Improved granzyme b variants

Abstract

The present invention relates to granzyme B variants having increased protease activity and / or increased resistance to inhibitors; polynucleotides encoding granzyme B variants; cells expressing granzyme B variants; pharmaceutical compositions containing cells expressing granzyme B variants; and pharmaceutical compositions containing granzyme B variants. In some embodiments, the pharmaceutical compositions can be used in combination with cells expressing chimeric receptors and / or antigen binding molecules.

Description

TECHNICAL FIELD

[0001] The present application relates to a Granzyme B variant having increased protease activity and / or increased resistance to inhibitors; a polynucleotide encoding the Granzyme B variant; a cell expressing the Granzyme B variant; a pharmaceutical composition comprising the cell expressing the Granzyme B variant; and a pharmaceutical composition comprising the Granzyme B variant. BACKGROUND

[0002] Granzymes are a group of proteases including a set of family members. Granzymes are expressed in T cells and NK cells (cytotoxic lymphocytes), and when these cells recognize a target antigen, granzymes are secreted into a target cell (a cell expressing the target antigen) along with perforin and the like, and exert cytotoxic activity. After being transferred to the target cell, granzymes cleave various substrates such as BID and caspases, and finally activate a signal transduction system involved in apoptosis of the target cell, thereby inducing cell death of the target cell (NPL 1).

[0003] Among the granzyme family, Granzyme A and Granzyme B are considered to have particularly large contributions to cytotoxic activity. Granzyme A exerts its action by forming a dimer, while Granzyme B exerts its action as a monomer. Therefore, it is easy to produce, express, and purify variants of Granzyme B using protein engineering, and research and development have been conducted with the aim of drug discovery. For example, various variants have been reported, including Granzyme B having an scFv that binds to a tumor-associated antigen fused thereto (NPL 2).

[0004] On the other hand, the use of Granzyme B as an anti-tumor agent is limited. The reasons include the limited protease activity of native Granzyme B and the inhibition of Granzyme B activity by the development of inhibitors of Granzyme B in target cells. NPL 3 indicates that PI-9, an inhibitor of Granzyme B activity, is highly expressed in tumors. In addition, NPL 4 indicates that heparin, which is known to enhance its production in tumors, inhibits Granzyme B activity. That is, in tumors in which these inhibitors are highly expressed, it is considered that even if Granzyme B is made to act on these tumors, it cannot exert sufficient cytotoxic activity, and therefore has only limited anti-tumor activity.

[0005] As a Granzyme variant having resistance to these inhibitors, PI-9-resistant (PTL 1 and NPL 5) and heparin-resistant (NPL 6) Granzyme B variants have been reported in previous studies. Further, a Granzyme B variant (PTL 2) in which rat Granzyme B was comprehensively modified to cleave VEGF and VEGF receptor has been reported.

[0006] On the other hand, when cytotoxic substances such as granzymes are used for treatment, it is preferable that they specifically exert cytotoxic effects on target cells (e.g., tumor cells). As one example of a technique to achieve this, a method is reported that uses a transgenic cell transfected with a gene encoding wild-type granzyme or the like and a gene encoding a chimeric antigen receptor (CAR), such that the cell is activated by binding of the CAR to a target antigen, thereby expressing granzyme or the like at an increased level (PTL 3).

[0007] BIBLIOGRAPHY

[0008] PATENT LITERATURE

[0009] [PTL 1] US9528101B

[0010] [PTL 2] WO2005100556A2

[0011] [PTL 3] Japanese Patent Application Kohyo Publication No. (JP-A) 2019-526285 (corresponding to unexamined Japanese national stage publication of non-Japanese international publication)

[0012] NON-PATENT LITERATURE

[0013] [NPL 1] How Do Cytotoxic Lymphocytes Kill Cancer Cells?

[0014] Luis Martinez-Lostao, Alberto Anel and Julian Pardo

[0015] Clin Cancer Res. 2015 Nov 15; 21(22): 5047-56

[0016] [NPL 2] Delivery and therapeutic potential of human granzyme B

[0017] Kurschus FC1, Jenne DE.

[0018] Immunol Rev. 2010 May; 235(1): 159-71

[0019] [NPL 3] Blockade of the granzyme B / perforin pathway through overexpression of the serine protease inhibitor PI-9 / SPI-6 constitutes a mechanism for immune escape by tumors

[0020] J. P. Medema, J. de Jong, L. T. C. Peltenburg, E. M. E. Verdegaal, A. Gorter, S. A. Bres, K. L. M. C. Franken, M. Hahne, J. P. Albar, C. J. M. Melief, and R. Offringa

[0021] Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11515-11520

[0022] [NPL 4] Cationic Sites on Granzyme B Contribute to Cytotoxicity by Promoting Its Uptake into Target Cells

[0023] Catherina H. Bird, Jiuru Sun, Kheng Ung, Diana Karambalis, James C. Whisstock, Joseph A. Trapani, and Phillip I. Bird

[0024] Mol Cell Biol. 2005 Sep;25(17):7854-7867

[0025] [NPL 5] Design of human granzyme B variants resistant to serpin B9

[0026] Losasso V, Schifer S, Barth S, Carloni P.

[0027] Proteins. 2012 Nov;80(11):2514-22

[0028] [NPL 6] Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosis

[0029] Kurschus FC, Fellows E, Stegmann E, Jenne DE.

[0030] Proc Natl Acad Sci U S A. 2008 Sep 16; 105(37): 13799-13804 [SUMMARY]

[0031] [PROBLEM TO BE SOLVED BY THE INVENTION]

[0032] The granule protease B variants reported in PTL 1 show a slight increase in protease activity compared to human wild-type granule protease B, and can not have sufficient cytotoxic activity. In PTL 2, although the rat granule protease B was changed comprehensively, there was no disclosure of resistance data to inhibitors, and it was not clear whether these variants exerted sufficient cytotoxic activity in the presence of inhibitors.

[0033] In addition, previous studies have proposed methods of using purified granule protease B or variants thereof by direct intravenous injection or the like. However, in such methods of use, there is a risk that cells that have taken up the administered granule protease B can undergo cell death and cause serious side effects. Therefore, it is also necessary to research techniques other than direct injection as a therapeutic method using granule protease B.

[0034] SOLUTION TO THE PROBLEM

[0035] Based on the above problems, the present inventors aimed to discover multiple changes that increase protease activity by changing and combining changes comprehensively, in order to improve the activity of granule protease B. As a result, the present inventors found that the activity of the granule protease B protease was enhanced by genetic changes, which had not been reported in previous literature. In addition, the present inventors showed that combining these changes can significantly increase the activity relative to wild-type granule protease B. Furthermore, the present inventors examined the resistance of the variants to granule protease B inhibitors to show that the variants exhibit high protease activity even in the presence of such inhibitors.

[0036] Accordingly, the present disclosure provides a Granzyme B variant whose protease activity and / or resistance to an inhibitor has been enhanced by genetic alteration, and further provides medical uses of the Granzyme B variant, pharmaceutical compositions comprising such variants, pharmaceutical compositions comprising cells expressing such variants, and pharmaceutical compositions comprising such variants for use in combination with receptors and / or therapeutic antibodies.

[0037] The present disclosure is based on the above findings, and specifically encompasses the following exemplary described embodiments.

