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

Improved Granzyme B Variants

[0001] The present invention relates to granzyme B variants with enhanced resistance to protease activity and / or inhibitors; polynucleotides encoding the granzyme B variants; cells expressing the granzyme B variants; pharmaceutical compositions comprising cells expressing the granzyme B variants; and pharmaceutical compositions comprising the granzyme B variants.

[0002] Granzymes are a family of proteases. They are expressed in cytotoxic lymphocytes such as T cells and NK cells. When these cells recognize a target antigen, they are secreted along with perforin and other proteins against the target cell (the cell expressing the target antigen) to exert cytotoxic activity. After granzymes are translocated into the target cell, they cleave various substrates, such as BID and caspases, and ultimately activate the signaling pathway involved in apoptosis in the target cell, thereby inducing cell death in the target cell (Non-Patent Document 1).

[0003] Of the granzyme family, granzyme A and granzyme B are thought to contribute significantly to cytotoxic activity. Granzyme A functions by forming a dimer, while granzyme B functions as a monomer. For this reason, it is easy to create modified granzyme B variants using protein engineering, and their expression and purification are easy, and research and development has been conducted with a view to drug discovery. For example, various modified granzyme B variants have been reported in which scFvs that bind to tumor-associated antigens are fused to granzyme B (Non-Patent Document 2).

[0004] On the other hand, there are limitations to the use of granzyme B as an antitumor agent. Reasons for this include the limited protease activity of natural granzyme B and the suppression of granzyme B activity due to the expression of granzyme B inhibitors in target cells. Non-Patent Document 3 shows that PI-9, an inhibitor of granzyme B activity, is highly expressed in tumors. Non-Patent Document 4 also shows that heparin, which is known to be produced at elevated levels in tumors, inhibits granzyme B activity. In other words, even if granzyme B is applied to tumors in which these inhibitors are highly expressed, sufficient cytotoxic activity is not exerted, and therefore, antitumor activity is also thought to be limited.

[0005] Previous studies have reported granzyme B variants that are resistant to these inhibitors, such as PI-9-resistant (Patent Document 1, Non-Patent Document 5) and heparin-resistant (Non-Patent Document 6). Additionally, a granzyme B variant in which rat granzyme B has been comprehensively modified to cleave VEGF and VEGF receptors has also been reported (Patent Document 2).

[0006] On the other hand, when a cytotoxic substance such as granzyme is used for treatment, it is preferable that it exerts a cytotoxic effect specifically on target cells (e.g., tumor cells). One example of a method for achieving this is a method using transfected cells into which a gene encoding a wild-type granzyme or the like and a gene encoding a chimeric antigen receptor (CAR) have been introduced, in which the cell is activated by binding of the CAR to the target antigen, thereby increasing the expression of granzyme or the like (Patent Document 3).

[0007] US9528101BWO2005100556A2 special table 2019-526285

[0008] How Do Cytotoxic Lymphocytes Kill Cancer Cells? Luis Martinez-Lostao, Alberto Anel and Julian PardoClin Cancer Res. 2015 Nov 15;21(22):5047-56Delivery and therapeutic potential of human granzyme B Kurschus FC1, Jenne DE. Immunol Rev. 2010 May;235(1):159-71Blockade of the granzyme B / perforin pathway through overexpression of the serine protease inhibitor PI-9 / SPI-6 constitutes a mechanism for immune escape by tumorsJ. 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. OffringaProc Natl Acad Sci U S A. 2001 Sep 25; 98(20): 11515-11520Cationic Sites on Granzyme B Contribute to Cytotoxicity by Promoting Its Uptake into Target CellsCatherina H. Bird, Jiuru Sun, Kheng Ung, Diana Karambalis, James C. Whisstock, Joseph A. Trapani, and Phillip I. BirdMol Cell Biol. 2005 Sep; 25(17): 7854-7867Design of human granzyme B variants resistant to serpin B9Losasso V, Schifer S, Barth S, Carloni P.Proteins. 2012 Nov;80(11):2514-22Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosisKurschus FC, Fellows E, Stegmann E, Jenne DE.Proc Natl Acad Sci US A. 2008 Sep 16; 105(37): 13799-13804.

[0009] The granzyme B variants reported in Patent Document 1 only slightly enhance protease activity compared to human wild-type granzyme B, and may not have sufficient cytotoxic activity. Patent Document 2 describes comprehensive modifications to rat granzyme B, but does not disclose data on inhibitor resistance, making it unclear whether these variants exert sufficient cytotoxic activity in the presence of inhibitors. Previous studies have proposed methods such as direct intravenous injection of purified granzyme B or its variants, but such use may result in cell death in cells that have taken up the administered granzyme B, potentially causing serious side effects. Therefore, investigation of methods other than direct injection as therapeutic methods using granzyme B is warranted.

[0010] Based on these challenges, the present invention has comprehensively identified multiple modifications that increase protease activity and combined them to increase the activity of granzyme B. As a result, it was found that granzyme B protease activity can be enhanced by genetic modifications not reported in prior literature. Furthermore, it was shown that combining these modifications can significantly increase activity compared to wild-type granzyme B. Furthermore, examination of the resistance of the modified forms to granzyme B inhibitors showed that the modified forms exhibited high protease activity even in the presence of the inhibitor.

