.05), and 100% TGI was observed in the 20 μg / mouse administration group (P < 0.05). On the final day of this experiment (day 50), complete tumor regression was observed in all mice in the 53L10 (20 μg / mouse) administration group.

[0469] The tumor regression caused by 53L10 administration was also replicated in another series of studies ( Fig.11 The proliferation of A549 subcutaneous tumors was significantly inhibited in a dose-dependent manner by the administration of 53L10, and complete tumor regression was observed in all mice in the 53L10 (20 μg / mouse) administration group on the final day (day 42).

[0470] The antitumor activity of 53D and 53G was also evaluated using the same model by comparing them with the control 53P triple antibody ( Fig.12 ). In the case of 53D, the 20 μg / mouse administration group showed enhanced tumor growth inhibition compared with 53P ( Fig.12). The tumor growth inhibition caused by 53G administration was enhanced in both the 2 μg / mouse and 20 μg / mouse administrations compared with 53P administration. In the 53G (20 μg / mouse) administration group, significant tumor regression was observed during the experimental period.

[0471] 2. Materials and Methods

[0472] Cell culture MDA-MB-231 breast cancer cells were cultured with Dulbecco's modified Eagle's medium (Gibco (trademark) DMEM, Life Technologies). A-549 lung cancer cells were cultured with Kaign's modified Ham's F-12 medium (F-12K, American Type Culture Collection (ATCC)). Cell lines were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO2. 10% (vol / vol) inactivated fetal bovine serum (FBS, Sigma) was added to the culture medium and 50U / mL penicillin, 50μg / mL streptomycin, and 2mM L-glutamine (all Gibco (trademark), Life Technologies) were added before use.

[0473] Antibodies and human recombinant proteins

[0474] Anti-PD-L1 antibodies (PD-L1_1 and 10.12), anti-PD-1 antibodies (PD-1_1), anti-LAG-3 antibodies (LAG3_1) and human IgG4 isotype control antibodies were purified as described above (Patent Document 2 and Non-Patent Document 10 described above). Tb535H tri-antibodies were prepared as described in Patent Document 1 described above. Recombinant human PD-L1 / Fc, PD-1 / Fc and human LAG3 / Fc proteins were purchased from R&D Systems. Human LAG-3 / His-GST and human HLA class II histocompatibility antigen-DRA were purchased from Cusabio. Human 5T4 / His was purchased from ACROBiosystems, and human CD3δ / ε heterodimer protein was purchased from AMSBio.

[0475] Production and purification of the new 53X triple antibody

[0476] In order to produce heterodimers in Expi293 cells, expression plasmids encoding L chain derivatives of each triantibody (53D, 53G, 53L1, 53L10 and 53P) and expression plasmids encoding H chain derivatives in combination were transiently transfected and expressed. Five days after transfection, each triantibody was purified from the cell culture supernatant by His tag affinity chromatography and subsequent gel filtration chromatography. In this process, the triantibody heterodimers were recovered while limiting the impurities such as variants produced. The purity of the triantibody was analyzed by SDS-PAGE, and the purity and freeze-thaw stability of the triantibody heterodimer were evaluated by size exclusion chromatography (SEC) analysis. >95% L / Fd chain triantibody heterodimers and low concentrations of endotoxin were confirmed by a LAL-based method. The triantibody was sterilized by 0.2μm filtration, aliquoted, and stored at -80°C.

[0477] Binding assay (ELISA)

[0478] In order to confirm the binding specificity of the newly generated tri-antibody, human chimeric recombinant target protein (5T4 / His, CD3 δ / ε ELISA test was performed using 1 μg / mL of PD-L1 / Fc, PD-1 / Fc or LAG-3 / Fc coated on a 96-well plate.

[0479] For the ELISA test of the coated recombinant target protein, the coated plate was blocked at 37°C for 1 hour by PBS containing 5% skim milk. The purified three antibodies were added to the plate in 3% BSA (bovine serum albumin) in PBS at different concentrations and incubated at room temperature for about 90 minutes with steady shaking. After being thoroughly washed with PBS, the plate was incubated with HRP-conjugated anti-human Igκ antibody (Southern Biotech) or anti-His-HRP-conjugated antibody (MBL) for 1 hour, washed again, incubated in TMB reagent (Sigma-Aldrich) for 10 minutes, and the reaction was stopped with an equal amount of 1N hydrochloric acid (HCl).

[0480] The absorbance at 450 nm was measured using Envision microplate reader (Perkin Elmer 2102 Waltham, MA, USA).

[0481] Competition ELISA assay

[0482] In order to investigate the competitive ability of the newly generated tri-antibodies in PD-L1 / PD-1 or LAG-3 / MHC II (HLA-DRA) binding compared to the corresponding parental mAbs, competition ELISA assays were performed by measuring the binding of each biotinylated chimeric protein (PD-L1 / Fc or MHCII) to PD-1 or LAG-3 / GST-His in the absence or presence of unlabeled competing tri-antibodies. For this purpose, PD-1 or LAG-3 proteins were coated on Nunc (trademark) flat-bottom 96-well plates. The coated plates were then pre-incubated with unlabeled anti-PD-L1, anti-PD-1 or anti-LAG-3 antibodies at saturating concentrations for 2 hours at room temperature (5:1 in the case of anti-PD-L1 or anti-PD-1 antibodies, 3:1 in the case of anti-LAG-3). Then, the biotinylated PD-L1 or MHCII chimeric proteins added to the plates at the same concentration of competing antibodies were further treated for 2 hours at room temperature. To detect bound biotinylated protein, HRP-conjugated streptavidin (Biorad) was added to the plate for 30 minutes, followed by absorbance measurement as described above.

[0483] Cell viability and cytotoxicity assay (LDH assay)

[0484] Human peripheral blood mononuclear cells (hPBMC) were isolated from the blood of healthy donors using Greiner Leucosep (registered trademark) tubes (Sigma-Aldrich) and cryopreserved according to the manufacturer's instructions. As previously reported, cryopreserved cell bottles were thawed, dormant cells were collected, and counted before use (Non-patent Document 11 described above).