[0038] [1] A Granzyme B variant comprising one or more amino acid residues selected from the group consisting of 1) to 20) below:

[0039] 1) T, E, N, or V at position 43; 2) L or F at position 44; 3) Q, L, or A at position 45; 4) I, E, F, or Q at position 46; 5) V at position 47; 6) F or K at position 48; 7) L at position 99; 8) A at position 106; 9) M at position 149; 10) L at position 151; 11) P at position 155; 12) L at position 172; 13) Q, E, or I at position 175; 14) P at position 183; 15) L at position 184; 16) R at position 200; 17) I at position 217; 18) F at position 219; 19) G or S at position 222; and 20) V at position 229.

[0040] [2] The Granzyme B variant of [1], wherein the variant comprises any one of the amino acid residue combinations of 1) to 12) below:

[0041] 1) L at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0042] 2) L at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0043] 3) F at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0044] 4) F at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0045] 5) L at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0046] 6) L at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0047] 7) L at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0048] 8) L at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0049] 9) F at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0050] 10) F at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0051] 11) F at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; and

[0052] 12) F at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0053] [3] The granzyme B variant of [1] or [2], wherein the protease activity is enhanced more than human wild-type granzyme B.

[0054] [4] The granzyme B variant of any one of [1] to [3], which is resistant to an inhibitor of human wild-type granzyme B.

[0055] [5] The granzyme B variant of [4], wherein the inhibitor is PI-9 or heparin.

[0056] [6] An isolated nucleic acid encoding the granzyme B variant of any one of [1] to [5].

[0057] [7] A vector comprising the isolated nucleic acid of [6].

[0058] [8] A cell transformed or transduced with the isolated nucleic acid of [6] or the vector of [7].

[0059] [9] A cell expressing the granzyme B variant of any one of [1] to [5].

[0060]

[10] A pharmaceutical composition comprising the isolated nucleic acid of [6], the vector of [7], or the cell of [8] or [9].

[0061]

[11] A pharmaceutical composition comprising the granzyme B variant of any one of [1] to [5].

[0062]

[12] A pharmaceutical composition for use in combination with administration of a cell expressing a receptor, wherein the composition comprises a granzyme B variant or a cell expressing a granzyme B variant,

[0063] wherein the receptor activates the cell expressing the receptor through its binding to a ligand, and

[0064] wherein the granzyme B variant has a higher protease activity than human wild-type granzyme B and is resistant to an inhibitor of human wild-type granzyme B.

[0065]

[13] The pharmaceutical composition of

[12] , wherein the receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and binds to the ligand through the extracellular binding domain.

[0066]

[14] The pharmaceutical composition of

[12] , wherein the receptor is a T cell receptor, and the ligand of the T cell receptor is a neoantigen.

[0067]

[15] The pharmaceutical composition of any one of

[12] to

[14] , wherein the granzyme B variant is the granzyme B variant in [1], [2], or [5].

[0068]

[16] The pharmaceutical composition of any one of

[12] to

[15] , comprising the cell expressing the receptor.

[0069]

[17] The pharmaceutical composition of

[16] , wherein the receptor and the granzyme B variant are expressed in the same T cell.

[0070]

[18] A pharmaceutical composition for use in combination with administration of an antigen-binding molecule and administration of a cell expressing a chimeric receptor, wherein the composition comprises a granzyme B variant or a cell expressing a granzyme B variant,

[0071] wherein the antigen-binding molecule has the ability to bind a target antigen,

[0072] wherein the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and is capable of binding a cell expressing the target antigen through the extracellular binding domain binding to the antigen-binding molecule, and

[0073] wherein the granzyme B variant has a higher protease activity than human wild-type granzyme B and is resistant to an inhibitor of human wild-type granzyme B.

[0074]

[19]

[18] pharmaceutical compositions,

[0075] The antigen-binding molecule contains a linker that can be cleaved by a protease, and

[0076] The extracellular binding domain can bind antigen-binding molecules after the linker is cleaved.

[0077]

[20]

[18] or

[19] pharmaceutical compositions wherein the granzyme B variant is the granzyme B variant in [1], [2] or [5].

[0078] A pharmaceutical composition comprising any one of

[21]

[18] to

[20] , wherein the composition comprises cells expressing a chimeric receptor.

[0079]

[22]

[21] pharmaceutical compositions wherein the chimeric receptor and granzyme B variant are expressed in the same T cells.

[0080] The pharmaceutical composition of any one of

[23]

[13] and

[15] to

[22] , wherein the chimeric receptor is a chimeric antigen receptor.

[0081] [1] A granzyme B variant comprising one or more amino acid residues selected from the following 1) to 20) relative to human wild-type granzyme B:

[0082] 1) 43T, 43E, 43N, or 43V; 2) 44L or 44F; 3) 45Q, 45L, or 45A; 4) 46I, 46E, 46F, or 46Q; 5) 47V; 6) 48F or K; 7) 99L; 8) 106A; 9) 149M; 10) 151L; 11) 155P; 12) 172L; 13) 175Q, 175E, or 175I; 14) 183P; 15) 184L; 16) 200R; 17) 217I; 18) 219F; 19) 222G or 222S; and 20) 229V.

[0083] Granase B variants of [A2][A1] that contain one or more amino acid residue mutations selected from the following 1) to 20) relative to human wild-type granase B:

[0084] 1) Q43T, Q43E, Q43N, or Q43V; 2) K44L or K44F; 3) S45Q, S45L, or S45A; 4) L46I, L46E, L46F, or L46Q; 5) K47V; 6) R48F; 7) A99L; 8) S106A; 9) Q149M; 10) A151L; 11) K155P; 12) K172L; 13) S175Q, S175E, or S175I; 14) S183P; 15) T184L; 16) K200R; 17) V217I; 18) Y219F; 19) N222G or N222S; and 20) A229V.

[0085] [A3] The granzyme B variant of [A1], wherein the amino acid residue is mutated to any of 1) to 12) below:

[0086] 1) 44L, 48K, 155P, 172L, 175I, and 200R;

[0087] 2) 44L, 48E, 155P, 172L, 175I, and 200R;

[0088] 3) 44F, 48K, 155P, 172L, 175I, and 200R;

[0089] 4) 44F, 48E, 155P, 172L, 175I, and 200R;

[0090] 5) 44L, 46I, 155P, 172L, 175I, and 200R;

[0091] 6) 44L, 46E, 155P, 172L, 175I, and 200R;

[0092] 7) 44L, 46F, 155P, 172L, 175I, and 200R;

[0093] 8) 44L, 46Q, 155P, 172L, 175I, and 200R;

[0094] 9) 44F, 46I, 155P, 172L, 175I, and 200R;

[0095] 10) 44F, 46E, 155P, 172L, 175I, and 200R;

[0096] 11) 44F, 46F, 155P, 172L, 175I, and 200R; and

[0097] 12) 44F, 46Q, 155P, 172L, 175I, and 200R.

[0098] [A4] The granzyme B variant of [A3], wherein the amino acid residue is mutated to any one of 1) to 12):

[0099] 1) K44L, R48K, K155P, K172L, S175I, and 200R;

[0100] 2) K44L, R48E, K155P, K172L, S175I, and 200R;

[0101] 3) K44F, R48K, K155P, K172L, S175I, and 200R;

[0102] 4) K44F, R48E, K155P, K172L, S175I, and 200R;

[0103] 5) K44L, L46I, K155P, K172L, S175I, and 200R;

[0104] 6) K44L, L46E, K155P, K172L, S175I, and 200R;

[0105] 7) K44L, L46F, K155P, K172L, S175I, and 200R;

[0106] 8) K44L, L46Q, K155P, K172L, S175I, and 200R;

[0107] 9) K44F, L46I, K155P, K172L, S175I, and 200R;

[0108] 10) K44F, L46E, K155P, K172L, S175I, and 200R;

[0109] 11) K44F, L46F, K155P, K172L, S175I, and 200R; and

[0110] 12) K44F, L46Q, K155P, K172L, S175I, and 200R.