[0011] Therefore, the present disclosure provides a granzyme B variant whose protease activity and / or inhibitor resistance have been enhanced by genetic modification, and also provides pharmaceutical compositions for the pharmaceutical use of this granzyme B variant, including a pharmaceutical composition containing the variant, a pharmaceutical composition containing cells expressing the variant, and a pharmaceutical composition comprising the variant in combination with a receptor and / or antibody drug.

[0012] The present disclosure is based on these findings and specifically includes the following exemplary embodiments: [1] A granzyme B variant comprising one or more amino acid residues selected from 1 to 20 below: 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], comprising a combination of any of the following 1 to 12 amino acid residues: 1) L (rank 44), K (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 2) L (rank 44), E (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 3) F (rank 44), K (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 4) F (rank 44), E (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 5) L (rank 44), I (rank 46), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 6) 44th L, 46th E, 155th P, 172nd L, 175th I and 200th R 7) 44th L, 46th F, 155th P, 172nd L, 175th I and 200th R 8) 44th L, 46th Q, 155th P, 172nd L, 175th I and 200th R 9) 44th F, 46th I, 155th P, 172nd L, 175th I and 200th R 10) 44th F, 46th E, 155th P, 172nd L, 175th I and 200th R 11) 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 according to [1] or [2], having enhanced protease activity compared to human wild-type granzyme B.[4] The granzyme B variant according to any one of [1] to [3], which is resistant to an inhibitor of human wild-type granzyme B. [5] The granzyme B variant according to [4], wherein the inhibitor is PI-9 or heparin. [6] An isolated nucleic acid encoding the granzyme B variant according to any one of [1] to [5]. [7] A vector comprising the isolated nucleic acid according to [6]. [8] A cell transformed or transduced with the isolated nucleic acid according to [6] or the vector according to [7]. [9] A cell expressing the granzyme B variant according to any one of [1] to [5].

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

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

[12] 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, wherein the receptor activates cells expressing the receptor by binding to a ligand, and the granzyme B variant has enhanced protease activity compared to human wild-type granzyme B and is resistant to inhibitors of human wild-type granzyme B.

[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 a ligand via the extracellular binding domain.

[14] The pharmaceutical composition of

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

[15] The pharmaceutical composition of any of

[12] to

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

[16] The pharmaceutical composition of any 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 administration of an antigen-binding molecule and administration of cells expressing a chimeric receptor, wherein the antigen-binding molecule has the ability to bind to a target antigen, the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, and is capable of binding to cells expressing the target antigen via binding of the extracellular binding domain to the antigen-binding molecule, and the granzyme B variant has enhanced protease activity compared to human wild-type granzyme B and is resistant to inhibitors of human wild-type granzyme B.

[19] The pharmaceutical composition of

[18] , wherein the antigen-binding molecule comprises a linker that is cleaved by a protease, and the extracellular binding domain is capable of binding to the antigen-binding molecule after linker cleavage.

[20] The pharmaceutical composition of

[18] or

[19] , wherein the granzyme B variant is one of [1], [2], or [5].

[21] The pharmaceutical composition according to any one of

[18] to

[20] , comprising a cell 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] The pharmaceutical composition according to any one of

[13] and

[15] to

[22] , wherein the chimeric receptor is a chimeric antigen receptor. [A1] A granzyme B variant comprising, relative to human wild-type granzyme B, one or more amino acid residue mutations selected from the following 1 to 20: 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 granzyme B variant according to [A1], which comprises, relative to human wild-type granzyme B, a mutation in one or more amino acid residues selected from 1 to 20 below.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] The granzyme B variant according to [A1], wherein the amino acid residue mutation is any one of 1 to 12 below: 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 granzyme B variant according to [A3], wherein the amino acid residue mutations are any of 1 to 12 below.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) 8) K44L, L46F, 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], which comprises mutations of one or more amino acid residues relative to human wild-type granzyme B set forth in SEQ ID NO: 1. [A6] A granzyme B variant according to any of [A1] to [A5], which has enhanced protease activity compared to human wild-type granzyme B. [A7] A granzyme B variant according to any of [A1] to [A6], which is resistant to an inhibitor of human wild-type granzyme B. [A8] The granzyme B variant according to [A7], wherein the inhibitor is PI-9 or heparin. [A9] An isolated nucleic acid encoding the granzyme B variant according to any of [A1] to [A8]. [A10] A vector comprising the isolated nucleic acid according to [A9]. [A11] A cell transformed or transduced with the isolated nucleic acid according to [A9] or the vector according to [A10]. [A12] A cell expressing the granzyme B variant described in any of [A1] to [A8]. [A13] A pharmaceutical composition comprising the isolated nucleic acid described in [A9], the vector described in [A10], or the cell described in [A11] or [A12]. [A14] A pharmaceutical composition comprising the granzyme B variant described in any of [A1] to [A8].[A15] The pharmaceutical composition according to any one of

[12] to

[14] , wherein the granzyme B variant is any one of [A1] to [A5]. [A16] The pharmaceutical composition according to [A15], comprising cells expressing a receptor. [A17] The pharmaceutical composition according to [A16], wherein the receptor and the granzyme B variant are expressed in the same T cell. [A18] The pharmaceutical composition according to

