Peptide tag and nucleic acid encoding same

Abstract

The present disclosure provides a peptide tag and a nucleic acid encoding the peptide tag. The peptide tag of the present disclosure can reduce protein aggregation properties in cells. More particularly, the peptide tag of the present disclosure can be a peptide tag in which 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acid, and (b) 20% or more of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N and A.

Description

Peptide tag and nucleic acid encoding said peptide tag

[0001] The present disclosure relates to peptide tags and nucleic acids encoding the peptide tags.

[0002] Antibodies that function within cells, or intrabodies, can affect cellular function by recognizing and binding to antigens (target molecules) within the cells of higher organisms. These antigens can also be important intracellular therapeutic targets that can be inactivated by binding to intracellular antibodies. Furthermore, the use of intracellular antibodies has emerged as a research technique for specifically inhibiting protein function directly by binding to antibodies within cells.

[0003] In the case of intracellular antibodies, it is common to first prepare a hybridoma that produces a monoclonal antibody that recognizes an antigen using a standard method, then construct an intracellular expression vector containing DNA encoding a single-chain antibody (single chain Fv: scFv) from the cDNA of the hybridoma, and use the complex of the heavy chain (VH) and light chain (VL) as the intracellular antibody. Recently, phage libraries displaying human scFv have been prepared from antibodies isolated from human B cells, and these libraries are sometimes used to isolate scFv that bind to intracellular antigens.

[0004] Antibodies typically circulate in extracellular spaces in the body, such as blood, and function by recognizing extracellular antigens, presumably acting in an extracellular environment. Therefore, when antibodies are expressed in the cytoplasm, folding and stability problems can arise, leading to reduced expression levels and limited half-lives of antibody domains. Problems with the stability of intracellular antibodies in the cytoplasm can lead to the formation of intracellular antibody aggregates in the cytoplasm. Aggregate formation can lead to reduced production of intracellular antibodies and inhibition of normal function. The same is true for proteins other than intracellular antibodies. Intracellular antibodies are particularly prone to aggregation, but proteins other than intracellular antibodies can also form aggregates in the cytoplasm when produced in the cytoplasm.

[0005] In contrast, it has been shown that peptide tags having an amino acid sequence containing 45% or more acidic amino acids improve the stability of intracellular antibodies (Patent Document 1, Non-Patent Document 1). Patent Document 1 and Non-Patent Document 1, when proposing the effectiveness of peptide tags having an amino acid sequence containing 45% or more acidic amino acids, point out the importance of designing peptide tags so that their electric potential and pI values ​​are sufficiently lower, based on the pH environment of the cytoplasmic surface of endosomes, than the pH environment of the cytoplasm. Non-Patent Document 3 adds a membrane localization signal of HRAS to the heavy chain variable region of an antibody.

[0006] WO2019 / 004213

[0007] Kabayama et al. , 2020, Nature Communication, 11, 336 Shubhada et al. , 2012, Biochemical genetics, Vol. 50, No. 7-8, pp. 625-41. Tanaka et al. , 2007, EMBO Journal, 26:3250-3259

[0008] The present disclosure provides peptide tags and nucleic acids encoding the peptide tags. The peptide tags of the present disclosure can reduce the aggregation of proteins in cells.

[0009] The present inventors have conducted extensive research into peptide tags with various amino acid sequences and have discovered peptide tags that have the effect of reducing intracellular protein aggregation.

[0010] The present disclosure provides the following inventions: [1] A peptide, for example, a peptide having an amino acid sequence of 600 amino acids or less, for example, 10 to 200 amino acids (for example, 10 to 90 amino acids), wherein (a) 5% to less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and (b) 20% or more, preferably 30% or more of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, preferably capable of reducing the intracellular aggregation of a protein to which the peptide is linked {wherein preferably 10% or 15% or more of the amino acids constituting the amino acid sequence are N or P}. [2] (c) The peptide according to [1] above, wherein 30% or less, preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L. [3] (d) The peptide according to [1] or [2] above, wherein A or G each accounts for less than 10% of the amino acids constituting the amino acid sequence. [4] The peptide according to any of [1] to [3] above, wherein: (a) 20% to less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids; (b) 30% to less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A; (c) 20% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L; and (d) A or G each accounts for less than 10% of the amino acids constituting the amino acid sequence. [5] A peptide having an amino acid sequence set forth in any of SEQ ID NOs: 2 to 11. [6] A nucleic acid encoding the peptide according to any of [1] to [5] above. [7] A protein expression vector comprising the nucleic acid according to [6] above operably linked to a regulatory sequence and a nucleic acid encoding a target protein in frame with the nucleic acid. [8] The protein expression vector according to the above-mentioned [7], wherein the target protein is an antibody or an antigen-binding fragment of the antibody. [9] The protein expression vector according to the above-mentioned [8], wherein the antigen-binding fragment of the antibody is a single-chain Fv (scFv).

[10] A fusion protein of the peptide according to any one of [1] to [5] above with a protein of interest.

[11] The fusion protein according to

[10] above, wherein the protein of interest is an antibody or an antigen-binding fragment of the antibody.

[12] The fusion protein according to

[11] above, wherein the antigen-binding fragment of the antibody is a single-chain Fv (scFv).

[13] A protein-producing cell comprising the nucleic acid according to [6] above operably linked to a regulatory sequence, and a nucleic acid encoding a protein of interest in frame with the nucleic acid.

[14] A method for selecting or identifying an amino acid sequence having a length of 600 amino acids or less, for example, 10 to 200 amino acids (for example, 10 to 90 amino acids), comprising: obtaining, from amino acid sequence(s) (which may include amino acid sequence(s) having a length of 10 to 200 amino acids (for example, 10 to 90 amino acids)), an amino acid sequence(s) in which: (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and (b) 20% or more of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A; selecting or identifying the amino acid sequence of a peptide tag that reduces the proportion of cells in which the fusion protein forms aggregates (for example, to a predetermined value or less) when a fusion protein of the peptide tag having the selected or identified amino acid sequence and a reference protein is expressed in mammalian cells (preferably human cells); and obtaining a peptide tag having the amino acid sequence or a nucleic acid encoding the peptide tag.

[15] The method according to claim 14, wherein the obtained group of amino acid sequences is a peptide according to any one of [1] to [5] above.

[16] The method according to

[14] or

[15] above, wherein the obtained group of amino acid sequences is a group of amino acid sequences encoded by a coding region of the human genome.

[17] The method according to any one of

[14] to

[16] above, wherein the obtained amino acid sequence comprises a neoantigen.

[0011]

[18] A peptide that satisfies one or more of the following conditions selected from the group consisting of (a) to (h) and that can reduce the intracellular aggregation of a protein to which the peptide is linked:

[19] A peptide that can reduce the intracellular aggregation of a protein to which the peptide is linked, selected from the group consisting of (A) to (AE) and (AF) to (AU) below.

[0012]

[20] A nucleic acid encoding the peptide according to

[18] above.

[21] A protein expression vector comprising the nucleic acid according to

[20] above operably linked to a regulatory sequence and a nucleic acid encoding a target protein in frame with the nucleic acid.

[22] A fusion protein of the peptide according to

[18] above and a target protein.

[23] The fusion protein according to

[22] above, wherein the target protein is an antibody or an antigen-binding fragment of an antibody.

[24] The fusion protein according to

[23] above, wherein the antigen-binding fragment of the antibody is a single-chain Fv (scFv).

[0013]

[25] A nucleic acid encoding the peptide according to

[19] above.

[26] A protein expression vector comprising the nucleic acid according to

[25] above operably linked to a regulatory sequence and a nucleic acid encoding a target protein in frame with the nucleic acid.

[27] A fusion protein of the peptide according to

[25] above and a target protein.

[28] The fusion protein according to

[26] above, wherein the target protein is an antibody or an antigen-binding fragment of an antibody.

[29] The fusion protein according to

[27] above, wherein the antigen-binding fragment of the antibody is a single-chain Fv (scFv).

[0014]

[30] The method according to

[14] above, wherein the amino acid sequence(s) comprises the peptide according to

[18] above.

[31] The method according to

[14] above, wherein the amino acid sequence(s) comprises the peptide according to

[19] above.

[0015]

[32] The method according to

[14] above, wherein the reference protein is an scFv and the predetermined value is 30% or less.

[33] The method according to

[14] above, wherein the reference protein is an scFv and the predetermined value is 20 or less.

[34] The method according to

[14] above, wherein the reference protein is an scFv and the predetermined value is 15% or less.

[35] The method according to

[14] above, wherein the reference protein is an scFv and the predetermined value is 10% or less.

[36] The method according to

[14] above, wherein the reference protein is an scFv and the predetermined value is 5% or less.

[0016]

[37] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (A) below, and preferably the obtained amino acid sequence further satisfies the condition described in (A) below.

[38] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (B) below, and preferably the obtained amino acid sequence further satisfies the condition described in (B) below.

[39] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (C) below, and preferably the obtained amino acid sequence further satisfies the condition described in (C) below.

[40] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (D) below, and preferably the obtained amino acid sequence further satisfies the condition described in (D) below.

[41] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (E) below, and preferably the obtained amino acid sequence further satisfies the condition described in (E) below.

[42] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (F) below, and preferably the obtained amino acid sequence further satisfies the condition described in (F) below.

[43] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (G) below, and preferably the obtained amino acid sequence further satisfies the condition described in (G) below.

[44] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (H) below, and preferably the obtained amino acid sequence further satisfies the condition described in (H) below.

[45] The method according to

[14] above, wherein the reference protein is an scFv, the group of amino acid sequences includes the peptides described in (I) below, and preferably, the obtained amino acid sequence further satisfies the condition described in (I) below.

[46] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (J) below, and preferably the obtained amino acid sequence further satisfies the condition described in (J) below.

[47] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (K) below, and preferably the obtained amino acid sequence further satisfies the condition described in (K) below.

[48] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (L) below, and preferably the obtained amino acid sequence further satisfies the condition described in (K) below.

[49] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (M) below, and preferably the obtained amino acid sequence further satisfies the condition described in (M) below.

[50] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (N) below, and preferably the obtained amino acid sequence further satisfies the condition described in (N) below.

[51] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (O) below, and preferably the obtained amino acid sequence further satisfies the condition described in (O) below.

[52] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (P) below, and preferably the obtained amino acid sequence further satisfies the condition described in (P) below.

[53] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (Q) below, and preferably the obtained amino acid sequence further satisfies the condition described in (Q) below.

[54] The method according to

[14] above, wherein the reference protein is an scFv, the group of amino acid sequences includes a peptide described in (R) below, and preferably, the obtained amino acid sequence further satisfies the condition described in (R) below.

[55] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (S) below, and preferably the obtained amino acid sequence further satisfies the condition described in (S) below.

[56] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (T) below, and preferably the obtained amino acid sequence further satisfies the condition described in (T) below.

[57] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (U) below, and preferably the obtained amino acid sequence further satisfies the condition described in (U) below.

[58] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (V) below, and preferably the obtained amino acid sequence further satisfies the condition described in (V) below.

[59] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (W) below, and preferably the obtained amino acid sequence further satisfies the condition described in (W) below.

[60] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (X) below, and preferably the obtained amino acid sequence further satisfies the condition described in (X) below.

[61] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (Y) below, and preferably the obtained amino acid sequence further satisfies the condition described in (Y) below.

[62] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (Z) below, and preferably the obtained amino acid sequence further satisfies the condition described in (Z) below.

[63] The method according to

[14] above, wherein the reference protein is an scFv, the group of amino acid sequences includes the peptides described in (AA) below, and preferably, the obtained amino acid sequence further satisfies the condition described in (AA) below.

[64] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (AB) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AB) below.

[65] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (AC) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AC) below.

[66] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (AD) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AD) below.

[67] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises a peptide described in (AE) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AE) below. [67A] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AF) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AF) below. [67B] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AG) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AG) below. [67C] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AH) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AH) below. [67D] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AI) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AI) below. [67E] The method described in

[14] above, wherein the reference protein is an scFv, the group of amino acid sequences includes the peptides described in (AJ) below, and preferably, the obtained amino acid sequence further satisfies the conditions described in (AJ) below.[67F] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AK) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AK) below. [67G] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AL) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AL) below. [67H] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AM) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AM) below. [67I] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptide described in (AN) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AN) below. [67J] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AO) below, and preferably the obtained amino acid sequences further satisfy the condition described in (AO) below. [67K] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AP) below, and preferably the obtained amino acid sequences further satisfy the condition described in (AP) below. [67L] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AQ) below, and preferably the obtained amino acid sequences further satisfy the condition described in (AQ) below. [67M] The method described in

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AR) below, and preferably the obtained amino acid sequences further satisfy the condition described in (AR) below. [67N] The method described in

[14] above, wherein the reference protein is an scFv, the group of amino acid sequences includes the peptides described in (AS) below, and preferably, the obtained amino acid sequence further satisfies the conditions described in (AS) below.[67O] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AT) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AT) below. [67P] The method according to

[14] above, wherein the reference protein is an scFv, and the group of amino acid sequences comprises the peptides described in (AU) below, and preferably the obtained amino acid sequence further satisfies the condition described in (AU) below.

[0017]

[68] The method according to any one of

[37] to

[67] and [67A] to [67N] above, wherein the reference protein is an scFv and the predetermined value is 15% or less.

[69] The method according to any one of

[37] to

[67] and [67A] to [67N] above, wherein the reference protein is an scFv and the predetermined value is 10% or less.

[70] The method according to any one of

[37] to

[67] and [67A] to [67N] above, wherein the reference protein is an scFv and the predetermined value is 5% or less.

[0018]

[71] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates exceeds 30%.

[72] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 30 to 40%.

[73] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 40 to 50%.

[74] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 50 to 60%.

[75] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 60 to 70%.

[76] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 70 to 80%.

[77] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 80 to 90%.

[78] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 90 to 95%.

[79] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 95 to 99%.

[80] The method according to any one of

[37] to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 99 to 99.9%.

[81] The method according to any one of

[37] to

[70] above, wherein the percentage of cells in which the reference protein forms aggregates is in the range of 99.9 to 100%.

[0019]

[82] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates exceeds 30%.

[83] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 30 to 40%.

[84] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 40 to 50%.

[85] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 50 to 60%.

[86] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 60 to 70%.

[87] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 70 to 80%.

[88] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 80 to 90%.

[89] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 90 to 95%.

[91] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 99 to 99.9%.

[92] The method according to

[69] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 99.9 to 100%.

[0020]

[93] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates exceeds 30%.

[94] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 30 to 40%.

[95] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 40 to 50%.

[96] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 50 to 60%.

[97] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 60 to 70%.

[98] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is in the range of 70 to 80%.

[99] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is a value in the range of 80 to 90%.

[100] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is a value in the range of 90 to 95%.

[101] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is a value in the range of 95 to 99%.

[102] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is a value in the range of 99 to 99.9%.

[103] The method according to

[70] above, wherein the proportion of cells in which the reference protein forms aggregates is a value in the range of 99.9 to 100%.

[0021]

[104] The peptide according to any one of [1] to [5] above, wherein the peptide tag is capable of reducing the aggregation tendency of an scFv having at least the amino acid sequence set forth in SEQ ID NO: 1.

[105] The nucleic acid according to [6] above, wherein the peptide tag is capable of reducing the aggregation tendency of an scFv having at least the amino acid sequence set forth in SEQ ID NO: 1.