[0111] [A5] The granzyme B variant of any one of [Al] to [A4], comprising one or more amino acid residue mutations relative to human wild-type granzyme B set forth in SEQ ID NO: 1.

[0112] [A6] The granzyme B variant of any one of [Al] to [A5], wherein the protease activity is enhanced more than human wild-type granzyme B.

[0113] [A7] The Granzyme B variant of any one of [A1] to [A6], which is resistant to an inhibitor of human wild-type Granzyme B.

[0114] [A8] The Granzyme B variant of [A7], wherein the inhibitor is PI-9 or heparin.

[0115] [A9] An isolated nucleic acid encoding the Granzyme B variant of any one of [A1] to [A8].

[0116] [A10] A vector comprising the isolated nucleic acid of [A9].

[0117] [A11] A cell transformed or transduced with the isolated nucleic acid of [A9] or the vector of [A10].

[0118] [A12] A cell expressing the Granzyme B variant of any one of [A1] to [A8].

[0119] [A13] A pharmaceutical composition comprising the isolated nucleic acid of [A9], the vector of [A10], or the cell of [A11] or [A12].

[0120] [A14] A pharmaceutical composition comprising the Granzyme B variant of any one of [A1] to [A8].

[0121] [A15] The pharmaceutical composition of any one of [A12] to [A14], wherein the Granzyme B variant is the Granzyme B variant of any one of [A1] to [A5].

[0122] [A16] The pharmaceutical composition of [A15], comprising a cell expressing a receptor.

[0123] [A17] The pharmaceutical composition of [A16], wherein the receptor and the Granzyme B variant are expressed in the same T cell.

[0124] [A18] The pharmaceutical composition of

[18] or

[19] , wherein the Granzyme B variant is the Granzyme B variant of any one of [A1] to [A5].

[0125] [A19] The pharmaceutical composition of [A18], comprising a cell expressing a chimeric receptor.

[0126] [A20] The pharmaceutical composition of [A19], wherein the chimeric receptor and the Granzyme B variant are expressed in the same T cell.

[0127] [A21] The pharmaceutical composition of any one of [A15] to [A20], wherein the receptor is a chimeric antigen receptor.

[0128] [B1] The granzyme B variant of [B3] or [A6] or the pharmaceutical composition of

[12] or

[18] , wherein the in vitro protease activity of the granzyme B variant is 1.5-fold or more of human wild-type granzyme B.

[0129] [B2] The granzyme B variant or pharmaceutical composition of [B1], wherein the in vitro protease activity of the granzyme B variant is 2-fold or more of human wild-type granzyme B.

[0130] [B3] The granzyme B variant of [4] or [A7] or the pharmaceutical composition of

[12] or

[18] , wherein the in vitro protease activity in the presence of the inhibitor is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more of human wild-type granzyme B.

[0131] [B4] The granzyme B variant or pharmaceutical composition of [B3], wherein the in vitro protease activity in the presence of the inhibitor is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, or 2-fold or more of human wild-type granzyme B.

[0132] [B5] The granzyme B variant or pharmaceutical composition of [B3] or [B4], wherein the inhibitor is PI-9 or heparin.

[0133] [B6] An isolated nucleic acid encoding the granzyme B variant of any one of [B1] to [B5].

[0134] [B7] A vector comprising the isolated nucleic acid of [B6].

[0135] [B8] A cell transformed or transduced with the isolated nucleic acid of [B6] or the vector of [B7].

[0136] [B9] A cell expressing the granzyme B variant of any one of [B1] to [B5].

[0137] [B10] A pharmaceutical composition comprising the isolated nucleic acid of [B6], the vector of [B7], or the cell of [B8] or [B9].

[0138] [B11] A pharmaceutical composition comprising the granzyme B variant of any one of [B1] to [B5].

[0139] [B12] The pharmaceutical composition of any one of [B1] to [B5],

[0140] wherein the receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and binds to a ligand through the extracellular binding domain.

[0141] [B13] The pharmaceutical composition of any one of [B1] to [B5], wherein the receptor is a T cell receptor, and a ligand of the T cell receptor is a neoantigen.

[0142] [B14] The pharmaceutical composition of any one of [B1] to [B5], [B12], and [B13], wherein the granzyme B variant is the granzyme B variant of any one of [1], [2], and [A1] to [A5].

[0143] [B15] The pharmaceutical composition of any one of [B1] to [B5] and [B12] to [B14], comprising a cell expressing the receptor.

[0144] [B16] The pharmaceutical composition of [B15], wherein the receptor and the granzyme B variant are expressed in the same T cell.

[0145] [B17] The pharmaceutical composition of any one of [B1] to [B5],

[0146] wherein the antigen binding molecule comprises a linker that is cleavable by a protease, and

[0147] wherein the extracellular binding domain is capable of binding the antigen binding molecule after cleavage of the linker.

[0148] [B18] The pharmaceutical composition of any one of [B1] to [B5] and [B17], wherein the granzyme B variant is the granzyme B variant of any one of [1], [2], and [A1] to [A5].

[0149] [B19] The pharmaceutical composition of any one of [B1] to [B5], [B17], and [B18], comprising a cell expressing the chimeric receptor.

[0150] [B20] The pharmaceutical composition of [B19], wherein the chimeric receptor and the granzyme B variant are expressed in the same T cell.

[0151] [B21] The pharmaceutical composition of any one of [B12] and [B14] to [B20], wherein the chimeric receptor is a chimeric antigen receptor.

[0152] [C1] The pharmaceutical composition of any one of

[10] to

[23] for use in the treatment or prevention of cancer.

[0153] [C2] The pharmaceutical composition of any one of

[10] to

[23] for use in the treatment or prevention of an inflammatory disease.

[0154] [C3] The granzyme B variant of any one of [1] to [5], [A1] to [A8], and [B1] to [B5] or the cell of [8] or [9] for use in the treatment or prevention of cancer or an inflammatory disease.

[0155] [C4] A method of treating or preventing cancer or an inflammatory disease, wherein the method comprises administering the granzyme B variant of any one of [1] to [5], [Al] to [A8], and [Bl] to [B5], or the cell of [8] or [9].

[0156] [C5] The method of [C4], further comprising administering a cell expressing a receptor, wherein binding by the receptor to a ligand activates the cell expressing the receptor.

[0157] [C6] The method of [C5], wherein the receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and binds to a ligand through the extracellular binding domain.

[0158] [C7] The method of [C6], further comprising administering an antigen-binding molecule, wherein the antigen-binding molecule has the ability to bind a target antigen, and wherein binding by the chimeric receptor to the antigen-binding molecule through the extracellular binding domain is capable of binding a cell expressing the target antigen.

[0159] [C8] The method of [C7], wherein the antigen-binding molecule comprises a linker that is cleavable by a protease, and wherein the extracellular binding domain is capable of binding to the antigen-binding molecule after cleavage of the linker.

[0160] [C9] The method of [C5], wherein the receptor is a T cell receptor, and the ligand of the T cell receptor is a neoantigen.

[0161] [C10] The method of any one of [C5] to [C9], wherein the administering is administering a T cell expressing the receptor and the granzyme B variant.

[0162] [C11] Use of the granzyme B variant of any one of [1] to [5], or the cell of [8] or [9] in the manufacture of a therapeutic or prophylactic agent for cancer or an inflammatory disease.

