Method for inhibiting formation of aggregates of polypeptide

Abstract

The present invention provides a polypeptide comprising a heavy-chain variable region (VH) domain and a light-chain variable region (VL) domain linked to each other through a peptide linker, the peptide linker comprising the amino acid sequence represented by SEQ ID NO: 1 or NO: 2.

Description

Method for inhibiting polypeptide aggregate formation

[0001] The present invention relates to a method for inhibiting aggregation of polypeptides, particularly single-chain antibodies, and further to a single-chain antibody in which aggregation is inhibited, and a method for producing the same.

[0002] Monoclonal antibodies are used as therapeutic agents in various clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, cardiology, and ophthalmology, as well as for various other applications such as diagnostic agents and sensor elements. The primary function of antibodies is to specifically bind to antigens (target molecules), which are recognized by the Fv domain.

[0003] The Fv domain of an antibody consists of an Fv domain (VH) derived from the heavy chain and an Fv domain (VL) derived from the light chain. Many antibodies retain their target binding ability even in Fv fragments, which are fragments obtained by cleaving the Fv domain from an antibody, and form the smallest unit of an antibody's antigen-binding function. A single-chain antibody (scFv: single-chain Fv) is formed by linking VH and VL with a peptide linker. The VH and VL contained in a single-chain antibody associate to form an antigen-binding site.

[0004] The stability of antibody molecules is evaluated from three perspectives: heat resistance or thermal stability, stability against proteases, and storage stability due to suppression of aggregate formation. Regarding storage stability, it has been reported that cyclizing single-chain antibodies suppresses intermolecular association and reduces the amount of aggregate formation (Patent Document 1). Furthermore, the design of heat-stable mutants of single-chain antibodies has been reported (Non-Patent Document 9).

[0005] A peptide linker is a synthetic sequence of amino acids commonly used to physically link polypeptide domains. Most peptide linkers consist of one or more repeat modules of the amino acids glycine and serine. For example, a single-chain antibody using the 15-amino acid (GGGGS)3 peptide linker is known, and such a peptide linker is thought not to interfere with the assembly and binding activity of the linked domains (Patent Document 2 and Non-Patent Document 1).

[0006] scFvs generally have the property of associating to form multimers, and dimers, trimers, and tetramers formed by scFvs have been reported (Non-Patent Documents 2 to 4). It is known that in single-chain antibodies, when the peptide linker is too short to allow intramolecular association of VH and VL, dimers are formed by intermolecular association of VH and VL (Non-Patent Document 8). Several reports have been published on peptide linkers that can be used in single-chain antibodies (Patent Document 2 and Non-Patent Documents 5 to 7, 9, and 10).

[0007] Generally, monoclonal antibodies are produced using eukaryotic cells such as CHO cells and hybridomas, which require significant production costs. scFvs have molecular weights of approximately 25 kDa, and diabodies have molecular weights of approximately 50 kDa, which are significantly smaller than full-length antibodies. This allows production using prokaryotes such as Escherichia coli as hosts (Non-Patent Document 7), and scFvs and diabodies have advantages over full-length antibodies in terms of production costs.

[0008] WO2020 / 013126 A1 WO2012 / 088461 A2 WO2006 / 116657 A2

[0009] Freund, C. et al. FEBS Letters 320 (1993) 1873-3468;PEI, XY, et al., Proc. Natl. Acad. Sci. USA. 1997, 94, 9637-9642;Todorovska, A. et al., Journal of Immunological Methods 248 (2001) 47-66;Lawrence, LJ et al., FEBS Letters 425 (1998) 479-484;Arslan, M. et al., Scientific Reports volume 12, Article number 5449 (2022);Tang., Y. et al., J Biol Chem, 1996, 271(26), 15682-6;Liu, C. et al., Journal of Biochemistry, 166, 6, 2019, 455-462;Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90, 6444-6448, 1993;Okazaki, K. et al., ACS Omega 2023, 8, 22945-22954Klement, M. et al., Journal of Biotechnology 199 (2015) 90-97

[0010] Polypeptides such as scFvs and diabodies, also known as small molecules, have characteristics such as tissue permeability, ease of production, use in conjugation, and the possibility of obtaining antagonist antibodies, and are widely studied as pharmaceuticals. However, there is a demand for small molecules that have favorable properties as active ingredients in pharmaceuticals, such as high stability and high binding activity.

[0011] The present inventors discovered that polypeptides designed to have specific sequences, particularly scFvs, have favorable properties as active ingredients of pharmaceuticals, such as high stability and high binding activity, and have thus completed the present invention.

[0012] The present invention provides the following: [1-1] A polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain connected by a peptide linker, wherein the peptide linker has the amino acid sequence: １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 1); or AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T (SEQ ID NO: 2), １２ ~X １８ and X ３４ ~X ４２ are each independently selected from naturally occurring amino acid residues.

[0013] [1-2] The polypeptide according to [1-1], wherein the peptide linker comprises the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3) or AAGGSGSGADGAT (SEQ ID NO: 4).

[0014] [1-3] The peptide linker has the amino acid sequence: １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5); or X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ SGX ３７ X３８ X ３９ X ４０ X ４１ X ４２ TX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6), １ ~X １８ and X ３１ ~X ４８ are each independently selected from natural amino acid residues.

[0015] [1-4] The peptide linker has the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GATAGAGQSWYG (SEQ ID NO: 7); or X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 8), １ ~X １１ , X ３１ ~X ３３ , and X ４３ ~X ４８ are each independently selected from natural amino acid residues.

[0016] [1-5] The polypeptide according to [1-1], wherein the peptide linker comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GPEEQYSAYATTGATAGAGQSWYG (SEQ ID NO: 9); or PGAAAGGSGSGADGATYTTTTPG (SEQ ID NO: 10).

[0017] [1-6] The polypeptide according to [1-1] or [1-2], wherein the peptide linker comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GGGGSGGGGGSGGGSTAGAGQSWYG (SEQ ID NO: 47).

[0018] [1-7] The polypeptide according to any one of [1-1] to [1-6], wherein the peptide linker comprises the amino acid sequence: GGGGS (SEQ ID NO: 48) or GGGS (SEQ ID NO: 49).

[0019] [1-8] The polypeptide according to any one of [1-1] to [1-7], wherein the peptide linker comprises the amino acid sequence: GGGGSGGGGGSGGGGS (SEQ ID NO: 50).

[0020] [1-9] The polypeptide according to any one of [1-1] to [1-8], wherein the peptide linker consists of 15 to 60 amino acids. [1-10] The polypeptide according to [1-8], wherein the peptide linker comprises a sequence consisting of 20 or more consecutive amino acids contained in the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 47.

[0021] [1-11] The polypeptide according to any one of [1-1] to [1-10], wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 47. [1-12] The polypeptide according to any one of [1-1] to [1-11], wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 1 and is linked to the C-terminus of the VH domain and the N-terminus of the VL domain.

[0022] [1-13] The polypeptide according to any one of [1-1] to [1-11], wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 2 and is linked to the C-terminus of the VL domain and the N-terminus of the VH domain.

[0023] [1-14] The polypeptide according to [1-12], wherein the peptide linker consists of 15 to 30 amino acids. [1-15] The polypeptide according to [1-13], wherein the peptide linker consists of 35 to 60 amino acids.

[0024] [1-16] VH domain and VL domain are identical and the peptide linker is (G ４ S) ３ The polypeptide according to any one of [1-1] to [1-15], which exhibits reduced aggregate formation compared to a single-chain antibody comprising the polypeptide.

[0025] [1-17] The polypeptide according to any one of [1-1] to [1-16], which is a single-chain antibody (scFv). [1-18] The polypeptide according to [1-12], wherein the VH domain and the VL domain have the following amino acid residues as specified by Kabat SEQ ID NOs: VL38(Q), 40(P), 41(G), 85(T), 87(Y), 95(P), 97(T), 98(F), and 101(G); and VH41(P), 42(G), 46(E), 62(S), and 91(Y).

[0026] [1-19] The polypeptide according to [1-13], wherein the VH domain and VL domain have the following amino acid residues as specified by Kabat SEQ ID NOs: VL38(Q), 40(P), 41(G), 445(K), 85(T), 87(Y), 98(F), 99(G), and 101(G); and VH2(V), 4(L), 41(P), 42(G), 43(K), 46(E), 91(Y), 102(Y), 104(G), 105(Q), and 106(G).

[0027] [2-1] A polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain linked by a linker, wherein the polypeptide has a peptide tag at its N-terminus, and the peptide tag has the amino acid sequence: １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 1); or AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T (SEQ ID NO: 2),１２ ~X １８ and X ３４ ~X ４２ A polypeptide, each independently selected from naturally occurring amino acid residues.

[0028] [2-2] The polypeptide according to [2-1], wherein the peptide tag comprises the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3) or AAGGSGSGADGAT (SEQ ID NO: 4).

[0029] [2-3] The peptide tag has the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5); or X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ TX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6), １ ~X １８ and X ３１ ~X ４８ are each independently selected from natural amino acid residues.

[0030] [2-4] The peptide tag has the amino acid sequence: X １ PX ２ X ３ X ４ X５ X ６ X ７ X ８ X ９ X １０ X １１ GATAGAGQSWYG (SEQ ID NO: 7); or X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 8), １ ~X １１ , X ３１ ~X ３３ , and X ４３ ~X ４８ are each independently selected from natural amino acid residues.

[0031] [2-5] The polypeptide according to [2-4], wherein the peptide tag comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GPEEQYSAYATTGATAGAGQSWYG (SEQ ID NO: 9); or PGAAAGGSGSGADGATYTTTTPG (SEQ ID NO: 10).

