Improved Granzyme B variant

Abstract

The present invention pertains to: a granzyme B variant having enhanced protease activity and / or enhanced tolerance against an inhibitory factor; 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. In some embodiments, the pharmaceutical composition can be used in combination with a cell expressing a chimeric receptor and / or an antigen-binding molecule.

Description

Technical Field

[0001] The present invention relates to a granzyme B variant with enhanced protease activity and / or resistance to inhibitors; a polynucleotide encoding the granzyme B variant; a cell expressing the granzyme B variant; a pharmaceutical composition containing the cell expressing the granzyme B variant; and a pharmaceutical composition containing the granzyme B variant.

Background Art

[0002] Granzymes are a group of proteases consisting of a family. Granzymes are expressed in cytotoxic lymphocytes such as T cells and NK cells, and when these cells recognize a target antigen, they are secreted together with perforin and the like to target cells (cells expressing the target antigen) to exert cytotoxic activity. After migrating into the target cell, granzyme cleaves various substrates such as BID and caspases, and finally induces cell death of the target cell by activating the signal transduction system related to apoptosis in the target cell (Non-Patent Document 1).

[0003] Among the granzyme family, granzyme A and granzyme B are particularly suggested to contribute greatly to cytotoxic activity. Among these, granzyme A forms a dimer and functions, while granzyme B functions as a monomer. Therefore, in the case of granzyme B, the production, expression, and purification of variants using protein engineering are easy, and research and development including drug discovery have been conducted. For example, various variants (Non-Patent Document 2) in which scFv binding to a tumor-associated antigen is fused to granzyme B have been reported.

[0004] On the other hand, there are limitations to the use of granzyme B as an antitumor agent. These limitations include the limited protease activity of naturally occurring granzyme B and the suppression of granzyme B activity due to the expression of inhibitors against granzyme B in target cells. Non-patent document 3 shows that PI-9, an inhibitor of granzyme B activity, is highly expressed in tumors. Furthermore, non-patent document 4 shows that heparin, which is known to be produced in increased amounts in tumors, inhibits granzyme B activity. In other words, even if granzyme B is applied to tumors that highly express these inhibitors, sufficient cytotoxic activity may not be exerted, and therefore its antitumor activity may be limited.

[0005] Previous studies have reported granzyme B variants that are resistant to these inhibitors, including PI-9 resistant (Patent Document 1, Non-Patent Document 5) and heparin resistant (Non-Patent Document 6). There are also reports of granzyme B variants (Patent Document 2) that have undergone comprehensive modifications to rat granzyme B to cleave VEGF and VEGF receptors.

[0006] On the other hand, when cytotoxic substances such as granzymes are used in therapy, it is preferable that they exert a specific cytotoxic effect on target cells (e.g., tumor cells). As an example of a method to achieve this, a method has been reported that uses gene-modified cells into which a gene encoding wild-type granzymes and a gene encoding a chimeric antigen receptor (CAR) have been introduced, and in which the cells are activated when the CAR binds to the target antigen, increasing the expression of granzymes (Patent Document 3). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] US9528101B [Patent Document 2] WO2005100556A2 [Patent Document 3] Special form 2019-526285

Non-licensed literature

[0008]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

[0009] The granzyme B variant reported in Patent Document 1 shows only a slight enhancement of protease activity compared to human wild-type granzyme B, and its cytotoxic activity may not be sufficient. Although Patent Document 2 describes comprehensive modifications to rat granzyme B, it does not disclose data on inhibitor resistance, and it is unclear whether these variants exhibit sufficient cytotoxic activity in the presence of inhibitors. Furthermore, previous studies have proposed methods of using purified granzyme B or its modified form by direct intravenous injection. However, such methods may cause serious side effects, as cells that take up the administered granzyme B may undergo cell death. Therefore, it is necessary to investigate methods other than direct injection as therapeutic methods using granzyme B. [Means for solving the problem]

[0010] Based on these challenges, the present invention comprehensively identified multiple modifications that increase protease activity and combined them to enhance granzyme B activity. As a result, it was found that granzyme B protease activity is enhanced by gene modifications not reported in prior literature. Furthermore, it was shown that a significant increase in activity compared to wild-type granzyme B is possible by combining these modifications. In addition, when the resistance of the modified organism to granzyme B inhibitors was investigated, it was shown that the modified organism exhibits high protease activity even in the presence of inhibitors.

[0011] Therefore, this disclosure provides a granzyme B variant in which protease activity and / or inhibitor resistance is enhanced by genetic modification, and also provides a pharmaceutical composition containing the granzyme B variant, a pharmaceutical composition containing cells expressing the variant, and a pharmaceutical composition obtained by using the variant in combination with a receptor and / or antibody drug.

[0012] This disclosure is based on such findings and specifically includes the embodiments described below as illustrative examples. [1] A modified granzyme B containing one or more amino acid residues selected from the following 1 to 20. 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, 20) V at position 229 〔2〕The granzyme B variant according to 〔1〕, which comprises any one of the following combinations of 1 to 12 amino acid residues. 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 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 〔3〕A granzyme B variant as described in 〔1〕 or 〔2〕, wherein the protease activity is enhanced compared to human wild-type granzyme B. 〔4〕A granzyme B variant as described in any one of 〔1〕 to 〔3〕, which has resistance to an inhibitor against human wild-type granzyme B. 〔5〕The granzyme B variant as described in 〔4〕, wherein the inhibitor is PI-9 or heparin. 〔6〕An isolated nucleic acid encoding the granzyme B variant as described in any one of 〔1〕 to 〔5〕. 〔7〕A vector containing the isolated nucleic acid as described in 〔6〕. 〔8〕A cell transformed or transfected with the isolated nucleic acid as described in 〔6〕 or the vector as described in 〔7〕. 〔9〕A cell expressing the granzyme B variant as described in any one of 〔1〕 to 〔5〕. 〔10〕A pharmaceutical composition comprising the isolated nucleic acid as described in 〔6〕, the vector as described in 〔7〕, or the cell as described in 〔8〕 or 〔9〕. 〔11〕A pharmaceutical composition comprising the granzyme B variant as described in any one of 〔1〕 to 〔5〕. 〔12〕A pharmaceutical composition, which is a cell expressing a granzyme B variant or a pharmaceutical composition comprising a granzyme B variant, for use in combination with administration of a cell expressing a receptor, wherein the receptor is activated by binding of a ligand to the cell expressing the receptor, and the granzyme B variant has protease activity enhanced compared to human wild-type granzyme B and has resistance to an inhibitor against human wild-type granzyme B. 〔13〕The pharmaceutical composition as described in 〔12〕, 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 via the extracellular binding domain. 〔14〕The pharmaceutical composition as described in 〔12〕, wherein the receptor is a T cell receptor having a neoantigen as a ligand. 〔15〕The pharmaceutical composition as described in any one of 〔12〕 to 〔14〕, wherein the granzyme B variant is as described in 〔1〕, 〔2〕 or 〔5〕.

