Protein l-binding polypeptide and use thereof

Abstract

To provide a polypeptide capable of binding to protein L.SOLUTION: A protein L-binding polypeptide comprising an amino acid sequence 1, that is, L1 sequence-antigen recognition sequence 1-A L2 sequence-antigen recognition sequence 2-A L3 sequence-antigen recognition sequence 3-A L4 sequence. The L1 sequence, L2 sequence, L3 sequence, and L4 sequence are each a specific amino acid sequence. The antigen-recognizing sequences 1, 2, and 3 may be an amino acid sequence encoding the light chain CDR1, an amino acid sequence encoding the light chain CDR2, and an amino acid sequence encoding the light chain CDR3, respectively, or an amino acid sequence encoding the heavy chain CDR1, an amino acid sequence encoding the heavy chain CDR2, and an amino acid sequence encoding the heavy chain CDR3, respectively.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a protein L-binding polypeptide and its use. [Background technology]

[0002] Immunoassays that utilize the molecular recognition ability of antibodies are increasingly using not only full-length antibodies (whole antibodies), but also antibodies other than full-length antibodies, such as single-chain antibodies (single chain Fv; scFv). Single-chain antibodies are generally known as fusion proteins in which the heavy chain variable region (VH) and light chain variable region (VL), which are the antigen recognition sites of full-length antibodies, are linked via a linker. They are smaller in size than full-length antibodies and can be produced inexpensively, for example, using Escherichia coli. For this reason, single-chain antibodies can be considered a new alternative to full-length antibodies.