[18] or

[19] , wherein the granzyme B variant is any one 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] The pharmaceutical composition according to any one of [A15] to [A20], wherein the receptor is a chimeric antigen receptor. [B1] The granzyme B variant according to [3] or [A6], or the pharmaceutical composition according to

[12] or

[18] , wherein the in vitro protease activity of the granzyme B variant is 1.5-fold or more that of human wild-type granzyme B. [B2] The granzyme B variant or pharmaceutical composition according to [B1], wherein the in vitro protease activity of the granzyme B variant is 2-fold or more that of human wild-type granzyme B. [B3] The granzyme B variant according to [4] or [A7], or the pharmaceutical composition according to

[12] or

[18] , wherein the in vitro protease activity in the presence of an 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 that of human wild-type granzyme B. [B4] A granzyme B variant or pharmaceutical composition according to [B3], whose in vitro protease activity in the presence of an 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 compared to human wild-type granzyme B. [B5] A granzyme B variant or pharmaceutical composition according to [B3] or [B4], wherein the inhibitor is PI-9 or heparin. [B6] An isolated nucleic acid encoding a granzyme B variant according to any of [B1] to [B5]. [B7] A vector comprising the isolated nucleic acid according to [B6]. [B8] A cell transformed or transduced with the isolated nucleic acid according to [B6] or the vector according to [B7].[B9] A cell expressing the granzyme B variant of any of [B1] to [B5]. [B10] A pharmaceutical composition comprising the isolated nucleic acid of [B6], the vector of [B7], or the cell of [B8] or [B9]. [B11] A pharmaceutical composition comprising the granzyme B variant of any of [B1] to [B5]. [B12] The pharmaceutical composition of any of [B1] to [B5], 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. [B13] The pharmaceutical composition of any of [B1] to [B5], wherein the receptor is a T cell receptor whose ligand is a neoantigen. [B14] The pharmaceutical composition of any of [B1] to [B5], [B12], or [B13], wherein the granzyme B variant is one of [1], [2], or [A1] to [A5]. [B15] The pharmaceutical composition according to any one of [B1] to [B5] or [B12] to [B14], comprising a cell expressing a receptor. [B16] The pharmaceutical composition according to [B15], wherein the receptor and the granzyme B variant are expressed in the same T cell. [B17] The pharmaceutical composition according to [B1] to [B5], wherein the antigen-binding molecule comprises a linker that is cleaved by a protease, and the extracellular binding domain is capable of binding to the antigen-binding molecule after cleavage of the linker. [B18] The pharmaceutical composition according to any one of [B1] to [B5] or [B17], wherein the granzyme B variant is one of [1], [2], or [A1] to [A5]. [B19] The pharmaceutical composition according to any one of [B1] to [B5], [B17], or [B18], comprising a cell expressing a chimeric receptor. [B20] The pharmaceutical composition according to [B19], wherein the chimeric receptor and the modified granzyme B are expressed in the same T cell. [B21] The pharmaceutical composition according to any one of [B12] and [B14] to [B20], wherein the chimeric receptor is a chimeric antigen receptor. [C1] The pharmaceutical composition according to any one of

[10] to

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

[10] to

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

[0013] 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. A pharmaceutical composition comprising a granzyme B variant of the present disclosure having such advantageous effects or cells expressing the same is more advantageous in inducing cell death in target cells than wild-type granzyme B and prior art granzyme B variants. Furthermore, by using the granzyme B variants of the present disclosure in combination with a pharmaceutical product using cells expressing a receptor and / or an antibody pharmaceutical product, it is possible to induce cell death specifically in target cells.

[0014]

[0039] Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of amino acid numbers 21-247 (amino acids 21 to 247 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). Within this sequence, the amino acids at the positions where modifications were performed in Example 2 are underlined.

[0040] Figure 2 shows the results of measuring the protease activity of granzyme B variants in Example 4-1. The vertical axis shows 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 shows the amino acid substitutions in the variants subjected to measurement.

[0050] Figure 2 shows the results of measuring the protease activity of granzyme B variants in Example 4-2. The vertical axis shows the relative value of the protease activity of each granzyme B variant, when the protease activity of wild-type granzyme B in (1) the absence of an inhibitor (Buffer), (2) the presence of heparin (Heparin), or (3) the presence of PI-9 (PI-9) is set to 1. The horizontal axis shows the amino acid substitutions of the variants subjected to measurement.

[0015] I. Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 those of ordinary skill in the art with a general guide to many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0016] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0017] As used herein, the term "granzyme B," unless otherwise indicated, 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). The term encompasses "full-length," unprocessed granzyme B, as well as 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 allelic variants. An exemplary amino acid sequence of human wild-type granzyme B is set forth in SEQ ID NO: 1 (NCBI Reference Sequence NP_004122.2), but is not necessarily limited thereto, and includes variants with partial amino acid sequence differences. Such variants include human wild-type granzyme B represented by an amino acid sequence having 90% or more, 95% or more, 97% or more, or 99% or more identity to SEQ ID NO: 1.