[106] The protein expression vector according to any one of [7] to [9] above, wherein the peptide tag is capable of reducing the aggregation tendency of an scFv having at least the amino acid sequence set forth in SEQ ID NO: 1.

[0022]

[107] The protein expression vector according to any one of [7] to [9] above, wherein the protein expression vector is a viral vector.

[108] The protein expression vector according to

[107] above, wherein the viral vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, vaccinia viral vectors, Sendai viral vectors, and vesicular stomatitis viral vectors.

[0023]

[109] The nucleic acid according to [6] above, wherein the nucleic acid is mRNA.

[110] The nucleic acid according to

[109] above, wherein the nucleic acid has a 5'-terminal Cap structure and a polyA tail in the 3'UTR.

[111] The nucleic acid according to

[109] or

[110] above, wherein the nucleic acid contains pseudouridine as U.

[112] A nanoparticle comprising the nucleic acid according to any of

[109] to

[111] above.

[113] The nanoparticle according to

[112] above, wherein the nanoparticle is a lipid nanoparticle.

[114] The method according to any of

[14] to

[17] and

[30] to

[103] above, wherein the reference protein has the amino acid sequence set forth in SEQ ID NO: 1.

[115] The method, peptide, fusion protein, nucleic acid, or vector according to any of the above, wherein the cell is a eukaryotic cell.

[116] The method, peptide, fusion protein, nucleic acid, or vector according to any of the above, wherein the cell is a human cell.

[117] Any of the above methods, peptides, fusion proteins, nucleic acids, or vectors, wherein the peptide tag does not interfere with the free localization of the target protein.

[0024] The peptide tags of the present disclosure can cause intracellular stabilization of the tagged protein, and therefore can be more biocompatible.

[0025] FIG. 1 shows the effect of the peptide tag Tag4-1 on the intracellular aggregation of single-chain Fv (scFv). FIG. 2A shows a schematic diagram of the intracellular accumulation model of α-synuclein, an amyloid. FIG. 2B shows fluorescence microscopy images showing the effect on intracellular synuclein fibrils of intracellular expression of an scFv-E6-CMA peptide fusion protein having Tag18-1, one of the peptide tags of the present disclosure. FIG. 2C shows the effect on synuclein fibril removal of intracellular expression of an scFv-E6-CMA peptide fusion protein having Tag4-8 or Tag18-1, one of the peptide tags of the present disclosure. FIG. 3A shows fluorescence microscopy images of scFv-C2 itself having Tag18-1, one of the peptide tags of the present disclosure, expressed intracellularly, showing its intracellular localization. FIG. 3B shows the stabilizing effect of scFv-C2 having Tag18-1, one of the peptide tags of the present disclosure. Specific Description of the Invention

[0026] In the present invention, a "subject" is a vertebrate, for example, a bird or a mammal, for example, a mammal, such as a mouse, rat, hamster, guinea pig, horse, cow, pig, goat, sheep, donkey, dog, and cat, as well as a primate, such as a monkey, chimpanzee, gorilla, orangutan, bonobo, or human, particularly a human. As used herein, the term "subject" includes humans, as described above, and the term "non-human" is used to exclude humans.

[0027] As used herein, the term "antibody" refers to an immunoglobulin, a protein consisting of two heavy chains (H chains) and two light chains (L chains) stabilized by disulfide bonds. The heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, and CH3, and a hinge region located between CH1 and CH2. The light chain consists of a light chain variable region VL (VL can be Vκ or Vλ) and a light chain constant region CL. Among these, the variable region fragment (Fv) consisting of VH and VL is directly involved in antigen binding and provides antibody diversity. The antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region. The regions of the variable regions that directly contact the antigen are particularly variable and are called complementarity-determining regions (CDRs). Portions other than the CDRs that show relatively little variation are called framework regions (FRs). The heavy and light chain variable regions each have three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus. Each CDR is incorporated into a framework region. The heavy chain variable region of an antibody has, in this order from the N-terminus to the C-terminus, heavy chain framework region 1, heavy chain CDR1, heavy chain framework region 2, heavy chain CDR2, heavy chain framework region 3, heavy chain CDR3, and heavy chain framework region 4. The light chain variable region of an antibody has, in this order from the N-terminus to the C-terminus, light chain framework region 1, light chain CDR1, light chain framework region 2, light chain CDR2, light chain framework region 3, light chain CDR3, and light chain framework region 4. The antibody may be a recombinant protein (recombinant antibody) and may be produced in animal cells such as Chinese hamster ovary cells (CHO cells). The origin of the antibody is not particularly limited, but examples include antibodies from non-human animals, non-human mammals (e.g., mouse antibodies, rat antibodies, camel antibodies), and human antibodies. The antibody may also be a chimeric antibody, a humanized antibody, or a fully humanized antibody. The antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.A "chimeric antibody" is an antibody in which the heavy chain variable region and light chain constant region of a different species are linked to the heavy chain variable region and light chain constant region, respectively. A humanized antibody refers to an antibody in which the corresponding positions in a human antibody are replaced with amino acid sequences characteristic of antibodies of non-human origin. For example, an antibody has heavy chain CDRs 1-3 and light chain CDRs 1-3 of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FRs) of the heavy and light chains, derived from a human antibody. Such antibodies are sometimes called CDR-grafted antibodies. A "humanized antibody" may also include human chimeric antibodies. A "human chimeric antibody" is an antibody in which the constant regions of a non-human antibody are replaced with human antibody constant regions. The antibody may be an isolated antibody or a purified antibody. The antibody may be, for example, an IgG.

[0028] The variable regions of immunoglobulin chains generally share the same overall structure, including relatively conserved framework regions (FRs) linked by three hypervariable regions (more often referred to as "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair are typically juxtaposed by the framework regions to form a structure that specifically binds to a specific epitope on a target protein (e.g., PCSK9). From the N-terminus to the C-terminus, naturally occurring light and heavy chain variable regions typically follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0029] As used herein, the term "antibody" includes antigen-binding fragments of antibodies. As used herein, non-fragmented antibodies may be referred to as "full antibodies." A full-length antibody may include the entire antibody excluding the signal sequence.

[0030] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody that retains its ability to bind to an antigen. The antigen-binding fragment may include the heavy chain variable region or the light chain variable region, or both, of the antibody of the present disclosure. The antigen-binding fragment may be chimerized or humanized. Examples of antigen-binding fragments include Fab, Fab', F(ab') and the like. ２ Antibody-binding fragments also include recombinantly produced conjugates or functional equivalents (e.g., scFv (single-chain Fv), diabodies, scDb, tandem scFv, leucine zipper type, sc(Fv) ２ (Single chain (Fv) ２ The antigen-binding fragment of such an antibody may include, but is not limited to, an antibody that is obtained by treating the antibody with an enzyme. For example, Fab can be obtained by digesting an antibody with papain. Alternatively, F(ab') can be obtained by digesting an antibody with pepsin. ２ can be obtained, which can be further reduced to obtain Fab'. Antigen-binding fragments of such antibodies can be used herein. In scFv, VL and VH are linked by an artificial polypeptide linker, allowing the same antigen specificity as the original antibody to be maintained. VL and VH can be linked in the order of VH and VL or VL and VH from the N-terminus. The linker can have a length of approximately 10 to 25 amino acids. The linker may be rich in glycine, or may contain amino acids such as serine and threonine to increase water solubility.

[0031] As used herein, an "intrabody" refers to an antibody expressed intracellularly (e.g., in the cytoplasm or nucleus). Intrabodies differ in that they are designed to be expressed and function intracellularly, whereas antibodies are secreted extracellularly and function. Intrabodies can affect the function of intracellular proteins and inhibit their function in the cytoplasm, nucleus, or secretory pathway. Oncogene products can be targets of intracellular antibodies (Biocca, S., Pierandrei-Amaldi, P., and Cattaneo, A. (1993) Biochem Biophys Res Commun 197:422-7; Biocca, S., Pierandrei-Amaldi, P., Campioni, N., and Cattaneo, A. (1994) Biotechnology (NY) 12:396-9; Cochet, O. et al. (1998) Cancer Res 58:1170-6). Intracellular antibodies bind directly to proteins with the aim of inhibiting their function. Binding may directly inhibit the function of the protein, or it may inhibit the protein from binding to other proteins.

[0032] Intracellular antibodies include various antibodies or antigen-binding fragments thereof, but are not particularly limited to scFvs, tandem scFvs, VHH antibodies (nanobodies), minibodies, and diabodies. An scFv is typically an antibody fragment having an antibody heavy chain variable region and a light chain variable region, which are linked via a linker. A tandem scFv is typically an antibody fragment having scFvs with two different antigen specificities, which are linked via a linker. A diabody is typically a dimer of scFvs. Diabodies are broadly classified into bivalent monospecific diabodies and bispecific diabodies. A minibody is typically formed by dimerizing two fusion proteins of a dimerization domain and an scFv via the dimerization domain. A VHH antibody is an antibody fragment consisting of the heavy chain variable domain of a heavy chain antibody. VHH antibodies are typically heavy chain variable domains of heavy chain antibodies derived from camelids (e.g., camels, llamas, alpacas, etc.). While conventional antibodies are expressed extracellularly and can only function extracellularly, intracellular antibodies are advantageous in that they can exert their antibody functions intracellularly. Intracellular antibodies can be used for various purposes, such as activating and inactivating target proteins within cells, and neutralizing or blocking protein-protein interactions. When expressed intracellularly, scFvs tend to aggregate. Therefore, in such cases, it is useful to link the peptide tag of the present disclosure to an intracellular antibody to obtain a fusion protein and reduce its aggregation. Reducing the aggregation tendency of a protein allows the protein to exert its intended function within the cell.

[0033] As used herein, a "peptide tag" refers to a peptide tag that is fused to a target protein to label the target protein or to alter the biochemical properties of the target protein. Examples of peptide tags include FLAG tags, 3xFLAG tags, Myc tags, HA tags, T7, 6xHis tags, PA tags, S tags, E tags, VSV-G, Glu-Glu, Strep-tag II, HSV tags, Chitin Binding Domain (CBD) tags, Calmodulin Binding Peptide (CBP) tags, V5 tags, GST tags, maltose-binding protein (MBP) tags, thioredoxin (Trx) tags, and mini-AID tags. Each of these tags can be used to affinity purify a target protein by utilizing its affinity for the tag, or to detect the target protein by producing an antibody against the tag. An antibody that recognizes a tag is generally called a tag antibody, and the tag sequence that serves as the epitope of the tag antibody is called an epitope tag. A tag can generally have a polypeptide chain ranging from a few amino acids to several tens of amino acids in length.

[0034] As used herein, a "target protein" refers to a protein expressed in a cell. The target protein may be an aggregating protein or a non-aggregating protein. In either case, the addition of the peptide tag of the present disclosure increases stability and makes the protein more robust against aggregation. However, the target protein may preferably be an aggregating protein, because the peptide tag of the present disclosure can reduce the aggregation tendency of aggregating proteins even when added to the aggregating protein. Even when the target protein is a secreted protein, the secreted protein may form aggregates within the cell before being secreted outside the cell. The peptide tag of the present disclosure can also be advantageously used for secreted proteins, preferably aggregating secreted proteins.

[0035] As used herein, "aggregating protein" refers to a protein that forms aggregates (especially insoluble aggregates) within cells. As used herein, "aggregating" refers to the property of forming aggregates, and "non-aggregating" refers to the property of not forming aggregates. Attenuation of aggregation can be promotion of non-aggregation, and promotion of aggregation can be attenuation of non-aggregation. As used herein, "non-aggregating" is used interchangeably with "stability." Aggregation can be observed, for example, as bright spots under a microscope using immunocytochemistry (IC). The aggregation rate can be calculated, for example, by the proportion of cells exhibiting aggregation among cells forced to express a protein. A decrease in the aggregation rate calculated in this way indicates an increase in protein-forced expression cells that are not affected by aggregation and can be an indicator of physiological suitability. A decrease in aggregation rate (e.g., a decrease in aggregation rate) is a different indicator from increased solubility. Increased solubility means an increase in the protein concentration available in aqueous solution, but does not directly correlate with the number of aggregates or the proportion of cells with aggregates. Therefore, increased solubility does not necessarily mean a decrease in aggregation rate (e.g., a decrease in aggregation rate).

[0036] As used herein, amino acid sequences are represented by single-letter abbreviations: A represents alanine, R represents arginine, N represents asparagine, D represents aspartic acid, C represents cysteine, Q represents glutamine, E represents glutamic acid, G represents glycine, H represents histidine, I represents isoleucine, L represents leucine, K represents lysine, M represents methionine, F represents phenylalanine, P represents proline, S represents serine, T represents threonine, W represents tryptophan, Y represents tyrosine, and V represents valine. The amino acids are typically the 20 L-amino acids listed above.

[0037] As used herein, the term "regulatory sequence" refers to a sequence that drives a gene operably linked thereto and has the activity of transcribing RNA from the gene. Examples of regulatory sequences include promoters. Examples of promoters include class I promoters (which can be used to transcribe rRNA precursors), class II promoters (which are composed of a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (which are further broadly divided into types I, II, and III).

[0038] The present invention provides a peptide tag that reduces the aggregation tendency of an aggregating protein. The present invention provides a protein expression vector comprising a gene operably linked to a regulatory sequence and encoding the peptide tag. The present invention provides a protein of interest fused with the peptide tag. The present invention provides a protein expression vector comprising a gene operably linked to a regulatory sequence and encoding the protein of interest fused with the peptide tag. In one aspect, the protein of interest may be an intracellular protein. In one aspect, the protein of interest may be an intracellular antibody. In one aspect, the protein of interest may be an scFv.

[0039] The peptide tag of the present disclosure that reduces the aggregation tendency of an aggregating protein is described in detail below. The peptide tag of the present disclosure can reduce the aggregation tendency of a target protein in eukaryotic cells, particularly human cells. When considering pharmaceutical applications, etc., it can be beneficial to reduce the aggregation tendency of a target protein in human cells.

[0040] The length of the peptide tag of the present disclosure is not particularly limited, and may be, for example, 600 amino acids or less, 500 amino acids or less, 400 amino acids or less, 300 amino acids or less, or 200 amino acids or less, for example, 5 to 100 amino acids, for example, 10 to 90 amino acids, 20 to 80 amino acids, 30 to 70 amino acids, 40 to 60 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, or 10 to 30 amino acids. In this aspect, the lower limit of the length of the peptide tag of the present disclosure may be 5 amino acids or more, 10 amino acids or more, 15 amino acids or more, 20 amino acids or more, 30 amino acids or more, 40 amino acids or more, 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, or 80 amino acids or more, and / or the upper limit may be 100 amino acids or less, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, or 20 amino acids or less.

[0041] (a) The peptide tag of the present disclosure may contain acidic amino acids (amino acids of element 1) in the following proportions: 45% or more of the amino acids constituting the amino acid sequence of the peptide tag of the present disclosure may be acidic amino acids. Less than 45% of the amino acids constituting the amino acid sequence of the peptide tag of the present disclosure may be acidic amino acids. In a preferred embodiment, 5% to less than 45% of the amino acids constituting the amino acid sequence of the peptide tag of the present disclosure may be acidic amino acids, more preferably 10% to less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, even more preferably 20% to less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, even more preferably 30% to less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, even more preferably 35% to less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, and particularly preferably 40% to less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids. The acidic amino acids are D or E. For example, the acidic amino acid content in the peptide tag of the present disclosure can be 44% or less, 43.5% or less, 43% or less, 42.5% or less, 42% or less, 41.5% or less, 41% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less, which, in one aspect, can reduce the risk of unexpected interactions with positively charged intracellular molecules or the like due to the high acidic amino acid ratio of the peptide tag.