[16] A pharmaceutical composition according to any one of

[12] to

[15] , comprising cells expressing a receptor.

[17] The pharmaceutical composition according to

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

[18] A pharmaceutical composition comprising cells expressing a granzyme B variant or a granzyme B variant for use in combination with the administration of an antigen-binding molecule and the administration of cells expressing a chimeric receptor, Antigen-binding molecules have the ability to bind to target antigens. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and can bind to cells expressing a target antigen via the binding of the extracellular binding domain to an antigen-binding molecule. A modified granzyme B is a pharmaceutical composition having enhanced protease activity compared to human wild-type granzyme B and possessing resistance to inhibitors of human wild-type granzyme B.

[19] The antigen-binding molecule contains a linker that is cleaved by a protease. The extracellular binding domain can bind to an antigen-binding molecule after linker cleavage.

[18] The pharmaceutical composition described above.

[20] The granzyme B modified product is the pharmaceutical composition described in [1], [2], or [5], as described in

[18] or

[19] .

[21] A pharmaceutical composition according to any one of

[18] to

[20] , comprising cells expressing a chimeric receptor.

[22] The pharmaceutical composition according to

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

[23] A pharmaceutical composition according to any one of

[13] ,

[15] to

[22] , wherein the chimeric receptor is a chimeric antigen receptor. [A1] A granzyme B variant that contains a mutation in one or more amino acid residues selected from the following 1 to 20, compared to human wild-type granzyme B. 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, 20) 229V [A2] A modified granzyme B according to [A1], comprising a mutation in one or more amino acid residues selected from the following 1 to 20, compared to human wild-type granzyme B. 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, 20) A229V [A3] A modified granzyme B as described in [A1], wherein the amino acid residue mutation is one of the following 1 to 12. 1) 44L, 48K, 155P, 172L, 175I and 200R 2) 44L, 48E, 155P, 172L, 175I and 200R 3) 44F, 48K, 155P, 172L, 175I and 200R 4) 44F, 48E, 155P, 172L, 175I and 200R 5) 44L, 46I, 155P, 172L, 175I and 200R 6) 44L, 46E, 155P, 172L, 175I and 200R 7) 44L, 46F, 155P, 172L, 175I and 200R 8) 44L, 46Q, 155P, 172L, 175I and 200R 9) 44F, 46I, 155P, 172L, 175I and 200R 10) 44F, 46E, 155P, 172L, 175I and 200R 11) 44F, 46F, 155P, 172L, 175I and 200R 12) 44F, 46Q, 155P, 172L, 175I and 200R [A4] A modified granzyme B as described in [A3], wherein the amino acid residue mutation is one of the following 1 to 12. 1) K44L, R48K, K155P, K172L, S175I and K200R 2) K44L, R48E, K155P, K172L, S175I and K200R 3) K44F, R48K, K155P, K172L, S175I and K200R 4) K44F, R48E, K155P, K172L, S175I and K200R 5) K44L, L46I, K155P, K172L, S175I and K200R 6) K44L, L46E, K155P, K172L, S175I and K200R 7) K44L, L46F, K155P, K172L, S175I and K200R 8) K44L, L46Q, K155P, K172L, S175I and K200R 9) K44F, L46I, K155P, K172L, S175I and K200R 10) K44F, L46E, K155P, K172L, S175I and K200R 11) K44F, L46F, K155P, K172L, S175I and K200R 12) K44F, L46Q, K155P, K172L, S175I and K200R [A5] A granzyme B variant according to any of [A1] to [A4], comprising one or more amino acid residue mutations compared to human wild-type granzyme B described in Sequence ID No. 1. [A6] A modified granzyme B according to any of [A1] to [A5], wherein the protease activity is enhanced compared to human wild-type granzyme B. [A7] A granzyme B variant according to any of [A1] to [A6] that has resistance to inhibitors of human wild-type granzyme B. [A8] A modified Granzyme B according to [A7], wherein the inhibitor is PI-9 or heparin. An isolated nucleic acid encoding a granzyme B variant as described in any of [A9], [A1], to [A8]. A vector containing the isolated nucleic acid described in [A10] and [A9]. Cells transformed or transduced with isolated nucleic acids described in [A11] or [A9] or the vector described in [A10]. Cells expressing a modified granzyme B described in any of [A12], [A1], to [A8]. A pharmaceutical composition comprising isolated nucleic acids as described in [A13] or [A9], a vector as described in [A10], or cells as described in [A11] or [A12]. A pharmaceutical composition comprising a modified Granzyme B described in any of [A14], [A1], to [A8]. [A15] A modified Granzyme B is a pharmaceutical composition described in any of

[12] to

[14] , which is described in any of [A1] to [A5]. [A16] The pharmaceutical composition according to [A15], comprising cells expressing a receptor. The pharmaceutical composition according to [A16], wherein the receptor and the granzyme B variant are expressed in the same T cell. [A18] A modified Granzyme B is a pharmaceutical composition as described in

[18] or

[19] , which is described in any of [A1] to [A5]. [A19] The pharmaceutical composition according to [A18], comprising cells expressing a chimeric receptor. [A20] The pharmaceutical composition according to [A19], wherein the chimeric receptor and the granzyme B variant are expressed in the same T cell. [A21] A pharmaceutical composition according to any of [A15] to [A20], wherein the receptor is a chimeric antigen receptor. [B1] A modified granzyme B according to [3] or [A6], wherein the in vitro protease activity of the modified granzyme B is 1.5 times or more that of human wild-type granzyme B, or a pharmaceutical composition according to

[12] or

[18] . [B2] The granzyme B modified or pharmaceutical composition according to [B1], wherein the in vitro protease activity of the granzyme B modified is twice or more that of human wild-type granzyme B. [B3] A modified granzyme B according to [4] or [A7], wherein the in vitro protease activity in the presence of an inhibitor is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more compared to human wild-type granzyme B, or a pharmaceutical composition according to