[0018] As used herein, the terms "mutation" and "modification" are used interchangeably and include the "addition" of an amino acid residue to an amino acid sequence, the "deletion" of an amino acid residue from an amino acid sequence, the "insertion" of an amino acid residue into an amino acid sequence, and / or the "substitution" of an amino acid residue in an amino acid sequence. Any combination of addition, deletion, insertion, and substitution can be introduced to obtain a variant (variant) with desired characteristics (e.g., protease activity or resistance to inhibitors). In one embodiment, the Granzyme B variant of the present disclosure contains a substitution of one or more amino acid residues.

[0019] When the position of each amino acid in granzyme B is indicated herein, the corresponding amino acid number in the amino acid sequence of human wild-type granzyme B exemplified by SEQ ID NO: 1 is designated (in the case of SEQ ID NO: 1, consecutive numbers are used, starting with 1 for the position of the N-terminal amino acid in the amino acid sequence). For example, when a granzyme B variant of the present disclosure is described as "containing an amino acid residue of F (Phe) at position 44," this means that the 44th amino acid residue from the N-terminus constituting the granzyme B variant is F (Phe). Furthermore, when an amino acid modification of granzyme B is described herein, the amino acid residues at that position before modification (i.e., wild-type granzyme B) and after modification (e.g., in single-letter notation) are indicated to the left and right, or left and right, of the amino acid number at the modified position, respectively. For example, when the amino acid residue at the position corresponding to the 44th amino acid residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is modified to F (Phe), the amino acid modification is referred to herein as K44F. Furthermore, when simply indicating that the 44th amino acid residue from the N-terminus of granzyme B is modified to F (Phe), it is represented as 44F. Even if the amino acid sequence of the granzyme B to be modified differs from the sequence shown in SEQ ID NO: 1 in part, those skilled in the art can appropriately determine (for example, by performing sequence alignment) which position of modification shown herein corresponds to which position in the granzyme B to be modified. In the present disclosure, a granzyme B variant contains one or more amino acid residues selected from 1 to 20 below. 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. In another aspect, the Granzyme B variant of the present disclosure comprises any combination of the following 1 to 12 amino acid residues.1) L (rank 44), K (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 2) L (rank 44), E (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 3) F (rank 44), K (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 4) F (rank 44), E (rank 48), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 5) L (rank 44), I (rank 46), P (rank 155), L (rank 172), I (rank 175), and R (rank 200) 6) 44th L, 46th E, 155th P, 172nd L, 175th I and 200th R 7) 44th L, 46th F, 155th P, 172nd L, 175th I and 200th R 8) 44th L, 46th Q, 155th P, 172nd L, 175th I and 200th R 9) 44th F, 46th I, 155th P, 172nd L, 175th I and 200th R 10) 44th F, 46th E, 155th P, 172nd L, 175th I and 200th R 11) F at 44th place, F at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 12) F at 44th place, Q at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place.

[0020] As used herein, "protease activity," particularly in the context of granzyme B, refers to the activity of granzyme B to cleave its substrate. Methods for assessing the protease activity of granzyme B are well known to those skilled in the art, and various activity assay kits and synthetic substrates are commercially available. Examples of such synthetic substrates include synthetic peptides containing a granzyme B recognition sequence (e.g., Ile-Glu-Pro-Asp (IEPD)) labeled with a detectable substance (e.g., p-nitroanilide (pNA)) (e.g., Ac-IEPD-pNA). Granzyme B cleavage of the synthetic substrate releases free detectable substances, which can be quantified using a fluorometer or spectrophotometer. As an example, the protease activity of granzyme B variants can be assessed using the method described in Example 4 of the present disclosure. For example, protease activity in the presence of an inhibitor can be assessed using the assay system described in Example 4 under the same granzyme B and inhibitor concentration conditions as in Example 4. The protease activity of the granzyme B variants of the present disclosure is preferably enhanced compared to the protease activity of wild-type granzyme B; for example, it is preferably at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, or at least 1.5 times higher than the protease activity of wild-type granzyme B, more preferably at least 2 times or at least 2.5 times higher, and particularly preferably at least 3 times 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 the present disclosure are preferably resistant to such inhibitors. As used herein, "resistance to an inhibitor" refers to the ability to exhibit higher protease activity than wild-type granzyme B in the presence of the inhibitor. The protease activity of the granzyme B variants of the present disclosure in the presence of such an inhibitor is preferably at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, or at least 1.5-fold higher than that of wild-type granzyme B, more preferably at least 2-fold, at least 2.5-fold, at least 3-fold, or at least 3.5-fold higher, and particularly preferably at least 4-fold, at least 4.5-fold, at least 5-fold, or at least 5.5-fold higher.

[0022] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0023] 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 vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0024] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" or "transductants" and "transduced cells," including the primary transformed or transduced cell and progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed or transduced cell was screened or selected are also included herein.