[0485] To determine the cytotoxic activity of the newly generated trio of antibodies, target tumor cells were co-cultured with hPBMCs (effector:target ratio 5:1) and treated with increasing concentrations of the trio of antibodies or the parental mAbs and corresponding combinations used in parallel experiments at the same concentrations. 4 The cells were plated at a density of 10 cells / well in a 96-well flat-bottom plate for 16 hours. Then, hPBMCs isolated from healthy donors were added in the absence or presence of the three antibodies or antibodies and incubated at 37°C for 48 hours. Untreated cells or cells treated with human IgG4 isotype control were used as negative controls.

[0486] The lysis of target cells was determined by measuring the concentration of lactate dehydrogenase (LDH) released into the supernatant of the co-culture using an LDH detection kit (Thermofisher Scientific) according to the manufacturer's recommendations. Cell lysis was analyzed by measuring the increase in LDH in the presence of each treatment relative to the amount present in the supernatant of co-cultures that were untreated or treated with a human IgG4 isotype control antibody, and is presented as a percentage (%).

[0487] Cytokine secretion assay

[0488] The secretion of human IL-2 and IFNγ in the supernatant of co-culture of tumor cells and hPBMC or hPBMC treated with three antibodies and the corresponding parental mAb was evaluated using DuoSet (registered trademark) ELISA kit. Briefly, the culture supernatant was centrifuged after treatment, and cytokines were quantified using IL-2 and IFNγ kits (R&D Systems) according to the manufacturer's recommendations. Concentration values ​​were converted to pg / mL and calculated as the average of at least 3 measurements.

[0489] T cell activation bioassay

[0490] TCR / CD3 activation in the presence of 5T4 expressing cells was performed using the T cell activation bioassay (NFAT) (Promega, catalog number J1621) according to the manufacturer's instructions. Briefly, a genetically modified Jurkat T cell line expressing a luciferase reporter gene driven by a NFAT response element (contained in the kit) was co-cultured with 5T4 expressing CHO-K1 cells in the presence of various concentrations of Tb535H and the new 53X tri-antibody. After incubation at 37°C for 4 hours, Bio-Glo (trademark) reagent (contained in the kit) was added and luminescence was measured using a photometer.

[0491] In vivo efficacy testing

[0492] All animal experiments in this example were permitted and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chiome Bioscience. Female NOD / ShiJic-scidJcl mice (NOD-SCID) (CLEA, Japan) aged 5 to 6 weeks were used for effectiveness studies. The A549 human alveolar lung adenocarcinoma cell line was collected from the cell culture dish by trypsin treatment, washed with PBS, and a cell suspension was prepared. For human PBMC (Cellular Technology), A549 cells were cultured in vitro for 4 days in the presence of Dynabeads Human T-Activator (CD3 / CD28) (Veritas) before inoculation into mice. 5×10 6 A cell suspension of A549 cells was mixed with an equal number of activated hPBMCs (the ratio of activated hPBMCs to A549 cancer cells transplanted was 1:1). The mixed cell suspension was transplanted subcutaneously into the right flank of NOD-SCID mice (day 0), and the indicated test articles were intravenously administered at the indicated dosages on days 0, 2, 4, 6, 8, and 10 after cell transplantation (a total of 6 doses). Regarding tumor growth, two perpendicular dimensions of the tumor were measured with a caliper, and the tumor volume (mm 3 ) with (width 2 The tumor volume was calculated by the formula of (× length)×3.14 / 6. The tumor volume is shown as mean±standard deviation (SD). The statistical significance of the difference was determined using Dunnett's test (relative to the vehicle-administered group). P<0.05.

[0493] Example 2

[0494] 1. Results

[0495] In vivo antitumor efficacy of the novel triple antibody 53L10 and Tb535H as single agents and Tb535H and pembrolizumab in combination Comparison of Fruit

[0496] Furthermore, the in vivo antitumor effects of 53L10 alone, Tb535H alone, and Tb535H in combination with pembrolizumab (anti-human PD-1 antibody, MSD Corporation) were compared using an A549 (human alveolar basal epithelial adenocarcinoma cell line) xenograft mouse model (Figure 13). 6A549 cells were mixed with an equal number of activated human PBMCs, and the resulting cell suspension was transplanted subcutaneously into the right flank of NOD-scid mice (day 0). From the day of transplantation, the tail vein was administered every other day for a total of 6 times (days 0, 2, 4, 6, 8, and 10), and solvent (PBS), Tb535H (6, 12, and 20 μg / mouse) or 53L10 (6, 12, and 20 μg / mouse) were administered. Pembrolizumab (200 μg / mouse) was administered intravenously for a total of 3 times on days 0, 4, and 8. The combination of Tb535H and pembrolizumab was a combination of Tb535H and pembrolizumab, respectively. Tumor volume was measured starting from 3 days after transplantation, and then twice a week until 50 days after transplantation.

[0497] Fig.13A The results of the comparison of the in vivo antitumor effects of Tb535H and 53L10 are shown. Fig. 13B The results of the comparison of the in vivo antitumor effects of Tb535H combined with pembrolizumab and 53L10 are shown. In the case of the Tb535H administration group, the 20 μg / mouse administration group was significantly inhibited from tumor growth compared with the solvent (PBS) administration group, and the TGI on the final day of the experiment (day 50) was 36.5% (*P<0.05) ( Fig.13A ).

[0498] On the other hand, the 53L10-administered group observed a tumor proliferation inhibition effect corresponding to the dosage, and all the administration groups observed significant tumor proliferation inhibition compared with the solvent (PBS)-administered group (*P<0.05). In addition, a significant reduction in tumor volume was observed in the 53L10-administered group compared with the Tb535H-administered groups (6μg / mouse, 12μg / mouse, and 20μg / mouse, respectively). The tumor proliferation inhibition (TGI (%) of the 53L10-administered group on the final day of the experiment (day 50) was 64.8% in the 6μg / mouse-administered group, 76.0% in the 12μg / mouse-administered group, and 95.7% in the 20μg / mouse-administered group. Complete tumor regression (TGI (%) was observed in 5 out of 8 individuals in the 53L10 (20μg / mouse)-administered group on the final day of the experiment (day 50). Fig.13A ).