[0003] Meanwhile, methods for purifying, detecting, and the like proteins such as antibodies using solid phases such as affinity columns and plates are known. For example, one method for purifying antibodies is to use proteins capable of specifically binding to antibodies (antibody-binding proteins), such as Protein L, Protein A, and Protein G, and antibody purification columns onto which antibody-binding proteins are immobilized are commercially available. Protein L is known to specifically bind to the light chain (κ light chain) of antibodies, and Proteins A and G are known to specifically bind to the Fc region of antibodies (Non-Patent Document 1). However, the binding affinity to antibody-binding proteins varies depending on the animal species and subclass of the antibody; for example, Protein L does not bind to all antibodies that have a light chain. Therefore, for example, even if an antibody purification column onto which Protein L is immobilized is used, antibodies that do not have binding affinity to Protein L cannot be purified. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Accelerating ScienceLearning at the Bench: Molecular Biology Experiments: What Everyone Wants to Know About Protein A / G / L | Everything You Need to Know About Protein Experiments, Part 14, by LATB Staff, 03.04.2020, Thermo Fisher Scientific website (https: / / www.thermofisher.com / blog / learning-at-the-bench / protein-basic14 / ) Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to provide polypeptides capable of binding to Protein L. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that a polypeptide having, in order from the N-terminus, amino acid sequence 1, i.e., an L1 sequence (such as the amino acid sequence represented by SEQ ID NO: 1 below), antigen recognition sequence 1 (such as CDR1 described below), L2 sequence (such as the amino acid sequence represented by SEQ ID NO: 2 below), antigen recognition sequence 2 (such as CDR2 described below), L3 sequence (such as the amino acid sequence represented by SEQ ID NO: 3 below), antigen recognition sequence 3 (such as CDR3 described below), and L4 sequence (such as the amino acid sequence represented by SEQ ID NO: 4 below) can bind to Protein L. The present inventors have also found that even when the amino acid sequences of antigen recognition sequences 1 to 3 located within the polypeptide are altered depending on the target antigen, the polypeptide can still bind to Protein L. Based on this finding, the present inventors have found that the L1 sequence, L2 sequence, L3 sequence, and L4 sequence contribute to the protein L-binding ability of the polypeptide. The present invention was completed through further research based on these findings, and the present disclosure encompasses, for example, the following representative inventions. Section 1. A protein L binding polypeptide comprising the following amino acid sequence: [amino acid sequence 1] L1 sequence-antigen recognition sequence 1-L2 sequence-antigen recognition sequence 2-L3 sequence-antigen recognition sequence 3-L4 sequence where: The L1 sequence consists of the following (L1-1) or (L1-2): (L1-1) the amino acid sequence represented by SEQ ID NO: 1; (L1-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1, and the isoelectric point of the amino acid sequence represented by SEQ ID NO: 1 is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 1; The L2 sequence consists of the following (L2-1) or (L2-2): (L2-1) the amino acid sequence represented by SEQ ID NO: 2; (L2-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, and the isoelectric point of the amino acid sequence represented by SEQ ID NO: 2 is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 2; The L3 sequence consists of the following (L3-1) or (L3-2): (L3-1) the amino acid sequence represented by SEQ ID NO: 3; (L3-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3, and the isoelectric point of the amino acid sequence represented by SEQ ID NO: 3 is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 3; The L4 sequence consists of the following (L4-1) or (L4-2): (L4-1) the amino acid sequence represented by SEQ ID NO: 4; (L4-2) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and which has an isoelectric point equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 4. Section 2. the amino acid sequence described in (L1-1) is an amino acid sequence represented by any one of SEQ ID NOs: 5 to 10, The amino acid sequence described in (L2-1) above is an amino acid sequence represented by any one of SEQ ID NOs: 11 to 16, the amino acid sequence described in (L3-1) is an amino acid sequence represented by any one of SEQ ID NOs: 17 to 22, and / or The amino acid sequence described in (L4-1) above is an amino acid sequence represented by any one of SEQ ID NOs: 23 to 28. Item 1. A protein L-binding polypeptide according to Item 1. Section 3. The antigen recognition sequences 1, 2, and 3 are, in order, an amino acid sequence encoding a light chain CDR1, an amino acid sequence encoding a light chain CDR2, and an amino acid sequence encoding a light chain CDR3; an amino acid sequence encoding a heavy chain CDR1, an amino acid sequence encoding a heavy chain CDR2, and an amino acid sequence encoding a heavy chain CDR3; Item 3. The Protein L-binding polypeptide according to Item 1 or 2. Section 4. Item 4. The protein L-binding polypeptide according to any one of Items 1 to 3, further comprising the following amino acid sequence 2 on the N-terminal or C-terminal side of the amino acid sequence 1: [Amino acid sequence 2] H1 sequence - antigen recognition sequence 4 - H2 sequence - antigen recognition sequence 5 - H3 sequence - antigen recognition sequence 6 - H4 sequence where: The H1 sequence consists of the following (H1-1) or (H1-2): (H1-1) the amino acid sequence represented by SEQ ID NO: 29; (H1-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 29, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 29; The H2 sequence consists of the following (H2-1) or (H2-2): (H2-1) the amino acid sequence represented by SEQ ID NO: 30; (H2-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 30, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 30; The H3 sequence consists of the following (H3-1) or (H3-2): (H3-1) the amino acid sequence represented by SEQ ID NO: 31; (H3-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 31, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 31; The H4 sequence consists of the following (H4-1) or (H4-2): (H4-1) the amino acid sequence represented by SEQ ID NO: 32; (H4-2) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 32, and which has an isoelectric point equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 32. Section 5. The amino acid sequence described in (H1-1) above is an amino acid sequence represented by any one of SEQ ID NOs: 33 to 38, the amino acid sequence described in (H2-1) above is the amino acid sequence represented by SEQ ID NO: 39 or 40, and / or The amino acid sequence described in (H3-1) above is an amino acid sequence represented by any one of SEQ ID NOs: 41 to 46. Item 5. A protein L-binding polypeptide according to Item 4. Section 6. Item 6. The protein L-binding polypeptide according to Item 4 or 5, wherein the antigen recognition sequences 1, 2, and 3 are, in order, an amino acid sequence encoding a light chain CDR1, an amino acid sequence encoding a light chain CDR2, and an amino acid sequence encoding a light chain CDR3, and the antigen recognition sequences 4, 5, and 6 are, in order, an amino acid sequence encoding a heavy chain CDR1, an amino acid sequence encoding a heavy chain CDR2, and an amino acid sequence encoding a heavy chain CDR3. Section 7. An antibody comprising the Protein L-binding polypeptide according to any one of Items 1 to 6. Section 8. The antibody according to item 7, which is used as an scFv, Fab, F(ab)2', scFv-Fc, scAb, sdAb or full-length antibody. [Effects of the Invention]