[0163] [D1] A method of making an isolated nucleic acid encoding the granzyme B variant of any one of [1] to [5], [Al] to [A8], and [Bl] to [B5].

[0164] [D2] A method of producing a vector comprising the isolated nucleic acid of [6], [A9], or [B6].

[0165] [D3] A method of producing a cell transformed or transduced with the isolated nucleic acid of [6], [A9], or [B6], or the vector of [7], [A10], or [B7].

[0166] [D4] A method of producing a cell expressing the granzyme B variant of any one of [1] to [5], [Al] to [A8], and [Bl] to [B5].

[0167] [Effects of the Invention]

[0168] In one non-limiting embodiment, the granzyme B variants of the present disclosure have higher protease activity and / or resistance to inhibitors compared to wild-type granzyme B. Pharmaceutical compositions comprising granzyme B variants having such advantageous effects or cells expressing the variants of the present disclosure are more advantageous than wild-type granzyme B and prior art granzyme B variants in inducing cell death of target cells. Furthermore, cell death specific to target cells can be induced by combining the granzyme B variants of the present disclosure with drugs using cells expressing receptors and / or therapeutic antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0169] [ Figure 1 ] represents the amino acid sequence of amino acids 21 to 247 (amino acids 21 to 247 from the N-terminal side) in the amino acid sequence of human wild-type granzyme B (NCBI Reference Sequence NP_004122.2; SEQ ID NO: 1). In this sequence, the amino acids at the positions where changes were made in Example 2 are underlined.

[0170] Figure 2 Results of measuring the protease activity of the granzyme B variants in Example 4-1 are shown. The vertical axis represents the relative value of the protease activity of each granzyme B variant when the protease activity of wild-type granzyme B is set to 1. The horizontal axis represents the amino acid substitution in the variant measured.

[0171] Figure 3 Results of measuring the protease activity of the granzyme B variants in Example 4-2 are shown. The vertical axis represents the relative value of the protease activity of each granzyme B variant when the protease activity of wild-type granzyme B is set to 1: (1) in the absence of an inhibitor (buffer), (2) in the presence of heparin (Heparin), or (3) in the presence of PI-9 (PI-9). The horizontal axis represents the amino acid substitution in the variant measured. DETAILED DESCRIPTION

[0172] I. DEFINITIONS

[0173] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th Ed., John Wiley & Sons (New York), New York, 1992, provide one of skill with a general guide to many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0174] For purposes of interpreting this specification, the following definitions will apply and whenever possible, the singular will be construed to include the plural and vice versa. It is further pointed out that, unless otherwise indicated, terms used herein are to be understood in the broadest possible sense unless otherwise indicated.

[0175] As used herein, the term "granzyme B" refers to any wild-type granzyme B from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed granzyme B as well as any form of granzyme B that results from processing in the cell. The term also encompasses naturally occurring variants of granzyme B, e.g., splice variants or allelic variants. An exemplary human wild-type granzyme B amino acid sequence is set forth in SEQ ID NO: 1 (NCBI Reference Sequence. NP_004122.2), but is not limited thereto and includes variants that differ in amino acid sequence in part. Such variants include human wild-type granzyme B represented by an amino acid sequence that has 90% or more, 95% or more, 97% or more, or 99% or more sequence homology with SEQ ID NO: 1.

[0176] As used herein, "mutation" and "alteration" are used interchangeably to include "addition" of amino acid residues to an amino acid sequence, "deletion" of amino acid residues from an amino acid sequence, "insertion" of amino acid residues into an amino acid sequence, and / or "substitution" of amino acid residues in an amino acid sequence. To obtain a variant (mutant) having a desired characteristic (e.g., protease activity or resistance to an inhibitor), any combination of additions, deletions, insertions, and substitutions can be introduced. In one embodiment, a granzyme B variant of the application comprises one or more amino acid residue substitutions.

[0177] When the position of each amino acid in the granzyme B is indicated herein, the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B exemplified in SEQ ID NO: 1 (in the case of SEQ ID No: 1, the consecutive number in the amino acid sequence in which the N-terminal amino acid position is 1) is designated. For example, when the granzyme B variant of the present application is indicated as "comprising the amino acid residue F (Phe) at position 44", it means that the amino acid residue at position 44 from the N-terminal side constituting the granzyme B variant is F (Phe).

[0178] Further, when the amino acid change in the granzyme B is indicated herein, the amino acid residue before the change (i.e., wild-type granzyme B) and after the change at the position are indicated as left and right, or left or right of the amino acid number at the position (for example, in one-letter notation), respectively. For example, when the amino acid residue at position 44 corresponding to the amino acid sequence shown in SEQ ID NO: 1 from the N-terminal side is changed to F (Phe) from K (Lys), the amino acid change is indicated herein as K44F. Further, when it is simply indicated that the amino acid residue at position 44 in the granzyme B from the N-terminal side is changed to F (Phe), it is indicated as 44F.

[0179] Even if the amino acid sequence of the granzyme B to be changed is partially different from the sequence shown in SEQ ID NO: 1, a person skilled in the art can appropriately determine what position in the granzyme B to be changed actually corresponds to the position of the change shown herein (for example, by performing sequence alignment).

[0180] In the present disclosure, the granzyme B variant comprises one or more amino acid residues selected from the following 1) to 20):

[0181] 1) T, E, N, or V at position 43; 2) L or F at position 44; 3) Q, L, or A at position 45; 4) I, E, F, or Q at position 46; 5) V at position 47; 6) F or K at position 48; 7) L at position 99; 8) A at position 106; 9) M at position 149; 10) L at position 151; 11) P at position 155; 12) L at position 172; 13) Q, E, or I at position 175; 14) P at position 183; 15) L at position 184; 16) R at position 200; 17) I at position 217; 18) F at position 219; 19) G or S at position 222; and 20) V at position 229.

[0182] In another aspect, the granzyme B variant of the present disclosure comprises any one of the amino acid residue combinations of the following 1) to 12):

[0183] 1) L at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0184] 2) L at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0185] 3) F at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0186] 4) F at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200;

[0187] 5) L at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0188] 6) L at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0189] 7) L at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0190] 8) L at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0191] 9) F at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0192] 10) F at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0193] 11) F at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200;

[0194] 12) F at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200.

[0195] "Protease activity" herein refers to, especially in the case of granzyme B, the activity of granzyme B to cleave its substrate. Methods to assess the protease activity of granzyme B are well known to the person skilled in the art and various activity measurement kits and synthetic substrates are commercially available. As an example of such a synthetic substrate, a synthetic peptide with the granzyme B recognition sequence (e.g. Ile-Glu-Pro-Asp (IEPD)) is labeled with a detectable substance known (e.g. p-nitroaniline (pNA) (e.g. Ac-IEPD-pNA). Cleavage of the synthetic substrate by granzyme B releases the free detectable substance which can be quantitatively determined with a fluorometer or a spectrophotometer. For example, the assessment of the protease activity of a granzyme B variant can be performed by the method described in Example 4 of the present disclosure. For example, the assessment of the protease activity in the presence of an inhibitor can be performed using the assessment system described in Example 4 and under the same conditions of granzyme B and inhibitor concentrations as in the Examples.

[0196] The protease activity of the granzyme B variants of the present disclosure is preferably enhanced over the wild-type granzyme B, for example 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more, more preferably two-fold or more or 2.5-fold or more, particularly preferably 3-fold or more, higher than the protease activity of wild-type granzyme B.