[0042] (b) The peptide tag of the present disclosure may contain basic amino acids (amino acids of element 2) at the following percentages: The peptide tag of the present disclosure preferably has 25% or less, 20% or less, more preferably 15% or less, even more preferably 10% or less, still more preferably 5% or less, and particularly preferably less than 3%, less than 2%, or less than 1% of the amino acids constituting its amino acid sequence. In the most preferred embodiment, the peptide tag of the present disclosure does not contain any basic amino acids in its amino acid sequence. Basic amino acids are K, R, or H.

[0043] (c) The peptide tag of the present disclosure may contain the following proportions of amino acids in element 3. The amino acids in element 3 may be F, P, Y, G, S, Q, N, and A. The peptide tag of the present disclosure may preferably have 10% or more, preferably 20% or more, more preferably 30% or more, or 40% or more of the amino acids constituting its amino acid sequence as amino acids in element 3. The peptide tag of the present disclosure may also have 50% or more, 60% or more, or 70% or more of the amino acids constituting its amino acid sequence as amino acids in element 3. The peptide tag of the present disclosure may preferably have 80% or less, more preferably 70% or less, and even more preferably 60% or less of the amino acids constituting its amino acid sequence as amino acids in element 3. The peptide tag of the present disclosure may also have 50% or less, 40% or less, 30% or less, or 20% or less of the amino acids constituting its amino acid sequence as amino acids in element 3. In a preferred embodiment, the peptide tag of the present disclosure may have 20% to 80% of the amino acids constituting its amino acid sequence, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 70%, 50% to 60%, 60% to 80%, or 60% to 70% of the amino acids of element 3.

[0044] In particularly preferred embodiments, in a peptide tag of the present disclosure, 5% or more, 10% or more, 15% or more, or 20% or more (preferably 21% or more, 25% or more, or 30% or more) of the amino acids constituting the amino acid sequence are either N or P. Also, for example, in a peptide tag of the present disclosure, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less of the amino acids constituting the amino acid sequence may be either N or P. In some embodiments, in a peptide tag of the present disclosure, 10% or more and 20% or less of the amino acids constituting the amino acid sequence may be either N or P. In some embodiments, in a peptide tag of the present disclosure, 55% or more and 90% or less of the amino acids constituting the amino acid sequence may be either N or P. In some embodiments, in a peptide tag of the present disclosure, more than 10% and 20% or less of the amino acids constituting the amino acid sequence may be either N or P. In certain aspects, a peptide tag of the present disclosure may have more than 20% but not more than 30% of the amino acids constituting its amino acid sequence being either N or P. In certain aspects, a peptide tag of the present disclosure may have more than 30% but not more than 40% of the amino acids constituting its amino acid sequence being either N or P. In certain aspects, a peptide tag of the present disclosure may have more than 40% but not more than 50% of the amino acids constituting its amino acid sequence being either N or P. In certain aspects, a peptide tag of the present disclosure may have more than 50% but not more than 60% of the amino acids constituting its amino acid sequence being either N or P.

[0045] For example, in a peptide tag of the present disclosure, 5% or more, 10% or more, 15% or more, or 20% or more (preferably 21% or more, 25% or more, or 30% or more) of the amino acids constituting the amino acid sequence are N. Also, for example, in a peptide tag of the present disclosure, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less of the amino acids constituting the amino acid sequence may be N. In some aspects, in a peptide tag of the present disclosure, 10% or more but 20% or less of the amino acids constituting the amino acid sequence may be N. In some aspects, in a peptide tag of the present disclosure, 55% or more but 90% or less of the amino acids constituting the amino acid sequence may be N. In some aspects, in a peptide tag of the present disclosure, more than 10% but not more than 20% of the amino acids constituting the amino acid sequence may be N. In some aspects, in a peptide tag of the present disclosure, more than 20% but not more than 30% of the amino acids constituting the amino acid sequence may be N. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 30% and not more than 40% N. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 40% and not more than 50% N. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 50% and not more than 60% N.

[0046] For example, in a peptide tag of the present disclosure, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more (preferably 21% or more, 25% or more, or 30% or more) of the amino acids constituting the amino acid sequence are P. Also, for example, in a peptide tag of the present disclosure, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less of the amino acids constituting the amino acid sequence may be P. In some aspects, in a peptide tag of the present disclosure, 55% or more but 90% or less of the amino acids constituting the amino acid sequence may be P. In some aspects, in a peptide tag of the present disclosure, 10% or more but 20% or less of the amino acids constituting the amino acid sequence may be P. In some aspects, in a peptide tag of the present disclosure, more than 10% but 20% or less of the amino acids constituting the amino acid sequence may be P. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 20% and not more than 30% P. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 30% and not more than 40% P. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 40% and not more than 50% P. In some aspects, the amino acid sequence of a peptide tag of the present disclosure may be comprised of more than 50% and not more than 60% P.

[0047] In a particularly preferred embodiment, 5% or less, 10% or less, 15% or less, or 20% or less of the amino acids constituting the amino acid sequence of the peptide tag of the present disclosure are F or Y. In a particularly preferred embodiment, 5% or less, 10% or less, 15% or less, or 20% or less of the amino acids constituting the amino acid sequence of the peptide tag of the present disclosure are F and / or Y. In a particularly preferred embodiment, 5% or less, 10% or less, 15% or more, or 20% or more of the amino acids constituting the amino acid sequence are either N or P, and 5% or less, 10% or less, 15% or less, or 20% or less are F and / or Y.

[0048] (d) The peptide tag of the present disclosure may contain the following proportions of amino acids in element 4. The amino acids in element 4 may be amino acids that are neither acidic amino acids, basic amino acids, nor amino acids in element 3. The amino acids in element 4 may be, for example, M, T, W, C, I, V, and L. The peptide tag of the present disclosure may preferably have 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting its amino acid sequence as amino acids in element 4. In a preferred aspect, the peptide tag of the present disclosure does not contain amino acids in element 4.

[0049] (e) The peptide tag of the present disclosure may preferably have 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting its amino acid sequence being G. In one aspect, the peptide tag of the present disclosure does not contain G.

[0050] (f) Preferably, the peptide tag of the present disclosure may have 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting its amino acid sequence be A. In one aspect, the peptide tag of the present disclosure does not contain A.

[0051] (g) Preferably, the peptide tag of the present disclosure has an amino acid sequence in which 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting the amino acid sequence are G, and 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting the amino acid sequence may be A.

[0052] (h) The peptide tag of the present disclosure may preferably contain S. The peptide tag of the present disclosure preferably does not contain S. The peptide tag of the present disclosure may preferably contain 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more of the amino acids constituting its amino acid sequence as S. The peptide tag of the present disclosure may preferably contain 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting its amino acid sequence as S.

[0053] (A) In the peptide tag of the present disclosure, less than 45% of the amino acids constituting its amino acid sequence may be acidic amino acids, and 20% to 80% (preferably 30% to 70%) of the amino acids constituting its amino acid sequence may be amino acids of element 3.

[0054] (B) The peptide tag of the present disclosure may have less than 45% of the amino acids constituting its amino acid sequence being acidic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids constituting its amino acid sequence being amino acids of element 3, and 10% or less of the amino acids constituting its amino acid sequence being A.

[0055] (C) The peptide tag of the present disclosure may have less than 45% of the amino acids constituting its amino acid sequence being acidic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids constituting its amino acid sequence being amino acids of element 3, and 10% or less of the amino acids constituting its amino acid sequence being G.

[0056] (D) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, in which 10% or less of the amino acids are A, and in which 10% or less of the amino acids are G.

[0057] (E) The peptide tag of the present disclosure may have less than 45% of the amino acids constituting its amino acid sequence being acidic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids constituting its amino acid sequence being amino acids of element 3, and 30% or less of the amino acids constituting its amino acid sequence being amino acids of element 4.

[0058] (F) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, and in which 10% or less of the amino acids are A.

[0059] (G) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, and in which 10% or less of the amino acids are G.

[0060] (H) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, in which 10% or less of the amino acids are A, and in which 10% or less of the amino acids are G.

[0061] (I) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, and 30% to 70% of the amino acids in the amino acid sequence may be amino acids of element 3.

[0062] (J) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, and in which 10% or less of the amino acids are A.

[0063] (K) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, and in which 10% or less of the amino acids are G.

[0064] (L) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are element 3 amino acids, in which 10% or less of the amino acids are A, and in which 10% or less of the amino acids are G.

[0065] (M) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, and in which 30% or less of the amino acids are amino acids of element 4.

[0066] (N) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, and in which 10% or less of the amino acids are A.

[0067] (O) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, and in which 10% or less of the amino acids are G.

[0068] (P) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, in which 10% or less of the amino acids are A, and in which 10% or less of the amino acids are G.

[0069] (Q) In the above-mentioned peptide tag, preferably, 20% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, more preferably, 30% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, even more preferably, 35% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and particularly preferably, 40% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids.

[0070] (R) In the above-mentioned peptide tag, preferably, 10% or less of the amino acids constituting the amino acid sequence are basic amino acids. More preferably, the peptide tag has 5% or less basic amino acids, and particularly preferably, less than 3%, less than 2%, or less than 1% basic amino acids. Alternatively, in a preferred embodiment, the peptide tag does not contain any basic amino acids.

[0071] (S) In the above peptide tag, preferably, 40% to 60% of the amino acids constituting the amino acid sequence are amino acids of element 3.

[0072] (T) In the above-mentioned peptide tag, preferably, 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids constituting the amino acid sequence may be amino acids of element 4. In a preferred embodiment, the above-mentioned peptide tag does not contain amino acids of element 4.

[0073] (U) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, 10% or less of the amino acids are basic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids are element 3 amino acids, 30% or less of the amino acids are element 4 amino acids, 10% or less of the amino acids are G, and 10% or less of the amino acids are A. In this embodiment, preferably, 5% or more but less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, more preferably, 10% or more but less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, even more preferably, 20% or more but less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, even more preferably, 3 ...5% or more but less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids, and particularly preferably, 40% or more but less than 45% of the amino acids constituting the amino acid sequence may be acidic amino acids.

[0074] (V) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, 5% or less of the amino acids are basic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, 30% or less of the amino acids are amino acids of element 4, 10% or less of the amino acids are G, and 10% or less of the amino acids are A. In this aspect, preferably, the peptide tag of the present disclosure may have an amino acid sequence in which less than 3%, less than 2%, or less than 1% of the amino acids are basic amino acids, or may contain no basic amino acids.

[0075] (W) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, in which 10% or less of the amino acids are basic amino acids, in which 30% to 70% of the amino acids are amino acids of element 3, in which 30% or less of the amino acids are amino acids of element 4, in which 10% or less of the amino acids are G, and in which 10% or less of the amino acids are A. In this aspect, the peptide tag of the present disclosure may have an amino acid sequence in which 40% to 60% of the amino acids are amino acids of element 3.

[0076] (X) The peptide tag of the present disclosure may have an amino acid sequence in which less than 45% of the amino acids are acidic amino acids, 10% or less of the amino acids are basic amino acids, 20% to 80% (preferably 30% to 70%) of the amino acids are amino acids of element 3, 30% or less of the amino acids are amino acids of element 4, 10% or less of the amino acids are G, and 10% or less of the amino acids are A. In this embodiment, the peptide tag of the present disclosure may have an amino acid sequence in which 20% or less, 15% or less, 10% or less, or 5% or less of the amino acids are amino acids of element 4. In a preferred embodiment, the peptide tag of the present disclosure does not include amino acids of element 4.

[0077] (Y) The peptide tag of the present disclosure may be such that 20% to less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids, 10% or less of the amino acids constituting its amino acid sequence are basic amino acids, 20% to less than 80% (preferably 30% to less than 70%) of the amino acids constituting its amino acid sequence are amino acids of element 3, 30% or less of the amino acids constituting its amino acid sequence are amino acids of element 4, 10% or less of the amino acids constituting its amino acid sequence are G, and 10% or less of the amino acids constituting its amino acid sequence are A. In this aspect, preferably 30% to less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids, more preferably 35% to less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids, and even more preferably 40% to less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids.

[0078] (Z) The peptide tag of the present disclosure may have an amino acid sequence in which 20% to less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, 10% or less of the amino acids constituting the amino acid sequence are basic amino acids, 30% to 70% of the amino acids constituting the amino acid sequence are amino acids of element 3, 30% or less of the amino acids constituting the amino acid sequence are amino acids of element 4, 10% or less of the amino acids constituting the amino acid sequence are G, and 10% or less of the amino acids constituting the amino acid sequence are A. In this embodiment, preferably 40% to 60% of the amino acids constituting the amino acid sequence are amino acids of element 3.

[0079] (AA) The peptide tag of the present disclosure may have an amino acid sequence in which 20% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, 10% or less of the amino acids constituting the amino acid sequence are basic amino acids, 20% or more but less than 80% (preferably 30% or more but less than 70%) of the amino acids constituting the amino acid sequence are amino acids of element 3, 20% or less of the amino acids constituting the amino acid sequence are amino acids of element 4, 10% or less of the amino acids constituting the amino acid sequence are G, and 10% or less of the amino acids constituting the amino acid sequence are A. In this embodiment, preferably, 15% or less of the amino acids constituting the amino acid sequence are amino acids of element 4, 10% or less of the amino acids constituting the amino acid sequence are amino acids of element 4, or 5% or less of the amino acids constituting the amino acid sequence are amino acids of element 4.

[0080] (AB) The peptide tag of the present disclosure may have 30% or more and less than 45% of the amino acids constituting its amino acid sequence being acidic amino acids, 10% or less of the amino acids constituting its amino acid sequence being basic amino acids, 30% or more and less than 70% of the amino acids constituting its amino acid sequence being amino acids of element 3, 30% or less of the amino acids constituting its amino acid sequence being amino acids of element 4, 10% or less of the amino acids constituting its amino acid sequence being G, and 10% or less of the amino acids constituting its amino acid sequence being A. In this embodiment, preferably, 40% or more and less than 60% of the amino acids constituting its amino acid sequence are amino acids of element 3. In this embodiment, more preferably, 35% or more and less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids, and even more preferably, 40% or more and less than 45% of the amino acids constituting its amino acid sequence are acidic amino acids. In this embodiment, preferably, 40% to 60% of the amino acids constituting the amino acid sequence are amino acids of element 3, and 35% to 45% of the amino acids constituting the amino acid sequence are acidic amino acids; more preferably, 40% to 60% of the amino acids constituting the amino acid sequence are amino acids of element 3, and 40% to 45% of the amino acids constituting the amino acid sequence are acidic amino acids.

[0081] (AC) The peptide tag of the present disclosure may have an amino acid sequence in which 30% to less than 45% of the amino acids are acidic amino acids, 10% or less of the amino acids are basic amino acids, 30% to 70% of the amino acids are element 3 amino acids, 20% or less of the amino acids are element 4 amino acids, 10% or less of the amino acids are G, and 10% or less of the amino acids are A. In this embodiment, preferably, 15% or less, 10% or less, or 5% or less of the amino acids are G. In one embodiment, the peptide tag of the present disclosure does not contain G.