[0007] It is possible to provide a polypeptide capable of binding to Protein L. It is also possible to provide an antibody comprising the polypeptide. By utilizing the Protein L-binding ability resulting from the L1, L2, L3, and L4 sequences of the polypeptide, the polypeptide and / or the antibody can be easily obtained by purification and detection means using a Protein L-supported solid phase, etc. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows the amino acid sequence (SEQ ID NO: 47) of the single-chain antibody of Comparative Example 1. [Figure 2] The amino acid sequence of the single-chain antibody of Example 1 (SEQ ID NO: 48) is shown. [Figure 3] The amino acid sequence of the single-chain antibody of Example 2 (SEQ ID NO: 49) is shown. [Figure 4] The amino acid sequence of the single-chain antibody of Example 3 (SEQ ID NO: 50) is shown. [Figure 5] 1 shows the results of Test Example 1 (antigen binding). [Figure 6] 1 shows the results of Test Example 1 (protein L binding). [Figure 7] 1 shows the results of Test Example 1 (protein L binding). [Figure 8] The amino acid sequences of Example 4 (SEQ ID NO: 51), Comparative Example 2 (SEQ ID NO: 52), and Comparative Example 3 (SEQ ID NO: 53) are shown below. [Figure 9] 1 shows the results of Test Example 2 (antigen binding). [Figure 10] The amino acid sequences of the single-chain antibodies of Example 5 (SEQ ID NO: 54) and Example 6 (SEQ ID NO: 55) are shown. [Figure 11]The amino acid sequences of the single-chain antibodies of Example 7 (SEQ ID NO: 56) and Example 8 (SEQ ID NO: 57) are shown. [Figure 12] 1 shows the results of Test Example 3 (protein L binding). [Figure 13] 1 shows the amino acid sequence of the single chain antibody of Example 10 (SEQ ID NO: 58). [Figure 14] 1 shows the amino acid sequence of the VHH antibody of Example 11 (SEQ ID NO: 59). [Figure 15] 1 shows the results of Test Example 4 (protein L binding). DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments included in the present disclosure will be described in more detail. In the present disclosure, the terms "contain" and "comprise" also encompass the meanings of "consist essentially of" and "consist of."

[0010] Protein L-binding polypeptide The present disclosure encompasses a protein L-binding polypeptide comprising the following amino acid sequence 1: In the present disclosure, the protein L-binding polypeptide may be referred to as the "polypeptide of the present disclosure." [amino acid sequence 1] L1 sequence-antigen recognition sequence 1-L2 sequence-antigen recognition sequence 2-L3 sequence-antigen recognition sequence 3-L4 sequence

[0011] L1 sequence The L1 sequence consists of the following (L1-1) or (L1-2): (L1-1) the amino acid sequence represented by SEQ ID NO: 1; (L1-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 1.

[0012] SEQ ID NO: 1 is as follows: ELX a1 X a2 TQTXa3 X a4 SVX a5 X a6 X a7 VGGTVTIX a8 CQX a9 (X a1 is D or V, and X a2 is L or M, and X a3 is P or A, and X a4 is A or S, and X a5 is E or S, and X a6 is A or V, and X a7 is A or S, and X a8 is K, R or S, and X a9 is A or S)

[0013] The amino acid sequence represented by SEQ ID NO: 1 is not limited thereto, but is preferably exemplified by the amino acid sequence represented by any one of SEQ ID NOs: 5 to 10. The amino acid sequences of SEQ ID NOs: 5 to 10 and their isoelectric points are shown in Table 1, together with the amino acid sequence represented by SEQ ID NO: 1.

[0014] [Table 1]

[0015] As shown in Table 1, the sequence with the lowest isoelectric point among SEQ ID NOS: 5 to 10 is SEQ ID NOS: 7 (with an isoelectric point of approximately 3.6). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NOS: 1 is preferably 3.6 or higher, more preferably 3.6 to 5, and even more preferably 3.6 to 4.6. In the present disclosure, the isoelectric point is calculated using the Expasy protparam tool (https: / / web.expasy.org / protparam / ) (Gasteiger E., et al., Protein Identification and Analysis Tools on the Expasy Server, in John M. Walker (ed.), The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607; https: / / web.expasy.org / docs / expasy_tools05.pdf). The tool calculates the isoelectric point by inputting the amino acid sequence into a specified screen. Below, the isoelectric point will be explained in a similar manner.