[0032] [2-6] The polypeptide according to [2-1] or [2-2], wherein the peptide tag comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GGGGSGGGGGSGGGSTAGAGQSWYG (SEQ ID NO: 47).

[0033] [2-7] The polypeptide according to any one of [2-1] to [2-6], wherein the peptide tag comprises the amino acid sequence: GGGGS (SEQ ID NO: 48) or GGGS (SEQ ID NO: 49).

[0034] [2-8] The polypeptide according to any one of [2-1] to [2-7], wherein the peptide tag comprises the amino acid sequence: GGGGSGGGGGSGGGGS (SEQ ID NO: 50).

[0035] [2-9] The polypeptide according to [2-5] or [2-6], wherein the peptide tag consists of 15 to 60 amino acids. [2-10] The polypeptide according to [2-5], wherein the peptide tag comprises a sequence consisting of 20 or more consecutive amino acids contained in the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0036] [2-11] The polypeptide according to any one of [2-1] to [2-10], wherein the peptide tag comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. [2-12] The polypeptide according to any one of [2-1] to [2-11], wherein the peptide tag comprises the amino acid sequence of SEQ ID NO: 1, a linker is attached to the C-terminus of the VH domain and the N-terminus of the VL domain, and the peptide tag is attached to the C-terminus of the VL domain.

[0037] [2-13] The polypeptide according to any one of [2-1] to [2-11], wherein the peptide tag comprises the amino acid sequence of SEQ ID NO: 2, the linker is bound to the C-terminus of the VL domain and the N-terminus of the VH domain, and the peptide tag is bound to the C-terminus of the VL domain.

[0038] [2-14] The polypeptide according to [2-12], wherein the peptide tag consists of 15 to 30 amino acids. [2-15] The polypeptide according to [2-10], wherein the peptide tag consists of 35 to 60 amino acids.

[0039] [2-16] The polypeptide according to any one of [2-1] to [2-15], wherein aggregate formation is suppressed compared to a polypeptide having the same linker, VH domain, and VL domain but no peptide tag. [2-17] The polypeptide according to any one of [2-1] to [2-16], wherein the linker is a peptide linker.

[0040] [2-18] The polypeptide according to any one of [2-1] to [2-17], which is a single-chain antibody (scFv). [2-19] The polypeptide according to [2-12], wherein the VH domain and the VL domain have the following amino acid residues as specified by Kabat SEQ ID NOs: VL38(Q), 40(P), 41(G), 85(T), 87(Y), 95(P), 97(T), 98(F), and 101(G); and VH41(P), 42(G), 46(E), 62(S), and 91(Y).

[0041] [2-20] A polypeptide according to [2-13], wherein the VH domain and VL domain have the following amino acid residues as specified by Kabat sequence numbers: VL38(Q), 40(P), 41(G), 45(K), 85(T), 87(Y), 98(F), 99(G), and 101(G); and VH2(V), 4(L), 41(P), 42(G), 43(K), 46(E), 91(Y), 102(Y), 104(G), 105(Q), and 106(G).

[0042] [3-1] A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the polypeptide according to any one of [1-1] to [1-19] and [2-1] to [2-20]. [3-2] A recombinant vector comprising the nucleic acid according to [3-1]. [3-3] A transformant into which the recombinant vector according to [3-2] has been introduced.

[0043] The present invention provides scFvs with high antigen-binding activity and high stability, particularly scFvs with reduced intermolecular aggregate formation, which can be used as analytical and diagnostic sensor elements, pharmaceuticals, and the like.

[0044] FIG. 1 shows the results of gel filtration chromatography purification of scFv (Tras-scFv-HL-Glue) and SDS-PAGE analysis. FIG. 2 shows the results of gel filtration chromatography purification of scFv (Tras-scFv-HL) and SDS-PAGE analysis. FIG. 3 shows a comparison of the results of gel filtration chromatography purification of scFv (Tras-scFv-HL and Tras-scFv-HL-Glue) with a control. FIG. 4 shows a comparison of the results of gel filtration chromatography analysis of scFv (Tras-scFv-HL and Tras-scFv-HL-Glue). FIG. 5 is a graph showing the results of evaluation of the aggregation stability of scFv (Tras-scFv-HL-Glue) by dynamic light scattering. FIG. 6 is a graph showing the results of surface plasmon resonance analysis of scFv (Tras-scFv-HL-Glue). FIG. 7 shows the results of purification of scFv (Y9-scFv-HL) by gel filtration chromatography and the results of SDS-PAGE. FIG. 8 shows the results of purification of scFv (Y9-scFv-HL-Glue) by gel filtration chromatography and the results of SDS-PAGE. FIG. 9 shows a comparison of the results of gel filtration chromatography analysis of scFv (Y9-scFv-HL-Glue) with a control. FIG. 10 is a graph showing the results of evaluation of the aggregation stability of scFv (Y9-scFv-HL-Glue) by dynamic light scattering. FIG. 11 shows a structural model of scFv (Tras-scFv-HL-Glue) obtained by molecular dynamics calculation. FIG. 12 shows the results of purification of scFv (Tras-scFv-LH-Glue) by gel filtration chromatography and the results of SDS-PAGE. FIG. 13 shows the results of purification of scFv (Tras-scFv-LH) by gel filtration chromatography and the results of SDS-PAGE. Figure 14 shows a comparison of the results of gel filtration chromatography analysis of scFv (Tras-scFv-LH and Tras-scFv-LH-Glue). Figure 15 is a graph showing the results of evaluating the aggregation stability of scFv (Tras-scFv-LH and Tras-scFv-LH-Glue) by dynamic light scattering.FIG. 16 shows the results of evaluating the antigen-binding activity of T scFv (Tras-scFv-LH and Tras-scFv-LH-Glue) by pull-down assay. FIG. 17 shows the results of purification of scFv (Y9-scFv-LH-Glue) by gel filtration chromatography and the results of SDS-PAGE. FIG. 18 shows the results of purification of scFv (Y9-scFv-LH) by gel filtration chromatography and the results of SDS-PAGE. FIG. 19 shows a comparison of the results of gel filtration chromatography analysis of scFv (Y9-scFv-LH and Y9-scFv-LH-Glue). FIG. 20 is a graph showing the results of evaluation of the aggregation stability of scFv (Y9-scFv-LH and Y9-scFv-LH-Glue) by dynamic light scattering. FIG. 21 shows a structural model of scFv (Tras-scFv-LH-Glue) obtained by molecular dynamics calculation. Figure 22 is a structural model diagram showing the binding surface of the HL-Glue core sequence of scFv (Tras-scFv-HL-Glue). The binding surface of the HL-Glue core sequence is shown in black, VH in light gray, and VL in dark gray. Figure 23 is a structural model diagram showing the binding surface of the LH-Glue core sequence of scFv (Tras-scFv-LH-Glue) obtained by molecular dynamics calculations. The binding surface of the LH-Glue core sequence is shown in black, VH in light gray, and VL in dark gray. Figure 24 shows a comparison of the analytical results of gel filtration chromatography of scFv (Tras-scFv-LH and GlueCore-Tras-scFv-LH). Figure 25 shows the analytical results of gel filtration chromatography of scFv (Tras-scFv-HL-Glue2) prepared in Example 6. Fig. 26 shows the results of SDS-PAGE after gel filtration chromatography purification of scFv (Tras-scFv-HL-Glue2) prepared in Example 6. Fig. 27 shows a comparison of the results of gel filtration chromatography purification of scFv (Tras-scFv-HL and Tras-scFv-HL-Glue2) with a control.

[0045] In one aspect of the present invention, there is provided a polypeptide comprising a peptide linker having a specific sequence, wherein the VH domain and VL domain contained in the polypeptide associate intramolecularly and have antigen-binding activity.

[0046] In one aspect of the present invention, the VH domain and VL domain are VH and VL, each consisting of approximately 100 to 120 amino acid residues, and have the same overall shape, called the "immunoglobulin fold," and are mainly composed of β-sheets. In one aspect of the present invention, an scFv may be arranged in the order of VH and VL domains, or VL and VH domains, from the N-terminus. In one aspect of the present invention, the VH domain and VL domain are VH and VL, respectively, or functional fragments thereof.

[0047] The regions corresponding to VH and VL in an antibody can be determined based on known findings. For example, references such as Martin, ACR Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133; and Elvin A. Kabat, Tai Te Wu, Carl Foeller, Harold M. Perry, and Kay S. Gottesman (1991) Sequences of Proteins of Immunological Interest can be consulted. In one aspect of the present invention, the VH region is defined as the amino acid sequence of Kabat SEQ ID NOs: 0 to 113, and the VL region is defined as the amino acid sequence of Kabat SEQ ID NOs: 0 to 109. In one embodiment, for antibodies in which the C-terminal 2 to 3 residues of framework 4 in both the VH and VL regions do not form a secondary structure, the region not including these residues can be defined as VH or VL.

[0048] In one aspect of the present invention, there is provided a polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain connected by a peptide linker, wherein the peptide linker comprises a sequence consisting of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more consecutive amino acids contained in the amino acid sequence GPEEQYSAYATTGATAGAGQSWYGE. In one aspect of the present invention, the peptide linker is a polypeptide consisting of 15 to 32, 15 to 31, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, or 15 to 25 amino acids.

[0049] In one aspect of the invention, the peptide linker has the amino acid sequence: X １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 1). In another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3). In yet another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5). In yet another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X７ X ８ X ９ X １０ X １１ It is a polypeptide comprising GATAGAGQSWYG (SEQ ID NO: 7).