[12] or

[18] . [B4] The granzyme B modified or pharmaceutical composition according to [B3], wherein the in vitro protease activity in the presence of an inhibitor is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 2 times or more compared to human wild-type granzyme B. [B5] A modified granzyme B or pharmaceutical composition according to [B3] or [B4], wherein the inhibitor is PI-9 or heparin. An isolated nucleic acid encoding a granzyme B variant as described in any of [B6], [B1], to [B5]. A vector containing isolated nucleic acids as described in [B7] and [B6]. Cells transformed or transduced with isolated nucleic acids described in [B8] or [B6] or the vector described in [B7]. Cells expressing a modified granzyme B described in any of [B9], [B1], to [B5]. A pharmaceutical composition comprising isolated nucleic acids as described in [B10] or [B6], a vector as described in [B7], or cells as described in [B8] or [B9]. A pharmaceutical composition comprising a modified Granzyme B described in any of [B11], [B1], to [B5]. [B12] The receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and binds to a ligand via the extracellular binding domain, as described in any of [B1] to [B5]. [B13] The receptor is a T cell receptor that uses a neoantigen as a ligand, as described in any of [B1] to [B5]. [B14] A modified Granzyme B is a pharmaceutical composition according to any of [B1] to [B5], [B12], or [B13], which is described in any of [1], [2], or [A1] to [A5]. A pharmaceutical composition according to any one of [B1] to [B5] or [B12] to [B14], comprising cells expressing the [B15] receptor. The pharmaceutical composition according to [B15], wherein the receptor [B16] and the granzyme B variant are expressed in the same T cell. [B17] The antigen-binding molecule contains a linker that is cleaved by a protease. The extracellular binding domain can bind to the antigen-binding molecule after linker cleavage. (Pharmaceutical composition as described in [B1] to [B5]) [B18] A modified Granzyme B is a pharmaceutical composition described in any of [1], [2], or [A1] to [A5], or any of [B1] to [B5], or any of [B17]. [B19] A pharmaceutical composition according to any one of [B1] to [B5], [B17], or [B18], comprising cells expressing a chimeric receptor. [B20] The pharmaceutical composition according to [B19], wherein the chimeric receptor and the granzyme B variant are expressed in the same T cell. [B21] A pharmaceutical composition according to any of [B12], [B14] to [B20], wherein the chimeric receptor is a chimeric antigen receptor. [C1] A pharmaceutical composition according to any one of

[10] to

[23] for use in the treatment or prevention of cancer. [C2] A pharmaceutical composition according to any one of

[10] to

[23] for use in the treatment or prevention of inflammatory diseases. [C3] A modified granzyme B according to any of [1] to [5], [A1] to [A8], or [B1] to [B5], or a cell according to [8] or [9], for use in the treatment or prevention of cancer or inflammatory diseases. A method for treating or preventing cancer or inflammatory disease, comprising administering a modified granzyme B described in any of [C4] [1] to [5], [A1] to [A8], or [B1] to [B5], or cells described in [8] or [9]. The method according to [C4], further comprising administering cells expressing the [C5] receptor, wherein the receptor activates the cells expressing the receptor by binding to a ligand. The method according to [C5], wherein the [C6] receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and binds to a ligand via the extracellular binding domain. The method according to [C6], further comprising administering an antigen-binding molecule, wherein 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 via the binding of its extracellular binding domain to the antigen-binding molecule. [C8] The method according to [C7], wherein the antigen-binding molecule contains a linker that is cleaved by a protease, and the extracellular binding domain can bind to the antigen-binding molecule after linker cleavage. The method described in [C5], wherein the [C9] receptor is a T cell receptor that uses a neoantigen as a ligand. [C10] The method according to any one of [C5] to [C9], wherein the administration is the administration of T cells expressing the receptor and a modified granzyme B. [C11] Use of a modified granzyme B according to any of [1] to [5] or the cells according to [8] or [9] in the manufacture of a therapeutic or prophylactic agent for cancer or inflammatory disease. A method for producing isolated nucleic acids encoding a granzyme B variant as described in any of [D1][1]-[5], [A1]-[A8], or [B1]-[B5]. A method for producing a vector containing isolated nucleic acids as described in [D2][6], [A9], or [B6]. A method for producing cells transformed or transduced with isolated nucleic acids as described in [D3][6], [A9], or [B6], or with vectors as described in [7], [A10], or [B7]. A method for producing cells expressing a modified granzyme B as described in any of [D4] [1] to [5], [A1] to [A8], or [B1] to [B5]. [Effects of the Invention]

[0013] In a non-limiting embodiment, the granzyme B variant of this disclosure has higher protease activity and / or resistance to inhibitors compared with wild-type granzyme B. A pharmaceutical composition comprising the granzyme B variant of this disclosure or cells expressing it having such advantageous effects is more advantageous than wild-type granzyme B and prior art granzyme B variants in inducing cell death in target cells. Furthermore, the granzyme B variant of this disclosure can be used in combination with pharmaceuticals and / or antibody drugs using receptor-expressing cells to specifically induce cell death in target cells. [Brief explanation of the drawing]

[0014] [Figure 1] The amino acid sequence (SEQ ID NO: 2) for amino acids 21-247 (the 21st to 247th amino acids from the N-terminus) in the amino acid sequence of human wild-type granzyme B (NCBI Reference Sequence. NP_004122.2: SEQ ID NO: 1) is shown. In this sequence, the amino acids at the positions where modifications were made in Example 2 are underlined. [Figure 2] The results of measuring the protease activity of the granzyme B variants in Example 4-1 are shown. The vertical axis shows the relative protease activity of each granzyme B variant, with the protease activity of wild-type granzyme B set to 1. The horizontal axis shows the amino acid substitutions of the variants used for measurement. [Figure 3]The results of measuring the protease activity of the granzyme B variants in Example 4-2 are shown. The vertical axis shows the relative protease activity of each granzyme B variant, with the protease activity of wild-type granzyme B set to 1 in (1) the absence of the inhibitor (Buffer), (2) the presence of heparin (Heparin), or (3) the presence of PI-9 (PI-9). The horizontal axis shows the amino acid substitutions of the variants used for measurement. [Modes for carrying out the invention]

[0015] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0016] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0017] 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 specified. The term encompasses both granzyme B that has not undergone “full-length” processing and any form of granzyme B resulting from processing in cells. The term also encompasses naturally occurring variants of granzyme B, such as splice variants and allele variants. An exemplary amino acid sequence of human wild-type granzyme B is shown in SEQ ID NO: 1 (NCBI Reference Sequence. NP_004122.2), but is not limited to this, and includes variants with some differences in amino acid sequence. Such variants include human wild-type granzyme B represented by amino acid sequences having 90% or greater homology, 95% or greater, 97% or greater, or 99% or greater homology to SEQ ID NO: 1.