[0197] It is known that the protease activity of wild-type granzyme B is inhibited by "inhibitors" such as PI-9 and heparin. The granzyme B variants of the present disclosure are preferably resistant to such inhibitors. "Resistance to inhibitors" herein refers to the ability to exert a higher protease activity than wild-type granzyme B in the presence of such inhibitors. The protease activity of the granzyme B variants of the present disclosure is preferably 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more, more preferably two-fold or more, 2.5-fold or more, 3-fold or more, or 3.5-fold or more, and particularly preferably 4-fold or more, 4.5-fold or more, 5-fold or more, or 5.5-fold or more, higher than the protease activity of wild-type granzyme B in the presence of such inhibitors.

[0198] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that typically contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0199] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0200] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" or "transductants" and "transduced cells," which include the primary transformed or transduced cell and progeny of the primary transformed or transduced cell that have been subjected to one or more further generations of cell division ex vivo. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical characteristics due to natural, accidental, or deliberate mutation. A mutant progeny that has the same function or biological activity as the originally transformed or transduced cell is included herein.

[0201] Herein, unless otherwise specified, "a cell expressing granzyme B" can be a cell expressing endogenous granzyme B or a cell expressing granzyme B by gene transfer. As a cell expressing endogenous granzyme B, for example, cytotoxic lymphocytes - T cells and NK cells - are known. On the other hand, a cell transfected to express granzyme B is not limited to T cells and NK cells, and recombinant granzyme B can be obtained by expressing recombinant granzyme B in various cells (e.g., purifying granzyme B from their culture supernatant). Furthermore, it is possible to introduce a gene expressing granzyme B into a cell (e.g., peripheral blood mononuclear cells (PBMCs)) derived from an individual (e.g., a healthy donor or a patient with a particular disease) and administer the transgenic cell to the same or a different individual. The transgenic cell expressing granzyme B can be treated to differentiate into a specific cell type (e.g., cytotoxic T cells) and then administered to an individual. As a method of introducing a gene encoding granzyme B into a cell, various gene transfer techniques known to those skilled in the art can be used.

[0202] A cell expressing granzyme B can be administered in combination with a cell expressing a chimeric receptor. Furthermore, a cell expressing both granzyme B and a chimeric receptor can be used, and such a cell can be produced by gene transfer of granzyme B and a chimeric receptor simultaneously or separately.

[0203] When a granzyme B variant is expressed in a cell expressing endogenous granzyme B (e.g., a T cell or an NK cell), endogenous wild-type granzyme B in the cell can or can not be knocked out.

[0204] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to the subject to which the formulation or composition is administered. The “active ingredient” can be constituted by an antibody, polypeptide, etc., or by a cell expressing an antibody, polypeptide, etc. (e.g., a granzyme B variant of the disclosure). For example, a formulation comprising a cell transformed or transduced with a nucleic acid encoding a granzyme B variant of the disclosure, or a vector comprising such a nucleic acid, can be referred to as a “pharmaceutical formulation” or “pharmaceutical composition” when the cell is administered to a patient for therapeutic or prophylactic purposes.

[0205] A “pharmaceutically acceptable carrier” refers to a component of a pharmaceutical formulation or pharmaceutical composition other than the active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0206] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0207] In one aspect, the present disclosure provides a granzyme B variant, a cell expressing a granzyme B variant, or a pharmaceutical composition comprising the same.

[0208] In one embodiment, a pharmaceutical composition of the disclosure can be used in combination with a cell expressing a receptor. A receptor is a receptor that activates a cell expressing the receptor by binding to a ligand, including, for example, but not limited to, a chimeric receptor whose extracellular binding domain binds to a ligand and a T cell receptor whose ligand is a neoantigen. A pharmaceutical composition of the disclosure can comprise a cell expressing a receptor and a cell expressing a granzyme B variant, and the receptor and the granzyme B variant can be expressed in the same cell (e.g., a T cell).

[0209] Such a pharmaceutical composition of the disclosure can be used in combination with an antigen-binding molecule capable of binding to the extracellular binding domain of the chimeric receptor. The antigen-binding molecule has the ability to bind to a target antigen, and the chimeric receptor can bind to a cell expressing the target antigen through the binding of the extracellular binding domain of the chimeric receptor to the antigen-binding molecule. In certain embodiments, the antigen-binding molecule comprises a linker that can be cleaved by a protease, and the extracellular binding domain can bind to the antigen-binding molecule after cleavage of the linker. A pharmaceutical composition of the disclosure can comprise a cell expressing a chimeric receptor and a cell expressing a granzyme B variant, and an antigen-binding molecule capable of binding to the extracellular binding domain of the chimeric receptor. The receptor and the granzyme B variant can be expressed in the same cell (e.g., a T cell).

[0210] When one or more Granzyme B variant, cells expressing Granzyme B variant, cells expressing a receptor, cells expressing both a receptor and a Granzyme B variant, and antigen binding molecules capable of binding to the extracellular binding domain of the chimeric receptor are used in combination, they can be used simultaneously, separately, or sequentially (e.g., administered to an individual).

[0211] In one embodiment, the pharmaceutical composition of the present disclosure is used to destroy a cell, induce cell death, inhibit cell proliferation, or treat or prevent a cancer or an inflammatory disease.

[0212] The term "chimeric receptor" refers to a recombinant polypeptide comprising at least an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, which provides specificity for a target cell, e.g., a cancer cell, and generates an intracellular signal when expressed in an immune effector cell. The term "chimeric antigen receptor" or "CAR" refers to a chimeric receptor whose extracellular binding domain binds to an antigen.

[0213] The term "extracellular binding domain" means any protein molecule or portion thereof that can specifically bind to a given molecule, e.g., an antigen, and includes, for example, a single chain antibody (scFv), in which the light (VL) and heavy (VH) chains of a monoclonal antibody variable region specific for a tumor antigen or the like are linked in tandem. The extracellular binding domain can be rewritten as extracellular recognition domain.

[0214] The term "transmembrane domain" includes a polypeptide located between the extracellular binding domain and the intracellular signaling domain and having the function of penetrating the cell membrane.

[0215] The term "intracellular signaling domain" refers to any oligopeptide domain or polypeptide domain known to have the function of transmitting a signal that leads to the activation or inhibition of a biological process within a cell, e.g., the activation of an immune cell such as a T cell or an NK cell, and this domain comprises at least one stimulatory molecule signaling domain derived from a T cell stimulatory molecule and at least one costimulatory molecule signaling domain derived from a T cell costimulatory molecule.

[0216] The term "T cell receptor whose ligand is a neoantigen" herein includes a T cell receptor designed to recognize a neoantigen, which is a mutated antigen resulting from a genetic mutation in a cancer cell.

[0217] Here, the term "antigen binding molecule" refers to a molecule that specifically binds to an antigenic determinant (epitope) in its broadest sense. In one embodiment, the antigen binding molecule is an antibody, an antibody fragment, or an antibody derivative. In one embodiment, the antigen binding molecule is a non-antibody protein, or a fragment thereof, or a derivative thereof.

[0218] Herein, an "antigen binding domain" refers to a region that specifically binds to and is complementary to all or part of an antigen. Herein, an antigen binding molecule comprises an antigen binding domain. When the molecular weight of an antigen is large, the antigen binding domain can bind only to a specific part of the antigen. The specific part is called an "epitope". In one embodiment, the antigen binding domain comprises an antibody fragment that binds to a specific antigen. The antigen binding domain can be provided from one or more antibody variable domains. In one non-limiting embodiment, the antigen binding domain comprises both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen binding domains include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv2)", "Fab", and "Fab'". In other embodiments, the antigen binding domain comprises a non-antibody protein that binds to a specific antigen or fragment thereof. In specific embodiments, the antigen binding domain comprises a hinge region.