[0016] In (L1-2), examples of plural numbers include 2 to 10 numbers, 2 to 9 numbers, 2 to 8 numbers, 2 to 7 numbers, 2 to 6 numbers, 2 to 5 numbers, 2 to 4 numbers, and 2 or 3 numbers.

[0017] In the present disclosure, the mutated amino acid may be, for example, either a natural amino acid or an artificial amino acid. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, and sulfur-containing amino acids. Specific examples of amino acids include 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). Techniques for deleting, substituting, inserting, and / or adding one or more amino acids in a specific amino acid sequence are known.

[0018] Preferred examples of mutations in the amino acid sequence of (L1-2) include amino acid substitutions. While not limiting the present disclosure, preferred examples of such substitutions include a substitution of K, a basic amino acid with a polar charged side chain, with R (i.e., K may be substituted with R, or R may be substituted with K, more preferably K with R), and a substitution of D, an acidic amino acid with a polar charged side chain, with E (i.e., D may be substituted with E, or E may be substituted with D, more preferably D with E).

[0019] Furthermore, although not limiting the present disclosure, a preferred example of such substitution is the substitution of an amino acid selected from nonpolar side chain amino acids (e.g., G, A, V, L, I, M, P, F, W) and polar uncharged side chain amino acids (e.g., S, T, N, Q, Y, C) with an amino acid selected from K, R, D, E, and H.

[0020] Furthermore, without limiting the present disclosure, preferred examples include substitution of an amino acid selected from nonpolar side chain amino acids with an amino acid selected from nonpolar side chain amino acids and polar uncharged side chain amino acids, and substitution of an amino acid selected from polar uncharged side chain amino acids with an amino acid selected from nonpolar side chain amino acids and polar uncharged side chain amino acids. More preferably, from the viewpoint of amino acid structural similarity, examples of such substitutions include substitution of N with Q (i.e., N may be substituted with Q, or Q may be substituted with N), substitution of S with T (i.e., S may be substituted with T, or T may be substituted with S), substitution of V with I with L (i.e., V may be substituted with I or L, or I may be substituted with V or L, or L may be substituted with V or I), and substitution of Y with W with F (i.e., Y may be substituted with W or F, or W may be substituted with Y or W).

[0021] Although not limiting the present disclosure, more preferred examples of the mutation include the substitution of K with R, the substitution of D with E, the substitution of N with Q, the substitution of S with T, the substitution of V with I with L, and the substitution of Y with W with F. Furthermore, from the viewpoint of obtaining a sequence with a higher isoelectric point, more preferred examples of the mutation include substitution of an amino acid selected from nonpolar side chain amino acids and polar uncharged side chain amino acids with an amino acid selected from K, R, and H.

[0022] The isoelectric point of the amino acid sequence of (L1-2) is preferably 3.6 or more, more preferably 3.6 to 5, and even more preferably 3.6 to 4.6.

[0023] The number of amino acid residues in the amino acid sequence of (L1-2) is, for example, 22 to 30, preferably 23, 24, 25, 26 or 27, and more preferably 25, for example.

[0024] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (L1-2) include amino acid sequences in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by any of SEQ ID NOs: 5 to 10, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before mutation). That is, preferred examples of the amino acid sequence of (L1-2) include the following (L1-2-5) to (L1-2-10).

[0025] (L1-2-5) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added (hereinafter sometimes referred to as "substitution, etc.") in the amino acid sequence represented by SEQ ID NO: 5, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 5 (hereinafter sometimes referred to as the "mutated sequence of SEQ ID NO: 5").

[0026] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 5 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 5 is preferably 4.1 or more, more preferably 4.1 to 5, and even more preferably 4.1 to 4.6.

[0027] (L1-2-6) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 6, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 6 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 6").

[0028] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 6 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 6 is preferably 4.1 or more, more preferably 4.1 to 5, and even more preferably 4.1 to 4.6.

[0029] (L1-2-7) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 7 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 7 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 7").

[0030] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 7 is approximately 3.6, the isoelectric point of the mutant sequence of SEQ ID NO: 7 is preferably 3.6 or more, more preferably 3.6 to 5, and even more preferably 3.6 to 4.6.

[0031] (L1-2-8) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 8, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 8 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 8").