[0499] Then, the Tb535H and pembrolizumab combination group and the 53L10 triple antibody single-dose group were compared ( Fig. 13B No significant antitumor effect was observed in the pembrolizumab (200 μg / mouse) administration group compared with the solvent (PBS) administration group ( Fig. 13BIn the case of the combined administration of Tb535H and pembrolizumab, no significant antitumor effect was observed in the Tb535H (6 μg / mouse) + pembrolizumab (200 μg / mouse) administration group and the Tb535H (12 μg / mouse) + pembrolizumab (200 μg / mouse) administration group compared with the solvent (PBS) administration group ( Fig. 13B ). Significant tumor growth inhibition was observed in the Tb535H (20 μg / mouse) + pembrolizumab (200 μg / mouse) administration group compared with the solvent (PBS) administration group. On the final day of the experiment (day 50), the TGI was 38.0% (*P<0.05) ( Fig. 13B ).

[0500] On the other hand, as described above, the 53L10-administered group observed a tumor proliferation effect corresponding to the dosage, and all the administration groups observed significant tumor proliferation inhibition relative to the solvent (PBS)-administered group (*P<0.05). In addition, a significant reduction in tumor volume was observed in the 53L10-administered group compared with the Tb535H+pembrolizumab-administered groups (Tb535H 6μg / mouse, 12μg / mouse, 20μg / mouse). Regarding the TGI of the 53L10-administered group on the final day of the experiment (day 50), the 6μg / mouse-administered group was 64.8%, the 12μg / mouse-administered group was 76.0%, and the 20μg / mouse-administered group was 95.7%. Complete tumor regression was observed in 5 out of 8 individuals in the 53L10 (20μg / mouse)-administered group on the final day of the experiment (day 50) ( Fig. 13B ).

[0501] Production of 53L10-M13

[0502] It is known that when using antibodies, antibody binding fragments, etc. to manufacture therapeutic antibodies that can be administered to humans, chemical modifications often occur during the manufacture of the antibodies after purification. It is important to correspond to such chemical modifications to ensure the quality and safety of purified antibodies, and this has been recognized in the art. Specifically, when there are amino acid sequence motifs that are susceptible to N-type sugar chain modification, protein cleavage, deamidation, racemization, oxidation, etc. in the sequence of the CDR region of the antibody, it is predicted that the quality of the purified antibody will decrease. As amino acid sequence motifs that are susceptible to modification, it is known that, for example, motifs containing asparagine residues and aspartic acid residues (e.g., Sydow et al., (2014). Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions. PLoS ONE, 9 (6), etc.) disclose that the method of changing these amino acid motifs while maintaining the activity of the antibody can further improve the usefulness of the antibody.

[0503] The aspartic acid residue (D) at position 62 of scFv (10.12) (SEQ ID NO: 56) was mutated to a serine residue (S) to produce scFv (10.12)-M13 (SEQ ID NO: 108). It is a substance in which the heavy chain CDR-H2 (SEQ ID NO: 59) contained in scFv (10.12) (SEQ ID NO: 56) is replaced with CDR-H2#2 (SEQ ID NO: 49). The 53L10_L chain derivative (SEQ ID NO: 107) containing scFv (10.12)-M13 (SEQ ID NO: 108) was co-expressed with the H chain derivative of SEQ ID NO: 1 in Expi293 cells by the above method, and the 53L10 variant (53L10-M13) was purified from the culture supernatant.

[0504] ELISA-based binding affinity of 53L10-M13 to recombinant 5T4

[0505] The binding affinity of purified 53L10-M13 to 5T4 antigen was investigated by ELISA ( Fig.14 ). Use with Figure 2 The recombinant human 5T4 protein (ACRO Biosystems) was immobilized on a 96-well plate in the same manner as in Example 1. The binding affinity of 53L10-M13 and 53L10 to human 5T4 was tested by increasing the concentration (0 to 500 nM) according to the described procedure. Fig.14As shown in A, 53L10-M13 (○) showed concentration-dependent binding to 5T4. The curve representing the binding affinity of 53L10-M13 to 5T4 overlaps with the curve representing the binding affinity of 53L10 (●) to 5T4. The EC of 53L10-M13 50 The value is 0.58 nM, and the EC of 53L10 50 The value is 0.53nM ( Fig.14 B).

[0506] ELISA-based binding affinity of 53L10-M13 to recombinant CD3

[0507] The binding affinity of purified 53L10-M13 to recombinant CD3 protein was investigated by ELISA. Fig.15 ). Use with Figure 3 The recombinant human CD3δ / ε heterodimer protein (ACRO Biosystems) was fixed to a 96-well plate in the same manner as in the method implemented in . The binding test of 53L10-M13 and 53L10 to human CD3 protein was carried out while increasing the concentration (0 to 500 nM) according to the procedure described in the "Materials and Methods" described below. Fig.15 As shown in A, 53L10-M13 (○) and 53L10 (●) showed concentration-dependent binding activity to CD3 protein. The EC of 53L10 50 The EC of 53L10-M13 was 39.9 nM. 50 65.5nM( Fig.15 B).

[0508] ELISA-based binding affinity of 53L10-M13 to recombinant PD-L1

[0509] The binding affinity of purified 53L10-M13 to recombinant PD-L1 protein was investigated by ELISA ( Fig.16 ). Use with Figure 4 The recombinant human PD-L1 / Fc protein (R&D systems) was fixed to a 96-well plate in the same manner as in the method implemented in . The binding test of 53L10-M13 and 53L10 to human PD-L1 protein was carried out by increasing the concentration (0 to 500 nM) according to the steps described in the "Materials and Methods" described below. Fig.16 As shown in A, 53L10-M13 (○) showed concentration-dependent binding activity to PD-L1. The curve representing the binding affinity of 53L10-M13 to PD-L1 overlaps with the curve representing the binding affinity of 53L10 (●) to PD-L1. The EC of 53L10-M13 50 The value is 36.9 nM, and the EC of 53L10 50 The value is 34.4nM ( Fig.16 B).

