Polypeptide and use thereof

Abstract

This polypeptide is for improving the antigen-binding properties of an antigen-binding polypeptide in a cell. The polypeptide comprises 3 to 20 (inclusive) amino acid residues, wherein 57.0% to 100.0% (exclusive) of amino acid residues constituting the polypeptide comprises acidic amino acid residues. Also provided is a fusion polypeptide comprising the polypeptide and an antigen-binding polypeptide.

Description

Polypeptides and their uses

[0001] This disclosure relates to polypeptides and uses thereof. This application claims priority to Japanese Patent Application No. 2024-011182, filed on January 29, 2024, the contents of which are incorporated herein by reference.

[0002] When selective antibodies against intracellular proteins are obtained by phage antibody display or animal immunization and then expressed intracellularly to act on target antigen proteins, it is known that in many cases the expressed antibodies themselves aggregate, preventing the desired effect from being fully exerted.

[0003] Furthermore, if antibodies aggregate intracellularly, not only will the desired effect not be exerted, but they may also cause cytotoxicity, suggesting concerns that their use for therapeutic purposes may pose a high safety risk (see, for example, Non-Patent Document 1). Therefore, in order to obtain antibodies that act on intracellular target antigen proteins, antibody engineering is required to enable them to function intracellularly, but this is generally not easy to accomplish.

[0004] In recent years, peptide fusion techniques capable of suppressing intracellular antibody self-aggregation have been reported, making it possible to obtain stable antibodies within cells (see, for example, Patent Document 1).

[0005] International Publication No. 2019 / 004213

[0006] H Kabayama et al., An ultra-stable cytoplasmic antibody engineered for in vivo applications, nature communications, Vol. 11, No. 336, pp. 1-20, 2020.

[0007] However, the inventors' studies have revealed that when an antibody fused with a peptide described in Patent Document 1 or the like is expressed intracellularly, the amount of antibody expressed intracellularly can be significantly reduced depending on the type of fused peptide, and there is a risk that the binding effect to the intended target antigen protein within the cell may not be fully exerted. In order to use an antigen-binding polypeptide such as an intracellular antibody for medical purposes, the expression level within the cell is likely to affect the level of medicinal efficacy, so it is required to express it more stably and to maintain a more excellent binding function to the target antigen protein within the cell, and there is a need for the development of a technology that can provide such effects.

[0008] The present disclosure has been made in view of the above circumstances, and provides a polypeptide that can improve the intracellular antigen-binding ability of an antigen-binding polypeptide.

[0009] That is, the present disclosure includes the following aspects: A polypeptide according to one aspect of the present disclosure is a polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic amino acids.

[0010] The polypeptide of the above aspect can improve the intracellular antigen-binding ability of an antigen-binding polypeptide.

[0011] FIG. 1 is a diagram showing the results of Experimental Example 1. FIG. 2 is a diagram showing the results of Experimental Example 2. FIG. 3 is a diagram showing the results of Experimental Example 3. FIG. 4 is a diagram showing the results of Experimental Example 5. FIG. 6 is a diagram showing the results of Experimental Example 6. FIG. 7 is a diagram showing the results of Experimental Example 8. FIG. 9 is a diagram showing the results of Experimental Example 10. FIG. 11 is a diagram showing the results of Experimental Example 12.

[0012] <Definition of Terms> As used herein, "polypeptide" is also referred to as "peptide" or "protein." "Polypeptide" refers to a compound in which two or more amino acids are linked by peptide bonds. A polypeptide may include a structure modified with, for example, sugars. The amino acids constituting a polypeptide may be naturally occurring amino acids, or may be known analogs of naturally occurring amino acids that can function in the same way as naturally occurring amino acids. The amino acids may be L-amino acids or D-amino acids.

[0013] As used herein, "acidic amino acid" refers to an amino acid having an isoelectric point of 3.9 or less. The acidic amino acid may be a naturally occurring amino acid or a known analogue of a naturally occurring amino acid. Examples of naturally occurring acidic amino acids include aspartic acid (D) and glutamic acid (E).

[0014] As used herein, the term "basic amino acid" refers to an amino acid having an isoelectric point of 7.4 or higher. A basic amino acid may be a naturally occurring amino acid or a known analogue of a naturally occurring amino acid. Examples of naturally occurring basic amino acids include histidine (H), lysine (K), and arginine (R).

[0015] As used herein, the term "neutral amino acid" refers to an amino acid other than the above-mentioned acidic amino acids and basic amino acids. That is, a neutral amino acid refers to an amino acid having an isoelectric point greater than 3.9 and less than 7.4. A neutral amino acid may be a naturally occurring amino acid or a known analogue of a naturally occurring amino acid. Examples of naturally occurring neutral amino acids include alanine (A), asparagine (N), cysteine ​​(C), phenylalanine (F), glycine (G), isoleucine (I), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

[0016] As used herein, the term "antigen" refers to any molecule to which an antigen-binding polypeptide can bind. Specific examples of antigens include proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleic acids, portions thereof, and combinations thereof. Antigens may be synthetically produced or derived from biological samples. Examples of such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, and liquids containing killed or inactivated whole cells or cell lysates.

[0017] As used herein, the term "antigen-binding polypeptide" refers to a polypeptide that specifically binds to an antigen (target molecule). An antigen-binding polypeptide has all or part of a region that has antigen-binding ability. Examples of antigen-binding polypeptides include, but are not limited to, antibodies and antigen-binding fragments thereof, peptide aptamers, and the like.

[0018] As used herein, the term "antibody" may refer to a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies may be natural antibodies or antibodies produced using genetic engineering. Natural antibodies may be derived from biological species, including, but not limited to, humans, mice, rats, monkeys, goats, rabbits, camels, llamas, cows, and chickens. Antibodies produced using genetic engineering include, but are not limited to, synthetic antibodies, recombinant antibodies, and mutated antibodies produced from natural antibodies. Antibodies produced using genetic engineering techniques also include antibodies that have been modified in the same manner as in the case of genetically modifying natural antibodies, as described above. Monoclonal antibodies include multispecific antibodies, single-chain antibodies, Fab fragments, Fab' fragments, F(ab') fragments, and F(ab') fragments. ２These include fragments, Fvs (variable fragments of antibodies), single-chain Fvs (scFvs), dsFvs (disulfide stabilized Fvs), dAbs (single domain antibodies), VHs, VLs, Intrabodies, minibodies, nanobodies (variable domains of heavy chain antibodies, VHHs), vNARs, diabodies, and full-length or partial anti-idiotype (anti-Id) antibodies.

[0019] As used herein, the term "intrabody" refers to an antibody that specifically binds to an antigen within a cell (intrabody).

[0020] As used herein, the term "nucleic acid" is also referred to as a "nucleotide" or a "polynucleotide." Unless otherwise specified, the term "nucleic acid" encompasses both DNA and RNA.

[0021] As used herein, the term "expression vector" refers to a vector that contains a regulatory sequence and a nucleic acid encoding a protein operably linked to the regulatory sequence, and that can transcribe mRNA encoding the protein in a cell or in a test tube. An expression vector may contain, for example, an origin of replication for replication in a cell, and a regulatory sequence and a nucleic acid encoding the protein operably linked to the regulatory sequence. An expression vector may have a selection marker, such as a drug resistance gene, for selecting cells into which it has been introduced.

[0022] The control sequence for expression in mammalian cells is not particularly limited, but examples thereof include promoters of RNA polymerase II, such as promoters of cytomegalovirus (CMV) immediate early (IE), HSV thymidine kinase, SV40 early, SV40 late, retroviral LTR, and metallothionein I.

[0023] Examples of promoters for expression in E. coli include lac, trp, lacI, lacZ, T3, T7, gpt, lambda PR, lambda PL, and 3-phosphoglycerate kinase promoters.

[0024] Promoters for in vitro expression are not particularly limited, but examples include SP6, T7, and T3 promoters.

[0025] Examples of expression vectors include plasmids, phages, phagemids, cosmids, fosmids, artificial chromosomes, retroviral vectors, lentiviral vectors, measles virus vectors, vaccinia virus vectors, adenoviral vectors, adeno-associated virus vectors, and Sendai virus vectors.

[0026] <Polypeptide for improving intracellular antigen-binding ability of an antigen-binding polypeptide> The polypeptide of this embodiment is a polypeptide for improving intracellular antigen-binding ability of an antigen-binding polypeptide. The polypeptide of this embodiment consists of 3 to 20 amino acids. More than 57.0% but less than 100.0% of the amino acids constituting the polypeptide of this embodiment are acidic amino acids.

[0027] As shown in Experimental Example 6 below, the inventors have found that a fusion polypeptide in which a polypeptide described in Patent Document 1, etc., which is known to increase intracellular antibody stability, is attached to the N-terminus of an antigenic polypeptide has an extremely low intracellular expression level, and as a result, the antigen-binding polypeptide is unable to fully exert its function within the cell.

[0028] On the other hand, it is known that the presence of "E" in the A site of the ribosome, "P," "G," or "D" in the P site, and "E" or "D" in the E site have a significant impact on ribosome stalling, and that the presence of a DE / P motif and / or a small amino acid at the N-terminus destabilizes ribosome translation (see Reference 1: "K Chyzynska et al., "Deep conservation of ribosome stall sites across RNA processing genes.", NAR Genomics and Bioinformatics, Vol. 3, Issue 2, 2021, pp.1-26.").

[0029] Based on the description in Reference 1, the inventor hypothesized that a polypeptide that contains consecutive acidic amino acids and is relatively long, i.e., 21 or more amino acids, destabilizes ribosomal translation, resulting in a significant decrease in the expression level of the antigen-binding polypeptide within cells. Therefore, although this is beneficial from the perspective of inhibiting intracellular antibody aggregation, it is not suitable for medical applications.

[0030] When antigen-binding polypeptides such as intrabodies are used for medical purposes, the expression level of the antigen-binding polypeptide within cells is related to its efficacy, and therefore there is a need to develop technologies that enable stable expression within cells and improve the ability to bind to target antigens.

[0031] As a result of intensive research based on the above-mentioned problems and hypotheses, the inventors discovered that by adding a polypeptide consisting of an amino acid sequence with acidic amino acids (E and D) as the main backbone and having a specific structure to the N-terminus of an antigen-binding polypeptide, it is possible to improve the binding ability to a target antigen in cells without significantly reducing the expression level of the antigen-binding polypeptide in cells, and thus completed the polypeptide of this embodiment.

[0032] By adding the polypeptide of this embodiment to the N-terminus of an antigen-binding polypeptide, when a fusion polypeptide of the polypeptide of this embodiment and an antigen-binding polypeptide is expressed in cells, the translation rate of ribosomes is slightly controlled, the folding efficiency of the antigen-binding polypeptide is improved, and the proportion of antigen-binding polypeptides that remain correctly folded in cells increases. As a result, it is presumed that the binding ability of the antigen-binding polypeptide to the antigen in cells is improved.

[0033] The polypeptide of this embodiment can also be said to be a polypeptide that controls the translation rate of an antigen-binding polypeptide in cells and improves the folding efficiency of the antigen-binding polypeptide, or to be a polypeptide that increases the proportion of antigen-binding polypeptides that maintain correct folding in cells.

[0034] However, the mechanism by which the polypeptide of this embodiment improves the antigen-binding activity of the antigen-binding polypeptide within cells is not limited to the above mechanism.

[0035] Improved intracellular antigen-binding ability of an antigen-binding polypeptide can be evaluated, for example, by the following method. First, an expression vector containing a nucleic acid encoding an antigen-binding polypeptide with the polypeptide of this embodiment attached to its N-terminus and an expression vector containing only a nucleic acid encoding the antigen-binding polypeptide without the polypeptide of this embodiment attached are prepared. These expression vectors are then introduced into cells and expressed intracellularly. The antigen-binding polypeptide is then recovered by immunoprecipitation, and the amount of the antigen-binding polypeptide and the intracellular target antigen bound to it are detected by Western blotting. The amount of intracellular target antigen per antigen-binding polypeptide detected is then compared between cells with and without the polypeptide of this embodiment attached, and whether the amount of intracellular target antigen bound to the polypeptide of this embodiment is increased compared to cells without the polypeptide of this embodiment attached can be evaluated to determine whether intracellular antigen-binding ability of the antigen-binding polypeptide is improved.

[0036] For example, the binding amount of an antigen-binding polypeptide having a polypeptide of this embodiment attached to its N-terminus to an intracellular target antigen can be, but is not limited to, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than the binding amount of an antigen-binding polypeptide not having a polypeptide of this embodiment attached to an intracellular target antigen.

[0037] Furthermore, as shown in Experimental Example 12 described below, cells expressing an antigenic polypeptide exhibit suppressed cell proliferation compared to cells not expressing the antigenic polypeptide, but a fusion polypeptide in which the polypeptide of this embodiment is affixed to the N-terminus of an antigen-binding polypeptide exhibits reduced cell proliferation suppression compared to when the antigenic polypeptide alone is expressed in cells. Furthermore, compared to when a fusion polypeptide in which a polypeptide described in Patent Document 1, etc., which is known to increase intracellular antibody stability, is affixed to the N-terminus of an antigen-binding polypeptide, the fusion polypeptide in which the polypeptide of this embodiment is affixed to the N-terminus of an antigen-binding polypeptide exhibits reduced cell proliferation suppression. In other words, it can be said that the polypeptide of this embodiment can impart the effect of reducing cell proliferation suppression when the antigen-binding polypeptide is expressed in cells.