[0032] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 8 is about 4.5, the isoelectric point of the mutant sequence of SEQ ID NO: 8 is preferably 4.5 or more, more preferably 4.5 to 5, for example.

[0033] (L1-2-9) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 9, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 9 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 9").

[0034] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 9 is about 4.5, the isoelectric point of the mutant sequence of SEQ ID NO: 9 is preferably 4.5 or more, more preferably 4.5 to 5, for example.

[0035] (L1-2-10) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 10, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 10 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 10").

[0036] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 10 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 10 is preferably 4.1 or more, more preferably 4.1 to 5, and even more preferably 4.1 to 4.6.

[0037] L2 array The L2 sequence consists of the following (L2-1) or (L2-2): (L2-1) Amino acid sequence represented by SEQ ID NO: 2 (L2-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 2.

[0038] SEQ ID NO:2 is as follows: LX b1 WYQQX b2 PGQX b3 PX b4 X b5 LIY (X b1 is A, G or S, X b2 is K or R, and X b3 is R or P, and X b4 is R or K, and X b5 is L or R)

[0039] The amino acid sequence represented by SEQ ID NO: 2 is not limited thereto, but is preferably exemplified by any of the amino acid sequences represented by SEQ ID NOs: 11 to 16. The amino acid sequences and their isoelectric points of SEQ ID NOs: 11 to 16 are shown in Table 2, along with the amino acid sequence represented by SEQ ID NO: 2.

[0040] [Table 2]

[0041] As shown in Table 2, the sequences with the lowest isoelectric points among SEQ ID NOs: 11 to 16 are SEQ ID NOs: 13, 15, and 16 (with an isoelectric point of approximately 9.5). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 2 is preferably 9.5 or higher, more preferably 9.5 to 11.5, and even more preferably 9.5 to 11.

[0042] In (L2-2), "plural" refers to 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. As in the explanation for (L1-2), preferred examples of mutations in the amino acid sequence of (L2-2) include amino acid substitutions, and the substitutions are as explained above. Preferred examples of the isoelectric point of the amino acid sequence of (L2-2) include 9.5 or more, more preferably 9.5 to 11.5, and even more preferably 9.5 to 11.

[0043] The number of amino acid residues in the amino acid sequence of (L2-2) is, for example, 15 to 22, preferably 16, 17, 18, 19 or 20, and more preferably 17, for example.

[0044] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (L2-2) include amino acid sequences represented by any of SEQ ID NOS: 11 to 16 in which one or more amino acids have been substituted, deleted, inserted, or added, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (L2-2) include the amino acid sequences shown in the following (L2-2-11) to (L2-2-16).

[0045] (L2-2-11) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 11 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 11 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 11").

[0046] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 11 is approximately 10.2, the isoelectric point of the mutant sequence of SEQ ID NO: 11 is preferably 10.2 or more, more preferably 10.2 to 11.5, and even more preferably 10.2 to 11, etc.

[0047] (L2-2-12) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 12, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 12 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 12").

[0048] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 12 is approximately 10.9, the isoelectric point of the mutant sequence of SEQ ID NO: 12 is preferably 10.9 or more, more preferably 10.9 to 11.5, and even more preferably 10.9 to 11, etc.

[0049] (L2-2-13) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 13, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 13 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 13").

[0050] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 13 is approximately 9.5, the isoelectric point of the mutant sequence of SEQ ID NO: 13 is preferably 9.5 or more, more preferably 9.5 to 11.5, and even more preferably 9.5 to 11, etc.

[0051] (L2-2-14) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 14 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 14 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 14").

[0052] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 11 is 11, the isoelectric point of the mutant sequence of SEQ ID NO: 14 is preferably 11 or more, more preferably 11 to 11.5, for example.

[0053] (L2-2-15) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 15 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 15 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 15").

[0054] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 15 is approximately 9.5, the isoelectric point of the mutant sequence of SEQ ID NO: 15 is preferably 9.5 or more, more preferably 9.5 to 11.5, and even more preferably 9.5 to 11.

[0055] (L2-2-16) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 16 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 16 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 16").

[0056] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 16 is approximately 9.5, the isoelectric point of the mutant sequence of SEQ ID NO: 16 is preferably 9.5 or more, more preferably 9.5 to 11.5, and even more preferably 9.5 to 11.