[0050] In one aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3). In one embodiment of this aspect, the peptide linker is a polypeptide consisting of 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, or 15-25 amino acids.

[0051] In one aspect of the invention, the peptide linker has the amino acid sequence: AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ In another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X (SEQ ID NO: 2). In another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: AAGGSGSGADGAT (SEQ ID NO: 4). In another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ TX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6). In yet another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X４６ X ４７ X ４８ (SEQ ID NO: 8).

[0052] In one aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: AAGGSGSGADGAT (SEQ ID NO: 4). In one embodiment of this aspect, the peptide linker is a polypeptide consisting of 15-60, 15-59, 15-58, 15-57, 15-56, 15-55, 15-54, or 15-53 amino acids.

[0053] In one aspect of the invention, X １ ~X １８ are each independently selected from the naturally occurring amino acid residues alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). １ ~X １８ are each independently selected from glycine (G), glutamic acid (E), glutamine (Q), tyrosine (Y), serine (S), alanine (A), and threonine (T).

[0054] In one aspect of the invention, the peptide linker is a polypeptide comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to the amino acid sequence: GPEEQYSAYATTGATAGAGQSWYGE.

[0055] In one aspect of the present invention, the amino acids constituting the peptide linker are not particularly limited as long as they are amino acids, and may be natural amino acids or unnatural amino acids. Examples of such amino acids include glycine, alanine, valine, isoleucine, leucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, and histidine, and specific examples thereof include glycine, alanine, serine, phenylalanine, glutamic acid, and arginine.

[0056] In one embodiment of the present invention, the polypeptide having a peptide linker may have a structure in which VH, the peptide linker, and VL are linked in this order from the N-terminus, or may have a structure in which VL, the peptide linker, and VH are linked in this order from the N-terminus. Herein, a single-chain antibody in which the C-terminus of VH is linked to VL via a peptide linker is referred to as scFv-HL, and a single-chain antibody in which the C-terminus of VL is linked to VH via a peptide linker is referred to as scFv-LH.

[0057] In one aspect of the present invention, the peptide linker is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to 4, and has a structure in which VH, the peptide linker, and VL are linked in this order from the N-terminus.

[0058] In one aspect of the present invention, there is provided a polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain linked by a linker, and having a peptide tag at the C-terminus. The linker is not particularly limited as long as it covalently links the VH domain and the VL domain, and examples thereof include peptide linkers. In one embodiment, the linker is a peptide linker having any sequence and a length of 12 residues or more, such as GSGGGGSGGGGGSGGGGS.

[0059] In one aspect of the invention, the peptide tag has the amino acid sequence: １２ X １３ X １４ GX １５GQX １６ WX １７ X １８ (SEQ ID NO: 1). In another aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3). In yet another aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5). In yet another aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: X １ PX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ It is a polypeptide comprising GATAGAGQSWYG (SEQ ID NO: 7).

[0060] In one aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: TAGAGQSWYG (SEQ ID NO: 3). In one embodiment of this aspect, the peptide tag is a polypeptide consisting of 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, or 15-25 amino acids.

[0061] In one aspect of the invention, the peptide tag has the amino acid sequence: AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X４１ X ４２ In another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: AAGGSGSGADGAT (SEQ ID NO: 4). In another aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ SGX ３７ X ３８ X ３９ X ４０ X ４１ X ４２ TX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6). In yet another aspect of the invention, the peptide linker is a polypeptide comprising the amino acid sequence: X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 8).

[0062] In one aspect of the invention, the peptide tag is a polypeptide comprising the amino acid sequence: AAGGSGSGADGAT (SEQ ID NO: 4). In one embodiment of this aspect, the peptide linker is a polypeptide consisting of 15-60, 15-59, 15-58, 15-57, 15-56, 15-55, 15-54, or 15-53 amino acids.

[0063] In one embodiment of the present invention, any host including E. coli, yeast, mammalian cells, insect cells, etc. can be used to produce the polypeptide, and any promoter including T7, Taq, lac, etc. can be used.

[0064] In one embodiment of the present invention, the polypeptide may be fused to another peptide or protein, or fragment thereof, that enhances solubility, such as maltose binding protein (MBP), thioredoxin, glutathione S-transferase (GST), or protein GB1 domain (GB1).

[0065] In one aspect of the present invention, the polypeptide comprising a peptide linker is a single-chain antibody. As used herein, the term "single-chain antibody (scFv)" refers to a single-chain Fv fragment (scFv) obtained by linking the Fv domain (VH) derived from the heavy chain and the Fv domain (VL) derived from the light chain of a full-length antibody via a peptide linker.

[0066] In one aspect of the present invention, a single-chain antibody may have either a structure in which the C-terminus of the VL domain and the N-terminus of the VH domain are linked by a peptide linker, or a structure in which the C-terminus of the VH domain and the N-terminus of the VL domain are linked by a peptide linker.

[0067] The scFv used in the present invention can be produced by the methods described in textbooks (e.g., Carl A.K. Borrebaeck (ed.), (1995) Antibody Engineering (Second Edition), Oxford University Press, New York; John McCafferty, Hennie Hoogenboom, Dave Chiswell (ed.), (1996) Antibody Engineering, A Practical Approach, IRL Press, Oxford, etc.) and literature (e.g., Biochim. Biophys. Acta - Protein Structure and Molecular Enzymology 1385, 17-32 (1998), Molecules. 2017 22:e1695, Journal of Biochemistry 161:37-43, etc.).

[0068] In one aspect of the present invention, polypeptides such as scFvs can be produced by culturing transformants prepared by genetic engineering techniques. Nucleic acids encoding the amino acid sequences of polypeptides such as single-chain antibodies can be synthesized by chemical synthesis, PCR, cassette mutagenesis, site-directed mutagenesis, or the like. For example, a nucleic acid of interest can be totally synthesized by chemically synthesizing multiple oligonucleotides of up to about 100 bases each having a complementary region of about 20 base pairs at its end, combining these, and performing overlap extension.

[0069] The recombinant vector of the present invention can be obtained by ligating (inserting) the above-mentioned nucleic acid into an appropriate vector. The vector used in the present invention is not particularly limited as long as it is replicable in a host or capable of integrating the target nucleic acid into the host genome. Examples include bacteriophages, plasmids, cosmids, and phagemids.

[0070] Examples of plasmid DNA include actinomycete-derived plasmids (e.g., pK4, pRK401, pRF31, etc.), Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, etc.), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.), and yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.). Examples of phage DNA include λ phage (λgt10, λgt11, λZAP, etc.). Furthermore, vectors derived from animal viruses such as retroviruses or vaccinia viruses, and insect viruses such as baculoviruses can also be used.

[0071] In one embodiment of the present invention, plasmid DNA can be prepared using pNMK (Patent Document 2 and Non-Patent Document 7) as a template. In another embodiment of the present invention, a similar construct can be prepared using vectors such as pET28 or pRSF-Duet (both from Millipore).

[0072] To insert a gene into a vector, a method may be employed in which purified DNA is first cleaved with an appropriate restriction enzyme, and the gene is inserted into an appropriate restriction enzyme site or multicloning site of the vector DNA, followed by ligation of the gene to the vector. The gene must be incorporated into the vector so that the improved protein of the present invention can be expressed. Therefore, in addition to a promoter and the nucleotide sequence of the gene, cis elements such as enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), start codons, and stop codons can be ligated to the vector of the present invention, as desired. A tag sequence can also be ligated to facilitate purification of the protein produced. The tag sequence can be a nucleotide sequence encoding a known tag, such as a His tag, a GST tag, or an MBP tag.

[0073] Whether or not a gene has been inserted into a vector can be confirmed using known genetic engineering techniques. For example, in the case of a plasmid vector, the vector can be subcloned using competent cells, the DNA extracted, and the nucleotide sequence determined using a DNA sequencer. Similar techniques can be used for other vectors that can be subcloned using bacteria or other hosts. Vector selection using a selection marker such as a drug resistance gene is also effective.

[0074] A transformant can be obtained by introducing the recombinant vector of the present invention into a host cell so that the improved protein of the present invention can be expressed. The host used for transformation is not particularly limited as long as it is capable of expressing a protein or polypeptide. Examples include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, plant cells, animal cells (COS cells, CHO cells, etc.), and insect cells.

[0075] When a bacterium is used as a host, it is preferable that the recombinant vector be capable of autonomous replication in the bacterium and comprise a promoter, a ribosome binding sequence, an initiation codon, a nucleic acid encoding the improved protein of the present invention, and a transcription termination sequence. Examples of Escherichia coli include Escherichia coli DH5α, and examples of Bacillus subtilis include Bacillus subtilis. The method for introducing the recombinant vector into bacteria is not particularly limited, as long as it is a method for introducing DNA into bacteria. Examples include a method using calcium ions and electroporation.

[0076] When synthesizing a polypeptide in a cell, a peptide involved in folding of the peptide may be co-expressed. Examples of co-expressed peptides include sulfhydryl oxidase (e.g., Erv1p), disulfide isomerase (e.g., DsbC, PDI), MarR-like regulatory factors (e.g., SlyA), TF (trigger factor), and peptidyl prolyl isomerase (PPIase, e.g., cyclophilin, FKBP, and Pin1, specifically FKBP12).

[0077] In one aspect of the present invention, the polypeptide having a peptide linker has a molecular weight of, for example, 10,000 to 60,000 Da, specifically 15,000 to 50,000 Da, more specifically 20,000 to 40,000 Da, and even more specifically 25,000 to 30,000 Da. In one aspect of the present invention, the molecular weight of the scFv has a molecular weight of, for example, 10,000 to 60,000 Da, specifically 15,000 to 50,000 Da, more specifically 20,000 to 40,000 Da, and even more specifically 25,000 to 30,000 Da.