[0082] (AD) A peptide tag of the present disclosure may have an amino acid sequence in which 35% to less than 45% of the amino acids are acidic amino acids, 10% or less of the amino acids are basic amino acids, 30% to 70% of the amino acids are element 3 amino acids, 10% or less of the amino acids are element 4 amino acids, 10% or less of the amino acids are G, and 10% or less of the amino acids are A.

[0083] (AE) The peptide tag of the present disclosure may have, for example, the amino acid sequence set forth in any of SEQ ID NOs: 2 to 11. The peptide tag of the present disclosure may preferably have the amino acid sequence of SEQ ID NO: 5.

[0084] (AF) Peptide tags of the present disclosure can have, for example, any of the amino acid sequences set forth in Tables 1-11, Table 12-1, Table 12-2, Table 13-1, Table 13-2, Table 14-1, and Table 14-2.

[0085] (AG) The peptide tag of the present disclosure may have one or more amino acid additions and insertions selected from the group consisting of N and P of any amino acid (e.g., amino acids of element 1, element 2, element 3, or element 4, e.g., amino acids of element 2, element 3, or element 4) of the amino acid sequence of any of (AE) and (AF), and substitutions of the above amino acids (e.g., substitutions of about 1 to 30% of amino acids, e.g., substitutions of 1 to 20, 10, or several amino acids). The peptide tag of the present disclosure may have one or more amino acid additions and insertions selected from the group consisting of S of any amino acid (e.g., amino acids of element 1, element 2, element 3, or element 4, e.g., amino acids of element 2, element 3, or element 4) of the amino acid sequence of any of (AF) and (AG), and substitutions of the above amino acids (e.g., substitutions of about 1 to 30% of amino acids, e.g., substitutions of 1 to 20, 10, or several amino acids). The peptide tag of the present disclosure may have a deletion of any amino acid in any of the amino acid sequences (AF) and (AG) above (e.g., an amino acid in element 1, element 2, element 3, or element 4, e.g., an amino acid in element 2, element 3 (particularly F and / or Y, an amino acid in element 4, A, or G).

[0086] (AH) The peptide tag of the present disclosure may preferably have 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more of the amino acids constituting its amino acid sequence be either N or P, or N and P. The peptide tag of the present disclosure may preferably have 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 25% or more, or 30% or more of the amino acids constituting its amino acid sequence be P. The peptide tag of the present disclosure may preferably have 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 25% or more, or 30% or more of the amino acids constituting its amino acid sequence being N.

[0087] (AI) In the peptide tag of the present disclosure, 45% or more of the amino acids constituting the amino acid sequence may be acidic amino acids, and 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 25% or more, or 30% or more may be N or P. In this aspect, less than 10% (preferably less than 5%, more preferably 0%) of the amino acids constituting the amino acid sequence may be G, less than 10% (preferably less than 5%, more preferably 0%) may be A, and / or less than 10% (preferably less than 5%, more preferably 0%) may be F and Y.

[0088] (AJ) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0089] (AK) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, and 10% or more of the amino acids constituting the amino acid sequence are N or P, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0090] (AL) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, and 10% or less of the amino acids constituting the amino acid sequence are F and / or Y, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0091] (AM) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, 10% or more of the amino acids constituting the amino acid sequence are N or P, and 10% or less of the amino acids constituting the amino acid sequence are F and / or Y, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0092] (AN) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 65% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, and 10% or less of the amino acids constituting the amino acid sequence are F and / or Y, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0093] (AN) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 65% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, and 10% or more of the amino acids constituting the amino acid sequence are N or P, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0094] (AO) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b) 30% or more but less than 65% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S, 10% or more of the amino acids constituting the amino acid sequence are N or P, and 10% or less of the amino acids constituting the amino acid sequence are F and / or Y, (c) 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L, and (d) A and G may each account for less than 10% of the amino acids constituting the amino acid sequence. In this embodiment, the amino acid sequence may have a length of 10 to 200 amino acids (e.g., 10 to 90 amino acids).

[0095] (AP) In the peptide tag of the present disclosure, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, (b1) 55% or more but less than 90% of the amino acids constituting the amino acid sequence are either N or P, and the remainder of the amino acids constituting the amino acid sequence may be other amino acids. In this embodiment, 20% or less (preferably 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%) of the amino acids constituting the amino acid sequence are neither acidic amino acids, N nor P.

[0096] (AQ) In the peptide tag of the present disclosure, (a) 45% or more of the amino acids constituting the amino acid sequence are acidic amino acids, (b1) 21% or more of the amino acids constituting the amino acid sequence are N and / or 7% or more of the amino acids constituting the amino acid sequence are P, and 20% or less of the amino acids constituting the amino acid sequence (preferably 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%) may be other amino acids.

[0097] (AR) In the above (AK) to (AQ), element 4 may preferably be 0%.

[0098] (AS) In the above (AK) to (AQ), A or G may be 0%. In the above (AK) to (AO), A and G are preferably 0%.

[0099] (AT) In the above (AK) to (AQ), preferably, element 4 is 0%, and A and G are 0%.

[0100] (AU) In the above (AR) to (AT), preferably, element 2 is 0%.

[0101] In the above (a), preferably, 10% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, more preferably, 15% or more but less than 45% are acidic amino acids, even more preferably, 20% or more but less than 45% are acidic amino acids, still more preferably, 25% or more but less than 45% are acidic amino acids, and particularly preferably, 30% or more but less than 45% are acidic amino acids. In these examples, the upper limit of the acidic amino acid content may be, for example, less than 45%, 40% or less, or 35% or less.

[0102] The peptide tag of the present disclosure can be attached to a protein (e.g., an intracellularly aggregating protein). Thus, the present disclosure provides a fusion protein of the peptide tag of the present disclosure and an intracellularly aggregating protein. The peptide tag of the present disclosure may also be attached to an intracellularly non-aggregating protein. Attaching the tag can increase the robustness of the non-aggregating protein with respect to non-aggregation. The protein may be, for example, an intracellular antibody. The intracellular antibody may be an antigen-binding fragment of an antibody. The peptide tag of the present disclosure may be attached to a fusion protein of an intracellular antibody and a degradation-inducing sequence. This can induce selective degradation of the target to which the intracellular antibody binds. Examples of the intracellular antibody include antibody fragments such as those described above. Intracellular antibodies also include antibodies that bind to α-synuclein, LRRK2, tau (Tau), β-amyloid, amyloid precursor protein (APP), C9orf72, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), fused in sarcoma (FUS), and prion proteins, as well as pathological forms thereof. Intracellular antibodies include antibodies that inhibit protein-protein interactions (PPI). Intracellular antibodies include those in the form of fusion proteins with degradation-inducing sequences that bind to targets. Intracellular antibodies include, for example, the intracellular antibodies described in Molecular Therapy, 29(2):859-872, 2021 (e.g., CP13 iB, PHF1 iB, and Tau5 iB) and intracellular antibodies having all of the CDR sequences of these intracellular antibodies. iB is an abbreviation for intrabody, i.e., an intracellular antibody. The present disclosure provides, for example, fusion proteins of these intracellular antibodies with the peptide tags of the present disclosure. The intracellular antibody may preferably further have a degradation-inducing sequence. Examples of the intracellular antibody include the intracellular antibodies described in Molecular Therapy, 30(4):1484-1499, 2022 (e.g., VHH E4-1 and VHHZ70) and intracellular antibodies having all of the CDR sequences of these intracellular antibodies. The intracellular antibody may preferably further have a degradation-inducing sequence.Examples of intracellular antibodies include the intracellular antibodies (e.g., M204-scFv) described in J. Biol. Chem., 295(31):10662-10676, 2020, and intracellular antibodies having all of the CDR sequences of this intracellular antibody. Preferably, the intracellular antibody may further have a degradation-inducing sequence. Examples of intracellular antibodies include the intracellular antibodies described in WO2018 / 231254 (e.g., BIIB092 antibody), the intracellular antibodies described in WO2016 / 207245, the intracellular antibodies described in WO2018 / 011073 (e.g., C10-2), and the intracellular antibodies described in WO2015 / 114538 (e.g., VHH tau A2, VHH tau A2-SH, VHH tau A2var-SH), intrabodies described in WO2014 / 059442 (e.g., F9T, D11C, D4G, G12C, H2A, and H7T), and JP2020 / 515233 (e.g., 1E4, 9B11, 3A9, 10F10, 11F11, AC8, AE8, AA9, DG5, AD2, AD7, DG11, DG8, DA9), and intrabodies having all of the CDR sequences of these intrabodies. Preferably, the intrabodies may further have a degradation-inducing sequence. Intrabodies also include intrabodies that can degrade and remove abnormal TDP-43 described in WO2019177138 (e.g., SEQ ID NOs: 21 to 24), and intrabodies having all of the CDR sequences of these intrabodies. Preferably, the intrabodies may further have a degradation-inducing sequence. Thus, intracellular antibodies (e.g., scFv or VHH) that bind to tau, intracellular antibodies (e.g., scFv or VHH) that bind to α-synuclein, and intracellular antibodies (e.g., scFv or VHH) against other amyloids that cause cytotoxicity within cells are preferred, and can be linked to a tag to form a fusion protein with the tag.

[0103] In some aspects, the peptide tag of the present disclosure does not have a CAAX motif (e.g., SEQ ID NO: 58: KLNPPDESGPGCMSCKCVLS). In some aspects, the peptide of the present disclosure does not have a membrane localization signal. In some aspects, the peptide tag of the present disclosure does not have a signal peptide sequence for extracellular secretion, with the aim of expressing a protein of interest intracellularly. In some aspects, the peptide tag of the present disclosure may have a signal sequence for extracellular secretion, with the aim of promoting extracellular secretion of a protein of interest. When a secreted protein is aggregating, a peptide tag of the present disclosure that includes a signal sequence in its sequence, or a peptide tag of the present disclosure that is linked in tandem to a signal sequence, may be advantageous. In some aspects, the peptide tag of the present disclosure may include a nuclear localization signal. In some aspects, the peptide tag of the present disclosure does not have a sequence that prevents the protein from being localized in the cytoplasm. In some aspects, the peptide tag of the present disclosure promotes the free distribution of a protein of interest within a cell. In some embodiments, the peptide tags of the present disclosure can promote binding of a target protein to its natural binding partner and co-localization with the binding partner within a cell. In some embodiments, the peptide tags of the present disclosure can have a sequence that imposes inherent constraints on the distribution of the target protein within a cell (or a sequence that prevents free distribution), but preferably do not have such a sequence.

[0104] In any embodiment, the peptide tag of the present disclosure does not include the following sequence (Enzymol. 326, 362-367 (2000)): S-tag: KETAAAKFERQHMDS (SEQ ID NO: 14). In some embodiments, the peptide tag of the present disclosure may have a sequence in which element 2 is 10% or less and / or A is 10% or less.

[0105] In any aspect, the peptide tag of the present disclosure does not have the sequence KLNPPDESGPGCMSCKCVLS (SEQ ID NO: 15) (Tanaka et al., 2007, EMBO Journal, 26:3250-3259). In some aspects, the peptide tag of the present disclosure does not have a sequence with 90% or more sequence identity thereto.

[0106] In any embodiment, the peptide tags of the present disclosure do not have the sequence EFGGAPEFPKPSTPPGSSGL (SEQ ID NO: 16) or a sequence having 90% or more sequence identity thereto (Paolo et al., 2003, Clinical Cancer Research, 9:2837-2848). In some embodiments, the peptide tags of the present disclosure do not have a sequence having 90% or more sequence identity thereto.

[0107] In any embodiment, a peptide tag of the present disclosure does not have any of the following sequences (Arimori et al., 2017, Structures, 25:1611-1622): hMst1: DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAK (SEQ ID NO: 17); hMST2: DFDFLKNLSLEELQMRLKALDPMMEREIEELRQRYTAKRQPILDAMDAK (SEQ ID NO: 18); hRaf1: GEVNWDAFSMPELHNFLRILQREEEEHLRQILQKYSYSRQKIQEALHAS (SEQ ID NO: 19); hRaf5: GEVEWDAFSIPELQNFLTILEKEEQDKIQQVQKKYDKFRQKLEEALRES (SEQ ID NO: 20); hSAV1: HILKWELFQLADLDTYQGMLKLLFMKELEQIVKMYEAYRQALLTELENR (SEQ ID NO: 21). In some aspects, the peptide tags of the present disclosure do not have a sequence that shares 90% or more sequence identity with any of these sequences.

[0108] In any embodiment, a peptide tag of the present disclosure does not have any of the following (Zhang et al., 2004, Protein Expression and Purification, 36(2):207-216): T7C: LEDPFQSGVMLGVASTVAASPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQ (SEQ ID NO: 22); T7B: LEDPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQ (SEQ ID NO: 23); T7B1: LEDPEEASVTSTEETLTPAQEAARTRPPNKARKEAELAAATAEQ (SEQ ID NO: 24); T7B2: T7B3: LEDPEEASVTSTEETLTPAQEAARTRGGNKARKEAELAAATAEQ (SEQ ID NO: 26); T7B4: LEDPEEASVTSTEETLTPAQEAARTRAANKARKEAELEAETAEQ (SEQ ID NO: 27); T7B5: LEDPEEASVTSTEETLTPAQEAARTRAAAAKARKEAELAAATAEQ (SEQ ID NO: 28); T7B6: LEDPEEASVTSTEETLTPAQEAARTRKEAELAAATAEQ (SEQ ID NO: 29); T7B7: T7B8: LEDPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAA (SEQ ID NO: 31); T7B9: LEDPEEASVTSTEETLTPAQEAARTRAANKARKEAELEAETAEQ (SEQ ID NO: 32); T7B10: LEDPTPAQEAARTRAANKARKEAELAAATAEQ (SEQ ID NO: 33); T7A: LEDPAANKARKEAELAAATAEQ (SEQ ID NO: 34); T7A1: LEDPERNKERKEAELAAATAEQ (SEQ ID NO: 35); T7A2: T7A3: LEDPERNKERKEAELEAATAEQ (SEQ ID NO: 37);T3: LEDPAVWEAGKVVAKGVGTADITATTSNGLIASCKVIVNAATS (SEQ ID NO: 38); T3A: LEDPAVWEAGKVVAKGVGTADITATTSNGLIASSEEADNAATS (SEQ ID NO: 39). In some aspects, the peptide tags of the present disclosure do not have a sequence that has 90% or more sequence identity with any of these sequences.

[0109] In any embodiment, the peptide tag of the present disclosure does not have any of the following (JP 2015-97519 A): Zif628: ERPYACPVESCDDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKIHLRQKD (SEQ ID NO: 40); HinR: GRPRAITKHEQEQISRLLEKGHPRQQLAIIFGIGVSTLYRYFPASSIKKRMN (SEQ ID NO: 41); and TrpR:MAQQSPYSAAMAEQRHXXQEWLRFVDLLKNAYQNXXDLHLPLLLNLMLTPDERXXEALGT RVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELRQWLEEVLLKSD (SEQ ID NO: 42).