[0038] The effect of reducing cell proliferation inhibition can be evaluated, for example, by the following method. First, an expression vector containing a nucleic acid encoding an antigen-binding polypeptide to which the polypeptide of this embodiment has been attached at the N-terminus, and an expression vector containing only a nucleic acid encoding the antigen-binding polypeptide without the polypeptide of this embodiment, are prepared. These expression vectors are then introduced into cells and expressed in the cells. Untreated cells without the vector are also prepared as a control. Next, using an Incucyte S3 (Sartorius), each cell is cultured while imaging over time. After 40 hours from the start of culture, the confluence (%) of cells in the culture vessel is calculated using the Incucyte analysis module. Next, the cell confluence (%) after 40 hours from the start of culture is compared between cells with and without the polypeptide of this embodiment attached. The presence or absence of the effect of reducing cell proliferation inhibition can be evaluated by determining whether the cell confluence (%) of cells with the polypeptide of this embodiment attached is higher than that of cells without the polypeptide of this embodiment attached. Furthermore, the presence or absence of an effect of reducing cell proliferation inhibition can be evaluated by checking whether the rate of reduction in cell confluence (%) of cells to which the polypeptide of this embodiment has been administered is smaller than the rate of reduction in cell confluence (%) of untreated cells to which no vector has been introduced, compared with cells to which the polypeptide of this embodiment has not been administered.

[0039] For example, the confluence (%) of cells in which a fusion polypeptide in which the polypeptide of this embodiment is attached to the N-terminus of an antigen-binding polypeptide is expressed can be, but is not limited to, 1.1-fold, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, or more than the confluence (%) of cells in which the antigenic polypeptide alone is expressed.

[0040] Furthermore, for example, in cells in which a fusion polypeptide in which the polypeptide of this embodiment is attached to the N-terminus of an antigen-binding polypeptide is expressed intracellularly, the rate of decrease in cell confluence Y (%) relative to the confluence X (%) of untreated cells {(1-Y / X) x 100(%)} can be, for example, but is not limited to, 40%, 30%, 20%, 10%, or less.

[0041] The polypeptide of this embodiment will be described in detail below.

[0042] The polypeptide of this embodiment consists of 3 or more amino acids, preferably 4 or more, more preferably 5 or more, even more preferably 6 or more, even more preferably 7 or more, still more preferably 8 or more, and particularly preferably 9 or more. On the other hand, the polypeptide of this embodiment consists of 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less amino acids.

[0043] When the number of amino acids constituting the polypeptide of this embodiment is at least the above-mentioned lower limit and is at most the above-mentioned upper limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target antigen can be improved without significantly reducing the intracellular expression level of the fusion polypeptide of this embodiment and the antigen-binding polypeptide. On the other hand, when the number of amino acids constituting the polypeptide of this embodiment is less than the lower limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target is reduced. Furthermore, when the number of amino acids constituting the polypeptide of this embodiment is greater than the upper limit, the risk of a reduction in the intracellular expression level of the fusion polypeptide of this embodiment and the antigen-binding polypeptide is increased. Therefore, when the number of amino acids constituting the polypeptide of this embodiment is within the above-mentioned numerical range, the fusion polypeptide of this embodiment and the antigen-binding polypeptide of this embodiment does not significantly reduce the intracellular expression level, and the presence of antigen-binding polypeptides that maintain correct folding in cells is increased, resulting in improved intracellular antigen-binding ability of the antigen-binding polypeptide.

[0044] That is, the polypeptide of this embodiment consists of, for example, 3 to 20 amino acids, 4 to 20 amino acids, 5 to 20 amino acids, 6 to 20 amino acids, 7 to 20 amino acids, 8 to 20 amino acids, or 9 to 20 amino acids.

[0045] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 19 amino acids, 4 to 19 amino acids, 5 to 19 amino acids, 6 to 19 amino acids, 7 to 19 amino acids, 8 to 19 amino acids, or 9 to 19 amino acids.

[0046] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 18 amino acids, 4 to 18 amino acids, 5 to 18 amino acids, 6 to 18 amino acids, 7 to 18 amino acids, 8 to 18 amino acids, or 9 to 18 amino acids.

[0047] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 17 amino acids, 4 to 17 amino acids, 5 to 17 amino acids, 6 to 17 amino acids, 7 to 17 amino acids, 8 to 17 amino acids, or 9 to 17 amino acids.

[0048] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 16 amino acids, 4 to 16 amino acids, 5 to 16 amino acids, 6 to 16 amino acids, 7 to 16 amino acids, 8 to 16 amino acids, or 9 to 16 amino acids.

[0049] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 15 amino acids, 4 to 15 amino acids, 5 to 15 amino acids, 6 to 15 amino acids, 7 to 15 amino acids, 8 to 15 amino acids, or 9 to 15 amino acids.

[0050] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 14 amino acids, 4 to 14 amino acids, 5 to 14 amino acids, 6 to 14 amino acids, 7 to 14 amino acids, 8 to 14 amino acids, or 9 to 14 amino acids.

[0051] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 13 amino acids, 4 to 13 amino acids, 5 to 13 amino acids, 6 to 13 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, or 9 to 13 amino acids.

[0052] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 12 amino acids, 4 to 12 amino acids, 5 to 12 amino acids, 6 to 12 amino acids, 7 to 12 amino acids, 8 to 12 amino acids, or 9 to 12 amino acids.

[0053] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 11 amino acids, 4 to 11 amino acids, 5 to 11 amino acids, 6 to 11 amino acids, 7 to 11 amino acids, 8 to 11 amino acids, or 9 to 11 amino acids.

[0054] Furthermore, the polypeptide of this embodiment consists of, for example, 3 to 10 amino acids, 4 to 10 amino acids, 5 to 10 amino acids, 6 to 10 amino acids, 7 to 10 amino acids, 8 to 10 amino acids, or 9 to 10 amino acids.

[0055] In particular, the polypeptide of this embodiment preferably consists of 9 to 20 amino acids, more preferably 9 to 18 amino acids, even more preferably 9 to 15 amino acids, particularly preferably 9 to 12 amino acids, and most preferably 9 amino acids.

[0056] The amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among these, the polypeptide of this embodiment is preferably composed of L-amino acids.

[0057] The polypeptide of this embodiment has a main backbone composed of acidic amino acids. Specifically, of the amino acids constituting the polypeptide of this embodiment, more than 57.0% but less than 100.0%, preferably 60.0% to 90.0%, more preferably 65.0% to 80.0%, even more preferably 66.0% to 78.0%, and particularly preferably 66.7% to 77.8% are acidic amino acids.

[0058] When the ratio of acidic amino acids to the amino acids constituting the polypeptide of this embodiment is above the above-mentioned lower limit or equal to or greater than the above-mentioned lower limit, the binding ability of a fusion polypeptide of this embodiment and an antigen-binding polypeptide to an intracellular target antigen can be improved. On the other hand, when the number of amino acids constituting the polypeptide of this embodiment is less than the above-mentioned upper limit or equal to or less than the above-mentioned upper limit, the binding ability of a fusion polypeptide of this embodiment and an antigen-binding polypeptide to an intracellular target can be improved without significantly reducing the expression level of the fusion polypeptide of this embodiment and an antigen-binding polypeptide. Therefore, when the ratio of acidic amino acids to the amino acids constituting the polypeptide of this embodiment is within the above-mentioned numerical range, the fusion polypeptide of this embodiment and an antigen-binding polypeptide of this embodiment does not significantly reduce the expression level of the fusion polypeptide of this embodiment and an antigen-binding polypeptide, and the presence of antigen-binding polypeptides that maintain correct folding in cells can be increased, resulting in improved antigen-binding ability of the antigen-binding polypeptide in cells.

[0059] In this specification, the ratio of a specific amino acid to the amino acids constituting a polypeptide is expressed as a percentage (%), calculated by dividing the number of specific amino acids contained in the polypeptide by the total number of amino acids constituting the polypeptide and multiplying the result by 100.

[0060] Acidic amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among these, the polypeptide of this embodiment preferably has glutamic acid at the N-terminus.

[0061] The polypeptide in this embodiment preferably consists of acidic amino acids and neutral amino acids.

[0062] Of the amino acids constituting the polypeptide of this embodiment, more than 0.0% and not more than 40.0%, preferably 10.0% to 40.0%, more preferably 20.0% to 35.0%, even more preferably 22.0% to 34.0%, and particularly preferably 22.2% to 33.3% are neutral amino acids.

[0063] When the ratio of neutral amino acids to the amino acids constituting the polypeptide of this embodiment is at least the above-mentioned lower limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target antigen can be improved without significantly reducing the intracellular expression level of the fusion polypeptide of this embodiment and the antigen-binding polypeptide. On the other hand, when the ratio of neutral amino acids to the amino acids constituting the polypeptide of this embodiment is not more than the above-mentioned upper limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target can be improved. Therefore, when the ratio of neutral amino acids to the amino acids constituting the polypeptide of this embodiment is within the above-mentioned numerical range, the presence of antigen-binding polypeptides that maintain correct folding in cells increases, and as a result, the intracellular binding ability of the antigen-binding polypeptide can be further improved.

[0064] Neutral amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among them, neutral amino acids are preferably those whose side chains are hydrophobic aliphatic groups or hydrophilic groups, more preferably proline, glycine, or serine, and particularly preferably proline.

[0065] Of the amino acids constituting the polypeptide of this embodiment, preferably 0.0% to 40.0%; more preferably 10.0% to 40.0%; even more preferably 20.0% to 35.0%; particularly preferably 22.0% to 34.0%; and most preferably 22.2% to 33.3%; are prolines.

[0066] When the ratio of proline to the amino acids constituting the polypeptide of this embodiment is at least the above-mentioned lower limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target antigen can be improved without significantly reducing the intracellular expression level of the fusion polypeptide of this embodiment and the antigen-binding polypeptide. On the other hand, when the ratio of proline to the amino acids constituting the polypeptide of this embodiment is not more than the above-mentioned upper limit, the binding ability of the fusion polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target can be improved. Therefore, when the ratio of proline to the amino acids constituting the polypeptide of this embodiment is within the above-mentioned numerical range, the presence of antigen-binding polypeptides that maintain correct folding in cells increases, and as a result, the intracellular binding ability of the antigen-binding polypeptide can be further improved.

[0067] Alternatively, in another preferred embodiment, the polypeptide further contains basic amino acids in addition to acidic amino acids. The polypeptide of this embodiment may consist of acidic amino acids and basic amino acids, or may consist of acidic amino acids, neutral amino acids and basic amino acids.

[0068] Acidic amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among these, the polypeptide of this embodiment preferably has glutamic acid at the N-terminus.

[0069] Neutral amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among them, neutral amino acids are preferably those whose side chains are hydrophobic aliphatic groups or hydrophilic groups, more preferably proline, glycine, or serine, and particularly preferably proline.

[0070] Examples of basic amino acids constituting the polypeptide of this embodiment include those exemplified in the definitions of terms above. Among these, lysine is preferred as the basic amino acid.

[0071] That is, the polypeptide of this embodiment preferably further comprises a basic amino acid, and the basic amino acid is lysine. Alternatively, the polypeptide of this embodiment preferably consists of an acidic amino acid, a neutral amino acid, and a basic amino acid, wherein the acidic amino acid is glutamic acid or aspartic acid, the neutral amino acid is proline, and the basic amino acid is lysine. Alternatively, the polypeptide of this embodiment preferably consists of an acidic amino acid, a neutral amino acid, and a basic amino acid, wherein the acidic amino acid is glutamic acid or aspartic acid, the neutral amino acid is proline, the basic amino acid is lysine, and the N-terminus is glutamic acid. Alternatively, the polypeptide of this embodiment preferably consists of an acidic amino acid and a basic amino acid, wherein the acidic amino acid is glutamic acid or aspartic acid, and the basic amino acid is lysine. Alternatively, the polypeptide of this embodiment preferably consists of an acidic amino acid and a basic amino acid, wherein the acidic amino acid is glutamic acid or aspartic acid, the basic amino acid is lysine, and the N-terminus is glutamic acid.

[0072] The total proportion of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide of this embodiment is preferably 10.0% or more and 40.0% or less, more preferably 20.0% or more and 35.0% or less, even more preferably 22.0% or more and 34.0% or less, and particularly preferably 22.2% or more and 33.3% or less.

[0073] When the total ratio of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide of this embodiment is at least the above-mentioned lower limit, the binding affinity of the polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target antigen can be improved without significantly reducing the intracellular expression level of the polypeptide. On the other hand, when the total ratio of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide of this embodiment is at most the above-mentioned upper limit, the binding affinity of a fusion polypeptide of the polypeptide of this embodiment and the antigen-binding polypeptide to an intracellular target is improved. Therefore, when the total ratio of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide of this embodiment is within the above-mentioned numerical range, the presence of antigen-binding polypeptides that maintain correct folding in cells increases, and as a result, the intracellular binding affinity of the antigen-binding polypeptide can be further improved.