[0018] In this specification, “mutation” and “modification” are used interchangeably and include the “addition” of amino acid residues to an amino acid sequence, the “deletion” of amino acid residues from an amino acid sequence, the “insertion” of amino acid residues into an amino acid sequence, and / or the “substitution” of amino acid residues within an amino acid sequence. Any combination of addition, deletion, insertion, and substitution may be introduced to obtain a variant (mutant) having a desired characteristic (e.g., protease activity or resistance to inhibitors). In one embodiment, the granzyme B variant of this disclosure includes the substitution of one or more amino acid residues.

[0019] When the position of each amino acid in granzyme B is indicated in this specification, the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B, as exemplified by Sequence ID No. 1, is specified (in the case of Sequence ID No. 1, a consecutive number starting from the N-terminal amino acid position 1 in the amino acid sequence). For example, when a granzyme B variant of this disclosure is described as "containing the amino acid residue F(Phe) at position 44," it means that the 44th amino acid residue from the N-terminus of the granzyme B variant is F(Phe). Furthermore, when an amino acid modification of granzyme B is indicated in this specification, the amino acid residues before modification (i.e., wild-type granzyme B) and after modification at the modified position are indicated (for example, in single-letter notation) to the left and right of the amino acid number of the modified position, or to either the left or right. For example, if the amino acid residue at the position corresponding to the 44th amino acid residue K(Lys) from the N-terminus in the amino acid sequence shown in Sequence ID No. 1 is modified to F(Phe), this specification will represent the amino acid modification as K44F. Also, if it is simply to indicate that the 44th amino acid residue from the N-terminus of granzyme B is modified to F(Phe), it will be represented as 44F. Even if the amino acid sequence of the granzyme B to be modified differs from the sequence shown in Sequence ID No. 1, a person skilled in the art can appropriately determine (for example, by performing sequence alignment) which position in the granzyme B to be modified corresponds to the modification position shown herein. In this disclosure, the Granzyme B variant contains one or more amino acid residues selected from the following 1 to 20. 1) T, E, N, or V at rank 43, 2) L or F at rank 44, 3) Q, L, or A at rank 45, 4) I, E, F, or Q at rank 46, 5) V at rank 47, 6) F or K at rank 48, 7) L at rank 99, 8) A at rank 106, 9) M at rank 149, 10) L at rank 151, 11) P at rank 155, 12) L at rank 172, 13) Q, E, or I at rank 175, 14) P at rank 183, 15) L at rank 184, 16) R at rank 200, 17) I at rank 217, 18) F at rank 219, 19) G or S at rank 222, 20) V at rank 229 In another context, the granzyme B variant of this disclosure comprises any combination of amino acid residues from 1 to 12 below. 1) L at 44th, K at 48th, P at 155th, L at 172nd, I at 175th, and R at 200th 2) L at 44th place, E at 48th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 3) F (44th), K (48th), P (155th), L (172nd), I (175th), and R (200th) 4) F (44th), E (48th), P (155th), L (172nd), I (175th), and R (200th) 5) L at 44th place, I at 46th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 6) L at 44th place, E at 46th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 7) L (44th), F (46th), P (155th), L (172nd), I (175th), and R (200th) 8) L (44th), Q (46th), P (155th), L (172nd), I (175th), and R (200th) 9) F (44th), I (46th), P (155th), L (172nd), I (175th), and R (200th) 10) F (44th), E (46th), P (155th), L (172nd), I (175th), and R (200th) 11) F at 44th, F at 46th, P at 155th, L at 172nd, I at 175th, and R at 200th 12) F (44th), Q (46th), P (155th), L (172nd), I (175th), and R (200th)

[0020] In this specification, "protease activity," particularly in relation to granzyme B, refers to the activity of granzyme B in cleaving its substrate. Methods for evaluating the protease activity of granzyme B are known to those skilled in the art, and various activity measurement kits and synthetic substrates are commercially available. An example of such a synthetic substrate is a synthetic peptide having the recognition sequence of granzyme B (e.g., Ile-Glu-Pro-Asp (IEPD)) labeled with a detectable substance (e.g., p-nitroanilide (pNA)) (e.g., Ac-IEPD-pNA). When granzyme B cleaves the synthetic substrate, free detectable substances are released, which can be quantified using a fluorometer or spectrophotometer. As an example, the protease activity of a modified granzyme B can be evaluated by the method described in Example 4 of this disclosure. For example, the protease activity in the presence of an inhibitor can be evaluated using the evaluation system described in Example 4, under the same granzyme B and inhibitor concentration conditions as in that example. The protease activity of the modified granzyme B disclosed herein is preferably enhanced compared to the protease activity of wild-type granzyme B. For example, it is preferably 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more higher than the protease activity of wild-type granzyme B, more preferably 2 times or more or 2.5 times or more higher, and particularly preferably 3 times or more higher.

[0021] The protease activity of wild-type granzyme B is known to be inhibited by "inhibitors" such as PI-9 and heparin. The granzyme B variants of this disclosure preferably have resistance to such inhibitors. In this specification, "resistance to inhibitors" means the ability to exhibit higher protease activity than wild-type granzyme B in the presence of such inhibitors. The protease activity of the granzyme B variants of this disclosure in the presence of such inhibitors is preferably 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more higher than that of wild-type granzyme B, more preferably 2 times or more, 2.5 times or more, 3 times or more, or 3.5 times or more higher, and particularly preferably 4 times or more, 4.5 times or more, 5 times or more, or 5.5 times or more higher.

[0022] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0023] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0024] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformers” and “transformed cells” or “transducers” and “transduced cells,” including primary transformed or transduced cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those used when the original transformed or transduced cells were screened or selected are also included herein.