[0110] In some preferred embodiments, the peptide tags of the present disclosure may be naturally occurring sequences found in non-human organisms. In some preferred embodiments, the peptide tags of the present disclosure may be non-naturally occurring sequences or portions thereof. In any embodiment, the peptide tags of the present disclosure are not any of the following (WO 2010 / 034183): NE-1: TKENPRSNQEESYDDNES (SEQ ID NO: 43); NE-8: TKENPRTNQEESYDDNES (SEQ ID NO: 44); NE-9: TKENPRSNQDESYDDNES (SEQ ID NO: 45); NE-10: TKENPRSNQPPSYDDNES (SEQ ID NO: 46).

[0111] In some aspects, a peptide tag of the present disclosure is not any of the following (WO 2011 / 034605): ACID. P1:GGSAQLEKELQALEKENAQLEWELQALEKELAQGAT (SEQ ID NO: 50).

[0112] In some aspects, a peptide tag of the present disclosure is not any of the following (WO 2009 / 023270): rPEG_K288-GFP: (GEGEGGGEG) ３２ (SEQ ID NO: 51).

[0113] In certain aspects, a peptide tag of the present disclosure is not any of the following (WO2020 / 059228): Hero7: MTRGNQRELARQKNMKKQSDSVKGKRRDDGLSAAARKQRDSEIMQQKQKK ANEKKEEPK (SEQ ID NO: 1038); Hero9: MSGPNGDLGMPVEAGAEGEEDGFGEAEYAAINSMLDQINSCLDHLEEKNDHLHARLQELLESNRQTRLEFQQQLGEAPSDASP (SEQ ID NO: 1039); Hero11: MAQGQRKFQAHKPAKSKTAAAAASEKNRGPRKGGRVIAPKKARVVQQQKLKKNLEVGIRKKIEHDVVMKASSSLPKKLALLKAPAKKKGAAATSSKTPS (SEQ ID NO: 1040).

[0114] In one aspect, the peptide tag of the present disclosure is not any of the following (Protein Engineering, Design & Selection, 26(8):490-501, 2013): PAS#1: ASPAAPAPASPAAPAPSAPAA (SEQ ID NO: 52); 1P2: ASAAAPAAAASAAAASAPSAAAA (SEQ ID NO: 53); PAS#5: AASPAAPSAPPAAAASPAAPSAPPAA (SEQ ID NO: 54); and repeat sequences thereof (e.g., the number of repeats is 200±20, 400±40, or 600±60, etc.).

[0115] Also, in one aspect, the peptide tag of the present disclosure is none of the following (Protein Engineering, Design & Selection, 17(11):779-786, 2004): Z(W): VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 55); Z(a1): VDNKFNKEQQNAEYEIEHLPNLNEEQENAFIQSLEDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 56); Z (a2): VDNKFNKEEEEAEEEIEHLPNLNEEQEEAFIESLEDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 57)

[0116] In certain aspects, the peptide tags of the present disclosure may have mutations selected from the group consisting of one or more, preferably two or more, amino acid substitutions, insertions, deletions, deletions, and deletions from any of the amino acid sequences set forth in SEQ ID NOs: 43 to 46, and 47 to 58. In certain aspects, the peptide tags of the present disclosure may have less than 90%, 85% or less, 80% or less, 75% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 44 to 47, and 47 to 58.

[0117] In any aspect, the peptide tag of the present disclosure does not include the following sequences (Protein Science (2019) 28, 823-836): PA12-tag: GVAMPGAEDDVV (SEQ ID NO: 47); PA14-tag: EGGVAMPGAEDDVV (SEQ ID NO: 48). Also, in any embodiment, a peptide tag of the present disclosure does not include the following sequence: DYKDDDDVEAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTTESVEEVYPGQVGYPGQVGYPGQV (SEQ ID NO: 49).

[0118] In certain aspects, the peptide tags of the present disclosure have less than 90%, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less sequence identity with any of SEQ ID NOs: 14-48, and 49-58.

[0119] In certain aspects, a peptide tag of the present disclosure may have a sequence that satisfies any one, two, or all of the following: (i) element 2 is 10% or less, (ii) As are 10% or less, and (iii) Gs are 10% or less.

[0120] In a preferred aspect, the peptide tag of the present disclosure does not have a sequence containing 5 or more consecutive As. In a preferred aspect, the peptide tag of the present disclosure does not have a sequence containing 5 or more consecutive Qs. In a preferred aspect, the peptide tag of the present disclosure does not have a sequence containing 5 or more consecutive Ss. In a preferred aspect, the peptide tag of the present disclosure does not have a sequence containing 5 or more consecutive Ns. In a preferred aspect, the content of a particular single amino acid in the amino acid sequence of the peptide tag does not exceed 50%, 40%, 35%, 30%, 25%, or 20%. In a preferred aspect, the peptide tag of the present disclosure does not include an amino acid sequence having a length of 3 to 8 amino acids listed in Table 1 of WO 2022 / 092132, or does not include consecutive repeats of such amino acid sequences (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more consecutive repeats).

[0121] For example, for intracellular antibodies, the peptide tags of the present disclosure may suppress aggregate formation, promoting uniform intracellular distribution of the antibody, and / or suppress aggregate formation, promoting intracellular binding and co-localization of the intracellular antibody with an antigen. In certain aspects, the peptide tags of the present disclosure may lack a sequence that imposes inherent constraints on the intracellular distribution of the intracellular antibody (or a sequence that hinders free distribution), and / or may lack intracellular binding and co-localization of the intracellular antibody with an antigen.

[0122] Any of the peptide tags disclosed herein can reduce, inhibit, or improve the aggregation tendency of the tagged protein; increase, promote, or improve non-aggregation; or enhance, promote, or improve stability. The peptide tags disclosed herein may lack sequences that inherently constrain the intracellular distribution of intracellular antibodies (or sequences that hinder free distribution) and / or may lack intracellular antibody binding and co-localization with antigens. When the target protein is an antigen-binding fragment of an antibody, the peptide tags disclosed herein can also promote co-localization of the protein with the antigen in the cytoplasm by binding to the antigen.

[0123] The peptide tag of the present disclosure may be, for example, a gene product or a fragment thereof encoded by a gene possessed by an organism, where the peptide tag may be a gene product or a fragment thereof encoded by a gene possessed by a non-human organism (e.g., a microorganism such as a bacterium, algae, or fungus, an animal such as a mammal, bird, or fish, or a plant). Alternatively, in a preferred embodiment, the peptide tag of the present disclosure may be a gene product or a fragment thereof encoded by a gene possessed by a human.

[0124] The peptide tag of the present disclosure, when fused to an aggregating protein, can alleviate, inhibit, or improve the aggregation tendency of the aggregating protein, or can increase, promote, or improve the non-aggregation property of the aggregating protein. Protein aggregation can have adverse effects on cells expressing the protein, and can also adversely affect the production yield and functionality of the protein. Therefore, alleviating, inhibiting, or improving the aggregation tendency can reduce the effects of aggregation on cells and can lead to reduced effects on the production yield and functionality of the protein. This can be beneficial in terms of improving the expression level and / or functionality of aggregating proteins expressed in cells, and therefore can be useful for forced expression of aggregating proteins in vivo.

[0125] Therefore, the peptide tag of the present disclosure may be fused to an aggregating protein. The aggregating protein may be, for example, a protein that forms aggregates in 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of cells when expressed in cells. The aggregating protein may be, for example, an aggregating protein that forms aggregates in 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less of cells when expressed in cells. Fusion may be performed, for example, at the N-terminus and / or C-terminus (preferably both the N-terminus and C-terminus) of the aggregating protein. Fusion may be achieved, for example, by linking a nucleic acid encoding the peptide tag of the present disclosure to a nucleic acid encoding the aggregating protein in frame (so that the codon reading frames are aligned). The peptide tag of the present disclosure may also be fused to a non-aggregating protein. This can further enhance the non-aggregating properties of the non-aggregating protein. A non-aggregating protein may be, for example, a protein that forms aggregates or does not form aggregates in 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of cells when expressed in cells.

[0126] The peptide tag of the present disclosure can be fused to a protein to enhance, promote, or improve the stability of the protein in an intracellular environment. Stabilization of a protein in an intracellular environment is beneficial for both aggregating and non-aggregating proteins.

[0127] In some embodiments, a target protein may be linked to a second peptide tag. The second peptide tag may be added to the target protein, for example, for detection or purification purposes. In this case, the peptide tag of the present disclosure can be used to reduce, inhibit, or improve the aggregation tendency of a fusion protein of the target protein and the second peptide tag; increase, promote, or improve non-aggregation; or enhance, promote, or improve stability. A conventional peptide tag, such as an HA tag, can be used as the second peptide tag.

[0128] The aggregating protein is not particularly limited, and may be, for example, an antigen-binding fragment of an antibody. The aggregating protein may preferably be a single-chain Fv (scFv) or a VHH antibody. An scFv is a fusion protein comprising the heavy chain variable region and light chain variable region of an antibody, with the heavy chain variable region and light chain variable region linked via a linker (preferably a flexible linker). For example, undraggable therapeutic targets exist within cells. This is particularly evident when, for example, no site to which a small molecule compound can bind is found on the therapeutic target. Antibodies can bind to targets with strong binding affinity based on a principle completely different from that of small molecule compounds, and can effectively act on therapeutic targets that have been considered undraggable by small molecule compounds. However, antibodies are usually secreted extracellularly and function extracellularly. Therefore, in order to express a secretory protein (a protein secreted extracellularly) within a cell, a gene can be designed to express a secretory protein (an intracellular antibody) within the cell. For example, to express a secretory protein (intracellular antibody), the signal sequence of the protein can be disrupted, preferably removed, etc. In particular, scFvs can exhibit aggregation in cells. Therefore, the tag of the present disclosure can be fused to an aggregating protein, particularly a secretory protein, particularly an antigen-binding fragment of an antibody, preferably scFv. Furthermore, secretory proteins may exhibit aggregation in cells. The tag of the present disclosure can be effective in stabilizing such secretory proteins in cells before secretion.

[0129] An antibody, or an antigen-binding fragment of an antibody, binds to its antigen, e.g., －５ M or less, 10 －６ M or less, 10 －７ M or less, 10 －８ M or less, 10 －９ M or less, 10 －１０ M or less, 10 －１１ M or less, or 10 －１２ The antibody may have a binding affinity (KD) of less than or equal to M. Binding and binding affinity studies can be performed, for example, in buffered saline.

[0130] Antigens include intracellular antigens, such as intracellular proteins. Intracellular proteins include cytoplasmic proteins (e.g., proteins in the cytoplasm outside intracellular vesicles), nuclear proteins (in which case the peptide tag or fusion protein may include a nuclear localization signal), nuclear transcription factors, proteins that bind to transcription factors, proteins that bind to genomic DNA, proteins that bind to proteins that bind to genomic DNA, proteins that constitute chromatin, proteins that bind to chromatin, the intracellular cytoskeleton, and proteins that bind to the intracellular cytoskeleton. Intracellular proteins also include, but are not limited to, gene products of cancer driver genes, proteins in activated signaling cascades (particularly in cancer cells or activated immune cells in patients with immune-related diseases), and gene products of tumor suppressor genes (particularly those under the control of binding partners that negatively regulate them). In one aspect, the antigen may be Kras.

[0131] Introduction of a protein into a cell can be achieved by introducing into the cell a protein expression vector containing a nucleic acid encoding a fusion protein (a fusion protein of a peptide tag and a protein of interest) operably linked to a regulatory sequence. The protein expression vector is introduced into a protein-producing cell or a mammalian cell, and can express the fusion protein in the cell.

[0132] The regulatory sequence may be a promoter capable of transcribing mRNA, and various Pol II promoters may be used. Pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1α promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, the CAG promoter, and the CBh promoter. Other promoters that may be used include promoters that drive bacteriophage-derived RNA polymerase, such as the T7 promoter, the T3 promoter, and the SP6 promoter, as well as Pol III promoters, such as the U6 promoter. For circular DNA, the T7 promoter is preferred, and for linear DNA, the SP6 promoter is preferred. The promoter may also be a viral promoter. The promoter may also be an inducible promoter. An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to the promoter only in the presence of an inducer that drives the promoter. Examples of inducible promoters include promoters that induce gene expression upon heating, such as heat shock promoters. Further, examples of inducible promoters include promoters in which the inducer that drives the promoter is a drug. Examples of such drug-inducible promoters include Cumate operator sequences, λ operator sequences (e.g., 12×λOp), tetracycline-inducible promoters, etc. Examples of tetracycline-inducible promoters include promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline), or reverse tetracycline-controlled transactivator (rtTA). Examples of tetracycline-inducible promoters include the TRE3G promoter.

[0133] Protein expression vectors may be, but are not limited to, viral vectors and plasmid vectors. Examples of viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, vaccinia viral vectors, Sendai viral vectors, and vesicular stomatitis viral vectors. These vectors may be pseudotyped to alter their infectivity to cells. These vectors may be derived from attenuated strains. Such vectors can be appropriately prepared using well-known techniques.

[0134] To simplify the preparation of a protein expression vector, the protein expression vector may comprise a control sequence and a nucleic acid encoding the peptide tag of the present disclosure operably linked to the control sequence, and may have a cloning site downstream of the nucleic acid for a nucleic acid encoding a target protein. The cloning site has a restriction enzyme cleavage site uniquely present within the vector and is suitable for introducing a fragment of a gene encoding a target protein. A gene encoding a fusion protein of a peptide tag and a target protein can be obtained by linking a gene encoding the peptide tag in frame with a gene encoding the target protein. Therefore, the present disclosure provides a protein expression vector comprising a control sequence and a nucleic acid encoding the peptide tag of the present disclosure operably linked to the control sequence. In a preferred embodiment, this vector has a cloning site downstream of the nucleic acid for a nucleic acid encoding a target protein. The present disclosure also provides a protein expression vector comprising a control sequence, a nucleic acid encoding the peptide tag of the present disclosure operably linked to the control sequence, and a nucleic acid encoding a target protein ligated in frame with the nucleic acid. In this manner, a fusion protein of a peptide tag and a target protein can be expressed in cells.

[0135] According to the present disclosure, there is provided a messenger RNA (mRNA) comprising a nucleic acid encoding the peptide tag of the present disclosure. The mRNA further comprises a nucleic acid encoding a protein of interest. The nucleic acid encoding the protein of interest is linked in-frame to the nucleic acid encoding the peptide tag. In one aspect, at least one uridine in the mRNA may be changed to pseudouridine. The pseudouridine may be 1-methylpseudouridine. The mRNA may be transcribed from cDNA, i.e., may not have an intron. The mRNA may also have a cap structure at the 5' end (Furuichi Y & Miura K. Nature. 1975;253(5490):374-5). The Cap0 structure can be added to mRNA by the Anti-Reverse Cap Analogues (ARCA) method using a Cap analog (Stepinski J et al. RNA. 2001 Oct;7(10):1486-95). Further treatment with 2'-O methyltransferase can convert the Cap0 structure of mRNA to the Cap1 structure. These can be performed by conventional methods, for example, using commercially available kits such as the ScriptCap m7G Capping System and ScriptCap 2'-O-Methyltransferase Kit, or the T7 mScript Standard mRNA Production System (AR Brown CO., LTD). The mRNA can have a polyA tail. The addition of the polyA tail can be performed by conventional methods, for example, using the A-Plus Poly(A) Polymerase Tailing Kit (AR Brown CO., LTD). Therefore, in one embodiment, the mRNA may be an mRNA having a Cap structure at the 5'-end and a polyA at the 3'-end, and preferably in which at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). The mRNA may be isolated or synthetic.