[0078] In one aspect of the present invention, the antigen-binding activity of a polypeptide such as scFv is expressed as an EC50 (i.e., 50% binding concentration) value. The EC50 value can be calculated from measurement results obtained by methods known in the art, such as ELISA, Western blot, or FACS. In one aspect of the present invention, the antigen-binding activity of a polypeptide such as scFv is expressed as a dissociation rate constant (kd), dissociation constant (Kd), or the like. These can be calculated from measurement results obtained by known methods, for example, using surface plasmon resonance.

[0079] The following examples illustrate the present invention but are not intended to limit the scope of the present invention.

[0080] Comparative Example 1: Preparation of Trastuzumab-Derived scFv (Tras-scFv-HL) The comparative scFv (Tras-scFv-HL) was prepared according to the method described in Non-Patent Document 9 (ACS Omega 2023, 8, 22945-22954). The coding sequence of trastuzumab VH-(GGGS)3-VL (Tras-scFv-HL, SEQ ID NO: 11) was synthesized using codons optimized for E. coli (Integrated DNA Technologies) (SEQ ID NO: 12, Patent Document 1) and cloned into the pET-28b(+) plasmid using the NcoI and NotI restriction enzyme sites. A hexahistidine tag was subsequently added to the Tras-scFv, which was then purified by affinity chromatography using Ni-NTA agarose resin (WAKO). The resulting plasmid was designated pET28-Tras-scFv.

[0081] Amino acid sequence of Tras-scFv-HL (SEQ ID NO: 11): MEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKSAAALEHHHHHH (GGGGSGGGGSGGGGS is the peptide linker).

[0082] Nucleic acid sequence encoding Tras-scFv-HL (SEQ ID NO: 12).

[0083] The nucleic acid sequence encoding Erv1p (SEQ ID NO: 13) was synthesized with codons optimized for E. coli and cloned into the first multicloning site of the plasmid pCDF-Duet-1. This plasmid was designated pCDF-Erv1p.

[0084] Erv1pをコードする nucleic acid sequence (sequence number 13) .

[0085] Erv1pのアフェックショックアフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフェフィーフェフェフェフェフェフェフェッシューフックPCNWCAKDFEKDFEKYIRENAPQVESREELGRWMCEAHNKVNKKLRGPKFDCNFWEKRWKDGWDE。

[0086] The coding sequence of Erv1p was amplified from the plasmid pCDF-Erv1p using primers Erv1p-Rv (5'-cgagatctcgatctcctttgttcgacttaagcattattcgtcc-3'; SEQ ID NO: 15) and Erv1p-Fw (5'-ggccacgatgcgtccctgacctcctatgcactcctgc-3'; SEQ ID NO: 16). The vector fragment of pET21-DsbC (see JP 2022-178934A) was amplified using primers pET21DsbC-Fw (5'-gaggatcgagatctcgatccgcgaaattaatacgactcac-3'; SEQ ID NO: 17) and pET21DsbC-Rv (5'-ggacgcatcgtggccggcatcaccggcc-3'; SEQ ID NO: 18). The two fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET21-Erv1p-DsbC, which was used to express both Erv1p and DsbC in the cytoplasm.

[0087] SHuffle T7 E. coli (NEB) was cotransformed with pET28-Tras-scFv and pET21-Erv1p-DsbC. Transformants were selected by plating on LB plates containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. A colony was inoculated into 50 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin and grown overnight at 30°C. Cells were harvested by centrifugation (6,000 rpm, 10 minutes, 25°C) and resuspended in 300 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. The medium was stirred at 30°C until the optical density at 600 nm reached 2.5-3.0. Trans-scFv, DsbC, and Erv1p were induced with 1 mM IPTG. After stirring at 15°C for more than 48 hours, the cells were harvested by centrifugation (6,000 rpm, 10 min, 4°C), washed with 30 ml of saline, and then harvested again by centrifugation (6,000 rpm, 20 min, 4°C). The harvested cells were suspended in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl buffer and disrupted by sonication on ice. After centrifugation (12,000 rpm, 40 min, 4°C), the supernatant was collected and loaded onto a column packed with Ni-NTA agarose (Fujifilm Wako Pure Chemicals). The column was washed with 10 bed volumes (20 mL) of 50 mM Tris-HCl (pH 8.0) containing both 500 mM NaCl and 20 mM imidazole. The scFv protein was eluted with a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole. The eluate was collected and centrifuged (12,000 rpm, 10 min, 4°C). The resulting protein was then subjected to gel filtration chromatography (HiLoad 16 / 600 Superdex 75 pg (prep grade) column (GE Healthcare) using a running buffer of 50 mM HEPES (pH 7.4) containing 150 mM NaCl and 5 mM EDTA. TM 16 / 60 SuperdexTM Purification was carried out using a 75 prep grade (Cytiva).

[0088] Example 1: Preparation of trastuzumab-derived scFv (Tras-scFv-HL-Glue) Plasmid pET28-Tras-scFv-HL-Glue was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, a vector fragment was amplified by PCR with the following primer set:

[0089] catggtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccg (pET-NEBuilder-Rv-v2; SEQ ID NO: 19) gcggccgcactcgagcaccaccacc (pET-NEBuilder-Fw; SEQ ID NO: 20)

[0090] A gene fragment encoding Tras-scFv-HL-Glue consisting of the following base sequence was purchased from IDT (SEQ ID NO: 21): CTTTAAGAAGGAGATATACCATG gaagtgcagctggttgaaagcggtggcggtctggtgcaaccgggcggtagcctgcgtctgagctgcgcggcgagcggttttaacatcaaagacac ctacattcactgggttcgtcaagcgccgggcaagggtctggagtgggttgcgcgtatctatccgaccaacggctacacccgttatgcggacagcgt gaaaggtcgttttaccattagcgcggataccagcaagaacaccgcgtacctgcagatgaacagcctgcgtgcggaagacaccgcggtttactattg cagccgttggggcggtgacggcttctacgcgatggattattggggccaaggcaccctggtgaccgttggcccggaagaacagtatagcgcgtatgc gaccaccggcgcgaccgcgggcgcggggccagagctggtatggcgaaatccagatgacccagagcccgagcagcctgagcgcgagcgtgggtgacc gtgttaccattacctgccgtgcgagccaagatgtgaacaccgcggttgcgtggtatcagcaaaagccgggcaaagcgccgaagctgctgatctaca gcgcgagcttcctgtatagcggtgtgccgagccgttttagcggcagccgtagcggcaccgacttcaccctgaccattagcagcctgcagccggaggattttgcgacctactattgccagcaacactataccacccccgccgacctttggtcaaggcaccaaagttgaaatcaagagcGCGGCCGCACTCGAGC

[0091] The two fragments thus obtained were ligated using NEBuilder HiFi DNA assembly (NEB) to obtain the plasmid pET28-Tras-scFv-HL-Glue.

[0092] Amino acid sequence of Tras-scFv-HL-Glue (SEQ ID NO: 22): MEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVGPEEQYSAYATTGATAGAGQSWYGEIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKSAAALEHHHHHH (GPEEQYSAYATTGATAGAGQSWYGE is the peptide linker).

[0093] Gene encoding the amino acid sequence of the underlined part (SEQ ID NO: 23) atggaagtgcagctggttgaaagcggtggcggtctggtgcaaccgggcggtagcctgcgtctgagctgcgcggcgagcggttttaacatcaaagacacctacattcactgggttcgtcaagcgccgggcaagggtctggagtgggttgcgcgtatctatccgaccaacggctacacccgttatgcggacagcgtgaaaggtcgttttaccattagcgcggataccagcaagaacaccgcgtacctgcagatgaacagcctgcgtgcggaagacaccgcggtttactattgcagccgttggggcggtgacggcttctacgcgatggattattggggccaaggcaccctggtgaccgttggcccggaagaacagtatagcgcgtatgcgaccaccggcgcgaccgcgggcgcgggccagagctggtatggcgaaatccagatgacccagagcccgagcagcctgagcgcgagcgtgggtgaccgtgttaccattacctgccgtgcgagccaagatgtgaacaccgcggttgcgtggtatcagcaaaagccgggcaaagcgccgaagctgctgatctacagcgcgagcttcctgtatagcggtgtgccgagccgttttagcggcagccgtagcggcaccgacttcaccctgaccattagcagcctgcagccggaggattttgcgacctactattgccagcaacactataccaccccgccgacctttggtcaaggcaccaaagttgaaatcaagagc

[0094] Tras-scFv-HL-Glue was prepared as follows. SHuffle T7 E. coli (NEB) was cotransformed with pET28-Tras-scFv-HL-Glue and pET21-Erv1p-DsbC. Transformants were selected by spreading on LB plates containing ampicillin (100 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L). A colony was inoculated into 50 ml of 2xYT medium containing ampicillin (100 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L) and cultured overnight at 30°C. Cells were harvested by centrifugation (6,000 rpm, 10 min, 25°C) and resuspended in 300 ml of 2xYT medium containing ampicillin (100 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L). The cells were stirred at 30°C until the optical density at 600 nm reached 2.5–3.0. Expression of Trans-scFv, DsbC, and Erv1p was induced with 1 mM IPTG. After stirring at 15°C for 48 hours or more, the cells were harvested by centrifugation (6,000 rpm, 10 min, 4°C), washed with 30 ml of saline, and then harvested again by centrifugation (6,000 rpm, 20 min, 4°C). The harvested cells were suspended in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl buffer and disrupted by sonication on ice. After centrifugation (12,000 rpm, 40 min, 4°C), the supernatant was collected and loaded onto a column packed with Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd.). The column was washed with 10 bed volumes (20 mL) of 50 mM Tris-HCl (pH 8.0) containing both 500 mM NaCl and 20 mM imidazole. The scFv protein was eluted with a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole.The eluate was collected and centrifuged (12,000 rpm, 10 min, 4°C) and subjected to gel filtration chromatography (HiLoad 16 / 600 Superdex75 pg (prep grade) column (GE Healthcare) using a running buffer of 50 mM HEPES (pH 7.4) containing 150 mM NaCl and 5 mM EDTA (HiLoad 16 / 600 Superdex75 pg (prep grade) column (GE Healthcare)). TM 16 / 60 Superdex TM Purification was carried out using a 75 prep grade (Cytiva).