[0025] In this specification, unless otherwise specified, "cells expressing granzyme B" may refer to cells expressing endogenous granzyme B or cells expressing granzyme B through gene transfer. Examples of cells expressing endogenous granzyme B include cytotoxic lymphocytes such as T cells and NK cells. On the other hand, cells that are transfected to express granzyme B are not limited to T cells and NK cells; recombinant granzyme B can be expressed in various cells, and recombinant granzyme B can be obtained (for example, by purifying granzyme B from the culture supernatant). Furthermore, a gene expressing granzyme B can be introduced into cells derived from an individual (for example, a healthy donor or a patient suffering from a specific disease) (e.g., peripheral blood mononuclear cells (PBMCs)), and these transfected cells can be administered to the same individual or another individual. Transfected cells expressing granzyme B may also be processed to differentiate them into a specific cell type (e.g., cytotoxic T cells) before being administered to an individual. Various gene transfer techniques well known to those skilled in the art can be used as methods for introducing the gene encoding granzyme B into cells. Cells expressing granzyme B may be administered in combination with cells expressing the chimeric receptor. Alternatively, cells expressing both granzyme B and the chimeric receptor may be used, and such cells can be produced by simultaneously or separately introducing the genes for granzyme B and the chimeric receptor. When expressing a modified granzyme B in cells that express endogenous granzyme B, such as T cells and NK cells, the endogenous wild-type granzyme B in those cells may or may not be knocked out.

[0026] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained herein can exert its effect, and which does not contain additional elements that are unacceptably toxic to the subject to which the preparation or composition is administered. The "active ingredient" may be composed of antibodies, polypeptides, etc., or may be composed of cells that express antibodies, polypeptides, etc. (e.g., the granzyme B variant of this disclosure). For example, when cells transformed or transduced with nucleic acids encoding the granzyme B variant of this disclosure or vectors containing such nucleic acids are administered to a patient for therapeutic or preventive purposes, the preparation containing such cells may be called a "pharmaceutical preparation" or "pharmaceutical composition."

[0027] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation or pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0028] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, 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.

[0029] In one aspect, this disclosure provides granzyme B variants, cells expressing granzyme B variants, or pharmaceutical compositions containing them. In one embodiment, the pharmaceutical composition of the present disclosure can be used in combination with cells expressing a receptor. Here, the receptor is one that activates the cells expressing the receptor by binding to a ligand, and includes, but is not limited to, chimeric receptors that bind to a ligand via an extracellular binding domain, and T cell receptors that use neoantigens as ligands. The pharmaceutical composition of the present disclosure may include cells expressing the receptor and cells expressing a granzyme B variant, and the receptor and the granzyme B variant may be expressed in the same cell (e.g., a T cell). Such pharmaceutical compositions of the present disclosure can be used in combination with an antigen-binding molecule that can bind to the extracellular binding domain of a chimeric receptor. Here, 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 via the binding of its extracellular binding domain to the antigen-binding molecule. In certain embodiments, the antigen-binding molecule includes a linker that is cleaved by a protease, and the extracellular binding domain can bind to the antigen-binding molecule after linker cleavage. The pharmaceutical compositions of the present disclosure may include cells expressing a chimeric receptor and cells expressing a granzyme B variant, as well as an antigen-binding molecule that can bind to the extracellular binding domain of a chimeric receptor, and the receptor and the granzyme B variant may be expressed in the same cell (e.g., a T cell). When using one or more of the following in combination: a granzyme B variant, cells expressing a granzyme B variant, cells expressing a receptor, cells expressing both the receptor and the granzyme B variant, and antigen-binding molecules capable of binding to the extracellular binding domain of a chimeric receptor, they can be used simultaneously, separately, or sequentially (for example, administered to an individual). In one embodiment, the pharmaceutical compositions of this disclosure are intended for use in the treatment or prevention of cancer or inflammatory diseases, including in the treatment of cell injury, induction of cell death, inhibition of cell proliferation, or in the treatment of cell death.

[0030] The term "chimeric receptor" refers to a recombinant polypeptide that, when expressed on immune effector cells, exhibits specificity towards target cells, such as cancer cells, and produces intracellular signals, and includes at least an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The term "chimeric antigen receptor" or "CAR" refers to a chimeric receptor in which the extracellular binding domain binds to an antigen.

[0031] The term "extracellular binding domain" refers to any protein molecule or part thereof that can specifically bind to a given antigen or other molecule. For example, it includes single-chain antibodies (scFv) in which the light chain (VL) and heavy chain (VH) of a monoclonal antibody variable region specific to tumor antigens are linked in series. The extracellular binding domain can also be referred to as the extracellular recognition domain.

[0032] The term "transmembrane domain" includes polypeptides that are located between the extracellular binding domain and the intracellular signaling domain and have the function of transmembrane.

[0033] The term "intracellular signaling domain" means any oligopeptide domain or polypeptide domain known to transmit signals that cause activation or inhibition of intracellular biological processes, such as activation of immune cells like T cells or NK cells, and includes at least one stimulatory signaling domain derived from a T cell stimulatory molecule and at least one co-stimulatory signaling domain derived from a T cell co-stimulatory molecule.

[0034] In this specification, the term “neoantigen-ligand T cell receptor” includes T cell receptors designed to recognize neoantigens, which are mutant antigens that arise as a result of genetic mutations in cancer cells.

[0035] In this specification, the term "antigen-binding molecule" refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In another embodiment, the antigen-binding molecule is a non-antibody protein, a fragment thereof, or a derivative thereof.

[0036] In this specification, “antigen-binding domain” means a region that specifically binds to and is complementary to a part or all of an antigen. In this specification, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of the antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. This specific portion 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 may be provided from the variable domains of one or more antibodies. In one non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include “scFv (single chain Fv)”, “single chain antibody”, “Fv”, “scFv2 (single chain Fv 2)”, “Fab”, or “Fab'”. In another embodiment, the antigen-binding domain comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the antigen-binding domain comprises a hinge region.

[0037] In this specification, "specifically binds" means that one molecule of a specifically binding molecule binds without showing any significant binding to any molecules other than the one or more molecules it binds to. It is also used when the antigen-binding domain is specific to a particular epitope among several epitopes contained in a given antigen. Furthermore, if the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing that epitope.