[0136] mRNA can be encapsulated in nanoparticles, such as lipid nanoparticles (LNPs). This prevents degradation of mRNA in vivo and improves the efficiency of mRNA delivery into cells. Thus, in one embodiment, the mRNA may have a Cap structure at the 5' end and a polyA at the 3' end, and preferably, at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). Lipid nanoparticles encapsulating such mRNA are also provided. Lipid nanoparticles that can be used include, but are not limited to, lipid nanoparticles described in US Pat. Nos. 9,364,435B, 8,822,668B, 8,802,644B, and 8,058,069B2. Alternatively, mRNA may be encapsulated in polyion complex micelles or polyion complex polymersomes (Miyata et al., Chem. Soc. Rev., 2012, 41, 2562-2574). Nanoparticles are particles with a diameter (e.g., in a hydrodynamic system) of less than 1 μm.

[0137] Thus, the present disclosure provides nanovesicles (e.g., lipid nanovesicles and polyion complex polymersomes) containing mRNA that contains at least a nucleic acid encoding the peptide tag of the present disclosure. The mRNA may contain a nucleic acid encoding a fusion protein of the peptide tag and a protein of interest.

[0138] According to the present disclosure, there is provided a method for reducing, inhibiting, or ameliorating the aggregation tendency of a protein, the method comprising fusing to the protein a peptide tag that reduces, inhibits, or ameliorates the aggregation tendency of the present disclosure. In this aspect, the protein may be an aggregating protein. The aggregation tendency may be a tendency to aggregate in an intracellular environment. The fusion is typically to the N-terminus and / or C-terminus of the protein. The method may be an in vitro method.

[0139] The present disclosure also provides a method for increasing, promoting, or improving the non-aggregation property of a protein, comprising fusing the protein with a peptide tag of the present disclosure that reduces, inhibits, or improves the aggregation tendency. In this aspect, the protein may be an aggregating protein. The non-aggregation property may be non-aggregation in an intracellular environment. The method may be an in vitro method.

[0140] The present disclosure also provides a method for enhancing, promoting, or improving the stability of a protein, comprising fusing to the protein a peptide tag that reduces, inhibits, or ameliorates the aggregation tendency of the present disclosure. In this aspect, the protein may be an aggregating protein. In this aspect, the protein may be a non-aggregating protein. The stability may be stability in an intracellular environment. The method may be an in vitro method.

[0141] The present disclosure provides a use of a peptide tag of the present disclosure for reducing, inhibiting, or improving the aggregation tendency of a protein. The present disclosure also provides a use of a peptide tag of the present disclosure for increasing, promoting, or improving the non-aggregation property of a protein. The present disclosure also provides a use of a peptide tag of the present disclosure for enhancing, promoting, or improving the stability of a protein. The use may be in vitro.

[0142] The extent to which the peptide tag of the present disclosure can alleviate, inhibit, or improve the aggregation tendency of a protein; increase, promote, or improve non-aggregation; or enhance, promote, or improve stability can be tested by in vitro assays. For example, a gene encoding a peptide tag of the present disclosure can be fused to the N-terminus or C-terminus of a gene encoding an aggregating protein, such as an scFv having the amino acid sequence of SEQ ID NO: 1, and the resulting gene can be introduced into cells to express the aggregating protein fused with the peptide tag of the present disclosure in the cells. Aggregates formed by the aggregating protein can be observed using an antibody against the aggregating protein, and the percentage (%) of cells with aggregates relative to cells expressing the aggregating protein can be calculated. This percentage allows the influence of the peptide tag of the present disclosure on the aggregation tendency, non-aggregation tendency, and stability of the protein to be evaluated.

[0143] According to the present disclosure, a composition including the peptide tag of the present disclosure may be provided. The peptide tag of the present disclosure can be linked to a target protein in a reaction solution, for example, by a click reaction. The click reaction may be a Fusgen reaction. One of the peptide tag and the target protein is modified with an alkyne, and the other is modified with an azide compound, thereby forming a 1,2,3-triazole ring, thereby achieving linkage between them.

[0144] According to the present disclosure, there is provided a composition comprising a fusion protein of a peptide tag of the present disclosure and a protein of interest. In one embodiment, the protein of interest may be an aggregating protein. In one embodiment, the protein of interest may be an antigen-binding fragment of an antibody. In one embodiment, the protein of interest may be an scFv.

[0145] The present disclosure provides mRNA containing a nucleic acid encoding a fusion protein of the peptide tag of the present disclosure and a protein of interest, and a composition containing the mRNA.The present disclosure also provides vesicles containing mRNA containing a nucleic acid encoding the fusion protein of the peptide tag of the present disclosure and a protein of interest, and a composition containing the vesicles.

[0146] According to the present disclosure, there are provided a protein expression vector comprising a nucleic acid encoding a fusion protein of the peptide tag of the present disclosure and a protein of interest operably linked to a regulatory sequence, and a composition comprising the protein expression vector.

[0147] In some embodiments, the composition may further comprise a pharmaceutically acceptable carrier, excipient, and / or additive. The composition may, in some embodiments, be a pharmaceutical composition.

[0148] In all aspects of the present disclosure, the fusion protein to which the peptide tag of the present disclosure is linked may be a non-naturally occurring protein.

[0149] In all aspects of the present disclosure, the peptides and proteins may be recombinant peptides and proteins, respectively.

[0150] In all aspects of the present disclosure, the peptide tags of the present disclosure can reduce, inhibit or ameliorate the aggregation tendency of a protein; increase, promote or improve non-aggregation; or enhance, promote or improve stability for at least an scFv having the amino acid sequence of SEQ ID NO:1.

[0151] Peptides, proteins, and nucleic acids can be isolated, enriched, or purified. Isolation refers to the separation of a component from at least one other component of a system. Purification refers to the increase in the relative concentration of a component relative to the concentration of at least one other component of a system. Enrichment refers to the increase in the concentration of a component.

[0152] In one aspect of the present disclosure, there is provided a method for obtaining (or selecting or identifying) an amino acid sequence (or a nucleic acid encoding the amino acid sequence), the method comprising obtaining an amino acid sequence in which: (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids; and (b) 20% or more of the amino acids constituting the amino acid sequence are selected from the group consisting of F, P, Y, G, S, Q, N, and A.

[0153] In one aspect of the present disclosure, there is provided a method for obtaining (or selecting or identifying) an amino acid sequence (or a nucleic acid encoding said amino acid sequence), the method comprising obtaining an amino acid sequence that satisfies any of the above conditions (A) to (AE) and (AF) to (AU), or any combination of these conditions.

[0154] In one aspect of the present disclosure, the obtaining method may further include selecting or identifying an amino acid sequence of a peptide tag that reduces (to a predetermined value or less) the proportion of cells in which the fusion protein forms aggregates (aggregation rate) when a fusion protein of a peptide having the selected or identified amino acid sequence and a reference protein (reference protein) is expressed in a mammalian cell (preferably a human cell).

[0155] In one aspect of the present disclosure, the obtaining method may further comprise obtaining a peptide having the amino acid sequence or a nucleic acid encoding the peptide.

[0156] In the above-mentioned obtaining method, the obtained amino acid sequence can be 10 to 200 amino acids in length (for example, 10 to 90 amino acids in length).

[0157] In one aspect of the present disclosure, there is provided a method for obtaining (or selecting or identifying) an amino acid sequence (or a nucleic acid encoding said amino acid sequence) having a length of 10 to 200 amino acids (10 to 90 amino acids), the method comprising: (a) obtaining an amino acid sequence in which at least 5% but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and (b) at least 20% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A; selecting or identifying the amino acid sequence of a peptide tag that reduces (to a predetermined value or less) the proportion of cells in which the fusion protein forms aggregates (aggregation rate) when a fusion protein of a peptide having the selected or identified amino acid sequence and a reference protein is expressed in mammalian cells (preferably human cells); and obtaining a peptide having the amino acid sequence or a nucleic acid encoding the peptide. In this method, by increasing the degree of reduction or decreasing the predetermined value, peptides exhibiting particularly excellent stabilizing activity can be selected from peptides having stabilizing activity. In one aspect, the method may further include expressing a fusion protein of a peptide having the selected or identified amino acid sequence and a reference protein (reference protein) in a mammalian cell (preferably a human cell). The method may further include obtaining a nucleic acid encoding the selected or identified amino acid sequence. The predetermined value may be a numerical value based on the ratio (%) of cells having aggregates to cells expressing the aggregating protein. For example, the predetermined value may be 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, or 0%. The predetermined value may also be a value in the range of 0% to 10%, a value in the range of 10% to 20%, or a value in the range of 20% to 30%. When a more effective peptide is desired, it is preferable to reduce the predetermined value.According to this aspect, for example, a peptide tag that exhibits a stronger effect can be selected from the peptide tags of the present disclosure (e.g., any of the peptide tags (A) to (Z), (AA) to (AE), and (AF) to (AU) above).

[0158] The reference protein can be, for example, a protein (aggregating protein, e.g., scFv) in which the percentage of cells in which the fusion protein forms aggregates is greater than 30%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 95%, 95% to 99%, 99% to 99.9%, or 99.9% or greater. The reference protein does not need to have any particular functionality or antigen binding ability, and is used simply for the purpose of evaluating the reduction in aggregation rate. Therefore, its CDR sequence may be any sequence. The aggregation rate of the scFv may vary depending on the amino acid sequence of the CDR. For scFvs with a varied aggregation rate, the amino acid sequence of a peptide tag that reduces the aggregation rate to a predetermined value or less can be searched for. In a preferred embodiment, the reference protein can be a protein having the amino acid sequence set forth in SEQ ID NO: 1.

[0159] In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (B) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (D) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (E) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (F) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (G) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (H) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (I) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (J) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (K) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (L) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (M) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (N) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (O) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (P) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (Q) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (R) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (S) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (T) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (U) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (V) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (W) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (X) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (Y) above.In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (Z) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AA) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AB) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AC) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AD) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AE) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AF) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AG) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AH) above. In some embodiments, the obtained amino acid sequence further satisfies the condition defined in (AI) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AJ) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AK) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AL) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AM) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AN) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AO) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AP) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AQ) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AR) above. In some embodiments, the obtained amino acid sequence further satisfies the conditions defined in (AS) above. In some embodiments, the obtained amino acid sequence further satisfies the condition (AT) above. In some embodiments, the obtained amino acid sequence further satisfies the condition (AU) above. In some embodiments, the obtained amino acid sequence satisfies any one or more of the conditions (A) to (Z), (AA) to (AE), and (AF) to (AU) above.

[0160] In one embodiment, the obtained amino acid sequence may be selected from a group of amino acid sequences. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (A) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (B) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (C) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (D) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (E) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (F) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (G) above. In one embodiment, the group of amino acid sequences may be or include the group of amino acid sequences defined in (H) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (I) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (J) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (K) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (L) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (M) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (N) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (O) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (P) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (Q) above. In one embodiment, the group of amino acid sequences may be or may include the group of amino acid sequences defined in (R) above.In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (S) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (T) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (U) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (V) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (W) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (X) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (Y) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (Z) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AA) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AB) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AC) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AD) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AE) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AF) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AG) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AH) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AI) above. In some embodiments, the group of amino acid sequences may be or include the group of amino acid sequences set forth in (AJ) above. In some embodiments, the group of amino acid sequences may be or include the group of amino acid sequences set forth in (AK) above.In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AL) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AM) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AN) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AO) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AP) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AQ) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AR) above. In some embodiments, the group of amino acid sequences may be or comprise the group of amino acid sequences defined in (AS) above. In some embodiments, the group of amino acid sequences may be or include the group of amino acid sequences defined in (AT) above. In some embodiments, the group of amino acid sequences may be or include the group of amino acid sequences defined in (AU) above.

[0161] In a preferred embodiment, the aggregation rate of the reference protein is 5 to 10%, and the aggregation rate of the fusion protein is lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 5 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 1% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 5 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 2% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 5 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 3% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 5 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 4% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 6 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 5% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 7 to 10%, and the aggregation rate of the fusion protein is not more than a predetermined value that is 6% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein is 8 to 10%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 7% or more lower than the aggregation rate of the reference protein. In a preferred embodiment, the aggregation rate of the reference protein is 9 to 10%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 8% or more lower than the aggregation rate of the reference protein.

[0162] In a preferred aspect, the ratio of the aggregation rate of the fusion protein to the aggregation rate of the reference protein is equal to or less than a predetermined value of 0.9, 0.8, 0.7, 0.6, 0.4, 0.3, 0.2, or 0.1. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.9. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.8. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.7. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.6. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is not more than a predetermined value of 0.5 or less. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is not more than a predetermined value of 0.4 or less. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is not more than a predetermined value of 0.3 or less. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is not more than a predetermined value of 0.2 or less. In a preferred aspect, the aggregation rate of the reference protein is 5 to 10%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is not more than a predetermined value of 0.1 or less.

[0163] In one aspect, the aggregation rate of the reference protein is less than 5%, and the aggregation rate of the fusion protein is lower than the aggregation rate. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.9 or less. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.8 or less. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.7 or less. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.6 or less. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.5 or less. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.4. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.3. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.2. In a preferred aspect, the aggregation rate of the reference protein is less than 5%, and the ratio of the aggregation rate of the fusion protein to the aggregation rate is equal to or less than a predetermined value of 0.1.

[0164] In a preferred embodiment, the aggregation rate of the reference protein exceeds 10%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 20%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 20%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 30%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 30%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate.

[0165] In a preferred embodiment, the aggregation rate of the reference protein exceeds 30%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 40%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 40%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 40%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 40%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate.

[0166] In a preferred embodiment, the aggregation rate of the reference protein exceeds 50%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 50%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 50%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 50%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 50%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 50% or more lower than the aggregation rate.

[0167] In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 50% or more lower than the aggregation rate.

[0168] In a preferred embodiment, the aggregation rate of the reference protein exceeds 60%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 60% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate.

[0169] In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 50% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 60% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 70%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 70% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate.

[0170] In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 50% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 60% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 70% or more lower than the aggregation rate.

[0171] In a preferred embodiment, the aggregation rate of the reference protein exceeds 80%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 80% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 10% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 20% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 30% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 40% or more lower than the aggregation rate.

[0172] In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 50% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 60% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 70% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 80% or more lower than the aggregation rate. In a preferred embodiment, the aggregation rate of the reference protein exceeds 90%, and the aggregation rate of the fusion protein is equal to or lower than a predetermined value that is 90% or more lower than the aggregation rate.

[0173] The resulting nucleic acid encoding the peptide tag can be linked in frame with a nucleic acid encoding a protein of interest to obtain a nucleic acid encoding a fusion protein of the peptide tag and the protein of interest. The fusion protein can be expressed from the nucleic acid encoding the fusion protein. A protein expression vector may be prepared containing the nucleic acid encoding the fusion protein operably linked to a regulatory sequence.