[0510] Bioassay of T cell activation by 53L10-M13 in the presence of 5T4 expressing target cells

[0511] Then, the T cell activation bioassay (NFAT) (Promega, catalog number J1621) was used to Figure 5 The same method as implemented in the investigation of 53L10-M13 T cell receptor (TCR) / CD3 activation on effector cells in the presence of 5T4 expressing target cells ( Fig.17 ). As a comparison object, 53L10 was also investigated at the same time. When the cell surface 5T4 on the target cells and the CD3 on the effector cells are cross-linked by 53L10-M13 or 53L10, TCR / CD3 transmits intracellular signals, resulting in luminescence mediated by NFAT-RE. The genetically modified Jurkat T cell line expressing a luciferase reporter gene driven by the NFAT response element (NFAT-RE) ("TCR / CD3 effector cells") was co-cultured with CHO-5T4 cells ("target cells") in the presence of 53L10-M13 and 53L10 as the concentration increased, as shown in the figure ( Fig.17 53L10-M13 (○) and 53L10 (●) both induced concentration-dependent luciferase activity, and the curves showing the concentration-dependent increase in luciferase activity of the two overlapped ( Fig.17 A), 53L10-M13 EC 50 The EC of 53L10 is 0.016 nM. 50 0.010nM( Fig.17 B).

[0512] The above results indicate that the antigen binding activity (5T4, CD3, and PD-L1) and the activation capacity of the TCR / CD3 complex on effector cells in the presence of 5T4-expressing target cells of 53L10-M13 are equivalent to those of 53L10.

[0513] Co-culture of A549 cells (5T4-positive PD-L1-positive human cancer cell line) and human peripheral blood mononuclear cells (hPBMC) T cell activation caused by 53L10-M13 and Tb535H

[0514] By measuring the expression of T cell activation markers (CD25, CD69) on CD3-positive cells, we investigated whether 53L10-M13 can efficiently activate T cells in the presence of target tumor cells. As a comparison object, Tb535H was also investigated at the same time. As target tumor cells, A549 cells, which are 5T4-positive PD-L1-positive human cancer cell lines, were first tested (Figure 18A). According to the procedures described in the "Materials and Methods" described below, the target cells were co-cultured with hPBMC (Cellular Technology Limited, lot. HHU20211125 and lot. HHU20220224, lot. HHU20220329, hereinafter referred to as PBMC1 and PBMC2, PBMC3) (effector:target ratio = 10:1), and 0.001 pM to 10 nM (10-fold common ratio) of 53L10-M13 or Tb535H was added. After 24 hours, the expression of CD3 and CD69 was measured by flow cytometry, and after 48 hours, the expression of CD3 and CD25 was measured by flow cytometry. The proportion of CD69-positive cells in the CD3-positive cell population (B in Figure 18) and the proportion of CD25-positive cells in the CD3-positive cell population (C in Figure 18) were calculated. As a result, both 53L10-M13 (●, ■, ▲) and Tb535H (○, □, △) induced the expression of CD25 and CD69 on effector cells in a concentration-dependent manner. For any hPBMC batch, 53L0-M13 strongly induced the expression of T cell activation markers compared to Tb535H at the same concentration (Figure 18B and Figure 18C). 50 When comparing 53L10-M13 and Tb535H, the CD69 expression of 53L10-M13 was 34.9-99.1pM, and that of Tb535H was 100.2-1548.0pM, and the value of 53L10-M13 was lower in any hPBMC batch. max) In terms of CD69 expression, 53L10-M13 was 34.9-57.1%, and Tb535H was 22.9-42.5%, and the value of 53L10-M13 was higher than that of Tb535H in any hPBMC batch. These results indicate that 53L10-M13 can enhance T cell activation compared with Tb535H.

[0515] T cell activation by 53L10-M13 and Tb535H against hPBMC (in the absence of human cancer cells)

[0516] On the other hand, T cell activation caused by 53L10-M13 and Tb535H was also investigated in the absence of target tumor cells. In the absence of target tumor cells, 0.01-1,000nM (10-fold common ratio) of 53L10-M13 or Tb535H was added to hPBMC. As a result, the marker expression of 53L10-M13 (●, ■, ▲) was slightly increased in the high concentration range above 10nM compared with Tb535H (○, □, △), but in the absence of target tumor cells, the expression of T cell activation markers (CD25, CD69) on CD3-positive cells was low compared with that in the presence of target tumor cells, indicating that T cells were almost not activated ( Fig.19 A and B).

[0517] The above results indicate that 53L10-M13 activated T cells in the presence of A549 cells as target tumor cells, and caused higher T cell activation in the presence of A549 cells than that of Tb535H.

[0518] MCF-7 cells (5T4-positive PD-L1-negative human cancer cell line) and hPBMC co-culture, 53L10-M13 and T cell activation induced by Tb535H

[0519] Then, the same method was used to investigate the T cell activation brought about by 53L10-M13 and Tb535H in the co-culture of MCF-7 cells and hPBMC as human breast cancer cell lines. MCF-7 cells were used as 5T4-positive PD-L1-negative target tumor cells (A in Figure 20). As a result, 53L10-M13 (●, ■, ▲) and Tb535H (○, □, △) both induced the expression of CD25 and CD69 on effector cells in a concentration-dependent manner, which was different from the use of A549 cells expressing both 5T4 and PD-L1 as target tumor cells. The curve indicating the increase in concentration-dependent expression of T cell activation markers overlapped with CD69 (B in Figure 20) and CD25 (C in Figure 20) in the case of 53L10-M13 and Tb535H. About EC 50 In terms of CD69 expression, 53L10-M13 was 0.10-2.85pM, and Tb535H was 2.63-5.49pM. In terms of CD25 expression, 53L10-M13 was 0.49-6.78pM, and Tb535H was 12.57-50.79pM. 53L10-M13 showed slightly lower values. maxIn terms of CD69 expression, 53L10-M13 was 41.85-50.68%, and Tb535H was 42.56-50.41%. In terms of CD25 expression, 53L10-M13 was 19.99-24.21%, and Tb535H was 27.30-29.49%, which were all at the same level. This shows that when 5T4-positive PD-L1-negative MCF-7 cells are used as target tumor cells, the T cell activation ability of 53L10-M13 and Tb535H is roughly equal (Figure 20B, C, and D).