[0095] The results of gel filtration chromatography purification of an scFv having a peptide linker containing the sequence of SEQ ID NO: 2 (Tras-scFv-HL-Glue) and an scFv containing a (G4S)3 (SEQ ID NO: 46) linker (Tras-scFv-HL:(G4S)3 linker, control) are shown in Figures 1 and 2 (yield: 2.6 mg / 300 mg medium). The gel filtration chromatography conditions are shown below.

[0096] System: AKTA TM explore (room temperature, Cytiva); Column: HiLoad TM 16 / 600 Superdex TM 75 prep grade (Cytiva); flow rate: 1 mL / min; fraction volume: 1.5 mL; buffer: 50 mM HEPES (pH 7.4 at 4°C), 150 mM NaCl, 5 mM EDTA; sample loop: 5 mL;

[0097] The scFv (Tras-scFv-HL-Glue) and the scFv containing a (G4S)3 linker (Tras-scFv-HL: (G4S)3 linker, control) prepared above were concentrated and analyzed by gel filtration chromatography. The results are shown in Figure 4. The gel filtration chromatography conditions are as follows:

[0098] System: AKTA TM explore (room temperature, Cytiva); Column: Superdex TM75 Increase 10 / 300 GL (Cytiva); flow rate: 0.5 mL / min; fraction volume: 0.5 mL; buffer: 50 mM HEPES (pH 7.4 at 4°C), 150 mM NaCl, 5 mM EDTA; sample loop: 0.1 mL; sample concentration: 30 μM.

[0099] It was confirmed that the association ability of Tras-scFv-HL-Glue was significantly reduced compared to the control (FIGS. 3 and 4).

[0100] The aggregation stability of the prepared scFv was confirmed by dynamic light scattering (DLS). DLS measurements were performed at a wavelength of 662 nm using a DynaPro NanoStar system (Wyatt Technology). Molecular size distribution was analyzed using DYNAMICS V7 software. Samples were concentrated to 3 mg / mL and incubated at 4°C. Immediately before measurement, they were centrifuged (15,000 rpm, 30 minutes, 4°C) and stored on ice until measurement. For each sample, 4 μL of solution was measured in a disposable cuvette at 25°C. The acquisition time for each measurement was set to 5 seconds, and the number of acquisitions was set to 10.

[0101] The results are shown in Figure 5. The smaller radius of gyration of Tras-scFv-HL-Glue compared to the control suggests that intramolecular assembly between the VH and VL domains is promoted. Furthermore, it was confirmed that the formation of intermolecular assembly over time is suppressed in Tras-scFv-HL-Glue.

[0102] The antigen-binding activity of the prepared scFv was confirmed by surface plasmon resonance (SPR). Measurements were performed using a Biacore T200 (Cytiva). Measurements were performed using a sensor chip immobilized with a fusion protein (GST-HER2) of a peptide fragment containing the trastuzumab-binding domain of HER2 and glutathione S-transferase (GST). The sensor chip used was a Series S Sensor Chip CM5 (Cytiva), and GST-HER2 was immobilized using a GST Capture Kit (Cytiva). Tras-scFv-HL-Glue and control Tras-scFv-HL were diluted to 70 nM in HBS-EP buffer at 25°C and then assayed. The results are shown in Table 1 and Figure 6. Tras-scFv-HL-Glue was confirmed to have antigen-binding activity equivalent to that of the control Tras-scFv-HL.

[0103]

[0104] Three-dimensional structural models of scFv (Trans-scFv-HL-Glue and Tras-scFv-LH-Glue) were obtained by molecular dynamics calculations. The calculation process is described below. The initial model structure and initial candidate sequence of Tras-scFv-HL-Glue were obtained using the structure prediction software Rosetta (https: / / www.rosettacommons.org / software), and molecular dynamics simulations were performed using the molecular dynamics software AMBER. Based on the results, mutant structural models were created using the molecular display software UCSF-chimera for residues with large fluctuations in the linker sequence. The mutants were created with the intention of strengthening the interaction between the Fv and the linker or the interaction within the linker. Molecular dynamics simulations were performed on this structure using AMBER to examine the effects of mutations. The effects of mutations were evaluated using Root Mean Square Fluctuation (RMSF), an index of conformational deviation. RMSF was calculated using cpptraj, a program included in AMBER. The linker region was designed to have a lower RMSF than the original sequence. We repeatedly created mutant structural models and examined the effects of mutations, ultimately obtaining several candidate sequences, including the Tras-scFv-HL-Glue shown in the Examples, where mutation refers to any of amino acid substitutions, insertions, and deletions.

[0105] The results are shown in Figures 11 and 21 to 23. Of the binding surfaces of the core sequence shown in Figure 22, the following residues in VL and VH were consistent between Tras-scFv-HL-Glue and Y9-scFv-HL-Glue: VL 38 (Q), 40 (P), 41 (G), 85 (T), 87 (Y), 95 (P), 97 (T), 98 (F), and 101 (G) in Kabat SEQ ID NOs: and VH 41 (P), 42 (G), 46 (E), 62 (S), and 91 (Y).

[0106] Of the binding surfaces of the core sequence shown in Figure 23, the following residues of VL and VH were consistent between Tras-scFv-LH-Glue and Y9-scFv-LH-Glue: VL 38 (Q), 40 (P), 41 (G), 445 (K), 85 (T), 87 (Y), 98 (F), 99 (G), and 101 (G) in Kabat sequence numbers; and VH 2 (V), 4 (L), 41 (P), 42 (G), 43 (K), 46 (E), 91 (Y), 102 (Y), 104 (G), 105 (Q), and 106 (G).

[0107] Comparative Example 2: Preparation of scFv (Y9-scFv-HL) derived from anti-GA-pyridine antibody (Y9) Plasmid pSAL-Y9-scFv was prepared by the method described in Patent Document 1 (WO2020 / 013126A1). PCR was performed using the following primer set and plasmid pSAL-Y9-scFv as a template to amplify the gene encoding Y9-scFv-HL.

[0108] caggggccccatatggcccaggtgaaactgcagcagtcaggacctag (Y9HL-RMH-Fw, SEQ ID NO: 24) ctcgagtgcggccgctatttccaactttgtcccccctccgaacgtg (Y9HL-RMH-Rv, SEQ ID NO: 25)

[0109] Using pCold-MK (Kobashigawa Y., et al., Biol. Pharm. Bull. 2021;44(1):125-130. doi:10.1248 / bpb.b20-00759) as a template, the vector fragment was amplified by PCR using the following primers (pCold-MK-NEBulder-Fw, pCold-MK-NEBulder-Rv): 5'-gcggccgcactcgagcaccaccacc-3' (pCold-MK-NEBulder-Fw, SEQ ID NO: 20) 5'-catatggggcccctggaacagaacttccagcg-3' (pCold-MK-NEBulder-Rv, SEQ ID NO: 26)

[0110] The two resulting fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pColdMK-Y9-scFv-HL.

[0111] SHuffle T7 E. coli (NEB) was cotransformed with pColdMK-Y9-scFv-HL and the above-mentioned pET21-Erv1p-DsbC. Transformants were selected by plating on LB plates containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. A colony was inoculated into 50 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin and grown overnight at 30°C. Cells were harvested by centrifugation (6,000 rpm, 10 minutes, 25°C) and resuspended in 300 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. The medium was stirred at 30°C until the optical density at 600 nm reached 2.5-3.0. Trans-scFv, DsbC, and Erv1p were induced with 1 mM IPTG. After stirring at 15°C for more than 48 hours, the cells were harvested by centrifugation (6,000 rpm, 10 minutes, 4°C), washed with 30 ml of saline, and then harvested again by centrifugation (6,000 rpm, 20 minutes, 4°C). The harvested cells were suspended in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mg of HRV3C protease, and then disrupted by sonication on ice. After centrifugation (12,000 rpm, 40 minutes, 4°C), the supernatant was collected and loaded onto a column packed with Ni-NTA agarose (Fujifilm Wako Pure Chemicals). The column was washed with 10 bed volumes (20 mL) of 50 mM Tris-HCl (pH 8.0) containing both 500 mM NaCl and 20 mM imidazole. The scFv protein was eluted with a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole. The eluate was collected and centrifuged (12,000 rpm, 10 min, 4°C). The scFv protein was then subjected to gel filtration chromatography (HiLoad 16 / 600 Superdex 75 pg (prep grade) column (GE Healthcare) using a running buffer of 50 mM HEPES (pH 7.4) containing 300 mM NaCl and 5 mM EDTA.TM 16 / 600 Superdex TM Purification was carried out using a 75 prep grade (Cytiva).

[0112] Amino acid sequence of Y9-scFv-HL (SEQ ID NO: 27): GPHMAQVKLQQSGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKFEYLGYISYSGRTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRPYYRYDYAIDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASLGEQVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSSSGSGTSYSLTISRMEAEDAATYYCQQSWKAPYTFGGGTKLEIAAALEHHHHHH (GGGGSGGGGSGGGGS is the peptide linker).