[0057] L3 sequence The L3 sequence consists of the following (L3-1) or (L3-2): (L3-1) Amino acid sequence represented by SEQ ID NO: 3 (L3-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 3.

[0058] SEQ ID NO: 3 is as follows: X c1 LASGVX c2 SRFX c3 X c4 SX c5 SGTX c6 X c7 TLTIX c8 DLX c9 X c10 X c11 DX c12 ATYYC (X c1 is T, K or N, and X c2 is S or P, and X c3 is K, R, T or E, and X c4 is G or S, and X c5 is G or R, and X c6 is D or E, and X c7 is F or Y, and X c8 is S or N, and X c9 is Q or E, and X c10 is C or S, and X c11 is D or A, and X c12 is A or G)

[0059] The amino acid sequence represented by SEQ ID NO: 3 is not limited thereto, but is preferably exemplified by the amino acid sequence represented by any one of SEQ ID NOs: 17 to 22. The amino acid sequences and their isoelectric points of SEQ ID NOs: 17 to 22 are shown in Table 3, along with the amino acid sequence represented by SEQ ID NO: 3.

[0060] [Table 3]

[0061] As shown in Table 3, the sequence with the lowest isoelectric point among SEQ ID NOs: 17 to 22 is SEQ ID NO: 19 (with an isoelectric point of approximately 4). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 3 is preferably 4 or more, more preferably 4 to 5.5, and even more preferably 4 to 5, etc.

[0062] In (L3-2), "plural" refers to 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. As in the explanation for (L1-2), preferred examples of mutations in the amino acid sequence of (L3-2) include amino acid substitutions, and the substitutions are as explained above. Preferred examples of the isoelectric point of the amino acid sequence of (L3-2) include 4 or more, more preferably 4 to 5.5, and even more preferably 4 to 5, etc.

[0063] The number of amino acid residues in the amino acid sequence of (L3-2) is, for example, 33 to 40, preferably 34, 35, 36, 37 or 38, and more preferably 36, for example.

[0064] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (L3-2) include amino acid sequences represented by any of SEQ ID NOS: 17 to 22 in which one or more amino acids have been substituted, deleted, inserted, or added, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (L3-2) include the amino acid sequences shown below in (L3-2-17) to (L3-2-22).

[0065] (L3-2-17) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 17 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 17 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 17").

[0066] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 17 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 17 is preferably 4.1 or more, more preferably 4.1 to 5.5, and even more preferably 4.1 to 5, etc.

[0067] (L3-2-18) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 18 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 18 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 18").

[0068] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 18 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 18 is preferably 4.1 or more, more preferably 4.1 to 5.5, and even more preferably 4.1 to 5, etc.

[0069] (L3-2-19) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 19 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 19 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 19").

[0070] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 19 is about 4, the isoelectric point of the mutant strain of SEQ ID NO: 19 is preferably 4 or more, more preferably 4 to 5.5, and even more preferably 4 to 5, etc.

[0071] (L3-2-20) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 20, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 20 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 20").

[0072] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 20 is approximately 4.7, the isoelectric point of the mutant sequence of SEQ ID NO: 20 is preferably 4.7 or more, more preferably 4.7 to 5.5, and even more preferably 4.7 to 5.

[0073] (L3-2-21) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 21 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 21 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 21").

[0074] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 21 is approximately 4.2, the isoelectric point of the mutant sequence of SEQ ID NO: 21 is preferably 4.2 or more, more preferably 4.2 to 5.5, and even more preferably 4.2 to 5, etc.

[0075] (L3-2-22) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 22, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 22 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 22").

[0076] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 22 is approximately 4.1, the isoelectric point of the mutant sequence of SEQ ID NO: 22 is preferably 4.1 or more, more preferably 4.1 to 5.5, and even more preferably 4.1 to 5, etc.

[0077] L4 sequence The L4 sequence consists of the following (L4-1) or (L4-2): (L4-1) the amino acid sequence represented by SEQ ID NO: 4; (L4-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 4.