[0025] As used herein, unless otherwise specified, "granzyme B-expressing cells" may refer to cells that express endogenous granzyme B or cells that express granzyme B through gene transfer. Examples of cells that express endogenous granzyme B include cytotoxic lymphocytes such as T cells and NK cells. Cells transfected with genes to express granzyme B are not limited to T cells and NK cells. Recombinant granzyme B can be expressed in various cells and then purified from the culture supernatant. Alternatively, a gene expressing granzyme B can be introduced into cells (e.g., peripheral blood mononuclear cells (PBMCs)) derived from an individual (e.g., a healthy donor or a patient suffering from a specific disease), and the transfected cells can be administered to the same or another individual. Granzyme B-expressing transfected cells may also be treated to differentiate into a specific cell type (e.g., cytotoxic T cells) before being administered to the individual. Various gene transfer techniques well known to those skilled in the art can be used to introduce a gene encoding granzyme B into cells. Cells expressing granzyme B may be administered in combination with cells expressing a chimeric receptor. Cells expressing both granzyme B and a chimeric receptor may also be used, and such cells can be prepared by simultaneous or separate gene transfer of granzyme B and a chimeric receptor. When expressing a granzyme B variant in cells that express endogenous granzyme B, such as T cells or NK cells, endogenous wild-type granzyme B in the cells may or may not be knocked out.

[0026] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation or composition is administered. The "active ingredient" can be configured as an antibody, polypeptide, etc., or as cells that express an antibody, polypeptide, etc. (e.g., a granzyme B variant of the present disclosure). For example, when a nucleic acid encoding a granzyme B variant of the present disclosure or cells transformed or transduced with a vector containing the nucleic acid are administered to a patient for therapeutic or preventive purposes, a preparation containing such cells can be called a "pharmaceutical formulation" or "pharmaceutical composition."

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

[0028] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 human.

[0029] In one aspect, the present disclosure provides granzyme B variants, cells expressing granzyme B variants, or pharmaceutical compositions comprising them. In one embodiment, the pharmaceutical composition of the present disclosure can be used in combination with cells expressing a receptor. Here, the receptor activates the cell expressing the receptor by binding to a ligand, and examples include, but are not limited to, chimeric receptors that bind to a ligand via an extracellular binding domain and T cell receptors that bind to a neoantigen. The pharmaceutical composition of the present disclosure may contain cells expressing the receptor and cells expressing a granzyme B variant, and the receptor and granzyme B variant may be expressed in the same cell (e.g., T cell). Such a pharmaceutical composition of the present disclosure can be used in combination with an antigen-binding molecule capable of binding to the extracellular binding domain of the 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 binding of its extracellular binding domain to the antigen-binding molecule. In certain embodiments, the antigen-binding molecule comprises a linker cleaved by a protease, and the extracellular binding domain can bind to the antigen-binding molecule after cleavage of the linker. The pharmaceutical composition of the present disclosure may comprise cells expressing a chimeric receptor and cells expressing a granzyme B variant, as well as an antigen-binding molecule capable of binding to the extracellular binding domain of the chimeric receptor, and the receptor and the granzyme B variant may be expressed in the same cell (e.g., T cell). When one or more of the granzyme B variant, cells expressing a granzyme B variant, cells expressing a receptor, cells expressing 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 together (used in combination), they can be used simultaneously, separately, or sequentially (e.g., administered to an individual). In one embodiment, the pharmaceutical composition of the present disclosure is for use in damaging cells, inducing cell death, inhibiting cell proliferation, or treating or preventing cancer or inflammatory diseases.

[0030] The term "chimeric receptor" refers to a recombinant polypeptide that contains at least an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, which, when expressed in an immune effector cell, confers specificity for a target cell, e.g., a cancer cell, and intracellular signal production. 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 proteinaceous molecule or a portion thereof that can specifically bind to a molecule such as a predetermined antigen, and includes, for example, a single-chain antibody (scFv) in which the light chain (VL) and heavy chain (VH) of the variable region of a monoclonal antibody specific to a tumor antigen or the like are linked in tandem. The extracellular binding domain can also be referred to as an extracellular recognition domain.

[0032] The term "transmembrane domain" includes a polypeptide that is located between the extracellular binding domain and the intracellular signaling domain and has the function of spanning a cell membrane.

[0033] The term "intracellular signaling domain" refers to any oligopeptide or polypeptide domain known to function to transmit a signal that causes the activation or inhibition of a biological process within a cell, for example, the activation of an immune cell such as a T cell or an NK cell, and includes 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.

[0034] As used herein, the term "T cell receptor with a neoantigen as a ligand" includes T cell receptors designed to recognize neoantigens, which are mutated antigens that arise as a result of genetic mutations in cancer cells.

[0035] As used herein, 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 one embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment or derivative thereof.

[0036] As used herein, the term "antigen-binding domain" refers to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When an antigen has a large molecular weight, the antigen-binding domain can bind only to a specific part of the antigen. This 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 by one or more antibody variable domains. In a 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 specific embodiment, the antigen-binding domain comprises a hinge region.

[0037] As used herein, the term "specifically binds" refers to a state in which one of the specifically binding molecules binds without showing any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies to cases in which an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0038] The term "antibody" is used herein in the broadest sense and encompasses a variety of 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.

[0039] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy 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 contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ).

[0040] 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 an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0041] In some embodiments, the present disclosure provides a method for damaging cells, inducing cell death, suppressing cell proliferation, treating inflammatory diseases, treating cancer, or preventing cancer, comprising administering an effective amount of a granzyme B variant of the present disclosure, a cell expressing a granzyme B variant, or a pharmaceutical composition containing them (hereinafter, these may be collectively referred to as the pharmaceutical composition of the present disclosure, etc.). In some embodiments, the term "effective amount" in the present invention refers to a dose of the pharmaceutical composition of the present disclosure, etc., that is effective in damaging cells, inducing cell death, suppressing cell proliferation, treating inflammatory diseases, treating cancer, or preventing cancer in an individual.