[0520] These results indicate that the enhanced T cell activation brought about by 53L10-M13 compared to Tb535H is a phenomenon observed under the condition that the target tumor cells express PD-L1, and that for tumors that do not express PD-L1, T cell activation at the same level as Tb535H, which has a divalent 5T4 binding site, can be induced. Therefore, it is clearly shown that 53L10 and 53L10-M13, which enhance tumor immunity by adding an anti-PD-L1 binding arm to b535H, are strategically appropriate for target tumor cells that express both 5T4 and PD-L1, and that 53L10 and 53L10-M13 have the same tumor immunity ability as Tb535H for tumor cells that do not express PD-L1.

[0521] In the presence of A549 cells (5T4-positive PD-L1-positive human cancer cell line) brought by 53L10-M13 and Tb535H, Cytokine production by hPBMCs in the absence of

[0522] Next, the cytokine production of hPBMCs brought about by 53L10-M13 and Tb535H in the presence and absence of A549 cells (hPBMC only) was measured. The culture supernatant obtained after 48 hours in the above-mentioned T cell activation experiment was recovered, and the concentrations of four cytokines secreted by T cells, namely human TNFα, human IL-2, human IL-6, and human interferon γ, in the culture supernatant were measured according to the steps described in the "Materials and Methods" described below. The results showed that in the absence of A549 cells (hPBMC only), neither 53L10-M13 nor Tb535H induced concentration-dependent cytokine production. On the other hand, in the presence of A549 cells, concentration-dependent IL-6 production was observed with Tb535H, while no concentration-dependent production was observed for other cytokines. In the case of 53L10-M13, cytokine production that depended on the concentration of 53L10-M13 added was observed for all four cytokines ( Fig.21A~D (A: human TNFα, B: human IL-2, C: human IL-6, D: human interferon γ)). Regarding the production of cytokines caused by 53L10-M13, similar to T cell activation (B and C in Figure 18), when 100 pM or more of 53L10-M13 was added, the production of all four cytokines was enhanced compared with the addition of Tb535H. This result also shows that 53L10-M13 induced stronger T cell activation than Tb535H in the presence of A549 cells.

[0523] In the co-culture of A549 cells (5T4-positive PD-L1-positive human cancer cell line) and hPBMC (without activation treatment), Cytotoxicity test of 53L10-M13 and Tb535H on A549 cells

[0524] Then, it was verified whether 53L10-M13 can efficiently induce cytotoxicity to target tumor cells. Under the same conditions as the T cell activation test, A549 cells were co-cultured with hPBMC (effector: target ratio = 10:1), and the cytotoxicity when 0.001pM~10nM (10-fold common ratio) of 53L10-M13 and Tb535H were added was investigated. According to the steps described in the "Materials and Methods" described below, hPBMCs were added to A549 cells whose cell membranes were previously labeled with PKH67, and the concentrations of 53L10-M13 and Tb535H were changed. After culturing for 48 hours, the cells were recovered. Dead cells were stained with propidium iodide (PI), and the number of PKH67-positive PI-negative cells was measured by flow cytometry to determine the cell killing power (%). The results showed that both 53L10-M13 (●, ■, ▲) and Tb535H (○, □, △) induced concentration-dependent cell killing. In any hPBMC batch, at the same concentration, 53L10-M13 induced higher cell killing than Tb535H ( Fig. 22 A). EC can be calculated for both 53L10-M13 and Tb535H 50 and E max PBMC3, 53L10-M13 EC 50 The EC of Tb535H is 11.5pM. 50 The EC of 53L10-M13 was 33.7 pM. 50 Low, 53L10-M13 E max 85.2%, Tb535H's E max 69.4%, 53L10-M13 E max High, indicating that 53L10-M13 has enhanced cell killing ability compared with Tb535H ( Fig. 22 B). Fig. 22 In B, “nc” means not calculated.

[0525] A549 cells (5T4-positive PD-L1-positive human cancer cell line) and activated hPBMC in co-culture, 53L10-M13 Cytotoxicity test of Tb535H and Tb535H on A549 cells

[0526] Furthermore, in the case of activated hPBMCs, the induction of cytotoxicity of 53L10-M13 and Tb535H on target cancer cells was also investigated. According to the steps described in the "Materials and Methods" described later, hPBMCs pre-activated with anti-CD3 antibody / anti-CD28 antibody solid phase magnetic beads were co-cultured with A549 cells (effector: target ratio = 1:1), and the cytotoxicity when 53L10-M13 (●, ▲) and Tb535H (○, △) were added was investigated. The results showed that in the case of activated hPBMCs, both 53L10-M13 and Tb535H induced cell killing in a concentration-dependent manner, with Tb535H inducing only 50% cell killing at most, while 53L10-M13 induced more than 90% cell killing at most ( Fig.23 A). EC can be calculated for both 53L10-M13 and Tb535H. 50 and E max PBMC3, 53L10-M13 EC 50 The EC of Tb535H is 15.3pM. 50 The EC of Tb535H is 10.4 pM. 50 Slightly lower, 53L10-M13 E max 90.0%, Tb535H E max 65.4%, 53L10-M13 E max This indicates that 53L10-M13 also enhances cell killing compared to Tb535H in the case of activated PBMCs ( Fig.23 B). Fig.23 In B, “nc” means not calculated.

[0527] These results indicate that 53L10-M13 enhances T cell activation in the presence of target tumor cells compared to Tb535H, and that the cell killing activity of activated T cells is also greatly enhanced. This clearly demonstrates that the strategy of 53L10-M13 to enhance tumor immunity against target tumor cells by adding an anti-PD-L1 binding arm to Tb535H is appropriate.