[0113] Gene encoding the underlined part of the above sequence (SEQ ID NO: 28): gcccaggtgaaactgcagcagtcaggacctagcctcgtgaaaccttctcagactctgtccctcacctgttctgtcactggcgactccatcaccagtggttactggaactggatccggaaattcccagggaataaatttgagtacttgggttacataagctacagtggtcgcacttactaca atccatctctcaaaagtcgaatctccatcactcgagacacatccaagaaccagtactacctgcagttgaattctgtgactactgaggacacagccacatattactgttcaagaccctactataggtacgactatgctatagactactggggccaagggaccacggtcaccgtctcctcaggt ggaggcggttcaggcggaggtggctctggcggtggcggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctctaggggaacaggtcaccatgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagcagaagcccaggatcctcccccaaac tctggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtagcagtggatctgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacctattactgccagcaaagttggaaggccccgtacacgttcggaggggggacaaagttggaaata

[0114] Example 2: Preparation of anti-GA-pyridine antibody (Y9)-derived scFv (Y9-scFv-HL-Glue) The plasmid pColdMK-Y9-scFv-HL-Glue was constructed by the following method: PCR was performed using the following primer set and the above-mentioned plasmid pColdMK-Y9-scFv-HL as a template to amplify the vector fragment.

[0115] gacggtgaccgtggtcccttggccccagtagtctatagc (Y9-LinkRep-Rv, SEQ ID NO: 29) atcgagctcactcagtctccagcaatcatgtctgcatctctagg (Y9-LinkRep-Fw, SEQ ID NO: 30)

[0116] Using the plasmid pET28-Tras-scFv-HL-Glue as a template, a gene fragment encoding the linker region was amplified by PCR with the following primer set:

[0117] gaccacggtcaccgtcggcccggaagaacagtatagcgcgtatgcg (Y9-LinkV3-Fw, SEQ ID NO: 31) gagactgagtgagctcgatttcgccataccagctctggcccgcgcc (Y9-LinkV3-Rv, SEQ ID NO: 32)

[0118] The two resulting fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was named pColdMK-Y9-scFv-HL-Glue.

[0119] SHuffle T7 E. coli (NEB) was cotransformed with pColdMK-Y9-scFv-HL-Glue and the above-mentioned pET21-Erv1p-DsbC. Transformants were selected by plating on LB plates containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. A colony was inoculated into 50 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin and grown overnight at 30°C. Cells were harvested by centrifugation (6,000 rpm, 10 minutes, 25°C) and resuspended in 300 ml of 2xYT medium containing 100 mg / L ampicillin, 50 mg / L kanamycin, and 50 mg / L streptomycin. The medium was stirred at 30°C until the optical density at 600 nm reached 2.5-3.0. Trans-scFv, DsbC, and Erv1p were induced with 1 mM IPTG. After stirring at 15°C for 48 hours or more, the cells were harvested by centrifugation (6,000 rpm, 10 minutes, 4°C), washed with 30 ml of saline, and then harvested again by centrifugation (6,000 rpm, 20 minutes, 4°C). The harvested cells were suspended in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mg of HRV3C protease, and disrupted by sonication on ice. After centrifugation (12,000 rpm, 40 minutes, 4°C), the supernatant was collected and loaded onto a column packed with Ni-NTA agarose (Fujifilm Wako Pure Chemicals). The column was washed with 10 bed volumes (20 mL) of 50 mM Tris-HCl (pH 8.0) containing both 500 mM NaCl and 20 mM imidazole. The scFv protein was eluted with a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole. The eluate was collected, centrifuged (12,000 rpm, 10 min, 4°C), and subjected to gel filtration chromatography (HiLoad) on a HiLoad 16 / 600 Superdex 75 pg (prep grade) column (GE Healthcare) using a running buffer of 50 mM HEPES (pH 7.4) containing 300 mM NaCl and 5 mM EDTA.TM 16 / 600 Superdex TM Purification was carried out using a 75 prep grade (Cytiva).

[0120] Amino acid sequence of Y9-scFv-HL-Glue (SEQ ID NO: 33): GPHMAQVKLQQSGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKFEYLGYISYSGRTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRPYYRYDYAIDYWGQGTTVTVGPEEQYSAYATTGATAGAGQSWYGEIELTQSPAIMSASLGEQVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSSSGSGTSYSLTISRMEAEDAATYYCQQSWKAPYTFGGGTKLEIAAALEHHHHHH (the underlined part is the peptide linker)

[0121] The results of gel filtration chromatography analysis of an scFv having a peptide linker containing the sequence of SEQ ID NO: 2 (Y9-scFv-HL-Glue) and an scFv containing a (G4S)3 linker (Y9-scFv-HL, (G4S)3 linker, control) are shown in Figures 7 and 8. The conditions for gel filtration chromatography are as follows:

[0122] System: AKTA TM explore (room temperature, Cytiva); Column: HiLoad TM 16 / 600 Superdex TM 75 prep grade (manufactured by Cytiva); flow rate: 0.5 mL / min; fraction volume: 0.5 mL; buffer: 50 mM HEPES (pH 7.4 at 4°C), 300 mM NaCl, 5 mM EDTA; sample loop: 5 mL.

[0123] It was confirmed that the association ability of Y9-scFv-HL-Glue was significantly reduced compared to the control (FIG. 8).

[0124] The aggregation stability of the prepared scFv was confirmed by dynamic light scattering (DLS). DLS measurements were performed at a wavelength of 662 nm using a DynaPro NanoStar system (Wyatt Technology). Molecular size distribution was analyzed using DYNAMICS V7 software. Samples were concentrated to 3 mg / mL and incubated at 4°C. Immediately before measurement, they were centrifuged (15,000 rpm, 30 minutes, 4°C) and stored on ice until measurement. For each sample, 4 μL of solution was measured in a disposable cuvette at 25°C. The acquisition time for each measurement was set to 5 seconds, and the number of acquisitions was set to 10.

[0125] The results are shown in Figure 9. The smaller radius of gyration of Y9-scFv-HL-Glue compared to the control suggests that intramolecular assembly between the VH and VL domains is promoted. Furthermore, it was confirmed that Y9-scFv-HL-Glue inhibits intermolecular assembly over time.

[0126] Comparative Example 3: Preparation of trastuzumab-derived scFv (Tras-scFv-LH) Plasmid pET28-Tras-scFv-LH was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, a vector fragment was amplified by PCR using the above-mentioned primer set (pET-NEBuilder-Rv-v2 (SEQ ID NO: 19) and pET-NEBuilder-Fw (SEQ ID NO: 20)).

[0127] The following gene sequence (SEQ ID NO: 34) encoding Tras-scFv-LH was amplified by PCR: CTTTAAGAAGGAGATATACCATG Gatatccagatgaccgagcccgagcagcctgagcgcgagcgtgggtgaccgtgttaccattacctgccgtgcgagccaagatgtgaacacc gcggttgcgtggtatcagcaaaagccggggcaaagcgccgaagctgctgatctacagcgcgagcttcctgtatagcggtgtgccgagccgttt agcggcagccgtagcggcaccgacttcaccctgaccattagcagcctgcagccggaggattttgcgacctactatttgccagcaacactataccacccgccgacctttggtcaaggcaccaaagttgaaatcggtagcggtggcggaggtagcggtggtggcggtagtggtggtggaggttccgaa gtgcagctggttgaaagcggtggcggtctggtgcaaccggggcggtagcctgcgtctgagctgcgcggcgagcggttttaacatcaaagacacc tacattcactgggttcgtcaagcgccgggcaagggtctggagtgggttgcgcgtatctatccgaccaacggctacacccgttatgcggacagc gtgaaaggtcgttttaccattagcgcggataccagcaagaacaccgcgtacctgcagatgaacagcctgcgtgcggaagacaccgcggtttactattgcagccgttggggcggtgacggcttctacgcgatggattattggggccaaggcaccctggtgaccgttagcagcGCGGCCGCACTCGAGC

[0128] The following primer set was used to amplify the gene: ctttaagaaggagatataccatggatatccagatgacccagagcccgagc (TrasLH-pET-NEB-Fw, SEQ ID NO: 35) gctcgagtgcggccgcgctgctaacggtcaccagggtgccttggc (TrasLH-pET-NEB-Rv, SEQ ID NO: 36)

[0129] The two resulting fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET28-Tras-scFv-LH. Using the pET28-Tras-scFv-LH plasmid, Tras-scFv-LH was prepared in the same manner as in Comparative Example 1.

[0130] Amino acid sequence of Tras-scFv-LH (SEQ ID NO: 37): MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHH (the underlined part is the peptide linker).

[0131] Example 3: Preparation of trastuzumab-derived scFv (Tras-scFv-LH-Glue) Plasmid pET28-Tras-scFv-LH-Glue was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, a vector fragment was amplified by PCR with the above-mentioned primer set (pET-NEBuilder-Rv-v2 (SEQ ID NO: 19) and pET-NEBuilder-Fw (SEQ ID NO: 20)).