[0038] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0039] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0040] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0041] In some embodiments, the Disclosure provides methods for injuring cells, inducing cell death, inhibiting cell proliferation, treating inflammatory diseases, treating cancer, or preventing cancer, comprising administering an effective amount of the Granzyme B variant of the Disclosure, cells expressing the Granzyme B variant, or pharmaceutical compositions containing them (hereinafter collectively referred to as the Pharmaceutical Compositions, etc. of the Disclosure). In some embodiments, “effective amount” as used in the Invention means a dose of the Pharmaceutical Compositions, etc. of the Disclosure that is effective in an individual for injuring cells, inducing cell death, inhibiting cell proliferation, treating inflammatory diseases, treating cancer, or preventing cancer.

[0042] In some embodiments, "treatment" in the present invention means that the number of cancer cells in an individual is reduced, the proliferation of cancer cells is suppressed, the size (volume and / or weight) of the tumor is reduced, tumor growth is suppressed, the invasion of cancer cells into peripheral organs is suppressed, the metastasis of cancer cells is suppressed, or various symptoms caused by cancer are improved by the pharmaceutical compositions of this disclosure. Also, in some embodiments, "prevention" in the present invention means preventing an increase in the number of cancer cells due to the regrowth of reduced cancer cells, preventing the regrowth of cancer cells whose proliferation has been suppressed, or preventing the size (volume and / or weight) of a reduced tumor from increasing again.

[0043] [Application Examples] The granzyme B variant of this disclosure can be used in combination with the administration of cells expressing a receptor. Examples of receptors include chimeric receptors that include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. Other examples include T cell receptors designed to recognize neoantigens that are not recognized by normal T cells. One embodiment of combining these receptor-based technologies with the granzyme B variant of this disclosure involves collecting T cells from a patient, introducing a gene encoding a chimeric receptor (e.g., a chimeric antigen receptor) and a gene encoding the granzyme B variant into these T cells, and then reintroducing them to the patient. (However, this is not limited to this method; the genes can also be introduced and the cells transferred separately.) The chimeric antigen receptor recognizes cell surface antigens such as cancer cells and activates T cells, and the enhanced cytotoxic activity of the granzyme B variant of this disclosure is exerted, leading to expectations of high therapeutic efficacy. Similarly, by introducing a gene encoding a neoantigen-ligand T cell receptor and a gene encoding a granzyme B variant into T cells collected from a patient, and then reintroducing them to the patient, a high therapeutic effect can be expected through this combination. Furthermore, the combination of the granzyme B variant and the chimeric receptor described herein can also be combined with an antigen-binding molecule (such as an antibody). In this case, the antigen-binding molecule has the ability to bind to the target antigen, and the chimeric receptor can bind to cells expressing the target antigen via the binding of its extracellular binding domain to the antigen-binding molecule. As one embodiment of the antigen-binding molecule, a linker that is cleaved by a protease can be included in the antigen-binding molecule beforehand, and the chimeric receptor can be designed to bind to the antigen-binding molecule after the linker has been cleaved. One embodiment of such a combination involves collecting T cells from a patient, introducing a gene encoding the chimeric receptor and a gene encoding the granzyme B variant into these T cells, reintroducing them to the patient, and separately administering a pharmaceutical composition containing the antigen-binding molecule to the patient. [Examples]

[0044] Examples of the construction, expression, purification, gene transfer, and use of modified granzymes for the treatment of diseases are described below, but the implementation of this patent is not limited to these examples.

[0045] [Example 1] Construction of a human wild-type granzyme B expression vector The sequence of human wild-type granzyme B (NCBI Reference Sequence. NP_004122.2) was synthesized. Of the ORF encoding granzyme B, the sequence encoding amino acids 21-247 (SEQ ID NO: 2) was fused to the 5' end with an artificial secretion signal sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG (SEQ ID NO: 3)) and an enterokinease recognition sequence (DDDDK (SEQ ID NO: 4)). A sequence encoding a histidine tag was added to the C-terminus (J Vis Exp. 2015 Jun 10;(100):e52911). The sequence encoding this sequence (SEQ ID NO: 5) was inserted into a mammalian cell expression vector.

[0046] In this specification, when an amino acid modification site of granzyme B is indicated, the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B shown in Sequence ID No. 1 (NCBI Reference Sequence. NP_004122.2) is specified.

[0047] [Example 2] Preparation of a base sequence encoding a modified Granzyme B Single amino acid residue substitutions and combinations thereof of multiple amino acid residue substitutions in human wild-type granzyme B were performed using PCR reactions by methods known to those skilled in the art. The amino acid positions intended for modification were selected by identifying amino acid residues close to the substrate binding site based on the crystal structure of human wild-type granzyme B (underlined amino acids in Figure 1). Primers were designed to encode substituted derivatives in which these amino acid residues are replaced with the original amino acids or any of the 18 amino acids excluding cysteine. A total of 1476 nucleotide sequences encoding granzyme B variants with single amino acid substitutions were generated using PCR and other methods known to those skilled in the art. Furthermore, using a similar method, nucleotide sequences encoding granzyme B variants with multiple amino acid substitutions were constructed.

[0048] [Example 3] Expression and purification of granzyme B variant The nucleotide sequence encoding granzyme B, prepared in Example 2, was transfected into 1 mL of Expi293 (invitrogen) culture medium using the method specified by the manufacturer. After 4 days, the culture supernatant was collected, and enterokinase at a final concentration of 0.3 μg / ml was added, and the mixture was reacted at 4°C for 16 hours. 1 / 10 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 equilibrium buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.5) was added. After reacting at 4°C for 1 hour, the reaction solution was added to a filter plate (Merck, MSGVS2210). Ni Sepharose Excel conjugated with granzyme B was washed five times with 200 μL of equilibrium buffer, and then reacted with elution buffer (25 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, pH 7.5) at 4°C for 15 minutes to elute granzyme B. The concentration of purified granzyme B was calculated using the absorbance at 280 nm of the eluted granzyme B solution and the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0049] [Example 4] Measurement of protease activity of modified Granzyme B (4-1) Granzyme B modified (single amino acid substitution) The expressed and purified granzyme B was suspended in a 2-fold 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 shows the relative protease activity of the granzyme B variants compared to wild-type granzyme B.