[0042] In some embodiments, "treatment" in the present invention means reducing the number of cancer cells in an individual, inhibiting cancer cell proliferation, reducing tumor size (volume and / or weight), inhibiting tumor growth, inhibiting cancer cell infiltration into peripheral organs, inhibiting cancer cell metastasis, or ameliorating various symptoms caused by cancer, by the pharmaceutical composition, etc. of the present disclosure. Also, in some embodiments, "prevention" in the present invention means preventing an increase in the number of cancer cells due to the re-proliferation of reduced cancer cells, preventing the regrowth of cancer cells whose proliferation has been inhibited, or preventing a re-increase in the size (volume and / or weight) of a reduced tumor.

[0043] [Application Example] The granzyme B variants of the present disclosure can be used in combination with the administration of cells expressing a receptor. One example of a receptor is a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. Another example is a T cell receptor designed to recognize neoantigens that cannot be recognized by normal T cells. One embodiment of combining these receptor-based technologies with the granzyme B variants of the present disclosure involves collecting T cells from a patient, transfecting them with a gene encoding a chimeric receptor (e.g., a chimeric antigen receptor) and a gene encoding a granzyme B variant, and then transferring them back into the patient. (Note: This is not a limitation; the genes can also be transfected separately and transferred into the patient.) The chimeric antigen receptor recognizes cell surface antigens, such as those on cancer cells, to activate T cells, which then exerts the enhanced cytotoxic activity of the granzyme B variants of the present disclosure, potentially resulting in a high therapeutic effect. Similarly, by introducing a gene encoding a T cell receptor that binds a neoantigen and a gene encoding a granzyme B variant into T cells collected from a patient and then transferring the resulting T cells back into the patient, a high therapeutic effect is expected. Furthermore, the combination of a granzyme B variant and a chimeric receptor disclosed herein can also be combined with an antigen-binding molecule (e.g., 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 binding of the extracellular binding domain to the antigen-binding molecule. In one embodiment of the antigen-binding molecule, the antigen-binding molecule is pre-incorporated with a linker that can be cleaved by a protease, and the chimeric receptor can be designed so that it can bind to the antigen-binding molecule after linker cleavage. In one embodiment of such a combination, T cells are collected from a patient, a gene encoding a chimeric receptor and a gene encoding a granzyme B variant are introduced into the T cells, the T cells are transferred back into the patient, and a pharmaceutical composition containing the antigen-binding molecule is separately administered to the patient.

[0044] Examples of the construction, expression, purification, gene transfer, and use of modified granzymes for the treatment of diseases and injuries are described below, but the practice of the 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. An artificial secretory 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 acids 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-terminus (J Vis Exp. 2015 Jun 10;(100):e52911.). The nucleotide sequence encoding this sequence (SEQ ID NO: 5) was inserted into a mammalian cell expression vector.

[0046] In addition, when the position of an amino acid modification in granzyme B is indicated in this specification, 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.

[0047] Example 2: Preparation of Nucleotide Sequences Encoding Granzyme B Variants Single amino acid residue substitutions and combinations 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 (the underlined amino acids in Figure 1). Primers were designed to encode substitutions of these amino acid residues with any of 18 amino acids excluding the original amino acid and cysteine. A total of 1,476 nucleotide sequences encoding granzyme B variants with single amino acid substitutions were prepared using PCR or other methods known to those skilled in the art. Nucleotide sequences encoding granzyme B variants with multiple amino acid substitutions were also prepared by a similar method.

[0048] Example 3: Expression and Purification of Granzyme B Variants The nucleotide sequence encoding granzyme B prepared in Example 2 was transfected into 1 mL of Expi293 (Invitrogen) culture medium according to the manufacturer's instructions. After 4 days, the culture supernatant was collected, and enterokinase was added to a final concentration of 0.3 μg / mL. The mixture was incubated at 4°C for 16 hours. One-tenth the volume of binding buffer (250 mM Tris, 3 M NaCl, pH 7.5) was added to the reaction solution, followed by 50 μL of Ni Sepharose Excel (GE Healthcare, 17371201) suspended in equilibrium buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.5). After incubation at 4°C for 1 hour, the reaction solution was applied to a filter plate (Merck, MSGVS2210). The Ni Sepharose Excel with bound granzyme B was washed five times with 200 μL of equilibrium buffer and then incubated 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 granzyme B variants (4-1) Granzyme B variants (single amino acid substitution) 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 (Enzo, BML-P133) at a final concentration of 0.5 mM. The absorbance of the reaction solution at 405 nm was measured. Figure 2 shows the relative protease activity of the granzyme B variants measured 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 granzyme B variant) / (protease activity of wild-type granzyme B).