[0174] In one aspect of the present disclosure, the method can be used to determine whether a peptide tag having an amino acid sequence 10 to 200 amino acids (e.g., 10 to 90 amino acids) in length has an effect of improving the aggregation properties of the tagged protein by a predetermined level or more. That is, in one aspect of the present disclosure, there is provided a method for determining whether a peptide tag having an amino acid sequence 10 to 90 amino acids in length has an effect of improving the aggregation properties of the tagged protein by a predetermined level or more, the method comprising: obtaining an amino acid sequence in which (α) at ​​least 5% but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and (β) at least 20% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A; and determining that an amino acid sequence of the peptide tag has an effect of improving aggregation properties (e.g., a predetermined level or more) when a fusion protein of a peptide having the selected or identified amino acid sequence with a reference protein is expressed in mammalian cells (preferably human cells), such that the proportion of cells in which the fusion protein forms aggregates (aggregation rate) is reduced (or is a predetermined value or less). In one aspect, the method may further comprise expressing a fusion protein of the peptide having the selected or identified amino acid sequence and the reference protein in a mammalian cell (preferably a human cell). In one aspect, the obtained amino acid sequence may be selected from a group of amino acid sequences. Details of the group of amino acids are as described above. In one aspect, the obtained amino acid sequence satisfies one or more additional conditions. The conditions are as described above. In one aspect, the aggregation rate of the reference protein and the predetermined value are as described above.

[0175] In one aspect, in the above-described method for selecting or identifying amino acid sequences 10 to 200 amino acids (e.g., 10 to 90 amino acids) in length, the group of amino acid sequences 10 to 200 amino acids (e.g., 10 to 90 amino acids) in length may be a group of amino acid sequences encoded by a coding region of the human genome. In one aspect, the group of amino acid sequences 10 to 90 amino acids in length may be a group of amino acid sequences encoded by a coding region of the genome of a non-human organism.

[0176] In some embodiments, a neoantigen may be an amino acid sequence of 10 to 200 amino acids (e.g., 10 to 90 amino acids) in length. Neoantigens were discovered as mutant antigens newly generated by unique genetic mutations in cancer cells. Neoantigens are not expressed in non-cancerous cells. Inducing immunity against neoantigens by administering peptides containing neoantigens is expected to induce cancer-specific immunity. Neoantigens may vary depending on the cancer cell. Neoantigens can be used, for example, to tag target proteins expressed intracellularly in cells containing the neoantigen. Since neoantigens are naturally expressed peptides, they are expected to have little or no adverse effect on the cells. Neoantigens may be naturally occurring mutants. For example, neoantigens have one or more mutations selected from the group consisting of addition, insertion, deletion, and substitution relative to their wild-type sequence. For example, a human neoantigen typically has one or more (e.g., 1 to 10, e.g., 1 to several, 1 to 5, 1 to 4, 1 to 3, 2, or 1) mutations selected from the group consisting of addition, insertion, deletion, and substitution relative to a human wild-type sequence. A human neoantigen can have, for example, 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity to a human wild-type sequence.

[0177] In one preferred embodiment, the reference protein is an scFv. In one embodiment, the scFv has the amino acid sequence of SEQ ID NO:1.

[0178] A further aspect of the present invention provides a method for modifying the amino acid sequence of a peptide tag, comprising: preparing a peptide tag for producing a fusion protein (which may be any of the peptide tags disclosed herein); and substituting one or more (preferably multiple) amino acids in element 2, 3, or 4 of the peptide tag (preferably, element 2, element 4, A, G, Y, or F) with either P or N to obtain a modified amino acid sequence. The method of this aspect may further comprise determining that an amino acid sequence of the peptide tag that, when a fusion protein of a peptide having a selected or identified amino acid sequence and a reference protein is expressed in mammalian cells (preferably human cells), reduces the proportion of cells in which the fusion protein forms aggregates (aggregation rate) in the cells (or is below a predetermined value), has the effect of improving aggregation properties (e.g., aggregation properties of a predetermined strength or greater). This enables modifications with a particularly strong aggregation-reducing effect.

[0179] Example 1 [Method] Construction of gene expression vector A gene fragment encoding a fusion protein containing a peptide tag and an aggregating protein was produced by Eurofins Genomics or Vector Builder Japan. The synthesized gene fragment was cloned into the pEF-BOS vector (Mizushima and Nagata, Nucleic Acids Res. 1990 Sep 11; 18(17): 5322.). The aggregating protein used was a protein (specifically, scFv) having the amino acid sequence set forth in SEQ ID NO: 1. This aggregating protein aggregates in the cytoplasm when expressed intracellularly. The tag sequences used and their SEQ ID NOs are shown in Table 1 below.

[0180]

[0181] The specific amino acid content ratios are as follows:

[0182]

[0183] In Table 2, element 1 is D and E; element 2 is H, K, and R; element 3 is F, P, Y, G, S, Q, N, and A; and element 4 is any other amino acid.

[0184] The peptide tag was fused to the N-terminus of the antibody fragment, and the HA tag of SEQ ID NO: 13 was linked to the C-terminus of the antibody fragment fused to the peptide tag for detection.

[0185] Measurement of intracellular aggregation rate of intracellular antibodies (Cell culture) HeLa cells derived from human cervical epithelioid carcinoma were prepared. HeLa cells were purchased from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition (JCRB9004) and cultured in DMEM (D-MEM, Fujifilm Wako Pure Chemical Industries, Ltd. 4548995066251) containing 10% FBS. (Transfection) 4 x 10 HeLa cells were cultured on a 35 mm glass-bottom tissue (IWAKI 3911-035, glass hole, inner diameter 12 mm) coated with poly-L-lysine (Sigma-Aldrich P1399 Poly-L-lysine hydrobromide mol. Wt. 150,000-300,000). ５The cells were seeded with 1000p-1000p-1000p-1000p-1000p-1000p, and 24 hours later, the antibody fragment gene was introduced into HeLa cells using Lipofectamine 3000 (Invitrogen, L3000-008) according to the manufacturer's instructions, resulting in intracellular expression of the tagged antibody fragment. 24 hours after transfection, the culture medium was removed, and the cells were fixed with 4% PFA. 20 minutes after fixation, the cells were washed with PBS(-). The intracellularly expressed antibody (hereinafter simply referred to as "intracellular antibody") was then observed by immunostaining, and its aggregation rate was measured. (Immunostaining) Immunostaining was performed using standard methods. The cells were treated with 0.3% Triton X-100 / PBS(-) for 2 minutes, and then incubated in blocking solution (1% BSA, 0.1% Triton X-100 / PBS(-)) at room temperature for 1 hour. The cells were incubated at room temperature for 2 hours with anti-HA antibody (rabbit anti-HA antibody: Sigma-Aldrich H6908) diluted in blocking solution. After washing the cells with blocking solution, the cells were incubated at room temperature for 2 hours with Alexa Fluor 488 Goat anti-rabbit IgG (H+L) (Invitrogen A11034) diluted in blocking solution. After washing the cells with blocking solution, they were incubated at room temperature for 15 minutes with a nuclear staining probe (NucBlue Fixed cell stain ReadyProbes, Invitrogen R37606), washed with PBS(-), and stored at 4°C until fluorescent images were acquired. (Fluorescence Image Acquisition) Fluorescence images were acquired using a Keyence BZ-X700 or BZ-X800 microscope with a 40x objective. Images containing 200 or more cells per dish were acquired, and the number of cells in which intracellular antibodies had aggregated (intracellular antibody-aggregated cells) was counted. The intracellular antibody aggregation rate was quantified by normalizing the number of intracellular antibody-aggregated cells by the total number of cells expressing intracellular antibodies.

[0186] [Results] Peptides having the amino acid sequences shown in Table 1 were fused to an antigen-binding fragment of an antibody (specifically, scFv), and the resulting fusion proteins were expressed in the cytoplasm of HeLa cells. The amino acid ratios of each peptide tag are shown in Table 2. The intracellular aggregation rates determined from fluorescence images were also shown in Table 2. In Table 2, a molecule tagged with a Myc tag was used as a control, and the effect of each peptide tag was evaluated based on the difference in aggregation rate from the Myc tag.

[0187] As shown in Table 2, when the proportion of element 3 was in the range of 40 to 75%, the aggregation rate tended to decrease; when the proportion of element 4 was smaller, the aggregation rate tended to decrease; when the proportion of alanine (A) exceeded 10%, the aggregation rate tended to increase; when the proportion of glycine (G) exceeded 10%, the aggregation rate tended to increase; and when the proportion of element 1 increased, the intracellular aggregation rate tended to decrease.

[0188] Serine (S) does not necessarily have to be present (see Tag-4-1), but even when present in large amounts, it did not adversely affect the reduction in aggregation rate (see Tag-1-1 to Tag-1-3).

[0189] In either case, peptide tags with an acidic amino acid content of less than 45% did not exhibit nonspecific adsorption to intracellular proteins (particularly nonspecific adsorption due to the negative charge of the peptide tag). This suggests the usefulness of the peptide tags of the present disclosure in that they do not impose tag-specific restrictions on the free intracellular localization of target proteins.

[0190] Furthermore, when comparing Myc tag (control) with Tag-4-1, it can be seen that reducing element 4 and increasing element 3 contribute strongly to the effect of reducing the aggregation rate of the peptide tag. Similarly, when comparing Tag-1-2 with Tag-1-1, it can be seen that reducing element 4 and increasing element 3 contribute strongly to the effect of reducing the aggregation rate of the peptide tag.

[0191] In particular, the aggregation rate was preferably reduced when the proportion of element 1 was 20% or more and less than 45%, the proportion of element 2 was 10% or less, the proportion of element 3 was 40 to 75%, the proportion of element 4 was 10% or less, the proportion of alanine was 10% or less, and the proportion of glycine was 10% or less.

[0192] HeLa cells expressing a fusion protein of an aggregating protein and a tag (Tag4-1) were observed under a fluorescent microscope. As a negative control, HeLa cells expressing an aggregating protein without the Tag4-1 tag were also observed under a fluorescent microscope. The results are shown in Figure 1. As shown in Figure 1, when tagged with Tag4-1 (fused with Tag-4-1), the aggregating protein was uniformly distributed in the cytoplasm, whereas the aggregating protein without the Tag4-1 tag formed aggregates in the cytoplasm of most cells.

[0193] Example 2: Experiment to add amino acids to tags Further analysis was carried out on the amino acid content in the tag and the aggregation rate of tagged proteins. Serine (S) was randomly inserted or added to Tag4-1 or Tag11-1, and the effect of the resulting modified tag on the protein aggregation rate was examined. The experiment was carried out as described in the above examples, except for the different tags. The results are shown in Table 3. In Table 3, the added or inserted amino acids are underlined.

[0194]

[0195] The results shown in Table 3 indicate that an increase in S in the tag did not significantly affect the aggregation rate of the tagged peptide. It is known that an increase in acidic amino acids in the tag significantly reduces the aggregation rate of the tagged peptide (US 2020 / 0157210A), but in this example, even a decrease in the proportion of acidic amino acids did not significantly reduce the aggregation rate.

[0196] Similarly, the content of amino acids other than serine was varied to determine the effect on the aggregation rate of the tagged peptides. In Tables 4-8, the added or inserted amino acids are underlined.

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] As described above, when the effects of tags obtained by randomly adding or inserting 10 or 20 specific amino acids on the aggregation rate of a protein were examined, the addition or insertion of F and Y tended to worsen the aggregation rate of the tagged protein. Furthermore, the addition or insertion of other amino acids had only a limited adverse effect on the aggregation rate of the tagged protein, despite a decrease in the D and E content.

[0203] Example 3: Effect of changing amino acid content by amino acid substitution in tag on protein aggregation rate

[0049] An attempt was made to identify amino acids that have an effect on protein aggregation rate by substituting N, P, S, Q, F, and Y with other amino acids. In Tables 9 and 10, the substituted amino acids are underlined.

[0204]

[0205]

[0206] Tables 9 and 10 show that the aggregation rate-reducing effect of tagged proteins was greatest when the tag had a high content of P or N, followed by S and Q contents, and then F and Y contents. The 45Tag1-m1 peptide tag, which was created by replacing all of element 3 with P in 45Tag1, which has an acidic amino acid content of approximately 51%, showed a significant improvement in its aggregation-inhibiting effect due to the amino acid substitution with P. Thus, it became clear that the increase in the aggregation rate-reducing effect due to the addition of P and N does not depend on the acidic amino acid content. Furthermore, these results also showed that it is possible for most of the constituent amino acids to be acidic amino acids and N or P.

[0207] Example 4: Sequence shuffling Two tags, Tag4-8(Scr1) and Tag4-8(Scr2), were synthesized by randomly shuffling the amino acid sequence of Tag4-8, and their aggregation-reducing effect on scFv was tested in the same manner as above. The results are shown in Table 11.

[0208]

[0209] As shown in Table 11, Tag4-8, Tag4-8(Scr1), and Tag4-8(Scr2) had the same effect of reducing the aggregation rate. Changing only the order of the amino acids without changing the content ratio or length of each amino acid did not affect the effect of reducing the aggregation rate.

[0210] Example 5: Use of human-derived peptide tags All peptides having a specific amino acid content ratio were extracted from the human proteome database (Proteome ID: UP000005640), and peptides were randomly selected and used as tags to examine the effect of reducing the aggregation rate of tagged proteins.

[0211] Extraction condition 1: Length 20-70 Amino acids Group [D, E] Content rate

[30] or more Group [D, E] Content rate less than

[45] Group [H, K, R] Content rate [5] or less Group [C, T, V, L, I, W, M] Content rate [5] or less Group [G] Content rate less than

[10] Group [A] Content rate less than

[10] Group [F, Y] Content rate [5] or less Group [N] Content rate

[15] or more *In the above extraction conditions, the unit of content rate is %. Amino acids are represented by single letters.

[0212]

[0213] In Table 12, the N content ratio of the amino acid sequences of the extracted tags was 15% or more, and the other amino acid content ratios were as described under extraction condition 1. To confirm that the aggregation-inhibiting effect was independent of the specific amino acid sequence, tags were randomly selected from those extracted under extraction condition 1. For some tags, additional amino acid sequences that met the same extraction conditions were selected from other locations on the same protein. The aggregation rates of scFvs tagged with tags having the selected amino acid sequences were tested, and the results shown in Table 12 were obtained. As shown in Table 12, the aggregation rates of tagged scFvs were generally low. Therefore, it is clear that the aggregation-inhibiting effect of peptide tags is highly dependent on their amino acid content ratios and only weakly dependent on the specific amino acid sequence itself.

[0214] The human-derived amino acid sequences that could be extracted under extraction condition 1 were as follows:

[0215] Extraction condition 2: Length 20 to 70 amino acids Group [D, E] Content rate

[30] or more Group [D, E] Content rate less than

[45] Group [P] Content rate

[10] or more Group [H, K, R] Content rate [5] or less Group [G] Content rate less than

[10] Group [A] Content rate less than

[10] Group [C, T, V, L, I, M, W] Content rate [0] Group [F, Y] Content rate [0] *In the above extraction conditions, the unit of content rate is %. Amino acids are represented by single letters.

[0216]

[0217] In Table 13, the P content ratio of the amino acid sequences of the extracted tags was 15% or more, and the other amino acid content ratios were as described under extraction condition 2. To confirm that the aggregation inhibition effect was independent of the specific amino acid sequence, tags were randomly selected from the tags extracted under extraction condition 2. For some tags, additional amino acid sequences that satisfied the same extraction conditions were selected from different locations of the same protein. The aggregation rates of scFv tagged with the selected amino acid sequences were tested, and the results are shown in Table 13. As shown in Table 13, the aggregation rates of tagged scFv were generally low.