[0219] Herein, "specifically binds" refers to a state of binding in which one of the molecules involved in the specific binding does not show any significant binding to molecules other than the single or multiple binding partner molecules. Also, it is used when the antigen binding domain has specificity for a particular epitope among multiple epitopes contained in an antigen. When the epitope bound by the antigen binding domain is contained in multiple different antigens, the antigen binding molecule comprising the antigen binding domain can bind to multiple antigens having the epitope.

[0220] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0221] A "native antibody" refers to an immunoglobulin molecule having different structures that exist in nature. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical disulfide-linked heavy chains. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of the constant domains, the light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l).

[0222] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6thEd., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, the VH or VL domain from an antibody that binds an antigen can be used to screen a library of complementary VL or VH domains, respectively, to isolate antibodies that bind the same antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0223] In several embodiments, the present disclosure provides a method of damaging cells, inducing cell death, inhibiting cell proliferation, treating an inflammatory disease, treating cancer, or preventing cancer, comprising administering an effective amount of a Granzyme B variant, a cell expressing the Granzyme B variant, or a pharmaceutical composition thereof of the present disclosure (hereinafter collectively referred to as "the pharmaceutical composition etc. of the present disclosure"). In some embodiments, the "effective amount" in the present invention means a dose of the pharmaceutical composition etc. of the present disclosure effective in damaging cells, inducing cell death, inhibiting cell proliferation, treating an inflammatory disease, treating cancer, or preventing cancer in an individual.

[0224] In several embodiments, "treatment" or "treating" or "therapeutic" according to the present invention means that the pharmaceutical composition etc. of the present disclosure reduces the number of cancer cells, inhibits the proliferation of cancer cells, reduces the size (volume and / or weight) of a tumor, inhibits the enlargement of a tumor, inhibits the infiltration of cancer cells into surrounding organs, inhibits the metastasis of cancer cells, or ameliorates various symptoms caused by cancer in an individual. Furthermore, in several embodiments, "prevention" or "preventing" or "prophylactic" according to the present invention means that it inhibits the increase in the number of cancer cells due to the re-proliferation of cancer cells that have been reduced, inhibits the re-proliferation of cancer cells whose proliferation has been inhibited, and inhibits the re-enlargement of a tumor whose size (volume and / or weight) has been reduced.

[0225] [Examples of the application]

[0226] The granzyme B variant of the present disclosure can be used in combination with the administration of cells expressing receptors. Examples of the receptors include chimeric receptors comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. Further, another example includes T cell receptors designed to recognize neoantigens that normal T cells cannot recognize. As one embodiment of the combination of techniques using these receptors and the granzyme B variant of the present disclosure, there is a method of collecting T cells from a patient, introducing a gene encoding a chimeric receptor (e.g., a chimeric antigen receptor) and a gene encoding a granzyme B variant into the T cells, and transferring them back into the patient. (But not limited to this, the genes can be introduced alone and the T cells can be transferred to the patient.) The chimeric antigen receptor recognizes a surface antigen of cells such as cancer cells to activate the T cells, and exerts enhanced cytotoxic activity by the granzyme B variant of the present disclosure, and thus a high therapeutic effect is expected.

[0227] Similarly, by introducing a gene encoding a T cell receptor for which a ligand is a neoantigen and a gene encoding a granzyme B variant into T cells collected from a patient, and then transferring them back into the patient, a high therapeutic effect of this combination is expected.

[0228] Further, in the combination of the granzyme B variant of the present disclosure and the chimeric receptor, a combination further combined with an antigen-binding molecule (e.g., an antibody) can also be used. In this case, the antigen-binding molecule has the ability to bind to a target antigen, and the chimeric receptor can bind to cells expressing the target antigen through the binding of the extracellular binding domain to the antigen-binding molecule. As one embodiment of the antigen-binding molecule, the antigen-binding molecule is prepared in advance to comprise a linker that can be cleaved by a protease, and the chimeric receptor can be designed to be able to bind to the antigen-binding molecule after the cleavage of the linker. As one embodiment of such a combination, there is a method of collecting T cells from a patient, introducing a gene encoding a chimeric receptor and a gene encoding a granzyme B variant into the T cells, transferring them back into the patient, and then administering a pharmaceutical composition comprising an antigen-binding molecule to the patient alone.

[0229] EMBODIMENT

[0230] The following describes embodiments of the construction, expression, purification, gene transfer, and therapeutic use of variant granzymes, but the methods of practicing the present patent are not limited to these embodiments.

[0231] [EMBODIMENT 1] Construction of a vector expressing human wild-type granzyme B

[0232] The sequence of human wild-type granzyme B (NCBI reference sequence.NP_004122.2) was genetically synthesized. Nucleotide sequences encoding an artificial secretion signal sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG (SEQ ID NO:3)) and an enterokinase recognition sequence (DDDDK (SEQ ID NO:4)) were fused to the 5' end of the nucleotide sequence encoding amino acid numbers 21-247 (SEQ ID NO:2) of the ORF encoding granzyme B, and a nucleotide sequence encoding a histidine tag was added to the C-terminal side (J Vis Exp. 2015 June 10; (100):e52911). The nucleotide sequence encoding this sequence (SEQ ID NO:5) was inserted into a mammalian cell expression vector.

[0233] Furthermore, when this document indicates an amino acid alteration in granzyme B, the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B shown in SEQ ID NO:1 (NCBI reference sequence.NP_004122.2) is specified.

[0234] [Example 2] Generation of the nucleotide sequence encoding the granzyme B variant

[0235] The substitution of single amino acid residues relative to human wild-type granzyme B, and the substitution of multiple amino acid residues in combination thereof, were performed using PCR reactions by methods known to those skilled in the art.

[0236] The amino acid position to be modified can be selected by identifying amino acid residues near the substrate binding site based on the crystal structure of human wild-type granzyme B. Figure 1 (The underlined amino acids). For these amino acid residues, primers encoding the substitution products were designed, wherein the amino acid residues were substituted by any one of all 18 amino acids except the original amino acid and cysteine. Using methods known to those skilled in the art, such as PCR using these primers, a total of 1476 nucleotide sequences were generated, encoding granzyme B variants in which a single amino acid was substituted.

[0237] Furthermore, using a similar method, nucleotide sequences encoding granzyme B variants with multiple amino acid substitutions were generated.

[0238] [Example 3] Expression and purification of granzyme B variant

[0239] The nucleotide sequence encoding the granule protease B produced in Example 2 was transfected into 1 mL of Expi293 (Invitrogen) medium according to the method specified by the supplier. After 4 days, the culture supernatant was collected, enterokinase was added to a final concentration of 0.3 μg / mL, and reacted at 4°C for 16 hours. One-tenth volume of binding buffer (250 mM Tris, 3 M NaCl, pH 7.5) was added to the reaction solution, and then 50 μL of Ni Sepharose Excel (GE healthcare, 17371201) suspended in equilibration buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.5) was added. After reacting at 4°C for one hour, the reaction solution was added to a filter plate (Merck, MSGVS2210). The granule protease B-bound Ni Sepharose Excel was washed five times with 200 μL of equilibration buffer, and then eluted with elution buffer (25 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5) at 4°C for 15 minutes to elute the granule protease B. The concentration of the purified granule protease B was calculated using the absorbance at 280 nm and the extinction coefficient calculated by the PACE method for the eluted granule protease B solution (Protein Science (1995) 4, 2411-2423).