[0528] 2. Materials and Methods

[0529] Cell culture

[0530] A549 human lung cancer cells were cultured with Kaign's modified Ham's F-12 medium (F-12K, Fujifilm-Wako). The cell line was purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was added with 10% (vol / vol) inactivated fetal bovine serum (FBS, Cytiva) and 50U / mL penicillin and 50μg / mL streptomycin before use. MCF-7 human breast cancer cells were cultured with minimal essential Eagle's medium (EMEM, Sigma-Aldrich). The cell line was purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was added with 10% (vol / vol) inactivated fetal bovine serum (FBS, Cytiva) and 50U / mL penicillin, 50μg / mL streptomycin, and 0.01mg / ml human recombinant insulin (Fujifilm-Wako) before use. hPBMCs were purchased from Cellular Technology Limited and cultured at 37°C in a humidified gas containing 5% CO2 using CTL-Test medium (Cellular Technology Limited) during overnight resting after thawing. The culture medium was added with 2mM L-glutamine (Thermo fisherscientific) before use. On the other hand, for activation induction, ImmunoCult-XF T Cell Expansion Medium (STEM CELL Technologies) was used and cultured at 37°C in a humidified gas containing 5% CO2. The culture medium was added with 3ng / mL human IL-2 (PeproTech) before use.

[0531] T cell activation marker assay

[0532] Human peripheral blood mononuclear cells (hPBMC) from healthy donors were thawed and rested overnight according to the instructions of the vendor (Cellular Technology Limited). The cells were recovered and counted before use. Target tumor cells were expressed as 8×10 4The cells were plated at a density of 10 cells / well in a 24-well flat-bottom plate for 4 hours. hPBMCs were added in the presence and absence of target tumor cells (effector: target ratio 10:1). Then, 53L10-M13 and Tb535H were added at concentrations varying from 0.001 pM to 10 nM in the presence of target tumor cells and 0.01 nM to 1,000 nM in the absence of target tumor cells, and incubated at 37°C for 24 or 48 hours. Regarding the staining of T cell activation markers, the suspended cells were recovered from the plate after 24 hours and 48 hours respectively. The cells recovered after 24 hours were stained with FITC anti-human CD3 antibody (Biolegend) and APC anti-human CD69 antibody (Biolegend), and the cells recovered after 48 hours were stained with FITC anti-human CD3 antibody (Biolegend) and APC anti-human CD25 antibody (Biolegend). In the measurement, FACSLyric (BD biosciences) was used to measure the expression of CD3 and CD69 after 24 hours and the expression of CD3 and CD25 after 48 hours. Flow jo 10.8.1 (BD biosciences) analysis software was used in the analysis to calculate the proportion of CD69-positive cells in the CD3-positive cell population after 24 hours and the proportion of CD25-positive cells in the CD3-positive cell population after 48 hours. Curve graph production and EC 50 、E max The calculation of 95% confidence interval (CI) was performed using the statistical software Prism (GraphPad Software).

[0533] Quantification of cytokine production

[0534] The secretion of human TNFα, human IL-2, human IL-6, and human interferon γ in the supernatant of co-culture of tumor cells and hPBMC in the presence of 53L10-M13 and Tb535H was evaluated by ELISA. Briefly, the culture supernatant after 48 hours of T cell activation experiment was centrifuged and human TNFα was evaluated by BD OptEIA TM Human TNF ELISA Set (BD bioscience), human IL-2 using BD OptEIA TMCytokines were quantified using Human IL-2 ELISA Set (BD bioscience), Human IL-6 ELISA Kit (R&D Systems) and Human IFN-gamma ELISA Kit (R&D Systems) according to the manufacturer's recommendations, and the concentration values ​​were converted to pg / mL.

[0535] Cell killing assay using flow cytometry

[0536] Human peripheral blood mononuclear cells (hPBMC) from healthy donors were thawed and rested overnight according to the instructions of the vendor (CTL), and the cells were collected and counted before use. In the case of activated PBMC, Dynabeads HumanT-activator CD3 / CD28 (Thermo Fisher Scientific) was further cultured for 3 days, the magnetic beads were removed, the cells were rested overnight, and the cells were collected and counted before use. Target tumor cells were labeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer's recommendations (labeling reaction at 25°C for 3 minutes) and plated in 48-well flat-bottom plates at 4×10 4 The cells were plated at a density of 10 cells / well for 4 hours. hPBMCs were added thereto, wherein the effector: target ratio was 10:1 when the hPBMCs were not activated and 1:1 when the hPBMCs were activated. Then, 53L10-M13 and Tb535H were added at different concentrations and incubated at 37°C for 48 hours. After 48 hours, the cells were collected, the dead cells were stained with PI (Thermo fisherscientific), and the number of PKH67-positive PI-negative cells was determined using FACSLyric (BD biosciences). In the analysis, Flow jo 10.8.1 (BD biosciences) analysis software was used to calculate the cytotoxicity (Cytotoxicity) (%) = (1-(the ratio of the PKH67-positive PI-negative cell population when activated PBMCs and each construct were added at each concentration) / (the ratio of the PKH67-positive PI-negative cell population when only activated PBMCs were added to A549). Curve graph preparation and EC 50 、E max The calculation of 95% confidence interval (CI) was performed using the statistical software Prism (GraphPad Software).

[0537] Industrial Applicability

[0538] According to the present invention, a fusion protein in the form of a multispecific tri-antibody with significantly increased anti-tumor effect compared to the conventional bispecific tri-antibody, i.e., fusion protein Tb535H, can be provided. The fusion protein of the present invention is a trispecific tri-antibody that has not only binding ability to 5T4 and CD3 receptor complex, but also binding ability to PD-1, PD-L1 or LAG3 (i.e., also has immune checkpoint inhibitory activity), and can exert excellent anti-tumor effects.

[0539] Sequence Listing Free Text

[0540] Sequence numbers 1 to 95 and 106 to 109: recombinant peptides.