[0074] Of the amino acids constituting the polypeptide of this embodiment, preferably 0.0% to 11.1% are neutral amino acids, particularly preferably proline. When the proportion of neutral amino acids, preferably proline, relative to the amino acids constituting the polypeptide of this embodiment is within the above range, the intracellular antigen-binding ability of the antigen-binding polypeptide can be further improved.

[0075] Preferably, 11.1% to 22.2% of the amino acids constituting the polypeptide of this embodiment are basic amino acids, particularly preferably lysine. When the ratio of basic amino acids, preferably lysine, to the amino acids constituting the polypeptide of this embodiment is within the above range, the intracellular antigen-binding ability of the antigen-binding polypeptide can be further improved.

[0076] In a more preferred embodiment, the polypeptide includes, for example, a polypeptide consisting of an amino acid sequence represented by the following general formula (I) (hereinafter, sometimes referred to as "polypeptide (I)").

[0077]

[0078] In general formula (I), X １ and X４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; n1 and n2 each independently represent an integer of 0 to 3, and 0≦n1+n2≦5.

[0079] X １ and X ４ Examples of the neutral amino acid in the above include those exemplified in the definition of the term.

[0080] X １ and X ４ Examples of the basic amino acid in the above include those exemplified in the definition of the term.

[0081] That is, X in general formula (I) １ and X ４ are each independently a neutral amino acid selected from the group consisting of alanine (A), asparagine (N), cysteine ​​(C), phenylalanine (F), glycine (G), isoleucine (I), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y), or a basic amino acid selected from the group consisting of histidine (H), lysine (K), and arginine (R). １ and X ４ are each independently preferably a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain, or K, more preferably G, P, S, or K.

[0082] In the general formula (I), when n1 is 2 or more, a plurality of X １ may be the same or different.

[0083] In the general formula (I), when n2 is 2 or more, a plurality of X ４ may be the same or different.

[0084] X ２ Examples of the neutral amino acid in the above include those exemplified in the definition of the term.

[0085] That is, X in general formula (I) ２ is D or a neutral amino acid selected from the group consisting of alanine (A), asparagine (N), cysteine ​​(C), phenylalanine (F), glycine (G), isoleucine (I), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y). ２ is preferably D or a neutral amino acid whose side chain is a hydrophobic aliphatic group or a hydrophilic group, more preferably D, G, P, or S, and even more preferably D.

[0086] In general formula (I), X ３ is preferably E.

[0087] n1 and n2 are each independently preferably an integer of 0 or more and 3 or less, and 0≦n1+n2≦4, more preferably an integer of 0 or more and 3 or less, and 1≦n1+n2≦4, and even more preferably an integer of 0 or more and 3 or less, and 2≦n1+n2≦4.

[0088] Preferred examples of the polypeptide (I) include those shown in the following table. Note that these are examples of the preferred polypeptide (I), and the polypeptide (I) is not limited to these.

[0089]

[0090] Among them, preferred polypeptides (I) include EX ２ X ３ A polypeptide consisting of the amino acid sequence １ DEX ２ X ３ A polypeptide or EX comprising the amino acid sequence ２ X ３ EX ４ or a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, and 72, and more preferably polypeptide (I) is a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, and 72.

[0091] Alternatively, in another more preferred embodiment, the polypeptide may be, for example, a polypeptide consisting of an amino acid sequence represented by the following general formula (II) (hereinafter, sometimes referred to as "polypeptide (II)").

[0092]

[0093] In general formula (II), X １ , X ２ , X ３ and X ４ is the same as in the above general formula (I), and X ５ represents a neutral amino acid or a basic amino acid; n3 and n4 each independently represent an integer of 0 or more and 1 or less, and satisfy the relation: 1≦n3+n4≦2.

[0094] X ５ Examples of the neutral amino acid in the above include those exemplified in the definition of the term.

[0095] X ５ Examples of the basic amino acid in the above include those exemplified in the definition of the term.

[0096] That is, X in general formula (II) ５ is a neutral amino acid selected from the group consisting of alanine (A), asparagine (N), cysteine ​​(C), phenylalanine (F), glycine (G), isoleucine (I), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y), or a basic amino acid selected from the group consisting of histidine (H), lysine (K), and arginine (R). ５ is preferably a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain or K, more preferably G, P, S or K.

[0097] Preferably, n3 and n4 are each independently 1, and n3+n4=2.

[0098]

[0099] Among these, the preferred polypeptide (II) is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:82.

[0100] Specific examples of polypeptides in more preferred embodiments include, but are not limited to, those shown in the table below.

[0101]

[0102] Among these, the polypeptide of this embodiment is preferably a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 23 to 25, 31, 33, 34, and 40 to 42. Furthermore, the polypeptide of this embodiment is particularly preferably a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 25.

[0103] Nucleic Acids Encoding Polypeptides In one embodiment, the present disclosure provides nucleic acids encoding the polypeptides.

[0104] The nucleic acid of this embodiment may be a DNA molecule, an RNA molecule, or a hybrid molecule of DNA and RNA. Furthermore, the nucleic acid of this embodiment may be single-stranded or double-stranded.

[0105] The nucleic acid of this embodiment can be prepared by known genetic engineering techniques, chemical synthesis methods, etc. The specific base sequence of the nucleic acid of this embodiment is the amino acid sequence of the above polypeptide, preferably EX ２ X ３ The amino acid sequence of EX １ DEX ２ X ３ or EX ２ X ３ EX ４ A person skilled in the art can easily design such a polypeptide from the amino acid sequence of D or the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, 72, and 82, more preferably the amino acid sequence represented by any one of SEQ ID NOs: 23 to 25, 31, 33, 34, and 40 to 42, for example, by referring to a codon table.

[0106] Specific examples of nucleic acids encoding preferred polypeptides include those shown in the table below.

[0107]

[0108] Among these, the nucleic acid encoding the polypeptide of this embodiment is preferably a nucleic acid consisting of the base sequence represented by any one of SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22. Furthermore, the nucleic acid encoding the polypeptide of this embodiment is particularly preferably a nucleic acid consisting of the base sequence represented by SEQ ID NO: 5.

[0109] <Fusion Polypeptide> The fusion polypeptide of this embodiment comprises the above-described polypeptide and an antigen-binding polypeptide. Alternatively, it can be said that the fusion polypeptide of this embodiment comprises an antigen-binding polypeptide and the above-described polypeptide attached to the N-terminus of the antigen-binding polypeptide.

[0110] In the fusion polypeptide of this embodiment, the above polypeptide is attached to the N-terminus of the antigen-binding polypeptide, thereby increasing the proportion of antigen-binding polypeptides that maintain correct folding within cells, and as a result, the antigen-binding ability of the antigen-binding polypeptide within cells can be further improved.

[0111] [Antigen-binding polypeptide] Antigen-binding polypeptides include those exemplified by the definitions of the terms above.

[0112] It is known that because the intracellular environment is more reductive than the extracellular environment, typical antibodies that require disulfide bond formation for structural formation are difficult to express. Therefore, the antigen-binding polypeptide is preferably one that is stably expressed and active even in the above-mentioned reductive intracellular environment. Therefore, among these, the antigen-binding polypeptide is preferably a monoclonal antibody against a specific intracellular antigen, and more preferably one that contains one or more complementarity-determining regions (CDRs) of an antibody against a specific intracellular antigen. Specific examples of such antibodies include, for example, one that contains at least one of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of any monoclonal antibody. Preferably, the antibody contains at least two, at least three, at least four, at least five, or all of these.

[0113] Among these, preferred antigen-binding polypeptides include scFv, VH, VL, VHH, vNAR, and Fab, which are directed against specific antigens.

[0114] [Other Polypeptides] The fusion polypeptide of this embodiment may further comprise another polypeptide at the C-terminus of the fusion polypeptide. The fusion polypeptide of this embodiment may comprise a linker sequence consisting of approximately one to ten amino acids between the antigen-binding polypeptide and the other polypeptide. Examples of linker sequences include, but are not limited to, linker sequences consisting of approximately one to ten, preferably two to five, glycine residues.

[0115] When the fusion polypeptide of this embodiment is used for pharmaceutical purposes, it is preferable to use a pharmaceutically acceptable polypeptide among other polypeptides, or to not contain any other polypeptides.

[0116] Examples of other polypeptides include purification tag peptides, detection peptides, degradation-promoting peptides, Neh2 domain peptides, stress-responsive degradation peptides, and drug-binding peptides.

[0117] Examples of purification tag peptides include known tag peptides such as HA tag and His tag.

[0118] The detection peptide is not particularly limited, and examples thereof include, but are not limited to, fluorescent proteins and enzymes that emit light or change color upon reaction.

[0119] Examples of fluorescent proteins include, but are not limited to, BFP, EBFP, CFP, ECFP, Cypet, AmCyan1, GFP, EGFP, YFP, Venus, mKO, mOrange, RFP, DsRed, tdTomato, mcherry, mStrawberry, Azalea, mPlum, mAG, Kaede, Dronpa, Keima, KikG, KikGR, and UnaG.

[0120] Examples of enzymes that undergo a reaction to emit light or change color include, but are not limited to, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, and acetylcholinesterase.

[0121] Degradation-promoting peptides include, but are not limited to, HSC70-binding peptides, XIAP RING domain peptides, and the like.

[0122] Examples of Neh2 domain peptides include, but are not limited to, peptides derived from Nrf2.

[0123] Examples of stress-responsive degradation peptides include, but are not limited to, oxygen-dependent degradation domains such as peptides derived from Hiflα.

[0124] Nucleic Acid Encoding the Fusion Polypeptide In one embodiment, the present disclosure provides a nucleic acid encoding the fusion polypeptide.

[0125] The nucleic acid encoding the fusion polypeptide may be a DNA molecule, an RNA molecule, or a hybrid molecule of DNA and RNA, and may be single-stranded or double-stranded.

[0126] A nucleic acid encoding the fusion polypeptide can be prepared by known genetic engineering techniques, chemical synthesis methods, etc. A specific base sequence of the nucleic acid of this embodiment can be easily designed by those skilled in the art from the amino acid sequence of the fusion polypeptide, for example, by referring to a codon table.

[0127] The nucleic acid encoding the fusion polypeptide may contain a non-coding nucleic acid consisting of one to several bases between the nucleic acid encoding the polypeptide and the nucleic acid encoding the antigen-binding polypeptide, or between the nucleic acid encoding the antigen-binding polypeptide and the nucleic acid encoding the other polypeptide. The number of bases contained in the non-coding nucleic acid may be, for example, 1 to 100, 1 to 50, 1 to 30, or 1 to 10.

[0128] <Expression Vector> The expression vector of this embodiment contains a nucleic acid encoding the polypeptide.

[0129] Alternatively, in other embodiments, the expression vector comprises a nucleic acid encoding the fusion polypeptide.

[0130] In this embodiment, examples of the expression vector into which the nucleic acid encoding the polypeptide or the nucleic acid encoding the fusion polypeptide is inserted include those exemplified in the definitions of the terms above.

[0131] The expression vector can be constructed, for example, by using known genetic engineering techniques.

[0132] <Pharmaceutical Composition> In one embodiment, the present disclosure provides a pharmaceutical composition comprising a nucleic acid encoding the fusion polypeptide or an expression vector comprising the nucleic acid, and a pharmaceutically acceptable carrier.

[0133] Examples of pharmaceutically acceptable carriers include, for example, when the pharmaceutical composition of the present embodiment is formulated as an aqueous solution, pure water such as sterilized water, physiological saline, phosphate-buffered physiological saline, etc. When the pharmaceutical composition of the present embodiment is formulated as another appropriate solution, examples thereof include organic esters that can be introduced into the body, such as glycol, glycerol, or olive oil.

[0134] The pharmaceutical composition of this embodiment may further contain other ingredients such as, for example, a lubricant, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, a buffer, a stabilizer, a preservative, an excipient, an antioxidant, a viscosity adjuster, a colorant, a flavoring, or a sweetener.

[0135] The pharmaceutical composition of the present embodiment may be contained in a container, pack, dispenser, or the like together with instructions for use.

[0136] [Medicinal Use] The pharmaceutical composition of this embodiment can be used, for example, to treat or prevent diseases associated with intracellular antigens.

[0137] In one embodiment, the pharmaceutical composition of the present disclosure is prepared by introducing a nucleic acid encoding the fusion polypeptide or an expression vector containing the nucleic acid into cells of a subject, thereby expressing the fusion polypeptide in the cells of the subject, and controlling, i.e., suppressing or activating, preferably suppressing, the function of an antigen associated with a disease by the antigen-binding polypeptide, thereby treating or preventing the disease.

[0138] One aspect of "treatment" includes alleviating or alleviating at least one symptom associated with a target disease, slowing the progression of the disease, curing the disease, etc. One aspect of "prevention" includes preventing the onset of at least one symptom associated with a target disease, etc.