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

[0052] (4-2) Granzyme B variants (multiple amino acid substitutions) In the comprehensive assay described above, 1,476 variants were assayed. As a result, 33 variants were found to have increased activity relative to wild-type granzyme B, while 1,443 variants had activity below that of the wild-type. For these 33 modifications that increase the protease activity of granzyme B, multiple variants of granzyme B were prepared by combining multiple mutations using the method described in Example 2. Among the variants prepared, examples that showed particularly significant increases in activity are shown in Table 1. In Table 1, the positions of amino acid substitutions in the variants refer to the corresponding amino acid numbers in the amino acid sequence of human wild-type granzyme B shown in SEQ ID NO: 1, and the amino acid sequences of the variants indicate the amino acid sequences of the variants corresponding to amino acids 21-247 of human wild-type granzyme B shown in SEQ ID NO: 1.

[0053]

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

[0055] Example 5: Expression of granzyme B variants in NK cell lines The granzyme B variants prepared in Example 4 and the amino acid sequence corresponding to amino acids 21-247 of wild-type granzyme B (SEQ ID NO: 1) are fused to the N-terminus with a secretory signal sequence and a cathepsin C / H recognition sequence derived from wild-type granzyme B, and fused to the C-terminus with a FLAG tag. The nucleotide sequences encoding these fusions are transfected into an NK cell line (NKL, ATCC No.) using the mammalian expression vector pGL4.30 (Promega, E8481). This NK cell line is selected with hygromycin B. The granzyme B variant cell line was selected using eBioscience TMThe cells were permeabilized using Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) according to a method specified by one skilled in the art, and fluorescently stained using an anti-FLAG antibody that recognizes the tag fused to the C-terminus of granzyme B and its isotype control antibody (Biolegend, 637310 and 400508). The stained cells were detected using a FACSverse (BD). As a result, a peak associated with granzyme B expression was observed in cells transfected with granzyme B, confirming the expression of the transfected granzyme B.

[0056] Example 6: Measurement of in vitro cytotoxic activity of cell lines expressing granzyme B variants. The established granzyme B variant-expressing cell lines and target cells (BxPC-3, HuCCT-1, or MCAS) were added to a 96-well plate, followed by dilutions of anti-EGFR antibodies CetuH0-H1076 / CetuL4-k0 / CetuH0-Kn125 / CetuL4-k0. After incubation at 37°C, LDH release associated with cell death was quantified using the Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific, 88954) according to the manufacturer's instructions. The results demonstrate that transfected cells expressing granzyme B variants exhibit stronger cytotoxic activity than transfected cells expressing wild-type granzyme B.

[0057] [Example 7] Preparation of viral vectors encoding granzyme B variants Retroviral vectors encoding human wild-type granzyme B and granzyme B variants were prepared using the pMCs-IRES-GFP Retroviral Vector (CELL BIOLABS, INC., RTV-040) according to the method specified by the manufacturer.

[0058] [Example 8] Genetic transfer of modified granzyme B into primary T cells HLA-A2 derived from a healthy donor +Peripheral blood mononuclear cells (PBMCs) (Biological Specialty Corp, Colmar, PA, USA) were isolated by Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation. PBMCs were cultured in 24-well tissue culture plates at 3 × 10 cells / well 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). 6 Cells were cultured at 1000 cells / well and activated with 50 ng / ml OKT3 (eBioscience, San Diego, CA, USA). Two days later, cells were harvested for retroviral transduction. For transduction, 24-well non-tissue culture-treated plates (BD Biosciences, Franklin Lakes, NJ, USA) were coated overnight at 4°C with 0.5 ml / well of 10 μg / ml recombinant human fibronectin fragment (RetroNectin; Takara Bio Inc., Otsu, Shiga Prefecture, Japan). After incubation, wells were blocked with 1 ml of Hank's Balanced Salt Solution (GIBCO brand; Invitrogen) supplemented with 2.5% human AB serum for 30 minutes at room temperature and washed with Hank's Balanced Salt Solution (GIBCO brand; Invitrogen) supplemented with 2.5% N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (GIBCO brand; Invitrogen). Transduction is performed as previously described (Johnson et al. Blood 114, 535-546 (2009)). Briefly, approximately 2.5 ml of retroviral supernatant is added to each coated well, followed by centrifugation at 2000 g for 2 hours at 32°C. 1.5 ml of viral supernatant is removed and 1 x 10 60.5 ml of activated PBMCs were added to each well in the presence of 100 U / ml IL-2. The plates were centrifuged at 1000 g for 10 minutes and then incubated overnight at 37°C. After transduction, the cells were washed, 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 was determined by flow cytometry after staining with an antibody recognizing the FLAG tag 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 transfected with granzyme B variants. Granzyme-expressing T cells prepared in Example 8 and target cells (BxPC-3, HuCCT-1, or MCAS) were added to a 96-well plate, followed by the addition of various diluted concentrations of anti-EGFR anti-CD3 bispecific antibodies CetuH0-F760nN17 / CetuL4-k0 / / TR01H113- F760nP17 / L0011-k0. After incubation at 37°C, LDH release associated with cell death was quantified using the Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific, 88954) according to the manufacturer's instructions. The results demonstrate that transfected cells expressing modified granzyme B exhibit stronger cytotoxic activity than transfected cells expressing wild-type granzyme B.