[0050] The activity of granzyme B, shown in Figure 2, is defined by the following formula. Fold change = (Protease activity of modified Granzyme B) / (Protease activity of wild-type Granzyme B)

[0051] Furthermore, the protease activity of granzyme B is defined by the following formula. Protease activity = (Change in absorbance of the reaction solution at 405 nm per unit time) / (Unit time)

[0052] (4-2) Granzyme B modified (multiple amino acid substitutions) In the comprehensive measurements described above, 1476 modified compounds were tested. As a result, 33 modified compounds showed increased activity compared to wild-type granzyme B, while 1443 modified compounds showed lower activity than wild-type granzyme B. For these 33 modifications that increased the protease activity of granzyme B, multiple modified granzyme B compounds were prepared by combining multiple mutations using the method described in Example 2. Table 1 shows examples of the prepared modified compounds that showed particularly significant increases in activity. In Table 1, the position of amino acid substitution in the modified compound refers to the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B shown in Sequence ID No. 1, and the amino acid sequence of the modified compound refers to the modified amino acid sequence corresponding to amino acid numbers 21-247 of human wild-type granzyme B shown in Sequence ID No. 1.

[0053] [Table 1]

[0054] When measuring protease activity in the presence of an inhibitor, wild-type granzyme B or a modified granzyme B was incubated at 37°C for 1 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 in a 1:1 volume ratio. All solutions were prepared using 2x diluted enzyme reaction buffer (2X Reaction buffer, Promokine, PK-CA577-1068-80) was used. For the measurement of the protease activity of the prepared modified product in the absence of the inhibitor, instead of the inhibitor suspension, the product was incubated with an enzyme reaction buffer containing 0.5 mM MDTT at 37°C for 1 hour, and then mixed with a 1 mM Ac-IEPD-pNA solution in a volume ratio of 1:1 for measurement. Absorbance was measured in the same manner as described in Example 4-1. The measurement results are shown in Figures 3(1) to (3). The results confirmed that the activity of granzyme B was increased compared to wild-type granzyme B, and that high protease activity was maintained even in the presence of the inhibitor.

[0055] [Example 5] Expression of granzyme B variant in NK cell line In Example 4, the granzyme B variant and wild-type granzyme B (SEQ ID NO: 1) were prepared, and the amino acid sequences corresponding to amino acid numbers 21-247 were fused with a secretory signal sequence and a Cathepsin C / H recognition sequence derived from wild-type granzyme B at the N-terminus, and a FLAG tag was fused to the C-terminus. The nucleotide sequences encoding these fusions were introduced into an NK cell line (NKL, ATCC No.) using the mammalian expression vector pGL4.30 (Promega, E8481). This NK cell line was selected using hygromycin B. The cell line for the granzyme B variant was selected using eBioscience. TMCells are permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (invitrogen, 00-5523-00) in a manner specified by those skilled in the art, and then fluorescently stained using an anti-FLAG antibody that recognizes the tag fused to the C-terminus of granzyme B and its isotype control antibodies (Biolegend, 637310 and 400508). The stained cells are detected by FACS verse (BD). As a result, a peak associated with granzyme B expression is observed in cells into which granzyme B has been gene-transfected, confirming that the gene-transfected granzyme B is being expressed.

[0056] [Example 6] Measurement of in vitro cytotoxic activity of cell lines expressing granzyme B variants The established granzyme B variant-expressing cell line and target cells (one of BxPC-3, HuCCT-1, or MCAS) are added to a 96-well plate, and then the anti-EGFR antibodies CetuH0-Hl076 / CetuL4-k0 / / CetuH0-Kn125 / CetuL4-k0, diluted to their respective concentrations, are added. After reaction at 37°C, LDH release associated with cell death is quantified using the Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific, 88954) according to the method specified by the manufacturer. The results show that transgenic cells expressing the granzyme B variant exhibit stronger cytotoxic activity than transgenic cells expressing wild-type granzyme B.

[0057] [Example 7] Preparation of a viral vector encoding a modified Granzyme B The sequences encoding human wild-type granzyme B and granzyme B variants are prepared as retroviral vectors using pMCs-IRES-GFP Retroviral Vector (CELL BIOLABS, INC., RTV-040) according to the method specified by the vendor.

[0058] [Example 8] Gene transfer of granzyme B variant into primary T cells HLA-A2 derived from a healthy donor + Peripheral blood mononuclear cells (PBMCs) (Biological Specialty Corp, Colmar, PA, USA) are isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA). PBMCs are then 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), in a ratio of 3 × 10⁶ units. 6 Cells are cultured in individual wells and activated with 50 ng / ml OKT3 (eBioscience, San Diego, CA, USA). After 2 days, cells are harvested for retroviral transduction. For transduction, 24-well non-tissue culture treated plates (BD Biosciences, Franklin Lakes, NJ, USA) are coated overnight at 4°C with 0.5 ml / well of 10 μg / ml recombinant human fibronectin fragment (RetroNectin; Takara Bio Inc., Otsu City, Shiga Prefecture, Japan). After incubation, the wells are blocked at room temperature for 30 minutes with 1 ml of Hanks' Solution (GIBCO brand; Invitrogen) supplemented with 2.5% human AB serum, and then washed with Hanks' Solution supplemented with 2.5% N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (GIBCO brand; Invitrogen). Transduction is performed using the previously described method (Johnson et al. Blood 114, 535-546 (2009)). Briefly, approximately 2.5 ml of retroviral supernatant is added to each coating well, followed by centrifugation at 2000 g at 32°C for 2 hours. 1.5 ml of viral supernatant is then removed and 1 × 10⁶ 6Add one (0.5 ml) of activated PBMC to each well in the presence of 100 U / ml of IL-2. Centrifuge the plate at 1000 g for 10 minutes, then incubate overnight at 37°C. After transduction, the cells are washed and maintained in the presence of IL-2 (100 U / ml) and used for experiments 5 days after transduction. Granzyme B expression in transduced human T cells is determined by flow cytometry after staining with an antibody that recognizes the FLAG tag labeled at the C-terminus of granzyme B (Biolegend, 637310) or its isotype control antibody (Biolegend, 400508).

[0059] [Example 9] Evaluation of cytotoxic activity of T cells genetically modified with Granzyme B variant. T cells expressing the granzyme prepared in Example 8 and target cells (either BxPC-3, HuCCT-1, or MCAS) were added to 96-well plates, and then the anti-EGFR anti-CD3 bispecific antibodies CetuH0-F760nN17 / CetuL4-k0 / / TR01H113-F760nP17 / L0011-k0, diluted to each concentration, were added. After reaction at 37°C, LDH release associated with cell death was quantified using the Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific, 88954) according to the method specified by the manufacturer. As a result, it was revealed that transgenic cells expressing modified granzyme B showed stronger cytotoxic activity than transgenic cells expressing wild-type granzyme B.