[0139] Living organisms that can be treated and prevented include, for example, humans and non-human animals. Specific examples of non-human animals include vertebrates such as fish, birds, and mammals. Mammals include laboratory animals such as mice, rats, rabbits, guinea pigs, and non-human primates; pet animals such as dogs and cats; and livestock such as pigs, cows, goats, sheep, and horses.

[0140] Examples of diseases to be treated or prevented include cancer, tumors, infectious diseases (viral infection, bacterial infection, etc.), autoimmune diseases or allergic diseases, inflammatory diseases, neurodegenerative diseases, and the like.

[0141] The administration route and administration method of the pharmaceutical composition of this embodiment are not particularly limited and can be selected appropriately depending on the target disease. It may be administered directly or indirectly to the affected area. Alternatively, cells expressing the fusion polypeptide may be administered. In one example, administration routes include oral, intrathecal, intracisternal, intraventricular, intravenous, intramuscular, subcutaneous, intratumoral, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine routes, as well as local administration and methods using a gene gun.

[0142] The dose and frequency of administration can be appropriately selected depending on the severity of symptoms, age, sex, body weight, administration form, specific type of disease, etc.

[0143] <Research Reagent> In one embodiment, the present disclosure provides a research reagent comprising a nucleic acid encoding the above-described polypeptide or an expression vector containing the nucleic acid. The research reagent may further contain other components in addition to the nucleic acid encoding the above-described polypeptide or the expression vector containing the nucleic acid. For such other components, reference can be made to those described above for the pharmaceutical composition.

[0144] The research reagent of this embodiment may be housed in a container, pack, dispenser, or the like together with instructions for use.

[0145] [Method of Use as a Research Tool] The research reagent of this embodiment can be used for functional analysis of intracellular antigens, such as polypeptides, etc. In one example, an antigen-binding polypeptide for the antigen to be functionally analyzed and a fusion polypeptide of the above polypeptide are expressed intracellularly, and the function of the antigen can be controlled, preferably inhibited, thereby allowing functional analysis of the antigen to be performed.

[0146] The target cells are not particularly limited. Examples of target cells include human cells and non-human animal cells. More specifically, non-human animal cells include vertebrate cells such as fish cells, bird cells, and mammalian cells. Mammalian cells include cells derived from laboratory animals such as mice, rats, rabbits, guinea pigs, and primates other than humans; pet animals such as dogs and cats; and livestock such as pigs, cows, goats, sheep, and horses.

[0147] The cells may be cultured cells or living cells, i.e., non-isolated cells present in a living body. Preferred examples of the cells include cultured human cells, living human cells, cultured cells of a non-human pathological model animal, and living cells of a non-human pathological model animal.

[0148] When introducing a viral vector into target cells, a method of suspending the viral vector in a cell culture medium can be used. Introduction of a non-viral vector into target cells can be achieved by, for example, electroporation, microinjection, lipofection, calcium phosphate, or DEAE-dextran.

[0149] Indicators of the results of inhibiting antigen function include phenotypic changes that appear in cells, tissues, or individuals, and antigen function can be analyzed based on these indicators. Indicators of cellular changes include changes in cellular phenotype, such as quantitative and / or qualitative changes in produced substances, changes in proliferation activity, changes in cell number, changes in morphology, changes in characteristics, and induction of apoptosis. Examples of produced substances that can be used include secreted proteins, surface antigens, intracellular proteins, mRNA, etc. Morphological changes include changes in process formation and / or the number of processes, changes in flatness, changes in elongation / aspect ratio, changes in cell size, changes in internal structure, heteromorphism / uniformity as a cell population, and changes in cell density. These morphological changes can be confirmed by observation under a microscope. Changes in characteristics that can be used include anchorage dependence, cytokine-dependent responsiveness, hormone dependence, drug resistance, cell motility, cell migration activity, pulsation, and changes in intracellular substances. Examples of cell motility include cell invasion activity and cell migration activity. Changes in intracellular substances include enzyme activity, mRNA amount, and Ca. ２＋ Alternatively, the amount of intracellular signaling substances such as cAMP, the amount of intracellular protein, etc. can be used. As an indicator of the tissue system, a functional change according to the tissue used can be used as a detection indicator. As an indicator of the biological system, changes in tissue weight, changes in the blood system, for example, changes in blood cell count, protein amount, enzyme activity, electrolyte amount, and changes in the circulatory system, for example, changes in blood pressure, heart rate, etc. can be used.

[0150] The method for measuring these detection indicators is not particularly limited, and methods such as absorbance, luminescence, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light scattering, and fluorescence resonance energy transfer can be used. These measurement methods are well known to those skilled in the art and can be appropriately selected depending on the purpose. For example, absorption spectrum can be measured using commonly used photometers or plate readers, luminescence can be measured using luminometers, and fluorescence can be measured using fluorometers. Mass can be measured using a mass spectrometer. Radioactivity can be measured using a measuring instrument such as a gamma counter depending on the type of radiation, fluorescence polarization can be measured using a BEACON (Takara Shuzo), surface plasmon resonance signal can be measured using a BIACORE (GE Healthcare), and time-resolved fluorescence, fluorescence resonance energy transfer, and the like can be measured using an ARVO (PerkinElmer). Furthermore, a flow cytometer or the like can also be used for measurement. These measurement methods may measure two or more detection indicators using a single method, and if convenient, two or more types of detection indicators can be measured simultaneously and / or consecutively to measure even more detection indicators. For example, fluorescence and fluorescence resonance energy transfer can be measured simultaneously in a fluorometer.

[0151] The research reagent of this embodiment can also be used for dynamic observation of an antigen, such as a polypeptide, within a cell. In one example, an antigen-binding polypeptide for the antigen to be subjected to dynamic observation and a fusion polypeptide of the above polypeptide are expressed within a cell, and the reaction between the antigen and the fusion polypeptide is detected, thereby enabling dynamic observation of the antigen. Dynamic observation of an antigen includes observation of antigen expression, antigen localization, etc. at a certain time or over time.

[0152] The reaction between the antigen and the fusion polypeptide can be detected, for example, by utilizing the fluorescence, luminescence, color development, etc. of the detection peptide contained in the fusion polypeptide.

[0153] <Method for expressing an antigen-binding polypeptide in a cell> In one embodiment, the present disclosure provides a method for expressing an antigen-binding polypeptide in a cell, comprising the step of expressing in the cell a nucleic acid encoding a fusion polypeptide of the above-mentioned polypeptide and the antigen-binding polypeptide.

[0154] Expression may be constitutive or transient. It may also be inducible. For constitutive expression, a nucleic acid in which a constitutively expressing promoter is linked upstream of the fusion polypeptide gene may be used. For transient expression, for example, RNA, rather than DNA, may be introduced into cells for expression. For inducible expression, a nucleic acid in which an inducible promoter is linked upstream of the fusion polypeptide gene may be used. Then, when expressing the antigen-binding polypeptide, an inducer, such as IPTG, may be introduced into the cells.

[0155] Target cells include the cells listed above as research reagents.

[0156] In the expression method of this embodiment, the antigen-binding polypeptide is expressed in a state fused with the above-mentioned polypeptide, i.e., as a fusion polypeptide. By containing the above-mentioned polypeptide, the fusion polypeptide increases the presence of antigen-binding polypeptides that maintain correct folding in cells compared to when the antigen-binding polypeptide is expressed alone. As a result, the antigen-binding ability of the antigen-binding polypeptide is improved in cells compared to when the antigen-binding polypeptide is expressed alone.

[0157] The expression method of this embodiment further comprises the step of introducing a nucleic acid encoding the above-described fusion polypeptide into a cell. The nucleic acid encoding the above-described fusion polypeptide may be introduced in the form of an expression vector. Specific introduction methods include the methods described above for research reagents. Therefore, in one embodiment, the present disclosure further provides a method for producing a cell that expresses an antigen-binding polypeptide within the cell. The production method comprises the step of introducing a nucleic acid encoding the above-described fusion polypeptide or an expression vector containing the nucleic acid into the cell. In addition, in one embodiment, the present disclosure provides a cell that expresses the above-described fusion polypeptide.

[0158] <Kit> In one embodiment, the present disclosure provides a kit for producing a nucleic acid encoding a fusion polypeptide comprising the above-mentioned polypeptide and an antigen-binding polypeptide, the kit comprising a nucleic acid encoding the polypeptide.

[0159] The kit of this embodiment can be a general-purpose kit that can easily prepare a nucleic acid encoding a fusion polypeptide for a desired antigen-binding polypeptide.

[0160] The nucleic acid encoding the above-mentioned polypeptide may be incorporated into an expression vector as described above. The nucleic acid may also contain factors necessary for protein expression, such as a promoter, a ribosome binding site, and a terminator, as well as a selection marker and a restriction enzyme recognition site.

[0161] The kit of this embodiment may further include at least one of buffers, restriction enzymes, other necessary reagents, instruments, and instructions for use.

[0162] <Additional Notes> The present disclosure can also be said to include the following aspects. (1) A polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic amino acids. (2) The polypeptide according to (1) above, wherein the polypeptide consists of 9 to 20 amino acids. (3) The polypeptide according to (1) or (2) above, wherein 60.0% to 90.0% of the amino acids constituting the polypeptide are acidic amino acids. (4) The polypeptide according to any one of (1) to (3) above, wherein 65.0% to 80.0% of the amino acids constituting the polypeptide are acidic amino acids. (4-1) The polypeptide according to any one of (1) to (4) above, wherein 66.0% to 78.0% of the amino acids constituting the polypeptide are acidic amino acids. (4-2) The polypeptide according to any one of (1) to (4-1) above, wherein 66.7% to 77.8% of the amino acids constituting the polypeptide are acidic amino acids. (5) The polypeptide according to any one of (1) to (4-2) above, wherein the N-terminus of the polypeptide is glutamic acid. (6) The polypeptide according to any one of (1) to (5) above, wherein the polypeptide consists of acidic amino acids and neutral amino acids. (7) The polypeptide according to (6) above, wherein 10.0% to 40.0% of the amino acids constituting the polypeptide are neutral amino acids. (8) The polypeptide according to (6) or (7) above, wherein 20.0% to 35.0% of the amino acids constituting the polypeptide are neutral amino acids. (8-1) The polypeptide according to any one of (6) to (8) above, wherein 22.0% to 34.0% of the amino acids constituting the polypeptide are neutral amino acids. (8-2) The polypeptide according to any one of (6) to (8-1) above, wherein 22.2% to 33.3% of the amino acids constituting the polypeptide are neutral amino acids. (9) The polypeptide according to any one of (6) to (8-2) above, wherein the side chains of the neutral amino acids are hydrophobic aliphatic groups or hydrophilic groups.(10) The polypeptide according to any one of (6) to (9), wherein the neutral amino acid is proline, glycine, or serine. (10-1) The polypeptide according to any one of (6) to (10), wherein the neutral amino acid is proline. (10-2) The polypeptide according to (10-1), wherein more than 0.0% and not more than 40.0% of the amino acids constituting the polypeptide are proline. (10-3) The polypeptide according to (10-1) or (10-2), wherein 10.0% or more and not more than 40.0% of the amino acids constituting the polypeptide are proline. (10-4) The polypeptide according to any one of (10-1) to (10-3), wherein 20.0% or more and not more than 35.0% of the amino acids constituting the polypeptide are proline. (10-5) The polypeptide according to any one of (10-1) to (10-4) above, wherein 22.0% to 34.0% of the amino acids constituting the polypeptide are proline. (10-6) The polypeptide according to any one of (10-1) to (10-5) above, wherein 22.2% to 33.3% of the amino acids constituting the polypeptide are proline. (11) The polypeptide according to any one of (1) to (5) above, further comprising a basic amino acid, and the basic amino acid is lysine. (11-1) The polypeptide according to (11) above, further comprising a neutral amino acid, and the total proportion of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide is 10.0% to 40.0%. (11-2) The polypeptide according to (11) or (11-1) above, wherein the total proportion of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide is 20.0% or more and 35.0% or less. (11-3) The polypeptide according to any one of (11) to (11-2) above, wherein the total proportion of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide is 22.0% or more and 34.0% or less. (11-4) The polypeptide according to any one of (11) to (11-3) above, wherein the total proportion of neutral amino acids and basic amino acids to the amino acids constituting the polypeptide is 22.2% or more and 33.3% or less.(11-5) The polypeptide according to any one of (11) to (11-4) above, wherein more than 0.0% and not more than 11.1% of the amino acids constituting the polypeptide are neutral amino acids. (11-6) The polypeptide according to any one of (11) to (11-5) above, wherein 0.0% to 11.1% of the amino acids constituting the polypeptide are proline. (11-7) The polypeptide according to any one of (11) to (11-6) above, wherein 11.1% to 22.2% of the amino acids constituting the polypeptide are basic amino acids. (11-8) The polypeptide according to any one of (11) to (11-7) above, wherein 11.1% to 22.2% of the amino acids constituting the polypeptide are lysine.