[0240] [Example 4] Measurement of protease activity of granule protease B variants

[0241] (4-1) Granule protease B variants (single amino acid substitution)

[0242] The expressed and purified granule protease B was suspended in twice-diluted enzyme reaction buffer (2x reaction buffer, Promokine, PK-CA577-1068-80) and mixed with the reaction substrate Ac-IEPD-pNA at a final concentration of 0.5 mM (Enzo, BML-P133). The absorbance of the reaction solution at 405 nm was measured. Figure 2 The relative protease activity of the granule protease B variants measured relative to wild-type granule protease B is shown.

[0243] Figure 2 The granule protease B activity shown is defined by the following formula.

[0244] Fold change = (protease activity of granule protease B variant) / (protease activity of wild-type granule protease B)

[0245] In addition, the protease activity of granule protease B is defined by the following formula.

[0246] Protease activity = (change in absorbance of reaction solution at 405 nm per unit time) / (unit time)

[0247] (4-2) Granule B variants (multiple amino acid substitutions)

[0248] As a result of measuring 1476 variants in the above-mentioned comprehensive assay, it was found that 33 variants had increased activity relative to wild-type granule B, and 1443 variants had lower activity than wild-type. For these 33 changes that increased the protease activity of granule B, multiple variants of granule B variants that combined multiple mutations were produced by the method described in Example 2. Among the generated variants, examples in which the activity increase was particularly significant are shown in Table 1. In Table 1, the amino acid substitution position in the variant refers to the corresponding amino acid number in the amino acid sequence of human wild-type granule B shown in SEQ ID NO: 1, and the amino acid sequence of the variant shows the variant amino acid sequence corresponding to amino acid numbers 21-247 of human wild-type granule B shown in SEQ ID NO: 1.

[0249] [Table 1]

[0250] Amino acid substitutions in variants Amino acid sequences of variants K44L, R48K, K155P, K172L, S175I, K200R SEQ ID NO: 6 K44L, R48E, K155P, K172L, S175I, K200R SEQ ID NO: 7 K44F, R48K, K155P, K172L, S175I, K200R SEQ ID NO: 8 K44F, R48E, K155P, K172L, S175I, K200R SEQ ID NO: 9 K44L, L46I, K155P, K172L, S175I, K200R SEQ ID NO: 10 K44L, L46E, K155P, K172L, S175I, K200R SEQ ID NO: 11 K44L, L46F, K155P, K172L, S175I, K200R SEQ ID NO: 12 K44L, L46Q, K155P, K172L, S175I, K200R SEQ ID NO: 13 K44F, L46I, K155P, K172L, S175I, K200R SEQ ID NO: 14 K44F, L46E, K155P, K172L, S175I, K200R SEQ ID NO: 15 K44F, L46F, K155P, K172L, S175I, K200R SEQ ID NO: 16 K44F, L46Q, K155P, K172L, S175I, K200R SEQ ID NO: 17

[0251] When measuring protease activity in the presence of an inhibitor, wild-type granule B or a granule B variant was incubated at 37°C for one hour in the presence of 40 μM heparin (heparin sodium salt from porcine intestinal mucosa, Sigma-Aldrich, H3393-50KU) or 0.5 μM PI-9 (Recombinant Human Serpin B9 / SERPINB9(C-6His) (Novoprotein, CJ32)) and 0.5 mM DTT, and then mixed with 1 mM Ac-IEPD-pNA solution at a volume ratio of 1:1. All solutions were prepared using twice-diluted enzyme reaction buffer (2x reaction buffer, Promokine, PK-CA577-1068-80). When measuring the protease activity of the generated variants in the absence of an inhibitor, they were incubated at 37°C for one hour with enzyme reaction buffer containing 0.5 mM DTT instead of the inhibitor suspension, and then mixed with 1 mM Ac-IEPD-pNA solution at a volume ratio of 1:1 and measured. The measurement of absorbance was performed in the same manner as described in Example 4-1. The results of the measurement are shown in Figure 3 -(1) to -(3). As a result of the measurement, it was confirmed that the granule B activity was increased compared to wild-type granule B and the variants maintained high protease activity even in the presence of an inhibitor.

[0252] [Example 5] Expression of granule B variants in NK cell lines

[0253] The secretion signal sequence derived from wild-type granzyme B and the cathepsin C / H recognition sequence were fused to the N-terminal side of the granzyme B variants produced in Example 4 as well as the amino acid sequence corresponding to the 21st to 247th amino acids of wild-type granzyme B (SEQ ID NO: 1), and a FLAG tag was fused to the C-terminal side thereof. The nucleotide sequence encoding these fusions was transfected into an NK cell line (NKL, ATCC No.) by a mammalian expression vector pGL4.30 (Promega, E8481). This NK cell line was selected with hygromycin B. The cell line of the granzyme B variants was subjected to eBioscience TM Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) was subjected to membrane permeabilization by a method designated by those skilled in the art, and fluorescent staining was performed using an anti-FLAG antibody that recognizes the tag fused to the C-terminal of granzyme B and an isotype control antibody thereof (Biolegend, 637310 and 400508). The stained cells were detected with FACS verse (BD). As a result, a peak associated with granzyme B expression was observed in cells transfected with granzyme B, and it was confirmed that the transfected granzyme B was expressed.

[0254] [Example 6] Measurement of in vitro cytotoxic activity of cell lines expressing granzyme B variants

[0255] The established cell lines expressing granzyme B variants and target cells (any one of BxPC-3, HuCCT-1, and MCAS) were added to a 96-well plate, and anti-EGFR antibodies CetuH0-Hl076 / CetuL4-k0 / / CetuH0-Kn125 / CetuL4-k0 diluted to each concentration were added. After reaction at 37°C, LDH release associated with cell death was quantitatively measured using a Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific, 88954) according to the method designated by the supplier. As a result, it was shown that the transgenic cells expressing granzyme B variants exhibited stronger cytotoxic activity than the transgenic cells expressing wild-type granzyme B.

[0256] [Example 7] Production of viral vectors encoding granzyme B variants

[0257] Retroviral vectors encoding human wild-type granzyme B and granzyme B variants were prepared by a method designated by the supplier using a pMCs-IRES-GFP retroviral vector (Cell Biolabs, Inc., RTV-040).

[0258] [Example 8] Gene transfer of granzyme B variants to primary T cells

[0259] HLA-A2 + Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation. PBMCs were cultured in 24-well tissue culture plates in AIM V medium (GIBCO brand; Invitrogen) supplemented with 5% human AB serum (Sigma-Aldrich), 1% MEM non-essential amino acids, 1% penicillin-streptomycin, and 100 U / mL recombinant human IL-2 (BioLegend, San Diego, CA, USA) at 3 x 10 6 cells per well and activated with 50 ng / mL OKT3 (eBioscience, San Diego, CA, USA). Two days later, cells were collected for retroviral transduction. For transduction, 24-well non-tissue culture treated plates (BD Biosciences, Franklin Lakes, NJ, USA) were coated with 10 pg / mL recombinant human fibronectin fragment (RetroNectin; Takara Bio Inc., Otsu, Shiga, Japan) at 0.5 mL / well, 4 °C overnight. After incubation, the wells were blocked with 1 mL of Hanks solution (GIBCO brand; Invitrogen) supplemented with 2.5% human AB serum for 30 min at room temperature and washed with Hanks solution supplemented with 2.5% N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (GIBCO brand; Invitrogen).