[0132] The following gene sequence (SEQ ID NO: 38) encoding Tras-scFv-LH-Glue was purchased from IDT. CTTTAAGAAGGAGATATACCATG gacattcagatgacccaaagtccatcaagtctttccgcgtcagtcggcgaccgcgttacaattacttgtcgcgcatcgcaggacgtaaacacggccgtggcttggtaccaacagaagcccggtaaagccccaaagctgttgatctactctgcctcattcttatatagcggagtcccttctcgcttctctggctcgcgcagtggtacagactttactttgacgatctctagcctgcaacctgaagacttcgcgacatactactgtcaacagcattatactaccccacctacttttggtcagggaacgaaggtagaaatcgtagaaatcggtcccgcagctgctgccactagcacttatacgtacgctactgccactgcgacaactggaaccacgtcctacacggctaacacaaccggacgcccgggtgctgccgcaggtggttccggaagcggtgctgacggcgcaacttacacgaccacgccaggtcaagtccagctggtggagtccggtggaggcctggtgcagcctggcggctccttacgtctgtcttgtgctgcctccggtttcaacattaaggacacctacatccactgggttcgccaggcacccggtaaaggtcttgagtgggtggcacgtatttacccaactaatggctacactcgctacgctgacagcgtaaaagggcgctttactatctccgcagataccagcaaaaacacggcatacctgcaaatgaattcacttcgtgctgaagacacggcagtgtattactgctcacgctggggcggagacggattctacgcaatggactattgggggcagggcacgttggtgacagttagttcaGCGGCCGCACTCGAGC

[0133] The two fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET28-Tras-scFv-LH-Glue. Using the pET28-Tras-scFv-LH-Glue plasmid, Tras-scFv-LH-Glue was prepared in the same manner as in Example 1.

[0134] Furthermore, Tras-scFv-LH and Tras-scFv-LH-Glue were analyzed by gel filtration chromatography and subjected to DLS measurement in the same manner as in Example 1.

[0135] Amino acid sequence of Tras-scFv-LH-Glue (SEQ ID NO: 39): MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIVEIGPAAAATSTYTYATATATTGTTSYTANTTGRPGAAAGGSGSGADGATYTTTPGQVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAAALEHHHHHH (the underlined part is the peptide linker).

[0136] The antigen-binding activity of Tras-scFv-LH and Tras-scFv-LH-Glue was evaluated by pull-down assay as follows: 50 mM HEPES (pH 7.4), 5 mM EDTA, and 150 mM NaCl were used as the washing buffer, and 50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 30 mM reduced glutathione were used as the elution buffer.

[0137] 50 μL of glutathione Sepharose 4B (GS4B) resin was placed in a 1.5 mL tube, washed three times with Milli-Q water, and equilibrated three times with wash buffer. 500 μL of the antigen GST-hHER2 (4 μM) was added to the resin and rotated at 4°C for 2 hours. The resin was washed twice with wash buffer, and 500 μL of Trans-scFv-LH or Trans-scFv-LH-Glue (4 μM) was added to the resin and rotated at 4°C for 2 hours. After centrifugation (4°C, 1000 rpm, 1 minute), the supernatant was removed, and the resin was transferred to a spin column and washed three times with wash buffer. Elution buffer was added to the column, and the column was centrifuged (4°C, 1000 rpm, 1 minute). The eluted sample obtained as the supernatant was subjected to 12.5% ​​SDS-PAGE. The analysis results are shown in FIG.

[0138] Comparative Example 4: Preparation of scFv (Y9-scFv-LH) derived from anti-GA-pyridine antibody (Y9) Plasmid pET28-Y9-scFv-LH was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, the vector fragment was amplified by PCR with the above-mentioned primer set (pET-NEBuilder-Rv-v2 (SEQ ID NO: 19) and pET-NEBuilder-Fw (SEQ ID NO: 20)).

[0139] The following gene sequence (SEQ ID NO: 40) encoding Y9-scFv-LH was purchased from IDT. CTTTAAGAAGGAGATATACCATG ggcagcagccatcatcatcatcatcactccagctcgctggaagttctgttccaggggccccatatggacatcgagctcactcagtctccagcaatcatgtctgcatctctaggggaacaggtcaccatgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtagcagtggatctgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacctattactgccagcaaagttggaaggccccgtacacgttcggaggggggacaaagttggaaataggcagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcgcaggtgaaactgcagcagtcaggacctagcctcgtgaaaccttctcagactctgtccctcacctgttctgtcactggcgactccatcaccagtggttactggaactggatccggaaattcccagggaataaatttgagtacttgggttacataagctacagtggtcgcacttactacaatccatctctcaaaagtcgaatctccatcactcgagacacatccaagaaccagtactacctgcagttgaattctgtgactactgaggacacagccacatattactgttcaagaccctactataggtacgactatgctatagactactggggccaagggaccacggtcaccgtctcctcataatGCGGCCGCACTCGAGC

[0140] The two resulting fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET28-Y9-scFv-LH. Y9-scFv-LH was prepared using the pET28-Y9-scFv-LH plasmid in the same manner as in Comparative Example 2.

[0141] Amino acid sequence of Y9-scFv-LH (SEQ ID NO: 41): MGSSHHHHHHSSSLEVLFQGPHMDIELTQSPAIMSASLGEQVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSSSGSGTSYSLTISRMEAEDAATYYCQQSWKAPYTFGGGTKLEIGSGGGGSGGGGSGGGGSQVKLQQSGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKFEYLGYISYSGRTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRPYYRYDYAIDYWGQGTTVTVSS (the underlined part is the peptide linker).

[0142] Example 4: Preparation of scFv (Y9-scFv-LH-Glue) derived from anti-GA-pyridine antibody (Y9) Plasmid pET28-Y9-scFv-LH-Glue was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, a vector fragment was amplified by PCR with the above-mentioned primer set (pET-NEBuilder-Rv-v2 (SEQ ID NO: 19) and pET-NEBuilder-Fw (SEQ ID NO: 20)).

[0143] The following gene sequence (SEQ ID NO: 42) encoding Y9-scFv-LH-Glue was purchased from IDT. CTTTAAGAAGGAGATATACCATG ggcagccatcaccatcaccatcattcatcatcattagaagtcttagagttcggtccacatatggctgatatcgaattaactcagtctcccgcaatcatgtcggccagcttaggtgagcaggtaacaatgacttgtaccgcttcttcgagtgtatcgtcgagttacctgcactggtaccaacaaaaaccaggttcttctcccaaactgtggatttattcaacctcaaaccttgcatctggcgtccccgctcgcttctcatcgtctggatcgggtacaagttattctcttacgatttctcgcatggaggcagaagacgcagctacctattattgccagcaatcttggaaggcgccttacacgttcggcggagggacaaaattggaaattggacccgccgctgcagcgacctcgacatacacctacgcgactgccacagcgacaacaggaacaactagctacacggcaaatacaacaggccgcccgggagctgccgccgggggttcaggaagtggagccgacggtgcaacttataccaccacacctgggcaagtcaaacttcaacagagcggcccgtcattggtgaaaccctcgcagacccttagtttgacctgttcggtgaccggagacagcattacatctggctattggaactggattcgcaaatttcctggtaacaaattcgagtatcttggctatattagctattctggacgtacctactacaatccatcccttaaatctcgtatttccatcacccgcgacacttcaaaaaatcagtattatttgcaactgaattcggtcactacagaagacacggctacctactattgttcacgtccgtattatcgctatgactatgctattgattactgggggcaggggacgaccgttactgtgtcatcttaatGCGGCCGCACTCGAGC。

[0144] The two fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET28-Y9-scFv-LH-Glue. Y9-scFv-LH-Glue was prepared using the pET28-Y9-scFv-LH-Glue plasmid in the same manner as in Example 2.

[0145] Furthermore, Y9-scFv-LH and Y9-scFv-LH-Glue were analyzed by gel filtration chromatography and subjected to DLS measurement in the same manner as in Example 2.

[0146] Amino acid sequence of Y9-scFv-LH-Glue (SEQ ID NO: 43): MGSHHHHHHSSSLEVLEFGPHMADIELTQSPAIMSASLGEQVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSSSGSGTSYSLTISRMEAEDAATYYCQQSWKAPYTFGGGTKLEIGPAAAATSTYTYATATATTGTTSYTANTTGRPGAAAGGSGSGADGATYTTTPGQVKLQQSGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKFEYLGYISYSGRTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRPYYRYDYAIDYWGQGTTVTVSS (the underlined part is the peptide linker).

[0147] Example 5: Preparation of trastuzumab-derived scFv (GlueCore-Tras-scFv-LH) Plasmid pET28-GlueCore-Tras-scFv-LH was constructed as follows: Using pET28-b(+) (Merck Millipore) as a template, a vector fragment was amplified by PCR with the above-mentioned primer set (pET-NEBuilder-Rv-v2 (SEQ ID NO: 19) and pET-NEBuilder-Fw (SEQ ID NO: 20)).

[0148] The following gene sequence (SEQ ID NO: 44) encoding GlueCore-Tras-scFv-LH was purchased from IDT. CTTTAAGAAGGAGATATACCATG ggttcccatcatcaccaccaccactcatcctcacttgaggtactggagtttcaggggccacatatgggttccaccgccggtgcaggacagtcctggtatggagagatccaaatgacgcagtcgccctcgtcgctttcggcctccgtgggagaccgcgtcacaattacgtgccgcgcgtcgcaagatgtaaacaccgccgttgcgtggtatcaacaga aaccaggtaaagcaccgaaacttcttatttactccgccagttttttatattcgggtgtcccatcccgtttttccggctcgcgctctggcacagacttcactctgacaatttcctccctgcagccggaagatttcgcgacttattattgccagcaacattatactaccccacctacgtttgggcaaggtactaaggtagaaatcggttcaggcggcggtggctcaggtggagggggttccggtgggggtggatcggaggtccagcttgtagaaagcggtggaggtttagtacaaccaggggggtctcttcgtctttcttgcgcggcgtcgggattcaatatcaaggatacttacattcactgggtacgccaagcgcccgggaaggggcttgagtgggtggcacgtatctaccccactaacggatatacccgttatgccgattctgtcaaagggcgctttaccatcagcgcagatacgagcaaaaatacggcgtaccttcaaatgaattcgctgcgtgctgaagacacagcagtttattactgttcccgctggggcggcgacggcttctacgctatggattactggggccaaggcacattggtcactgtgtcatcctaatGCGGCCGCACTCGAGC

[0149] The two resulting fragments were ligated using NEBuilder HiFi DNA assembly (NEB), and the resulting plasmid was designated pET28-GlueCore-Tras-scFv-LH. GlueCore-Tras-scFv-LH was prepared using the pET28-GlueCore-Tras-scFv-LH plasmid in the same manner as in Example 1.