[0163] (12) The polypeptide according to any one of (1) to (11-8) above, which consists of an amino acid sequence represented by the following general formula (I):

[0164]

[0165] In general formula (I), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; n1 and n2 each independently represent an integer of 0 to 3, and 0≦n1+n2≦5.

[0166] (12-1) A polypeptide for improving the intracellular binding ability of an antigen-binding polypeptide to an antigen, the polypeptide consisting of an amino acid sequence represented by the following general formula (I):

[0167]

[0168] In general formula (I), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３is D or E; n1 and n2 each independently represent an integer of 0 to 3, and 0≦n1+n2≦5.

[0169] (12-2) The X １ and the X ４ and each independently represent a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain, or represent lysine. １ and the X ４ and each independently represent glycine, proline, serine, or lysine. ２ is aspartic acid or a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain. ２ (12-6) The polypeptide according to any one of (12) to (12-4) above, wherein X is aspartic acid, glycine, proline, or serine. ２ (12-7) The polypeptide according to any one of (12) to (12-5) above, wherein X is aspartic acid. ３ is glutamic acid. (12-8) The polypeptide according to any one of (12) to (12-7) above, wherein n1 and n2 satisfy 0≦n1+n2≦4. (12-9) The polypeptide according to any one of (12) to (12-8) above, wherein n1 and n2 satisfy 1≦n1+n2≦4. (12-10) The polypeptide according to any one of (12) to (12-9) above, wherein n1 and n2 satisfy 2≦n1+n2≦4. (12-11) The polypeptide is an EX ２ X ３ A polypeptide consisting of the amino acid sequence １ DEX ２ X ３ A polypeptide or EX comprising the amino acid sequence ２ X ３ EX ４The polypeptide according to any one of (12) to (12-10) above, which is a polypeptide consisting of the amino acid sequence of D or a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, and 72. (12-12) The polypeptide according to any one of (12) to (12-11) above, which is a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, and 72.

[0170] (13) The polypeptide according to any one of (1) to (11-8) above, which consists of an amino acid sequence represented by the following general formula (II):

[0171]

[0172] In general formula (II), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; X ５ represents a neutral amino acid or a basic amino acid; n3 and n4 each independently represent an integer of 0 or more and 1 or less, and satisfy the relation: 1≦n3+n4≦2.

[0173] (13-1) A polypeptide for improving the intracellular binding ability of an antigen-binding polypeptide to an antigen, the polypeptide consisting of an amino acid sequence represented by the following general formula (II):

[0174]

[0175] In general formula (II), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; X ５ represents a neutral amino acid or a basic amino acid; n3 and n4 each independently represent an integer of 0 or more and 1 or less, and satisfy the relation: 1≦n3+n4≦2.

[0176] (13-2) The X １ and the X４ and each independently represent a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain, or represent lysine. １ and the X ４ and each independently represent glycine, proline, serine, or lysine. ２ is aspartic acid or a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain. ２ (13-6) The polypeptide according to any one of (13) to (13-4), wherein X is aspartic acid, glycine, proline, or serine. ２ (13-7) The polypeptide according to any one of (13) to (13-5), wherein X is aspartic acid. ３ (13-8) The polypeptide according to any one of (13) to (13-6), wherein X is glutamic acid. ５ (13-9) The polypeptide according to any one of (13) to (13-7), wherein X is a neutral amino acid having a hydrophobic aliphatic group or a hydrophilic group in the side chain, or is lysine. ５ is glycine, proline, serine, or lysine. (13-10) The polypeptide according to any one of (13) to (13-9) above, wherein n3 and n4 are 1. (13-11) The polypeptide according to any one of (13) to (13-10) above, wherein n3 and n4 are 1.

[0177] (14) The polypeptide according to any one of (1) to (13-11) above, consisting of an amino acid sequence represented by any one of SEQ ID NOs: 23 to 25, 31, 33, 34, and 40 to 42. (14-1) A polypeptide for improving the intracellular binding of an antigen-binding polypeptide, comprising an amino acid sequence represented by any one of SEQ ID NOs: 23 to 25, 31, 33, 34, and 40 to 42. (14-2) The polypeptide according to any one of (1) to (14-1) above, consisting of the amino acid sequence represented by SEQ ID NO: 25. (14-3) A polypeptide for improving the intracellular binding of an antigen-binding polypeptide, comprising the amino acid sequence represented by SEQ ID NO: 25.

[0178] (15) A fusion polypeptide comprising the polypeptide according to any one of (1) to (14-3) above and an antigen-binding polypeptide. (16) The fusion polypeptide according to (15) above, wherein the antigen-binding polypeptide comprises one or more complementarity determining regions of an antibody against a specific antigen. (17) The fusion polypeptide according to (15) or (16) above, wherein the antigen-binding polypeptide is Fab, VH, VL, VHH, vNAR, or scFv. (17-1) The fusion polypeptide according to any one of (15) to (17) above, wherein the antigen-binding polypeptide is VH, VHH, vNAR, or scFv.

[0179] (18) A nucleic acid encoding the polypeptide according to any one of (1) to (14-3) above. (18-1) The nucleic acid according to (18) above, consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22. (18-2) A nucleic acid encoding a polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, comprising a nucleotide sequence represented by any one of SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22. (18-3) The nucleic acid according to any one of (18) to (18-2) above, consisting of a nucleotide sequence represented by SEQ ID NO: 5. (18-4) A nucleic acid encoding a polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, comprising a nucleotide sequence represented by SEQ ID NO: 5. (19) An expression vector comprising the nucleic acid according to any one of (18) to (18-4) above. (19-1) The expression vector according to (19) above, comprising a nucleic acid consisting of any of the nucleotide sequences represented by SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22. (19-2) An expression vector comprising a nucleic acid encoding a polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, wherein the nucleic acid consists of any of the nucleotide sequences represented by SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22. (19-3) The expression vector according to any one of (19) to (19-2) above, comprising a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 5. (19-4) An expression vector according to any one of (19) to (19-2) above, comprising a nucleic acid encoding a polypeptide for improving the intracellular antigen-binding ability of an antigen-binding polypeptide, wherein the nucleic acid consists of the nucleotide sequence represented by SEQ ID NO: 5. (20) A nucleic acid encoding the fusion polypeptide according to any one of (15) to (17) above. (21) An expression vector comprising the nucleic acid according to (20) above.

[0180] (22) A method for improving the antigen-binding ability of an antigen-binding polypeptide in a cell, comprising expressing a nucleic acid encoding a fusion polypeptide in the cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (22-1) A method for improving the antigen-binding ability of an antigen-binding polypeptide in a cell, comprising expressing a nucleic acid encoding a fusion polypeptide in the cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0181] (23) A method for improving the folding efficiency of an antigen-binding polypeptide in a cell, comprising expressing a nucleic acid encoding a fusion polypeptide in a cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (23-1) A method for improving the folding efficiency of an antigen-binding polypeptide in a cell, comprising expressing a nucleic acid encoding a fusion polypeptide in a cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0182] (24) A method for improving the presence of antigen-binding polypeptides that maintain correct folding in cells, comprising expressing a nucleic acid encoding a fusion polypeptide in cells, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (24-1) A method for improving the presence of antigen-binding polypeptides that maintain correct folding in cells, comprising expressing a nucleic acid encoding a fusion polypeptide in cells, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0183] (25) A method for treating or preventing a disease associated with an intracellular antigen, comprising expressing a nucleic acid encoding a fusion polypeptide in a cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (25-1) A method for treating or preventing a disease associated with an intracellular antigen, comprising expressing a nucleic acid encoding a fusion polypeptide in a cell, wherein the fusion polypeptide comprises the antigen-binding polypeptide and a polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide. (25-2) The method according to any one of (22) to (25-1) above, further comprising introducing a nucleic acid encoding the fusion polypeptide into a cell prior to the expression.

[0184] (26) A fusion polypeptide used in the treatment or prevention of a disease associated with an intracellular antigen, comprising an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (26-1) A fusion polypeptide used in the treatment or prevention of a disease associated with an intracellular antigen, comprising an antigen-binding polypeptide and the polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0185] (27) Use of a fusion polypeptide for the manufacture of a pharmaceutical composition for the treatment or prevention of a disease associated with an intracellular antigen, wherein the fusion polypeptide comprises an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (27-1) Use of a fusion polypeptide for the manufacture of a pharmaceutical composition for the treatment or prevention of a disease associated with an intracellular antigen, wherein the fusion polypeptide comprises the antigen-binding polypeptide and the polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0186] (28) A nucleic acid used in the treatment or prevention of a disease associated with an intracellular antigen, wherein the nucleic acid encodes a fusion polypeptide, the fusion polypeptide comprising an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, the polypeptide consisting of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (28-1) A nucleic acid used in the treatment or prevention of a disease associated with an intracellular antigen, wherein the nucleic acid encodes a fusion polypeptide, the fusion polypeptide comprising an antigen-binding polypeptide and the polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0187] (29) Use of a nucleic acid for the manufacture of a pharmaceutical composition for treating or preventing a disease associated with an intracellular antigen, wherein the nucleic acid encodes a fusion polypeptide, the fusion polypeptide comprising an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, the polypeptide consisting of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic. (29-1) Use of a nucleic acid for the manufacture of a pharmaceutical composition for treating or preventing a disease associated with an intracellular antigen, wherein the nucleic acid encodes a fusion polypeptide, the fusion polypeptide comprising the antigen-binding polypeptide and the polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide. (30) Use according to any one of (28) to (29-1) above, wherein the nucleic acid is incorporated into an expression vector.

[0188] (31) A pharmaceutical composition used for the treatment or prevention of a disease associated with an intracellular antigen, comprising: a nucleic acid encoding a fusion polypeptide or an expression vector comprising the nucleic acid; and a pharmaceutically acceptable carrier, wherein the fusion polypeptide comprises an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, the polypeptide consisting of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic amino acids. (31-1) A pharmaceutical composition used for the treatment or prevention of a disease associated with an intracellular antigen, comprising: a nucleic acid encoding the fusion polypeptide according to any one of (15) to (17) above or an expression vector comprising the nucleic acid; and a pharmaceutically acceptable carrier. (31-2) A pharmaceutical composition used for the treatment or prevention of a disease associated with an intracellular antigen, comprising: a nucleic acid encoding a fusion polypeptide or an expression vector comprising the nucleic acid; and a pharmaceutically acceptable carrier, wherein the fusion polypeptide comprises an antigen-binding polypeptide and the polypeptide according to any one of (1) to (14-3) above linked to the N-terminus of the antigen-binding polypeptide.

[0189] (32) A research reagent used for functional analysis of an antigen in a cell, comprising a nucleic acid encoding the polypeptide according to any one of (1) to (14-3) above or an expression vector containing the nucleic acid.

[0190] (33) A method for producing a cell that expresses an antigen-binding polypeptide in the cell, comprising expressing a nucleic acid encoding a fusion polypeptide in the cell, wherein the fusion polypeptide comprises an antigen-binding polypeptide and a polypeptide linked to the N-terminus of the antigen-binding polypeptide, the polypeptide consisting of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic amino acids. (33-1) The method according to (33) above, further comprising introducing a nucleic acid encoding the fusion polypeptide into the cell prior to the expression.

[0191] (34) A kit for producing a nucleic acid encoding a fusion polypeptide, comprising a nucleic acid encoding the polypeptide according to any one of (1) to (14-3) above or an expression vector containing the nucleic acid.

[0192] Hereinafter, the present disclosure will be described with reference to examples, but the present disclosure is not limited to the following examples.

[0193] [Antigen-binding polypeptide] As the antigen-binding polypeptide, an anti-KRAS antibody which is an scFv (reference: Werge TM, et al., Intracellular immunization Cloning and intracellular expression of a monoclonal antibody to the p21 ras The intrabody used was an intrabody (hereinafter referred to as "Y13-259-HA") based on an HA-tagged Y13-259 protein (Y13-259-HA, FEBS Letters 274, 193-198, 1990) (nucleotide sequence: SEQ ID NO: 1; amino acid sequence: SEQ ID NO: 2).

[0194] Furthermore, an intrabody (nucleotide sequence: SEQ ID NO: 83; amino acid sequence: SEQ ID NO: 84) (hereinafter referred to as "scFv13-HA") was used, which is an anti-β-galactosidase antibody (scFv) (reference: P. Martineau et al., Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm, J. Mol. Biol. 280, 117-127, 1998) tagged with an HA tag at the C-terminus.

[0195] Furthermore, as an antigen-binding polypeptide in a format different from scFv, an Intrabody (nucleotide sequence: SEQ ID NO: 85; amino acid sequence: SEQ ID NO: 86) (hereinafter referred to as "iDab#6-HA") was used, which was an anti-HRAS (G12V mutant) antibody (VH) (reference: T Tanaka et al., Tumor prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS, EMBO J. 26, 3250-3259, 2007) tagged with an HA tag at the C-terminus.