Claims

1. A fusion protein comprising two different chains, a first chain and a second chain, The first chain contains two antibody variable regions VH1 and VH2, one antibody variable region VL2, and one antibody constant region CH1 or CL. The second chain contains two antibody variable regions VL1 and VL3, one antibody variable region VH3, and one antibody constant region CL or CH1. The first chain and the second chain have a heterodimer interaction. The VH1 / VL1 binding domain, VH2 / VL2 binding domain, and VH3 / VL3 binding domain as the three combined VH / VL binding domains formed in the fusion protein have a structure selected from: (i) Binding ability to 5T4, (ii) binding capacity to the CD3 receptor complex, and (iii) Binding ability to PD-1, PD-L1 or LAG3 Any combination of The above three combinations of VH / VL binding domains have mutually different binding abilities.

2. The fusion protein according to claim 1, wherein The above-mentioned fusion protein is a heterodimeric protein comprising the above-mentioned first chain and the above-mentioned second chain.

3. The fusion protein according to claim 1, wherein The two different chains mentioned above contain: a) Chain 1: VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or VH(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and Chain 2: VL(5T4)-CL-L1-VH(X)-L2-VL(X) or Combination of VL(5T4)-CL-L1-VL(X)-L2-VH(X); b) Chain 1: VH(5T4)-CH1-L1-VH(X)-L2-VL(X) or VH(5T4)-CH1-L1-VL(X)-L2-VH(X), and Chain 2: The combination of VL(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or VL(5T4)-CL-L1-VL(CD3)-L2-VH(CD3); c) Chain 1: VL(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) or VL(5T4)-CH1-L1-VL(CD3)-L2-VH(CD3), and the second chain: A combination of VH(5T4)-CL-L1-VH(X)-L2-VL(X) or VH(5T4)-CL-L1-VL(X)-L2-VH(X); d) Chain 1: VL(5T4)-CH1-L1-VH(X)-L2-VL(X) or VL(5T4)-CH1-L1-VL(X)-L2-VH(X), and the second chain: The combination of VH(5T4)-CL-L1-VH(CD3)-L2-VL(CD3) or VH(5T4)-CL-L1-VL(CD3)-L2-VH(CD3); e) Chain 1: VH(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or VH(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and the second chain: A combination of VL(CD3)-CL-L1-VH(X)-L2-VL(X) or VL(CD3)-CL-L1-VL(X)-L2-VH(X); f) Chain 1: VH(CD3)-CH1-L1-VH(X)-L2-VL(X) or VH(CD3)-CH1-L1-VL(X)-L2-VH(X), and the second chain: A combination of VL(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or VL(CD3)-CL-L1-VL(5T4)-L2-VH(5T4); g) Chain 1: VL(CD3)-CH1-L1-VH(5T4)-L2-VL(5T4) or VL(CD3)-CH1-L1-VL(5T4)-L2-VH(5T4), and the second chain: A combination of VH(CD3)-CL-L1-VH(X)-L2-VL(X) or VH(CD3)-CL-L1-VL(X)-L2-VH(X); h) First chain: VL(CD3)-CH1-L1-VH(X)-L2-VL(X) or VL(CD3)-CH1-L1-VL(X)-L2-VH(X), and the second chain: A combination of VH(CD3)-CL-L1-VH(5T4)-L2-VL(5T4) or VH(CD3)-CL-L1-VL(5T4)-L2-VH(5T4); i) Chain 1: VH(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or VH(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and the second chain: A combination of VL(X)-CL-L1-VH(CD3)-L2-VL(CD3) or VL(X)-CL-L1-VL(CD3)-L2-VH(CD3); j) Chain 1: VH(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or VH(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and the second chain: A combination of VL(X)-CL-L1-VH(5T4)-L2-VL(5T4) or VL(X)-CL-L1-VL(5T4)-L2-VH(5T4); k) Chain 1: VL(X)-CH1-L1-VH(5T4)-L2-VL(5T4) or VL(X)-CH1-L1-VL(5T4)-L2-VH(5T4), and the second chain: A combination of VH(X)-CL-L1-VH(CD3)-L2-VL(CD3) or VH(X)-CL-L1-VL(CD3)-L2-VH(CD3); or, l) Chain 1: VL(X)-CH1-L1-VH(CD3)-L2-VL(CD3) or VL(X)-CH1-L1-VL(CD3)-L2-VH(CD3), and the second chain: A combination of VH(X)-CL-L1-VH(5T4)-L2-VL(5T4) or VH(X)-CL-L1-VL(5T4)-L2-VH(5T4), Here, in the above a) to l), VH and VL are antibody variable regions. VH(5T4) and VL(5T4) are the variable regions of the antibody against 5T4. VH(CD3) and VL(CD3) are antibody variable regions targeting CD3 receptor complex, VH(X) and VL(X) are antibody variable regions targeting PD-1, PD-L1 or LAG3, CH1 and CL are antibody constant regions, L1 and L2 are connectors.

4. The fusion protein according to claim 1, wherein The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2 and CDR3 in VH1, VH2 and VH3 are respectively composed of the following sequences: The amino acid sequences shown in SEQ ID NOs: 3, 4 and 5, The amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or 27, The amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, The amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 48 or 49 and SEQ ID NO: 50, or The amino acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59 or 49 and SEQ ID NO: 60, and The amino acid sequences of CDR1, CDR2 and CDR3 in VL1, VL2 and VL3 are respectively composed of the following sequences: The amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, The amino acid sequences shown in SEQ ID NOs: 14, 15 and 16, The amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, 32 or 33, The amino acid sequences shown in SEQ ID NOs: 41, 42 and 43, The amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, or The amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO:

64.

5. The fusion protein according to claim 1, wherein The amino acid sequences of VH1, VH2 and VH3 are different from each other, and consist of, or contain, or have at least 90% identity with the amino acid sequence of SEQ ID NO: 88, 90, 8, 92, 23, 78, 46, 81, 57, 109, 82, 83, 84 or 36, and The amino acid sequences of VL1, VL2 and VL3 are different from each other, and are composed of the amino acid sequence shown in sequence numbers 89, 91, 13, 93, 28, 79, 80, 51, 61, 85, 86, 87 or 40, or contain the amino acid sequence, or have at least 90% identity with the amino acid sequence.

6. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH(5T4) are respectively composed of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL(5T4) are composed of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

7. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of VH (CD3) are respectively composed of the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL (CD3) are composed of the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

8. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining region (CDR) 1, CDR2 and CDR3 of VH(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or 27, wherein X is PD-1, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, 32 or 33, wherein X is PD-1.

9. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining region (CDR) 1, CDR2, and CDR3 of VH(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 47, SEQ ID NO: 48 or 49, and SEQ ID NO: 50, wherein X is PD-L1, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, wherein X is PD-L1.

10. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining region (CDR) 1, CDR2, and CDR3 of VH(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59 or 49, and SEQ ID NO: 60, wherein X is PD-L1, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, wherein X is PD-L1.

11. The fusion protein according to claim 3, wherein The amino acid sequences of the complementarity determining region (CDR) 1, CDR2, and CDR3 of VH(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, wherein X is LAG3, and The amino acid sequences of CDR1, CDR2 and CDR3 of VL(X) are respectively composed of the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, wherein X is LAG3.

12. The fusion protein according to claim 3, wherein The amino acid sequence of VH(5T4) consists of the amino acid sequence shown in SEQ ID NO: 88 or 90, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and The amino acid sequence of VL(5T4) consists of the amino acid sequence shown in SEQ ID NO: 89 or 91, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

13. The fusion protein according to claim 3, wherein The amino acid sequence of VH (CD3) consists of the amino acid sequence shown in SEQ ID NO: 8 or 92, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and The amino acid sequence of VL (CD3) consists of the amino acid sequence shown in SEQ ID NO: 13 or 93, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence.

14. The fusion protein according to claim 3, wherein The amino acid sequence of VH(X) consists of, comprises, or has at least 90% identity with the amino acid sequence of SEQ ID NO: 23 or 78, wherein X is PD-1, and The amino acid sequence of VL(X) consists of the amino acid sequence shown in SEQ ID NO: 28, 79 or 80, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence, wherein X is PD-1.

15. The fusion protein according to claim 3, wherein The amino acid sequence of VH(X) consists of, comprises, or has at least 90% identity with the amino acid sequence of SEQ ID NO: 46 or 81, wherein X is PD-L1, and The amino acid sequence of VL(X) consists of the amino acid sequence shown in SEQ ID NO: 51, or includes the amino acid sequence, or has at least 90% identity with the amino acid sequence, wherein X is PD-L1.

16. The fusion protein according to claim 3, wherein The amino acid sequence of VH(X) consists of, comprises, or has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 57, 109, 82, 83 or 84, wherein X is PD-L1, and The amino acid sequence of VL(X) consists of the amino acid sequence shown in SEQ ID NO: 61, 85, 86 or 87, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence, wherein X is PD-L1.

17. The fusion protein according to claim 3, wherein The amino acid sequence of VH(X) consists of, comprises, or has at least 90% identity with the amino acid sequence of SEQ ID NO: 36, wherein X is LAG3, and The amino acid sequence of VL(X) consists of the amino acid sequence shown in SEQ ID NO: 40, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, wherein X is LAG3.

18. The fusion protein according to claim 1, wherein The first chain consists of the amino acid sequence of SEQ ID NO: 1, or comprises the amino acid sequence, or has at least 90% identity with the amino acid sequence, and The second chain consists of the amino acid sequence shown in SEQ ID NO: 17, 34, 44, 55 or 107, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence.

19. The fusion protein according to claim 3, wherein Chain 1: VH(5T4)-CH1-L1-VH(CD3)-L2-VL(CD3) consists of the amino acid sequence of SEQ ID NO: 1, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence, and Chain 2: VL(5T4)CL-L1-VH(X)-L2-VL(X) is composed of the amino acid sequence shown in sequence number 17, 34, 44, 55 or 107, or contains the amino acid sequence, or has at least 90% identity with the amino acid sequence.

20. The fusion protein according to claim 1 or 3, which is a trispecific antibody against (a) 5T4, (b) CD3 receptor complex, and (c) PD-1, PD-L1 or LAG3. The fusion protein according to claim 1 or 3, which has anti-tumor activity.

22. The fusion protein according to claim 21, wherein The tumor is one that expresses 5T4 in tumor cells or cancer cells.

23. The fusion protein according to claim 22, wherein The tumor is at least one selected from the group consisting of human mesothelioma, human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human uterine end cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental site trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor. A polynucleotide encoding the fusion protein of claim 1 or 3. A vector comprising the polynucleotide of claim 24 .

26. A transformant obtained by introducing the vector according to claim 25 into a host cell.

27. A method for producing the fusion protein according to claim 1 or 3, comprising: The transformant according to claim 26 is cultured, and the fusion protein is recovered from the obtained culture.

28. A pharmaceutical composition comprising the fusion protein of claim 1 or 3.

29. The pharmaceutical composition according to claim 28, which is used in the treatment, prevention or diagnosis of tumors.

30. The pharmaceutical composition according to claim 29, wherein The tumor is one that expresses 5T4 in tumor cells or cancer cells.

31. The pharmaceutical composition according to claim 30, wherein The tumor is at least one selected from the group consisting of human mesothelioma, human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human uterine end cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental site trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

32. A method for treating, preventing or diagnosing a tumor, comprising: The pharmaceutical composition of claim 28 is administered to a subject.

33. The method of claim 32, wherein: The tumor is a tumor that expresses 5T4 in tumor cells or cancer cells.

34. The method of claim 33, wherein: The tumor is at least one selected from the group consisting of human mesothelioma, human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human uterine end cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental site trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

35. A kit for treating, preventing or diagnosing a tumor, comprising the fusion protein of claim 1 or 3.

36. The kit according to claim 35, wherein The tumor is a tumor that expresses 5T4 in tumor cells or cancer cells.

37. The kit according to claim 36, wherein The tumor is at least one selected from the group consisting of human mesothelioma, human lung cancer, human gastric cancer, human colorectal cancer, human esophageal cancer, human uterine end cancer, human ovarian cancer, human cervical cancer, human choriocarcinoma, human placental site trophoblastic tumor, human bladder cancer, human breast cancer, human pancreatic cancer, human prostate cancer, human kidney cancer, human head and neck cancer, and human non-seminoma germ cell tumor.

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