[0260] Transduction was performed by previously described methods (Johnson et al., Blood 114, 535-546 (2009)). Briefly, approximately 2.5 mL of retroviral supernatant was added to each coated well, followed by centrifugation at 2000 g for 2 h at 32 °C. 1.5 mL of viral supernatant was removed and 1 x 10 6 activated PBMCs (0.5 mL) were added to each well. The plates were centrifuged at 1000 g for 10 min, followed by incubation at 37 °C overnight.

[0261] After transduction, cells were washed and maintained in the presence of IL-2 (100 U / mL) and used for experiments five days after transduction. Expression of granzyme B in transduced human T cells was determined by flow cytometry after staining with an antibody recognizing a FLAG tag at the C-terminal end of granzyme B (Biolegend, 637310) or its isotype control antibody (Biolegend, 400508).

[0262] [Example 9] Evaluation of cytotoxic activity of T cells transfected with granzyme B variants

[0263] T cells expressing granzyme prepared in Example 8 and target cells (any of BxPC-3, HuCCT-1, MCAS) were added to a 96-well plate, respectively, and anti-EGFR anti-CD3 bispecific antibody CetuH0-F760nN17 / CetuL4-k0 / / TR01H113-F760nP17 / L0011-k0 diluted to each concentration was added. After reaction at 37°C, LDH release associated with cell death was quantitatively determined using a Pierce LDH Cytotoxicity Assay Kit (Thermofisher scientific, 88954) according to the method specified by the supplier. The results showed that transgenic cells expressing granzyme B variants exhibited stronger cytotoxic activity than transgenic cells expressing wild-type granzyme B.

[0264] [Example 10] Evaluation of cytotoxic activity of T cells transfected with granzyme B variants in vitro

[0265] The cytotoxic activity of granzyme-transfected T cells produced in Example 8 was also evaluated using a BD FACSVerse TM (BD Biosciences). Cancer cells BxPC-3, HuCCT-1, or MCAS were prepared as target cells. Target cells were seeded at 1 x 10 5 cells or 3 x 10 5 cells in a 6-well plate, respectively. T cells expressing granzyme B variants or wild-type granzyme B were used as effector cells and mixed so that the ratio of effector cells to target cells (E:T) was 1:1 or 1:3. Next, anti-EGFR anti-CD3 bispecific antibody CetuH0-F760nN17 / CetuL4-k0 / / TR01H113-F760nP17 / L0011-k0 was added at 10 μg per well. After 48 hours of addition, granzyme B-transfected T cells and target cells were collected. The number of live cells was quantitatively determined using a Zombie Aqua TMFixable Viability Kit (BioLegend, 423102) was used to stain dead cells in the collected cells, and an anti-human CD45 antibody (BioLegend, 304039) was used to stain T cells expressing granzyme B.

[0266] Cytotoxic activity was evaluated by the proportion of residual cancer cells. The proportion of residual cancer cells was calculated as the proportion of CD45- part cells among live cells. These results indicate that granzyme-expressing T cells have enhanced cytotoxic activity in vitro.

[0267] [Industrial applicability]

[0268] The granzyme B variant of the present disclosure exhibits higher protease activity than wild-type granzyme B, and thus is more useful than wild-type granzyme B in treatments that induce cell death of target cells (e.g., cancer cells). In addition, the granzyme B variant of the present disclosure exhibits high protease activity even in the presence of an inhibitor, and thus is more useful than wild-type granzyme B in treatments that induce cell death in tumors that highly express such an inhibitor.

Claims

1. A granzyme B variant of human wild-type granzyme B relative to SEQ ID NO:1, wherein the amino acid residue mutation of said variant consists of any one of the following combinations of amino acid residues 1) to 12): 1) L at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200; 2) L at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200; 3) F at position 44, K at position 48, P at position 155, L at position 172, I at position 175, and R at position 200; 4) F at position 44, E at position 48, P at position 155, L at position 172, I at position 175, and R at position 200; 5) L at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 6) L at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 7) L at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 8) L at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 9) F at position 44, I at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 10) F at position 44, E at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; 11) F at position 44, F at position 46, P at position 155, L at position 172, I at position 175, and R at position 200; as well as 12) F at position 44, Q at position 46, P at position 155, L at position 172, I at position 175, and R at position 200.

2. An isolated nucleic acid encoding the granzyme B variant of claim 1.

3. A vector comprising the isolated nucleic acid as described in claim 2.

4. Cells transformed or transduced using the isolated nucleic acid as described in claim 2 or the vector as described in claim 3.

5. Cells expressing the granzyme B variant of claim 1.

6. A pharmaceutical composition comprising the isolated nucleic acid of claim 2, the carrier of claim 3, or the cell of claim 4 or 5.

7. A pharmaceutical composition comprising the granzyme B variant of claim 1.

8. A pharmaceutical composition for administration in combination with cells expressing a receptor, wherein the composition comprises the granzyme B variant of claim 1 or cells expressing the granzyme B variant. The receptor activates cells expressing the receptor through its binding to a ligand, and The granzyme B variant described herein has higher protease activity than the human wild-type granzyme B shown in SEQ ID NO:1 and is resistant to inhibitors of the human wild-type granzyme B.

9. The pharmaceutical composition of claim 8, wherein the receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain and an intracellular signal transduction domain, and binds to a ligand through the extracellular binding domain.

10. The pharmaceutical composition of claim 8, wherein the receptor is a T-cell receptor and the ligand of the T-cell receptor is a neoantigen.

11. The pharmaceutical composition of claim 8 or 9, wherein the composition comprises cells expressing the receptor.

12. The pharmaceutical composition of claim 10, wherein the composition comprises cells expressing the receptor.

13. The pharmaceutical composition of claim 11, wherein the receptor and the granzyme B variant are expressed in the same T cells.

14. The pharmaceutical composition of claim 12, wherein the receptor and the granzyme B variant are expressed in the same T cells.

15. The pharmaceutical composition of claim 9, wherein the chimeric receptor is a chimeric antigen receptor.

16. The pharmaceutical composition of claim 13, wherein the receptor is a chimeric antigen receptor.

17. The pharmaceutical composition of claim 11, wherein the receptor is a chimeric antigen receptor.

18. A pharmaceutical composition for administration of an antigen-binding molecule and for administration of cells expressing a chimeric receptor, wherein the composition comprises the granzyme B variant of claim 1 or cells expressing the granzyme B variant. The antigen-binding molecule described herein has the ability to bind to the target antigen. The chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain, and is capable of binding to cells expressing the target antigen via the binding of the antigen-binding molecule through the extracellular binding domain. The granzyme B variant described herein has higher protease activity than the human wild-type granzyme B shown in SEQ ID NO:1 and is resistant to inhibitors of the human wild-type granzyme B.

19. The pharmaceutical composition of claim 18, The antigen-binding molecule contains a linker that can be cleaved by a protease, and The extracellular binding domain is capable of binding the antigen-binding molecule after the linker is cleaved.

20. The pharmaceutical composition of claim 18 or 19, comprising cells expressing the chimeric receptor.

21. The pharmaceutical composition of claim 20, wherein the chimeric receptor and the granzyme B variant are expressed in the same T cells.

22. The pharmaceutical composition of any one of claims 18, 19 and 21, wherein the chimeric receptor is a chimeric antigen receptor.

23. The pharmaceutical composition of claim 20, wherein the chimeric receptor is a chimeric antigen receptor.

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