[0196] Additionally, antigen-binding polypeptides that recognize antigens other than KRAS, HRAS, and β-galactosidase and have a format different from that of scFv and VH were used, including an intrabody (nucleotide sequence: SEQ ID NO: 87; amino acid sequence: SEQ ID NO: 88) (hereinafter referred to as "BsG73-HA") in which an HA tag was attached to the C-terminus of an anti-EGFP shark antibody (vNAR; Reference Patent Document: WO 2023 / 077287), and an intrabody (nucleotide sequence: SEQ ID NO: 89; amino acid sequence: SEQ ID NO: 90) (hereinafter referred to as "enhancer-HA") in which an HA tag was attached to the C-terminus of an anti-EGFP camelid antibody (VHH (also known as nanobody); Reference PDB ID: 3K1K).

[0197] [Polypeptides that improve the intracellular antigen-binding ability of antigen-binding polypeptides] The nucleotide sequences (SEQ ID NOs: 3 to 22) and amino acid sequences (SEQ ID NOs: 23 to 42) of peptides 1 to 20 used in this example are shown in the table below. Vectors expressing antibodies in which these peptides were added to the N-terminus of an antibody consisting of the amino acid sequence of SEQ ID NO: 1 were prepared and used in various studies.

[0198]

[0199]

[0200] [Antigen Protein] The antigen protein recognized by Y13-259-HA was KRAS tagged with a His tag at the C-terminus (nucleotide sequence: SEQ ID NO: 43, amino acid sequence: SEQ ID NO: 44) (hereinafter referred to as "KRAS-His").

[0201] Furthermore, the antigen protein recognized by scFv13-HA was β-galactosidase tagged with a FLAG tag at the C-terminus (nucleotide sequence: SEQ ID NO: 91; amino acid sequence: SEQ ID NO: 92) (hereinafter referred to as "β-Gal-FLAG")

[0202] The antigen protein recognized by iDab#6-HA was HRAS(G12V) tagged with a FLAG tag at the C-terminus (nucleotide sequence: SEQ ID NO: 93; amino acid sequence: SEQ ID NO: 94) (hereinafter referred to as "HRAS(G12V)-FLAG").

[0203] Furthermore, EGFP (nucleotide sequence: SEQ ID NO: 95; amino acid sequence: SEQ ID NO: 96) was used as the antigen protein recognized by BsG73-HA and enhancer-HA.

[0204] [Construction of Expression Vectors] First, Y13-259-HA (nucleotide sequence: SEQ ID NO: 1), peptide3 (nucleotide sequence: SEQ ID NO: 5)-Y13-259-HA (nucleotide sequence: SEQ ID NO: 1), and KRAS-His (nucleotide sequence: SEQ ID NO: 43) were each prepared by artificial gene synthesis and inserted into the multicloning site of pcDNA3.4 (Invitrogen) using genetic engineering techniques to construct each expression vector. Next, using the pcDNA3.4 expression vector into which Y13-259-HA had been inserted as a template, gene fragments of Y13-259-HA conferred with each peptide other than peptide3 were amplified by PCR using the primers shown in the table below, and each expression vector was constructed by the infusion method. The sequence cloned into each expression vector was a combination of the nucleotide sequence represented by any of SEQ ID NOs: 3 to 22 and the nucleotide sequence represented by SEQ ID NO: 1.

[0205]

[0206] Expression vectors other than those mentioned above were constructed using the pcDNA3.4 vector by GeneArt service (Thermo Fisher Scientific).

[0207] [Immunoprecipitation] In Experiments 1 to 6, 293A cells cultured in a 96-well plate were cotransfected with an antigen protein expression vector and an antigen-binding polypeptide expression vector. Plasmid DNA transfection was performed using FuGENE HD Transfection Reagent (Promega) according to the attached protocol. Three days after transfection, the cells were harvested and lysed using 40 μL of Radioimmunoprecipitation (RIPA) buffer (Nacalai Tesque). The lysed cell solution was centrifuged at 4°C and 10,000 × g for 10 minutes to prepare approximately 40 μL of cell lysate. 15 μL of each cell lysate was added to 75 μL of phosphate-buffered saline (PBS) buffer and mixed with 5 μL of anti-HA-tag Monoclonal antibody-Magnetic Agarose (Medical and Biological Laboratories), reacted at 4 °C for 1 hour, and the intracellularly expressed antibody was collected. After the reaction, the supernatant was removed using a magnet, and the magnetic agarose was washed four times with PBS-T buffer (0.1 v / v% Tween 20). After washing, 12 μL of Western blotting buffer was added to the magnetic agarose, heat-treated at 100 °C for 10 minutes, and the supernatant was collected using a magnet.

[0208] In Experimental Example 7 (immunoprecipitation of endogenous KRAS) and Experimental Example 9 (immunoprecipitation of HRAS (G12V)), cells were cultured in 6-well plates. Cells were lysed with 200 μL of RIPA buffer, and 60 μL of each prepared cell lysate was added to 160 μL of PBS buffer, and the mixture was mixed with 20 μL of anti-HA-tag magnetic agarose. After washing, in Experimental Example 8, 24 μL of Western blotting buffer was added to the magnetic agarose.

[0209] In Experiments 9 to 11, the transfection reagent was changed to FuGENE 4K Transfection Reagent (Promega), and reverse transfection was performed. The washing buffer was also changed to PBS-T buffer (0.01 v / v% Tween 20).

[0210] In Experimental Example 8 (immunoprecipitation of β-Gal-FLAG) and Experimental Examples 10 and 11 (immunoprecipitation of EGFP), immunoprecipitation was carried out by adding 80 μL of PBS-buffer to 20 μL of the prepared cell lysate.

[0211] In Experimental Example 7 (immunoprecipitation of endogenous KRAS), transfection was performed using the pcDNA3.4_Y13-259-HA expression vector alone, with or without peptide added, or with pep3 added. In Experimental Examples 8 to 11, cotransfection was performed using various pcDNA3.4_intrabody-HA expression vectors with or without peptide added, and pcDNA3.4 vectors of the corresponding target antigens.

[0212] In Experimental Example 8, the test was carried out with N=6. In Experimental Examples 1 to 7 and 9 to 11, the test was carried out with N=3.

[0213] [Western blotting] 10 μL of each cell lysate prepared by immunoprecipitation was diluted with 4 to 20 wt% Mini-Protean TGX Stain-Free. ＴＭThe immunoprecipitation was run on a gel (BioRad) at 280 V for 15-20 minutes. Similarly, 10 μL of the supernatant recovered after immunoprecipitation was run on 4-20 wt% Mini-PROTEAN TGX Precast Protein Gels (BioRad). After electrophoresis, the immunoprecipitation was transferred to a low-fluorescence Polyvinylidene Difluoride (PVDF) membrane (BioRad) using a Transblot Turbo (BioRad) and blocked with EveryBlot blocking buffer (BioRad). After blocking, to detect each Y13-259-HA and KRAS-His, the cells were reacted for 1 hour with 2500-fold diluted HA-Tag (C29F4) Rabbit Monoclonal Antibody (Cell Signaling Technology) and 5000-fold diluted 6xHis, His-Tag Monoclonal Antibody (Proteintech Group Inc.) as primary antibodies. After the reaction, the sections were washed with Tris Buffered Saline (TBS)-T buffer (0.1 v / v% Tween 20) and then reacted with 5000-fold diluted Goat Anti-Rabbit IgG (H+L), Highly Cross-Adsorbed (Biotium), and 2500-fold diluted StarBright Blue 700 Goat Anti-Mouse IgG (BioRad). After the reaction, the sections were washed with TBS-T buffer and imaged using ChemiDoc MP (BioRad).

[0214] In Experiments 6 to 11, the detection antibodies were changed to increase the detection sensitivity. For intrabody detection, a 2,000-fold diluted anti-HA high affinity rat antibody (MERCK) was used as the primary antibody, and a 2,000-fold diluted goat anti-rat IgG (H+L) cross-adsorbed secondary antibody, DyLight, was used as the secondary antibody. ＴＭ 800 (Thermo Fisher Scientific) was used. For detection of various target antigens, the following primary antibodies and 2,500-fold diluted StarBright Blue 700 Goat Anti-Rabbit IgG (BioRad) were used as the secondary antibody.

[0215] For the detection of the target antigen KRAS-His in Experimental Example 6, 1,000-fold diluted Anti-His-tag pAb (Medical and Biological Laboratories) was used as the primary antibody. For the detection of intracellular KRAS, the target antigen in Experimental Example 7, 10,000-fold diluted KRAS Polyclonal antibody 12063-1-AP (Proteintech) was used as the primary antibody. For the detection of β-Gal-FLAG, the target antigen in Experimental Example 8, and HRAS(G12V)-FLAG, the target antigen in Experimental Example 9, 500-fold diluted anti-FLAG （Ｒ） A rabbit antibody (MERCK) was used. For detection of EGFP, the target antigen in Experimental Examples 10 and 11, a 2,000-fold diluted eGFP Polyclonal Antibody (CAB4211, Thermo Fisher Scientific) was used.

[0216] Only in the Western blotting of Experimental Example 8, the amount of the supernatant sample after immunoprecipitation to be electrophoresed on the gel was set to 3 μL.

[0217] [Cell proliferation assay] In Experimental Example 12, 293A cells cultured in a 96-well half-area plate were cotransfected with the pcDNA3.4_KRAS-His expression vector and the pcDNA3.4_Y13-259-HA expression vector containing each peptide. Plasmid DNA transfection was performed by reverse transfection using FuGENE 4K Transfection Reagent (Promega). After transfection, cells in each well were incubated at 37°C while being imaged over time using an Incucyte S3 (Sartorius). Furthermore, the confluence (%) of 293A cells in each well was calculated using the Incucyte analysis module. The test was carried out with N=3.

[0218] Experimental Example 1 (Investigation of the amino acid backbone in polypeptides that improve intracellular antigen-binding ability of antigen-binding polypeptides) To identify polypeptides that improve the intracellular binding function of antigen-binding polypeptides to their target antigens, we first designed peptides consisting of nine amino acids, each composed mainly of acidic amino acids (peptide 3), basic amino acids (peptide 7), and neutral amino acids (peptide 8) as the polypeptide backbone, and constructed vectors expressing Intrabodies in which each peptide was fused to the N-terminus of Y13-259A-HA. The three constructed Intrabody expression vectors and the pcDNA3.4_KRAS-His expression vector were co-transfected into 293A cells, and each Intrabody expressed in the cells was recovered by immunoprecipitation. The amounts of each Intrabody and KRAS contained in the recovered samples were compared by Western blotting.

[0219] The upper panel of Figure 1 shows photographs and graphs illustrating the results of analysis of cell lysates (samples before immunoprecipitation). The upper left panel of Figure 1 shows photographs illustrating the results of Western blotting analysis of the expression of each Intrabody (solid arrows) and KRAS (dotted arrow) contained in the cell lysates before immunoprecipitation.

[0220] The graph in the upper center of Figure 1 shows the results of comparing the ratio of expression level of each Intrabody based on the results of Western blotting, where the brightness value of each band was normalized by the total protein amount, the logarithmically transformed values ​​were averaged, and the value for Y13-259-HA to which no peptide had been attached was used as the standard. The vertical axis of the graph shows the expression level (relative value) of the Intrabody. As a result, compared to Y13-259-HA to which no peptide was attached (in the figure, indicated as "non-pep"), Y13-259-HA to which peptide 3 consisting of an acidic amino acid backbone was attached (in the figure, indicated as "Peptide3" or "pep3") and Y13-259-HA to which peptide 8 consisting of a neutral amino acid backbone was attached (in the figure, indicated as "Peptide8" or "pep8") showed a tendency for the expression level of Intrabody to increase. On the other hand, Y13-259-HA to which peptide 7 consisting of a basic amino acid backbone was attached (in the figure, indicated as "Peptide7" or "pep7") showed no difference in expression level with Y13-259-HA to which no peptide was attached.

[0221] The upper right panel of Figure 1 shows a graph comparing the expression levels of KRAS based on the results of Western blotting, where the brightness values ​​of each band were normalized by the total protein amount, and the logarithmically transformed values ​​were averaged. The graph's vertical axis represents the expression level (relative value) of KRAS. As a result, co-expression with Intrabody tended to increase the amount of KRAS accumulated in the cytoplasm, and in particular, the amount of KRAS accumulated in the cytoplasm tended to be greater in the case of peptide addition compared to the case of no peptide addition.

[0222] The lower panel of Figure 1 shows photographs and graphs illustrating the results of analyzing the samples after immunoprecipitation. The lower left panel of Figure 1 shows photographs illustrating the results of Western blotting analysis of the amounts of each Intrabody (solid arrow) and KRAS (dotted arrow) in the samples after immunoprecipitation.

[0223] The graph in the lower center of Figure 1 shows the results of comparing the amount of each Intrabody based on the results of Western blotting, where the brightness values ​​of each Intrabody after immunoprecipitation were logarithmically transformed and then averaged, relative to the value for Y13-259-HA to which no peptide was attached. The vertical axis of the graph shows the amount of Intrabody (relative value). As a result, the amount of each Intrabody after immunoprecipitation showed a ratio equivalent to that of the cell lysate, indicating that immunoprecipitation had been carried out without any problems.