[0060] [Example 10] Evaluation of in vitro cytotoxic activity of T cells introduced with modified granzyme B. The cytotoxic activity of granzyme-transformed T cells produced in Example 8 was measured by BD FACSVerse. TM (Also evaluated by BD Biosciences). Cancer cells such as BxPC-3, HuCCT-1, or MCAS are prepared as target cells. Target cells are placed in a 6-well plate at a rate of 1 × 10⁶ 5 cells or 3 × 105 The cells are seeded separately. T cells expressing modified granzyme B or wild-type granzyme B are used as effector cells, and the effector cells to target cells (E:T) are mixed in a ratio of 1:1 or 1:3. Next, the anti-EGFR anti-CD3 bispecific antibody CetuH0-F760nN17 / CetuL4-k0 / / TR01H113-F760nP17 / L0011-k0 is added at a rate of 10 μg per well. 48 hours after addition, the granzyme B-transformed T cells and target cells are harvested. The harvested cells are then placed in Zombie Aqua. TM Dead cells are stained using the Fixable Viability Kit (BioLegend, 423102), and granzyme B-expressing T cells are stained using the anti-human CD45 antibody (BioLegend, 304039). Cytotoxic activity is evaluated by the percentage of surviving cancer cells. The percentage of surviving cancer cells is calculated as the percentage of CD45- fraction cells among viable cells. This result demonstrates the enhancement of cytotoxic activity of granzyme-expressing T cells in vitro. [Industrial applicability]

[0061] The granzyme B variant of this disclosure exhibits higher protease activity compared to wild-type granzyme B, and is therefore more useful than wild-type granzyme B in therapies that induce cell death in target cells (e.g., cancer cells). Furthermore, the granzyme B variant of this disclosure exhibits high protease activity even in the presence of inhibitors of wild-type granzyme B, and is therefore more useful than wild-type granzyme B in therapies that induce cell death in tumors that highly express such inhibitors.

Claims

1. A granzyme B variant having a modified amino acid sequence of human wild-type granzyme B as described in Sequence ID No. 2, wherein the modification is a modification to any combination of amino acid residues from 1 to 12 listed below in the amino acid sequence of human wild-type granzyme B (with the first amino acid of Sequence ID No. 2 being at position 21), and the granzyme B variant having enhanced protease activity compared to human wild-type granzyme B. 1) L at 44th, K at 48th, P at 155th, L at 172nd, I at 175th, and R at 200th 2) L at 44th place, E at 48th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 3) F (44th), K (48th), P (155th), L (172nd), I (175th), and R (200th) 4) F (44th), E (48th), P (155th), L (172nd), I (175th), and R (200th) 5) L at 44th place, I at 46th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 6) L at 44th place, E at 46th place, P at 155th place, L at 172nd place, I at 175th place, and R at 200th place 7) L (44th), F (46th), P (155th), L (172nd), I (175th), and R (200th) 8) L (44th), Q (46th), P (155th), L (172nd), I (175th), and R (200th) 9) F (44th), I (46th), P (155th), L (172nd), I (175th), and R (200th) 10) F (44th), E (46th), P (155th), L (172nd), I (175th), and R (200th) 11) F at 44th, F at 46th, P at 155th, L at 172nd, I at 175th, and R at 200th 12) F (44th), Q (46th), P (155th), L (172nd), I (175th), and R (200th)

2. A granzyme B variant according to claim 1, which has resistance to inhibitors of human wild-type granzyme B.

3. The granzyme B variant according to claim 2, wherein the inhibitor is PI-9 or heparin.

4. An isolated nucleic acid encoding a granzyme B variant according to any one of claims 1 to 3.

5. A vector comprising the isolated nucleic acid described in claim 4.

6. Cells transformed or transduced with the isolated nucleic acid described in claim 4 or the vector described in claim 5.

7. Cells expressing the granzyme B variant described in any one of claims 1 to 3.

8. A pharmaceutical composition comprising the isolated nucleic acid described in claim 4, the vector described in claim 5, or the cells described in claim 6 or 7.

9. A pharmaceutical composition comprising a modified granzyme B according to any one of claims 1 to 3.

10. A pharmaceutical composition comprising cells expressing a granzyme B variant or a granzyme B variant, for use in combination with the administration of cells expressing a receptor, Receptors activate cells that express the receptor upon binding to a ligand. The granzyme B modified is the granzyme B modified according to claim 2, wherein the granzyme B modified has enhanced protease activity compared to human wild-type granzyme B and is resistant to inhibitors of human wild-type granzyme B, and is a pharmaceutical composition.

11. The pharmaceutical composition according to claim 10, wherein the receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and which binds to a ligand via the extracellular binding domain.

12. The pharmaceutical composition according to claim 10, wherein the receptor is a T cell receptor that uses a neoantigen as a ligand.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein the modified Granzyme B is as described in claim 3.

14. A pharmaceutical composition according to any one of claims 10 to 13, comprising cells expressing a receptor.

15. The pharmaceutical composition according to claim 14, wherein the receptor and the granzyme B variant are expressed in the same T cell.

16. A pharmaceutical composition comprising cells expressing a granzyme B variant or a granzyme B variant, for use in combination with the administration of an antigen-binding molecule and the administration of cells expressing a chimeric receptor, Antigen-binding molecules have the ability to bind to target antigens. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and can bind to cells expressing a target antigen via the binding of the extracellular binding domain to an antigen-binding molecule. The granzyme B modified is the granzyme B modified according to claim 2, wherein the granzyme B modified has enhanced protease activity compared to human wild-type granzyme B and is resistant to inhibitors of human wild-type granzyme B, and is a pharmaceutical composition.

17. Antigen-binding molecules contain linkers that are cleaved by proteases. The pharmaceutical composition according to claim 16, wherein the extracellular binding domain can bind to an antigen-binding molecule after linker cleavage.

18. The pharmaceutical composition according to claim 16 or 17, wherein the modified Granzyme B is as described in claim 3.

19. A pharmaceutical composition according to any one of claims 16 to 18, comprising cells expressing a chimeric receptor.

20. The pharmaceutical composition according to claim 19, wherein the chimeric receptor and the granzyme B variant are expressed in the same T cell.

21. The pharmaceutical composition according to any one of claims 11, 16 to 20, wherein the chimeric receptor is a chimeric antigen receptor.

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