[0150] Furthermore, GlueCore-Tras-scFv-LH was analyzed by gel filtration chromatography in the same manner as in Example 1 ( FIG. 24 ). The trastuzumab-derived scFv (Tras-scFv-LH) prepared in [Comparative Example 3] was used as a control.

[0151] Amino acid sequence of Tras-scFv-LH-Glue (SEQ ID NO: 45) MGSHHHHHH 1 SSSLEVLEFQGPHMGSTAGAGQSWYG 2 EIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIGSGGGGSGGGGSGGGGS 3 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (underlined part 1 indicates His-tag, and the underlined part indicates 2 indicates the core sequence of the peptide tag, and the underlined part indicates the 3 is a peptide linker).

[0152] Example 6 Preparation of Trastuzumab-Derived scFv (Tras-scFv-HL-Glue2) A trastuzumab-derived scFv having the following amino acid sequence (Tras-scFv-HL-Glue2, the underlined part is the linker) was prepared by constructing the following plasmid, pET28-Tras-scFv-HL-Glue2, and following the procedure described in Example 1 (yield: 3.7 mg / 300 mL medium).

[0153] Amino acid sequence of Tras-scFv-HL-Glue2 (SEQ ID NO: 51): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVGGGGSGGGGSGGGSTAGAGQSWYGEIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKS

[0154] The following gene sequence (SEQ ID NO: 52) encoding Tras-scFv-HL-Glue2: CTTTAAGAAGGAGATATACCATG gaagtgcagctggttgaaagcggtggcggtctggtgcaaccgggcggtagcctgcgtctgagctgcgcggcgagcggttttaacatcaaagacac ctacattcactgggttcgtcaagcgccgggcaagggtctggagtgggttgcgcgtatctatccgaccaacggctacacccgttatgcggacagcgt gaaaggtcgttttaccattagcgcggataccagcaagaacaccgcgtacctgcagatgaacagcctgcgtgcggaagacaccgcggtttactattg cagccgttggggcggtgacggcttctacgcgatggattattggggccaaggcaccctggtgaccgttgggggaggggttcgggtggggggggttc ggggggaggcagcactgcaggagctggtcaatcctggtatggggaaatccagatgacccagagcccgagcagcctgagcgcgagcgtgggtgacc gtgttaccattacctgccgtgcgagccaagatgtgaacaccgcggttgcgtggtatcagcaaaagccgggcaaagcgccgaagctgctgatctaca gcgcgagcttcctgtatagcggtgtgccgagccgttttagcggcagccgtagcggcaccgacttcaccctgaccattagcagcctgcagccggaggattttgcgacctactattgccagcaacactataccacccccgccgacctttggtcaaggcaccaaagttgaaatcaagagcGCGGCCGCACTCGAGC

[0155] A comparison of the results of gel filtration chromatography purification of scFv (Tras-scFv-HL and Tras-scFv-HL-Glue2) with the control is shown in FIG.

Claims

1. A polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain connected by a peptide linker, wherein the peptide linker has the amino acid sequence: １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 1); or AX ３４ X ３５ X ３６ S.G.X. ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T (SEQ ID NO: 2), １２ ~X １８ and X ３４ ~X ４２ are each independently selected from naturally occurring amino acid residues.

2. The polypeptide of claim 1, wherein the peptide linker comprises the amino acid sequence: TAGAGQSWYG (SEQ ID NO:3) or AAGGSGSGADGAT (SEQ ID NO:4).

3. The peptide linker has the amino acid sequence: １ P.X. ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5); or X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ S.G.X. ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T.X. ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6), １ ~X １８ and X ３１ ~X ４８ The polypeptide of claim 1, wherein each of the following is independently selected from naturally occurring amino acid residues:

4. The peptide linker has the amino acid sequence: １ P.X. ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GATAGAGQSWYG (SEQ ID NO: 7); or X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 8), １ ~X １１ , X ３１ ~X ３３ , and X ４３ ~X ４８ The polypeptide of claim 1, wherein each of the following is independently selected from naturally occurring amino acid residues:

5. The polypeptide of claim 1, wherein the peptide linker comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GPEEQYSAYATTGATAGAGQSWYG (SEQ ID NO: 9); PGAAAGGSGSGADGATYTTTPG (SEQ ID NO: 10); or GGGGSGGGGSGGGGSTAGAGQSWYG (SEQ ID NO: 47).

6. A polypeptide according to any one of claims 1 to 5, wherein the peptide linker consists of 15 to 60 amino acids.

7. The polypeptide of claim 5, wherein the peptide linker comprises a sequence consisting of 20 or more consecutive amino acids contained in the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

8. A polypeptide according to any one of claims 1 to 7, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

9. A polypeptide according to any one of claims 1 to 8, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:1 and links the C-terminus of the VH domain to the N-terminus of the VL domain.

10. A polypeptide according to any one of claims 1 to 8, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:2 and links the C-terminus of the VL domain to the N-terminus of the VH domain.

11. The polypeptide of claim 9, wherein the peptide linker consists of 15 to 30 amino acids.

12. The polypeptide of claim 10, wherein the peptide linker consists of 35 to 60 amino acids.

13. The VH and VL domains are identical and the peptide linker is (G ４ S) ３ The polypeptide according to any one of claims 1 to 12, which has suppressed aggregation formation as compared to a single-chain antibody comprising the polypeptide 14. A polypeptide according to any one of claims 1 to 13, which is a single chain antibody (scFv).

15. A polypeptide comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain connected by a linker, the polypeptide having a peptide tag at the N-terminus, the peptide tag having the amino acid sequence: １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 1); or AX ３４ X ３５ X ３６ S.G.X. ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T (SEQ ID NO: 2), １２ ~X １８ and X ３４ ~X ４２ each independently selected from naturally occurring amino acid residues.

16. The polypeptide of claim 15, wherein the peptide tag comprises the amino acid sequence: TAGAGQSWYG (SEQ ID NO:3) or AAGGSGSGADGAT (SEQ ID NO:4).

17. The peptide tag has the amino acid sequence: X １ P.X. ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GX １２ X １３ X １４ GX １５ GQX １６ WX １７ X １８ (SEQ ID NO: 5); or X ３１ X ３２ X ３３ AX ３４ X ３５ X ３６ S.G.X. ３７ X ３８ X ３９ X ４０ X ４１ X ４２ T.X. ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 6), １ ~X １８ and X ３１ ~X ４８ 16. The polypeptide of claim 15, wherein each is independently selected from naturally occurring amino acid residues.

18. The peptide tag has the amino acid sequence: X １ P.X. ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ GATAGAGQSWYG (SEQ ID NO: 7); or X ３１ X ３２ X ３３ AAGGSGSGADGATX ４３ X ４４ X ４５ X ４６ X ４７ X ４８ (SEQ ID NO: 8), １ ~X １１ , X ３１ ~X ３３ , and X ４３ ~X ４８ 16. The polypeptide of claim 15, wherein each is independently selected from naturally occurring amino acid residues.

19. The polypeptide of claim 18, wherein the peptide tag comprises a sequence consisting of 15 or more consecutive amino acids contained in the amino acid sequence: GPEEQYSAYATTGATAGAGQSWYG (SEQ ID NO: 9); or PGAAAGGSGSGADGATYTTTPG (SEQ ID NO: 10).

20. The polypeptide of claim 19, wherein the peptide tag consists of 15 to 60 amino acids.

21. The polypeptide of claim 19, wherein the peptide tag comprises a sequence consisting of 20 or more consecutive amino acids contained in the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

22. A polypeptide according to any one of claims 15 to 21, wherein the peptide tag comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

23. A polypeptide according to any one of claims 15 to 22, wherein the peptide tag comprises the amino acid sequence of SEQ ID NO:1, the linker is attached to the C-terminus of the VH domain and the N-terminus of the VL domain, and the peptide tag is attached to the C-terminus of the VL domain.

24. A polypeptide according to any one of claims 15 to 22, wherein the peptide tag comprises the amino acid sequence of SEQ ID NO:2, the linker is attached to the C-terminus of the VL domain and the N-terminus of the VH domain, and the peptide tag is attached to the C-terminus of the VL domain.

25. The polypeptide of claim 23, wherein the peptide tag consists of 15 to 30 amino acids.

26. The polypeptide of claim 24, wherein the peptide tag consists of 35 to 60 amino acids.

27. A polypeptide according to any one of claims 15 to 26, which has suppressed aggregation formation compared to a polypeptide having the same linker, VH domain and VL domain and no peptide tag.

28. A polypeptide according to any one of claims 15 to 27, wherein the linker is a peptide linker.

29. A polypeptide according to any one of claims 15 to 28, which is a single chain antibody (scFv).

30. A nucleic acid comprising a base sequence encoding the amino acid sequence of a polypeptide according to any one of claims 1 to 29.

31. A recombinant vector containing the nucleic acid of claim 30.

32. A transformant incorporating the recombinant vector according to claim 31.

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