[0224] The graph on the lower right of Figure 1 shows the results of Western blotting, where the intensity values ​​of KRAS bound to each Intrabody after immunoprecipitation were logarithmically transformed and then averaged, and the amount of KRAS bound to each Intrabody was evaluated based on the amount of KRAS bound to Y13-259-HA to which no peptide had been attached. The vertical axis of the graph indicates the amount of KRAS bound to each Intrabody (relative value). As a result, KRAS could not be detected in Y13-259-HA to which no peptide had been attached, but the amount of KRAS dramatically increased in Y13-259-HA to which peptide 3, consisting of an acidic amino acid backbone, had been attached. It was also shown that Y13-259-HA to which peptide 8 consisting of a neutral amino acid backbone was attached also showed a tendency for the amount of KRAS to increase, although it was less than that of Y13-259-HA to which peptide 3 was attached, but the tendency for the increase in KRAS amount was found to be even smaller in Y13-259-HA to which peptide 7 consisting of a basic amino acid backbone was attached. These results suggest that the attached peptide improves the binding function of the antibody expressed in the cell to the target antigen, and it was found that a peptide consisting of an acidic amino acid backbone is important as a peptide that improves the binding function of the antibody to the target antigen in the cell.

[0225] [Experimental Example 2] (Study on peptide length capable of maintaining improved intracellular antibody binding function to target antigen) Considering the use of intracellular antibodies in gene therapy, it is desirable that the peptide length be as short as possible. Therefore, in order to confirm the range of peptide lengths consisting of an acidic amino acid backbone that can exhibit the intracellular antibody binding function to a target antigen, peptides of different lengths and consisting of an acidic amino acid backbone were designed: peptide 1 (20 amino acids), peptide 2 (15 amino acids), peptide 3 (9 amino acids), peptide 16 (3 amino acids), and peptide 17 (6 amino acids), and the effect of improving the intracellular antibody binding function to the target antigen was evaluated by immunoprecipitation.

[0226] The upper left of Figure 2 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). As a result, it was confirmed that each Intrabody and KRAS were expressed in the cell lysate. The lower left of Figure 2 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 2, "KRAS Only" indicates the results of expressing KRAS only, "non-pep" indicates the results of expressing Y13-259-HA without peptide, "pep1" indicates the results of expressing Y13-259-HA with peptide 1 attached, and so on.

[0227] The right side of Figure 2 is a graph showing the results of calculating the brightness values ​​of each Intrabody and KRAS contained in the sample after immunoprecipitation, logarithmically transforming the ratio of the amount of KRAS to the amount of Intrabody (ratio = amount of KRAS / amount of Intrabody) and averaging the values, and comparing the ratio of the amount of KRAS to the amount of each Intrabody with the ratio of the amount of KRAS to the amount of Y13-259-HA to which no peptide was attached as the standard. The vertical axis of the graph shows the ratio (relative value) of the amount of KRAS to the amount of each Intrabody.

[0228] As a result, it was found that Y13-259-HA to which peptides 1 to 3 had been attached had a stable improved binding function to the target antigen of the antibody in cells compared to Y13-259-HA to which no peptide had been attached.

[0229] On the other hand, although the effect of improving the intracellular binding function of an antibody to a target antigen was also observed in Y13-259-HA to which peptide 16 and peptide 17 were attached, it was shown that Y13-259-HA to which other peptides were attached is more likely to be able to exert this effect more stably. These results demonstrate that when the length of the acidic amino acid backbone peptide is 9 to 20 amino acids, it is possible to particularly stably improve the intracellular binding function of an antibody to a target antigen.

[0230] Experimental Example 3 (Study on the content of other amino acids in the acidic amino acid backbone) The study in Experimental Example 2 demonstrated that peptides with an acidic amino acid backbone containing 22.2% to 26.6% of the neutral amino acid proline (P) as the other amino acid can still retain the function of improving the binding functionality of an antibody to a target antigen in cells. Furthermore, to determine the range of other amino acid content allowable in an acidic amino acid backbone, peptides (peptides 12 to 15) containing 33.3%, 44.4%, 55.5%, or 66.6% other amino acids (the neutral amino acid P was used in this study) in the acidic amino acid backbone peptide were designed, and the effect of improving the binding functionality of an antibody to a target antigen in cells was evaluated by immunoprecipitation.

[0231] The upper left panel of Figure 3 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left panel of Figure 3 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. The right panel of Figure 3 is a graph showing the results of logarithmic transformation of the intensity values ​​of KRAS bound to each Intrabody contained in the post-immunoprecipitation sample, followed by averaging, and comparing the amount of KRAS bound to each Intrabody with the amount of KRAS bound to Y13-259-HA to which peptide 15 had been added. In Figure 3, "Peptide 12" indicates the results of expressing Y13-259-HA to which peptide 12 had been added, and the same applies below. The vertical axis of the graph indicates the amount of KRAS bound to each Intrabody (relative value).

[0232] As a result, all peptides expressed approximately the same levels of intrabody and KRAS in the cell lysate. However, in the post-immunoprecipitation samples, although relatively stable binding to KRAS was confirmed for Y13-259-HA to which peptide 12 was added, stable binding to KRAS was not confirmed for the other peptides.

[0233] These results demonstrate that by keeping the content of other amino acids in the acidic amino acid backbone at 33.3% or less, it is possible to particularly stably improve the intracellular binding function of an antibody to a target antigen.

[0234] [Experimental Example 4] (Study of neutral amino acid species in acidic amino acid backbone) With regard to the design of a polypeptide that improves the intracellular binding function of an antigen-binding polypeptide to a target antigen, Experimental Example 3 demonstrated that proline (hydrophobic / imino acid), a type of neutral amino acid, is an acceptable amino acid species that can be introduced into an acidic amino acid backbone. Furthermore, to verify whether there are neutral amino acid species that can be introduced into an acidic amino acid backbone, glycine (G), which belongs to the hydrophobic / aliphatic group, phenylalanine (F), which belongs to the hydrophobic / aromatic group, and serine (S), which belongs to the hydrophilic group, were selected as representative amino acids of each property, and peptides (peptides 9 to 11) were designed in which the proline in peptide 3 was replaced with each of these, and the effect of improving the intracellular binding function of an antibody to a target antigen was evaluated by immunoprecipitation.

[0235] The upper left of Figure 4 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 4 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 4, "non-pep" indicates the results of expressing Y13-259-HA without peptide, "Peptide 3" indicates the results of expressing Y13-259-HA with peptide 3, and so on. As a result, it was confirmed that each intrabody and KRAS were expressed in the cell lysate in all samples.

[0236] The right side of Figure 4 is a graph showing the results of a comparative evaluation of the amount of KRAS bound to each Intrabody, where the intensity values ​​of KRAS bound to each Intrabody in the post-immunoprecipitation samples were logarithmically transformed and averaged, and the amount of KRAS bound to each Intrabody was measured using the amount of KRAS bound to Y13-259-HA without peptide as the standard. The vertical axis of the graph shows the amount of KRAS bound to each Intrabody (relative value).

[0237] As a result, after immunoprecipitation, a tendency for increased binding to KRAS was confirmed in Y13-259-HA conjugated with peptide 9 and Y13-259-HA conjugated with peptide 11. A slight tendency for increased binding to KRAS was also observed in Y13-259-HA conjugated with peptide 10, but the tendency for increased binding was lower than in Y13-259-HA conjugated with peptide 9 and Y13-259-HA conjugated with peptide 11.

[0238] These results suggest that amino acids other than proline may be acceptable as neutral amino acids that can be introduced into the acidic amino acid backbone of a polypeptide to improve the intracellular binding function of the antigen-binding polypeptide to its target antigen, but that hydrophobic / aliphatic or hydrophilic neutral amino acids are particularly effective.

[0239] [Experimental Example 5] (Investigation of basic amino acid species in acidic amino acid backbone) In designing a polypeptide that improves the intracellular binding function of an antigen-binding polypeptide to a target antigen, it was shown in Experimental Example 4 that the introduction of a neutral amino acid into an acidic amino acid backbone is permissible. In Experimental Example 1, the effect of a basic amino acid backbone on improving the intracellular binding function of an antibody to a target antigen was significantly reduced. However, to verify whether the introduction of a neutral amino acid into an acidic amino acid backbone is permissible, peptides 18 to 20 were designed in which the proline in peptide 3 was replaced with lysine (K), and the effect of improving intracellular target binding function was evaluated by immunoprecipitation.

[0240] The upper left of Figure 5 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 5 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 5, "non-pep" indicates the results of expressing Y13-259-HA without peptide, "Peptide 3" indicates the results of expressing Y13-259-HA with peptide 3, and so on. As a result, it was confirmed that each intrabody and KRAS were expressed in the cell lysate in all samples.

[0241] The right side of Figure 5 shows the results of a comparative evaluation of the amount of KRAS bound to each Intrabody, calculated by logarithmically transforming and averaging the intensity values ​​of KRAS bound to each Intrabody contained in the post-immunoprecipitation sample, using the amount of KRAS bound to Y13-259-HA (denoted as "pep0" in Figure 5) to which no peptide had been attached as the standard. The vertical axis of the graph indicates the amount of KRAS bound to each Intrabody (relative value). The results confirmed that Y13-259-HA attached with peptides 18 to 20 bound to KRAS at a level equivalent to that of peptide 3.

[0242] These results demonstrate that the introduction of a basic amino acid into the acidic amino acid backbone is permissible in order to improve the intracellular binding function of an antigen-binding polypeptide to its target antigen.

[0243] Experimental Example 6 (Comparative study of the effect of improving the binding functionality of antibodies to target antigens in cells) A comparative evaluation was performed by immunoprecipitation and Western blotting of the intracellular expression and binding function to the target antigen (KRAS-His) of Y13-259-HA to which peptide 4 (SEQ ID NO: 26, s3Flag), peptide 5 (SEQ ID NO: 27, DE2.0), and peptide 6 (SEQ ID NO: 28, DE5.0) (all see Patent Document 1 (WO 2019 / 004213)), which have been reported to have the effect of improving the aggregation tendency of antibodies expressed in cells, and Y13-259-HA to which peptide 2 and peptide 3, which were found in this study to have the effect of improving the binding functionality of antibodies to target antigens in cells, have been added to the N-terminus.

[0244] The upper left of Figure 6 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 6 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 6, "KRAS only" indicates the results of expressing only KRAS-His, "Y13-259-HA" indicates the results of expressing Y13-259-HA without peptide, "pep2-Y13-259-HA" indicates the results of expressing Y13-259-HA with peptide 2, and so on. As a result, a significant decrease in expression level was observed only for Y13-259-HA with peptide 6. In the other samples, it was confirmed that each intrabody and KRAS were co-expressed.

[0245] The right side of Figure 6 is a graph showing the results of calculating the brightness values ​​of each intrabody and KRAS-His contained in the sample after immunoprecipitation and comparing the ratio of the amount of KRAS-His to the amount of intrabody (ratio = amount of KRAS-His / amount of intrabody). The vertical axis of the graph shows the ratio of the amount of KRAS-His to the amount of each intrabody. As a result, it was confirmed that Y13-259-HA to which peptide 2, peptide 3, peptide 4, peptide 5, or peptide 6 was attached had improved binding function with KRAS-His compared to Y13-259-HA to which no peptide was attached. Furthermore, it was confirmed that Y13-259-HA to which peptide 2 or peptide 3 was attached had significantly improved intracellular KRAS-His binding function compared to Y13-259-HA to which peptide 4, peptide 5, or peptide 6 was attached.

[0246] These results confirm that, compared to conventional polypeptides that have been reported to be effective in improving the aggregation properties of antibodies expressed in cells, the polypeptide of this embodiment stably maintains the expression level of antibodies and the like in cells and significantly improves the binding function of antigen-binding polypeptides to target antigens.

[0247] [Experimental Example 7] (Investigation of the ability of antigen-binding polypeptides to improve binding function for endogenous target antigens) In previous studies, intracellularly overexpressed KRAS-His was used as the target antigen. To confirm whether the improving effect of the polypeptide binding function found in this study also applies to endogenous target antigens, the improving effect of the polypeptide binding function for endogenous KRAS was evaluated by immunoprecipitation.

[0248] The upper left of Figure 7 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 7 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 7, "Y13-259-HA" indicates the result of expressing Y13-259-HA without peptide, "pep3-Y13-259-HA" indicates the result of expressing Y13-259-HA with peptide 3, and "mock" indicates the result of transfection with pcDNA3.4 empty vector as a negative control. As a result, it was confirmed that each intrabody was expressed in the cell lysate of Y13-259-HA and pep3-Y13-259-HA, and that intracellular KRAS was present. The band indicated by the arrow marked with "*" in the lower left of Figure 7 was thought to be a nonspecific band derived from the beads used for immunoprecipitation, and the band indicated by the arrow marked with "**" was thought to be a degradation product of KRAS.

[0249] The right side of Figure 7 is a graph showing the results of calculating the brightness value of endogenous KRAS bound to each intrabody contained in the sample after immunoprecipitation and comparing the ratio of endogenous KRAS amount to intrabody amount (ratio = endogenous KRAS amount / intrabody amount). The vertical axis of the graph shows the ratio of endogenous KRAS amount to each intrabody amount. As a result, it was confirmed that Y13-259-HA to which peptide 3 was added had significantly improved binding function with intracellular KRAS compared to that to which no peptide was added.