[0218] The human-derived amino acid sequences that could be extracted under extraction condition 2 were as follows:

[0219] Extraction condition 3: Length 20 to 70 Group [D, E] Content rate

[45] or more Group [G] Content rate less than

[10] Group [A] Content rate less than

[10] Group [F, Y] Content rate [0] Group [C, M, L, I, W, T, V] Content rate [0] Group [P] Content rate

[15] or more

[0220]

[0221] In Table 14, the amino acid sequence of the extracted tag had an acidic amino acid ratio of 45% or more, a P content ratio of 15% or more, and other amino acid content ratios were as described under extraction condition 3. To confirm that the aggregation-inhibiting effect was independent of the specific amino acid sequence, tags were randomly selected from those extracted under extraction condition 3. For some tags, additional amino acid sequences that met the same extraction conditions were selected from different locations on the same protein. The aggregation rates of scFvs tagged with selected amino acid sequences were tested, and the results shown in Table 14 were obtained. As shown in Table 14, the aggregation rates of tagged scFvs were generally low. Table 14-1 demonstrates that the strategy of reducing the G, A, F, Y, C, M, L, I, W, T, and V content and increasing the P content works even for peptide tags with a high acidic amino acid content.

[0222] The human-derived amino acid sequences that could be extracted under extraction condition 3 were as follows:

[0223] As shown by the above examples, the protein aggregation rate reducing effect of peptide tags was less dependent on the specific amino acid sequence, less dependent on the protein from which they were derived, and more dependent on the amino acid content.

[0224] Example 6: Addition to various proteins and aggregation-inhibitory effect In the above examples, Y13-259 was used as the scFv. In this example, a VHH antibody (heavy chain variable domain of a heavy chain antibody) was used. iDab#6, which binds to Ras, was used as the VHH antibody. Tag4-8 was used as the tag. Other conditions were the same as in Example 1. As a result, the aggregation rate of the VHH antibody without a tag was 57.89%, while the aggregation rate of the VHH antibody having Tag4-8 at the C-terminus was 8.77%, demonstrating that tagging exerts a strong aggregation-inhibitory effect.

[0225] Furthermore, when SHSY5Y (human dopamine-like cells), a neuroblastoma cell line, was used as the cells, scFv-6E (6E) was used as the scFv, and Tag4-8 or Tag18-1 was added as a tag to the C-terminus and N-terminus, the aggregation rate of Tag4-8-added scFv was 0.96%, and the aggregation rate of Tag18-1-added scFv was 1.14%, indicating that tagging contributed to the low aggregation rate.

[0226] Furthermore, when D4 (SEQ ID NO: 1032: QVQLQQSGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLCISSSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADDLRCGSNWSSYFRGSWGQGTQVTVSS) which binds to botulinum toxin type A was used as the VHH antibody and Tag4-8 was added as a tag to the C-terminus of the above VHH antibody, the aggregation rate of Tag4-8-added D4 was 7.8%, while the aggregation rate of tag-free D4 was 81.5%. These results showed that tagging contributed to a decrease in the aggregation rate of VHH antibodies.

[0227] Example 7: Enhanced Effect by Intracellular Antibody Stabilization (Enhancing Effect of Antibody Action on Amyloid Accumulation) It is known that central nervous system disorders can occur due to the accumulation of amyloid within neurons. When human α-synuclein fibrils are extracellularly introduced into neurons, they engulf synuclein with normal intracellular structure to form synuclein fibrils. When GFP-tagged synuclein is expressed intracellularly, it combines with the introduced human α-synuclein fibrils to form synuclein fibrils, which can be observed by GFP fluorescence. In this example, GFP-tagged synuclein was expressed in SHSY-5Y cells, and α-synuclein fibrils (Cosmobio, SYNO3) were extracellularly introduced into the SHSY-5Y cells. In this example, the ability of antibodies to reduce these synuclein fibrils was tested. Specifically, the antibody is scFv-6E (SEQ ID NO: 1033: AEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYIASGGDTTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG) that binds to fibrillated synuclein. The scFv-6E antibody used was ASAFDYWGQGTLVTVSSGGGGSGGGGGSGGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSNDPYTFGQGTKVEIKR. scFv-6E is an antibody that does not exhibit significant binding to synuclein monomers or oligomers, and such a synuclein fibril-selective or specific antibody is suitable for selectively removing synuclein fibrils. A tag (Tag4-8 or Tag18-1) with an aggregation-reducing effect and a degradation-inducing sequence (CMA (SEQ ID NO: 1034): MARVKKDQAEPLHRKFERQPPG) were added to scFv-6E. The expression plasmid vectors for the above proteins were introduced into cells using X-tremGENE9, and synuclein fibrils were introduced into cells using Multifectam (Merck).Antibodies were tagged with HA or myc and detected with an anti-HA tag antibody. Synuclein fibrils were detected with an anti-phosphorylated α-synuclein antibody. These antibodies were specifically detected with Alexa 555- and Alexa 633-labeled antibodies. Fluorescently stained cells were observed using a Keyence BZ-X800 microscope.

[0228] The tags were added to the N-terminus and C-terminus. The aggregation rate of the scFv-E6 with Tag4-8 added to the N-terminus and C-terminus was 0.96% in SHSY-5Y cells, and the aggregation rate of the scFv-E6 with Tag18-1 added to the N-terminus and C-terminus was 1.14% in SHSY-5Y cells. Considering that the aggregation rate of untagged scFv-E6 was 41.6% in HeLa cells, these results are considered to have shown good inhibition of aggregation.

[0229] When scFv binds to synuclein fibrils, it targets the synuclein fibrils to lysosomes due to the degradation-inducing sequence, and is expected to degrade the synuclein fibrils, resulting in a decrease in the amount of intracellular synuclein fibrils. We evaluated whether the intracellular amount of functional scFv increases by reducing the aggregation rate of scFv with a tag, thereby increasing the amount of synuclein fibrils reduced.

[0230] The above antibodies were expressed in cells that had formed synuclein fibrils, and the number of synuclein fibril-positive cells was counted. The synuclein fibril-positive rates were compared between cells that expressed the antibody and cells that did not. The results were calculated by dividing the ratio of synuclein fibril-positive cells among antibody-positive cells by the ratio of synuclein fibril-positive cells among antibody-negative cells (P / N).

[0231] As shown in Figure 2, phosphorylated synuclein disappeared in cells transfected with tagged scFv-E6. The P / N ratio was as shown in Figure 3. As shown in Figure 3, both scFv-E6 tagged with Tag4-8 and scFv-E6 tagged with Tag18-1 significantly reduced the rate of synuclein-positive cells.

[0232] Example 8: Enhancement of Intracellular Antibody Activity by Stabilization (Enhancing Effect of Antibody Restoring CFTR Function) The cystic fibrosis transmembrane conductance regulator (CFTR; UniProtKB / Swiss-Prot: P13569.3) is an anion channel expressed in epithelial membrane cells throughout the body, and its abnormality causes cystic fibrosis. The CFTR F508 deletion mutation (CFTRΔF508) is known to be the most common mutation of CFTR, resulting in the deletion of phenylalanine at position 508 due to a deletion of three nucleotides. As a result, CFTRΔF508 does not fold normally, exhibits a strong tendency to form aggregates, and is thought to translocate to membranes at a lower level than the wild-type. scFv-C2 (SEQ ID NO: 1035: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMRLGLFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQ SPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRGDVPPTFGQGTKVEIKAAA) binds to the NBD1 domain of CFTR and can inhibit NBD1 aggregate formation (Lovato et al., Protein Engineering, Design and Selection, 20(12):607-614, 2007). It also has the effect of increasing the amount of CFTRΔF508 translocation to the membrane. In this example, we evaluated whether adding a tag (Tag18-1) to scFv-C2 could inhibit aggregation of the NBD1 domain mutant ΔF508 in HeLa cells. Plasmid vectors expressing these proteins were transfected into HeLa cells using Lipo3000™. The antibodies were myc-tagged and detected with a rabbit anti-myc tag antibody, while NBD1 was His-tagged and detected with a mouse anti-His tag antibody. The mouse anti-His tag antibody was detected with an Alexa 633-labeled anti-mouse IgG antibody, and the rabbit anti-myc tag antibody was detected with an Alexa 488-labeled anti-rabbit IgG antibody.The fluorescently stained cells were observed using a Keyence BZ-X800.

[0233] First, the aggregation rate of scFv-C2 in HeLa cells was 82%. In contrast, the aggregation rate of scFv-C2 bearing Tag18-1 at the N-terminus was 31%, and the aggregation rate of scFv-C2 bearing Tag18-1 at both the N-terminus and C-terminus was 4.6% (see Figures 3A and 3B). The aggregation rate of the wild-type NBD1 domain was 74% in scFv-C2-expressing cells, and the aggregation rate of the NBD1 ΔF508 mutant was 85% in scFv-C2-expressing cells. However, in scFv-C2-expressing cells bearing Tag18-1 at both the N-terminus and C-terminus, the aggregation rate of the wild-type NBD1 domain was 32%, and the aggregation rate of the NBD1 ΔF508 mutant was 43%. This indicates that adding a tag to scFv-C2 suppresses the aggregation of scFv-C2 itself, thereby suppressing the formation of aggregates by NBD1. It is expected that the suppression of NBD1 aggregation will contribute to improving the expression level of NBD1 on the cell membrane.

[0234] Example 9: Effect of Amino Acid Substitution on Existing Tags The above examples demonstrated that amino acid substitutions that meet the conditions that the peptide tag of the present disclosure should have, particularly amino acid substitutions to P or N, enhance the aggregation rate-reducing effect of the tag. In this example, some amino acids in the PEST sequence were substituted with N, and the aggregation rates of scFv (Y14-259) bearing tags before and after the substitution were examined.

[0235]

[0236] When each was expressed in cells and the aggregation rate of the scFv was evaluated in the same manner as in Example 1, the aggregation rate of the scFv having the tag before substitution was 50.3%, while the aggregation rate of the scFv having the tag after substitution was 18.0%. Thus, it was revealed that amino acid substitutions that meet the conditions that the peptide tag of the present disclosure should have, particularly amino acid substitutions to P or N, enhance the protein aggregation-inhibiting effect of the tag.

[0237] As described above, various proteins, including antibodies, form aggregates within cells, which can partially or completely impair their function. Protein tags that suppress aggregate formation can suppress the aggregation of these proteins, thereby allowing the proteins to exert their intended functions. Tags with a relatively low ratio of acidic amino acids can be useful in situations where tags with a high ratio of acidic amino acids are difficult to use.

Claims

1. 1. A method for selecting or identifying an amino acid sequence, comprising: (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids; (b) 20% or more of the amino acids constituting the amino acid sequence are selected from the group consisting of F, P, Y, G, S, Q, N, and A; Obtaining an amino acid sequence; selecting or identifying an amino acid sequence of a peptide tag that, when a fusion protein of a peptide tag having the selected or identified amino acid sequence and a reference protein is expressed in a mammalian cell (preferably a human cell), reduces the percentage of cells in which the fusion protein forms aggregates within the cell, or reduces the percentage to a predetermined value or less; obtaining a peptide tag having the amino acid sequence or a nucleic acid encoding the peptide tag; Including, method.

2. The method described in claim 1, wherein the aggregation rate of the reference protein exceeds 30% and the specified value is 10% or more lower than the aggregation rate.

3. The method of claim 1, wherein the obtained amino acid sequences are a group of amino acid sequences encoded by the coding region of the human genome.

4. The method of claim 1, wherein the obtained amino acid sequence comprises a neoantigen.

5. A method for selecting or identifying an amino acid sequence, comprising: (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids; (b) 30% or more but less than 70% of the amino acids constituting the amino acid sequence are selected from the group consisting of F, P, Y, G, S, Q, N, and A, and 60% or less of the amino acids constituting the amino acid sequence are S; (c) 10% or less of the amino acids constituting the amino acid sequence are selected from the group consisting of M, T, W, C, I, V, and L; and (d) A and G each account for less than 10% of the amino acids constituting the amino acid sequence; Obtaining a candidate amino acid sequence; selecting or identifying an amino acid sequence of a peptide tag that, when a fusion protein of an intracellular antibody and a peptide tag having the selected or identified candidate amino acid sequence is expressed in mammalian cells, causes the proportion of cells in which the fusion protein forms aggregates (aggregate formation rate) to be equal to or less than a predetermined value, wherein the aggregate formation rate of the intracellular antibody exceeds 10% and the predetermined value is 10% or more lower than the aggregation rate; obtaining a peptide tag having the amino acid sequence or a nucleic acid encoding the peptide tag; method.

6. The method described in claim 5, wherein the agglutination rate of the intracellular antibody exceeds 30% and the specified value is 10% or more lower than the agglutination rate.

7. The candidate amino acid sequence 5% or less of the amino acids that make up the amino acid sequence are basic amino acids The method of claim 5 further comprising:

8. The method described in claim 5, wherein the agglutination rate of the intracellular antibody exceeds 40% and the specified value is 20% or more lower than the agglutination rate.

9. The method described in claim 5, wherein the mammalian cells are human cells.

10. The candidate amino acid sequence is an amino acid sequence encoded by the coding region of the human genome. The method of claim 5, wherein the group is

11. The method described in claim 5, wherein the intracellular antibody is an scFv.

12. The method described in claim 5, further comprising obtaining the amino acid sequence of a fusion protein of the selected or identified peptide tag and the intracellular antibody or a nucleic acid sequence encoding the amino acid sequence.

13. A peptide, (a) 5% or more but less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids; (b) 20% or more, preferably 30% or more, of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A; The intracellular aggregation of a protein linked to the peptide can be reduced. peptide.

14. (c) The peptide according to claim 13, wherein 30% or less, preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of M, T, W, C, I, V, and L.

15. (d) A or G each accounts for less than 10% of the amino acids constituting the amino acid sequence.

16. The peptide of claim 13, (a) 20% or more and less than 45% of the amino acids constituting the amino acid sequence are acidic amino acids, and (b) 30% or more and less than 70% of the amino acids constituting the amino acid sequence are amino acids selected from the group consisting of F, P, Y, G, S, Q, N, and A, (c) 20% or less of the amino acids constituting the amino acid sequence are selected from the group consisting of M, T, W, C, I, V, and L; (d) A or G each accounts for less than 10% of the amino acids constituting the amino acid sequence; peptide.

17. A peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 11.

18. A nucleic acid encoding the peptide described in claim 13.

19. A protein expression vector comprising the nucleic acid of claim 18 operably linked to a regulatory sequence and a nucleic acid encoding a target protein in frame with the nucleic acid.

20. The protein expression vector according to claim 7 , wherein the target protein is an antibody or an antigen-binding fragment of an antibody.

21. The protein expression vector of claim 20, wherein the antigen-binding fragment of an antibody is a single-chain Fv (scFv).

22. A fusion protein of the peptide described in claim 13 and a target protein.

23. The fusion protein of claim 22, wherein the target protein is an antibody or an antigen-binding fragment of an antibody.

24. The fusion protein of claim 23 , wherein the antigen-binding fragment of an antibody is a single-chain Fv (scFv).

25. A protein-producing cell comprising the nucleic acid of claim 18 operably linked to a regulatory sequence and a nucleic acid encoding a target protein in frame with said nucleic acid.