[0060] [Example 10] Evaluation of in vitro cytotoxic activity of T cells transfected with modified granzyme B The cytotoxic activity of the granzyme-transfected T cells prepared in Example 8 was evaluated using BD FACSVerse. TM The target cells are BxPC-3, HuCCT-1, or MCAS cancer cells. The target cells are placed in a 6-well plate at 1 × 10 5 cells or 3 × 10 5T cells expressing modified granzyme B or wild-type granzyme B are used as effector cells, and are mixed at an effector cell to target cell (E:T) 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 10 μg per well. 48 hours after addition, the granzyme B-introduced T cells and target cells are harvested. The harvested cells are then stored in Zombie Aqua TM Dead cells were stained using a Fixable Viability Kit (BioLegend, 423102), and granzyme B-expressing T cells were stained using an anti-human CD45 antibody (BioLegend, 304039). Cytotoxic activity was assessed by the percentage of remaining cancer cells, which was calculated as the percentage of CD45- cells among viable cells. These results demonstrate enhanced cytotoxic activity of granzyme B-expressing T cells in vitro.

[0061] The granzyme B variants of the present disclosure exhibit higher protease activity than wild-type granzyme B, and are therefore more useful in therapies for inducing cell death in target cells (e.g., cancer cells) than wild-type granzyme B. Furthermore, the granzyme B variants of the present disclosure exhibit higher protease activity even in the presence of an inhibitor of wild-type granzyme B, and are therefore more useful than wild-type granzyme B in therapies for inducing cell death in tumors that highly express the inhibitor.

Claims

1. A granzyme B variant comprising one or more amino acid residues selected from the following 1 to 20: 1) R at 200th place, 2) P at 155th place, 3) I, Q, or E at 175th place, 4) L at 172nd place, 5) L or F at 44th place, 6) I, E, F, or Q at 46th place, 7) K or F at 48th place, 8) T, E, N, or V at 43rd place, 9) Q, L, or A at 45th place, 10) V at 47th place, 11) L at 99th place, 12) A at 106th place, 13) M at 149th place, 14) L at 151st place, 15) P at 183rd place, 16) L at 184th place, 17) I at 217th place, 18) F at 219th place, 19) G or S at 222nd place, 20) V at 229th place

2. The granzyme B variant according to claim 1, comprising any combination of the following 1 to 12 amino acid residues: 1) L at 44th place, K at 48th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 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 at 44th place, K at 48th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 4) F at 44th place, E at 48th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 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 at 44th place, F at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 8) L at 44th place, Q at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 9) F at 44th place, I at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 10) F at 44th place, E at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 11) F at 44th place, F at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place 12) F at 44th place, Q at 46th place, P at 155th place, L at 172nd place, I at 175th place and R at 200th place

3. The granzyme B variant according to claim 1 or 2, which has enhanced protease activity as compared to that of human wild-type granzyme B.

4. The granzyme B variant according to any one of claims 1 to 3, which has resistance to an inhibitor of human wild-type granzyme B.

5. The granzyme B variant according to claim 4, wherein the inhibitor is PI-9 or heparin.

6. An isolated nucleic acid encoding a Granzyme B variant according to any one of claims 1 to 5.

7. A vector comprising the isolated nucleic acid of claim 6.

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

9. A cell expressing the granzyme B variant according to any one of claims 1 to 5.

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

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

12. A pharmaceutical composition comprising a cell expressing a Granzyme B variant or a Granzyme B variant for use in combination with the administration of a cell expressing a receptor, A receptor activates a cell expressing the receptor by binding to a ligand; A pharmaceutical composition comprising a granzyme B variant having enhanced protease activity as compared to human wild-type granzyme B and resistance to inhibitors of human wild-type granzyme B.

13. The pharmaceutical composition of claim 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 according to claim 12, wherein the receptor is a T cell receptor whose ligand is a neoantigen.

15. The pharmaceutical composition according to any one of claims 12 to 14, wherein the granzyme B variant is that according to claim 1, 2 or 5.

16. A pharmaceutical composition according to any one of claims 12 to 15, comprising a cell expressing the receptor.

17. The pharmaceutical composition according to claim 16, wherein the receptor and the modified granzyme B are expressed in the same T cell.

18. A pharmaceutical composition comprising a cell expressing a granzyme B variant or a granzyme B variant for use in combination with administration of an antigen-binding molecule and administration of a cell expressing a chimeric receptor, The antigen-binding molecule has a binding ability to a target antigen, The chimeric receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signaling domain, and is capable of binding to a cell expressing a target antigen via binding of the extracellular binding domain to an antigen-binding molecule; A pharmaceutical composition comprising a granzyme B variant having enhanced protease activity as compared to human wild-type granzyme B and resistance to inhibitors of human wild-type granzyme B.

19. The antigen-binding molecule comprises a linker that is cleaved by a protease, The pharmaceutical composition according to claim 18, wherein the extracellular binding domain is capable of binding to the antigen-binding molecule after cleavage of the linker.

20. 20. The pharmaceutical composition according to claim 18 or 19, wherein the granzyme B variant is that according to claim 1, 2 or 5.

21. 21. The pharmaceutical composition of any one of claims 18 to 20, comprising a cell expressing the chimeric receptor.

22. The pharmaceutical composition according to claim 21, wherein the chimeric receptor and the modified granzyme B are expressed in the same T cell.

23. The pharmaceutical composition according to any one of claims 13, 15 to 22, wherein the chimeric receptor is a chimeric antigen receptor.