[0250] These results demonstrate that the effect of improving the binding functionality of the polypeptide of this embodiment is also effective in binding to intracellular target antigens.

[0251] [Experimental Example 8] (Investigation of the effect of antigen-binding polypeptides on improving binding functionality for different target antigens) In previous studies, KRAS was used as the target antigen. To confirm whether the improving effect of polypeptide binding functionality found in this study can be seen with other target antigens, the improving effect of anti-β-galactosidase antibody scFv13 on the binding functionality of β-galactosidase was evaluated by immunoprecipitation.

[0252] The upper left of Figure 8 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 8 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 8, "scFv13-HA" indicates the result of expressing scFv13-HA without peptide, "pep3-scFv13-HA" indicates the result of expressing scFv13-HA with peptide 3, "β-Gal-FLAG only" indicates the result of expressing only β-Gal-FLAG as a negative control, and "mock" indicates the result of transfection with pcDNA3.4 empty vector as a negative control. As a result, it was confirmed that each intrabody and β-Gal-FLAG were expressed in the cell lysate of scFv13-HA and pep3-scFv13-HA.

[0253] The right side of Figure 8 is a graph showing the results of calculating the brightness value of β-Gal-FLAG bound to each intrabody contained in the sample after immunoprecipitation and comparing the ratio of the amount of β-Gal-FLAG to the amount of intrabody (ratio = amount of β-Gal-FLAG / amount of intrabody). The vertical axis of the graph shows the ratio of the amount of β-Gal-FLAG to the amount of each intrabody. As a result, it was confirmed that scFv13-HA to which peptide 3 was added had significantly improved binding function to β-Gal-FLAG compared to scFv13-HA to which peptide was not added.

[0254] These results demonstrate that the effect of improving the binding functionality of the polypeptide of this embodiment is also effective in binding to target antigens other than KRAS.

[0255] [Experimental Example 9] (Investigation of the effect of improving the binding functionality of antigen-binding polypeptides (VH) of different formats against different target antigens) In previous studies, scFv was used as the antigen-binding polypeptide. To confirm whether the improving effect of polypeptide binding functionality found in this study can also be observed for antigen-binding polypeptides of other formats, the improving effect of binding functionality of iDab#6, an anti-HRAS (G12V mutant) antibody VH, was evaluated by immunoprecipitation.

[0256] The upper left of Figure 9 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 9 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 9, "iDab#6-HA" indicates the result of expressing iDab#6-HA without peptide, "pep3-iDab#6-HA" indicates the result of expressing iDab#6-HA with peptide 3, "HRAS-FLAG only" indicates the result of expressing only HRAS(G12V)-FLAG as a negative control, and "mock" indicates the result of transfection with pcDNA3.4 empty vector as a negative control. As a result, it was confirmed that each intrabody and HRAS(G12V)-FLAG were expressed in the cell lysate of iDab#6-HA and pep3-iDab#6-HA.

[0257] The right side of Figure 9 is a graph showing the results of calculating the brightness value of HRAS(G12V)-FLAG bound to each intrabody contained in the sample after immunoprecipitation and comparing the ratio of HRAS(G12V)-FLAG amount to intrabody amount (ratio = HRAS(G12V)-FLAG amount / intrabody amount). The vertical axis of the graph shows the ratio of HRAS(G12V)-FLAG amount to each intrabody amount. As a result, it was confirmed that iDab#6-HA to which peptide 3 was attached had significantly improved binding function to HRAS(G12V)-FLAG compared to that to which no peptide was attached.

[0258] These results demonstrate that the effect of improving the binding functionality of the polypeptide of this embodiment is effective not only for scFv format, but also for antigen-binding polypeptides in VH format.

[0259] [Experimental Example 10] (Investigation of the effect of antigen-binding polypeptides of different formats (shark antibody vNAR) on improving binding functionality to different target antigens) Following the VH format of Experimental Example 10, to confirm whether the effect can also be seen with antigen-binding polypeptides in the shark antibody vNAR format, the effect of improving binding functionality of the anti-EGFP antibody vNAR, BsG73, was evaluated by immunoprecipitation.

[0260] The upper left of Figure 10 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 10 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 10, "BsG73-HA" indicates the result of expressing BsG73-HA without peptide, "pep3-BsG73-HA" indicates the result of expressing BsG73-HA with peptide 3, "EGFP only" indicates the result of expressing only EGFP as a negative control, and "mock" indicates the result of transfection with pcDNA3.4 empty vector as a negative control. As a result, it was confirmed that each intrabody and EGFP were expressed in the cell lysate in BsG73-HA and pep3-BsG73-HA.

[0261] The right side of Figure 10 is a graph showing the results of calculating the brightness value of EGFP bound to each intrabody contained in the sample after immunoprecipitation and comparing the ratio of EGFP amount to intrabody amount (ratio = EGFP amount / intrabody amount). The vertical axis of the graph shows the ratio of EGFP amount to each intrabody amount. As a result, it was confirmed that BsG73-HA to which peptide 3 was added had significantly improved binding function to EGFP compared to BsG73-HA to which peptide was not added.

[0262] These results demonstrate that the effect of improving the binding functionality of the polypeptide of this embodiment is also effective in antigen-binding polypeptides in the shark antibody vNAR format.

[0263] [Experimental Example 11] (Investigation of the effect of antigen-binding polypeptides of different formats (camelid antibody VHH) on improving binding functionality for different target antigens) To confirm whether antigen-binding polypeptides in the camelidae antibody VHH format are also effective, the effect of an "enhancer," an anti-EGFP antibody VHH, on improving binding functionality was evaluated by immunoprecipitation.

[0264] The upper left of Figure 11 is a photograph showing the results of Western blotting of cell lysate (sample before immunoprecipitation). The lower left of Figure 11 is a photograph showing the results of Western blotting of the sample after immunoprecipitation. In Figure 11, "enhancer-HA" indicates the result of expressing enhancer-HA without peptide, "pep3-enhancer-HA" indicates the result of expressing enhancer-HA with peptide 3, "EGFP only" indicates the result of expressing only EGFP as a negative control, and "mock" indicates the result of transfection with pcDNA3.4 empty vector as a negative control. As a result, it was confirmed that each intrabody and EGFP were expressed in the cell lysate in enhancer-HA and pep3-enhancer-HA.

[0265] The right side of Figure 11 is a graph showing the results of calculating the brightness value of EGFP bound to each intrabody contained in the sample after immunoprecipitation and comparing the ratio of the amount of EGFP to the amount of intrabody (ratio = EGFP amount / intrabody amount). The vertical axis of the graph shows the ratio of the amount of EGFP to the amount of each intrabody. As a result, it was confirmed that enhancer-HA to which peptide 3 was added had significantly improved binding function to EGFP compared to enhancer-HA to which no peptide was added.

[0266] These results demonstrate that the effect of improving the binding functionality of the polypeptide of this embodiment is also effective for antigen-binding polypeptides in the camelid antibody VHH format.

[0267] The results thus far have demonstrated that the effect of improving the binding functionality of the polypeptide of this embodiment is effective for antigen-binding polypeptides in any antibody format and for any target antigen.

[0268] [Experimental Example 12] (Comparative study of the effect of reducing cell proliferation inhibition) In addition to Experimental Example 6, peptide4 (SEQ ID NO: 26, s3Flag), peptide5 (SEQ ID NO: 27, DE2.0), peptide6 (SEQ ID NO: 28, DE5.0) (all of which have been reported to improve the aggregation of antibodies expressed in cells) (see Patent Document 1 (WO 2019 / 004213)) were added to Y13-259-HA, and peptide2 and peptide3, which have been found to have an effect of improving the binding functionality of antibodies to target antigens in cells in this study, were added to the N-terminus of Y13-259-HA. A comparative evaluation of the effect on cell proliferation when expressed in 293A cells was carried out. After transfection with each vector, the cells were imaged over time, and the confluence (%) of 293A cells was calculated based on these images.

[0269] The left side of Figure 12 is a graph showing cell proliferation after transfection over time, using confluence (%) as an index. The vertical axis of the graph represents the confluence (%) of 293A cells, and the horizontal axis represents the time elapsed after transfection. The right side of Figure 12 is a bar graph with the vertical axis representing the confluence (%) 40 hours after transfection. In Figure 12, "Y13-259-HA" indicates the result of expressing Y13-259-HA without peptide, "pep2-Y13-259-HA" indicates the result of expressing Y13-259-HA with peptide 2, and so on. Furthermore, "untreated" indicates the result of not performing transfection.

[0270] As a result, it was confirmed that cell proliferation was suppressed in cells expressing Y13-259-HA compared to untreated cells. Furthermore, it was confirmed that when Y13-259-HA to which peptide 2, peptide 3, peptide 4, or peptide 6 was added was expressed, cell proliferation suppression was significantly reduced.

[0271] In addition, it was confirmed that Y13-259-HA to which peptide 3 was added had a significantly improved effect of reducing the inhibition of intracellular proliferation compared to Y13-259-HA to which peptide 4, peptide 5, or peptide 6 was added.

[0272] It was confirmed that Y13-259-HA to which peptide 2 was added had a significantly improved effect of reducing cell proliferation inhibition compared to Y13-259-HA to which peptide 5 or peptide 6 was added. Furthermore, a tendency for intracellular proliferation inhibition to be further reduced was observed even compared to Y13-259-HA to which peptide 4 was added.

[0273] From the above results, it was confirmed that the polypeptide of the present embodiment significantly reduces cell growth inhibition compared to conventional polypeptides that have been reported to have the effect of improving the aggregation properties of antibodies expressed in cells.

[0274] The polypeptide of this embodiment can improve the intracellular binding ability of an antigen-binding polypeptide to an antigen.

Claims

1. A polypeptide for improving the intracellular binding ability of an antigen-binding polypeptide to an antigen, wherein the polypeptide consists of 3 to 20 amino acids, and more than 57.0% but less than 100.0% of the amino acids constituting the polypeptide are acidic amino acids.

2. The polypeptide of claim 1, wherein the polypeptide consists of at least 9 and at most 20 amino acids.

3. The polypeptide according to claim 1, wherein 60.0% to 90.0% of the amino acids constituting said polypeptide are acidic amino acids.

4. The polypeptide according to claim 3, wherein 65.0% to 80.0% of the amino acids constituting said polypeptide are acidic amino acids.

5. The polypeptide of claim 1, wherein the polypeptide is N-terminally glutamic acid.

6. The polypeptide of claim 1, wherein the polypeptide consists of acidic and neutral amino acids.

7. The polypeptide according to claim 6, wherein 10.0% to 40.0% of the amino acids constituting said polypeptide are neutral amino acids.

8. The polypeptide according to claim 7, wherein 20.0% to 35.0% of the amino acids constituting said polypeptide are neutral amino acids.

9. The polypeptide of claim 6, wherein the side chain of the neutral amino acid is a hydrophobic aliphatic group or a hydrophilic group.

10. The polypeptide of claim 9, wherein the neutral amino acid is proline, glycine, or serine.

11. The polypeptide of claim 1, wherein the polypeptide further comprises a basic amino acid, and the basic amino acid is lysine.

12. The polypeptide according to claim 1, wherein the polypeptide consists of an amino acid sequence represented by the following general formula (I) or (II): In general formula (I), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; n1 and n2 each independently represent an integer of 0 to 3, and 0≦n1+n2≦5. In general formula (II), X １ and X ４ are each independently a neutral amino acid or a basic amino acid; ２ is a D or neutral amino acid; X ３ is D or E; X ５ represents a neutral amino acid or a basic amino acid; n3 and n4 each independently represent an integer of 0 or more and 1 or less, and satisfy the relation: 1≦n3+n4≦2.

13. The polypeptide according to claim 12, wherein the polypeptide consists of the amino acid sequence represented by any one of SEQ ID NOs: 64 to 66, 72, and 82.

14. The polypeptide of claim 1, consisting of the amino acid sequence represented by any one of SEQ ID NOs: 23 to 25, 31, 33, 34 and 40 to 42.

15. A fusion polypeptide comprising a polypeptide according to any one of claims 1 to 14 and an antigen-binding polypeptide.

16. The fusion polypeptide of claim 15, wherein the antigen-binding polypeptide comprises one or more complementarity-determining regions of an antibody against a specific antigen.

17. A nucleic acid encoding a polypeptide according to any one of claims 1 to 14.

18. The nucleic acid according to claim 17, which consists of a base sequence represented by any one of SEQ ID NOs: 3 to 5, 11, 13, 14, 19 and 20 to 22.

19. An expression vector comprising the nucleic acid of claim 17.

20. The expression vector according to claim 19, wherein the nucleic acid consists of a base sequence represented by any one of SEQ ID NOs: 3 to 5, 11, 13, 14, 19, and 20 to 22.

21. A nucleic acid encoding the fusion polypeptide of claim 15.

22. An expression vector comprising the nucleic acid of claim 21.

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