[0078] SEQ ID NO: 4 is as follows: FGGTX d1 VX d2 VX d3 X d4 S (X d1 is E or G, and X d2is V, E, or D, and X d3 is K, R or T, and X d4 is R or G)

[0079] The amino acid sequence represented by SEQ ID NO:4 is not limited thereto, but is preferably exemplified by any of the amino acid sequences represented by SEQ ID NOs:23 to 28. The amino acid sequences of SEQ ID NOs:23 to 28 and their isoelectric points are shown in Table 4, along with the amino acid sequence represented by SEQ ID NO:4.

[0080] [Table 4]

[0081] As shown in Table 4, the sequence with the lowest isoelectric point among SEQ ID NOs: 23 to 28 is SEQ ID NO: 25 (with an isoelectric point of approximately 4.5). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 4 is preferably 4.5 or higher, more preferably 4.5 to 11, and even more preferably 4.5 to 10, for example.

[0082] In (L4-2), "plural" refers to, for example, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. As in the explanation for (L1-2), preferred examples of mutations in the amino acid sequence of (L4-2) include amino acid substitutions, and the substitutions are as explained above. Preferred examples of the isoelectric point of the amino acid sequence of (L4-2) include 4.5 or more, more preferably 4.5 to 11, and even more preferably 4.5 to 10.

[0083] The number of amino acid residues in the amino acid sequence of (L4-2) is, for example, 10 to 15, preferably 11, 12, 13 or 14, and more preferably 12, for example.

[0084] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (L4-2) include amino acid sequences in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by any one of SEQ ID NOS: 23 to 28, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (L4-2) include the amino acid sequences shown below in (L4-2-23) to (L4-2-28).

[0085] (L4-2-23) An amino acid sequence in which the above-mentioned substitution or the like has been made in the amino acid sequence represented by SEQ ID NO: 23, and in which the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 23 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 23").

[0086] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 23 is approximately 8.7, the isoelectric point of the mutant sequence of SEQ ID NO: 23 is preferably 8.7 or higher, more preferably 8.7 to 11, and even more preferably 8.7 to 10, etc.

[0087] (L4-2-24) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 24 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 24 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 24").

[0088] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 24 is approximately 9.6, the isoelectric point of the mutant sequence of SEQ ID NO: 23 is preferably 9.6 or higher, more preferably 9.6 to 11, and even more preferably 9.6 to 10, etc.

[0089] (L4-2-25) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 25 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 25 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 25").

[0090] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 25 is approximately 4.5, the isoelectric point of the mutant sequence of SEQ ID NO: 23 is preferably 4.5 or more, more preferably 4.5 to 11, and even more preferably 4.5 to 10, etc.

[0091] (L4-2-26) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 26 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 26 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 26").

[0092] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 26 is approximately 6.1, the isoelectric point of the mutant sequence of SEQ ID NO: 26 is preferably 6.1 or more, more preferably 6.1 to 11, and even more preferably 6.1 to 10, etc.

[0093] (L4-2-27) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 27 and which has been subjected to the above-mentioned substitution, etc., and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 27 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 27").

[0094] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 27 is approximately 6, the isoelectric point of the mutant sequence of SEQ ID NO: 27 is preferably 6 or more, more preferably 6 to 11, and even more preferably 6 to 10, etc.

[0095] (L4-2-28) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 28 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 28 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 28").

[0096] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 28 is approximately 5.8, the isoelectric point of the mutant sequence of SEQ ID NO: 28 is preferably 5.8 or more, more preferably 5.8 to 11, and even more preferably 5.8 to 10, etc.

[0097] In the present disclosure, the L1, L2, L3, and L4 sequences are not limited as described above. While not limiting the present disclosure, the isoelectric point of an amino acid sequence consisting of only the L1, L2, L3, and L4 sequences, in which these four sequences are directly linked, is preferably 4.3 or higher, more preferably 4.3 to 10, and even more preferably 4.3 to 9. The isoelectric points of the amino acid sequences consisting of only the L1, L2, L3, and L4 sequences used in the test examples described below are shown in the "VL frame" in Table 9.

[0098] The total number of amino acid residues in the L1, L2, L3 and L4 sequences is 85 to 100, preferably 88 to 95, and more preferably 90, for example.

[0099] Each of the sequences in L1, L2, L3, and L4 is usually selected from the aforementioned sequences and used in combination, and although there is no limitation thereto, preferred examples include combinations selected from the following (A) to (C). (A) [L1] SEQ ID NO:5, SEQ ID NO:6, a variant of SEQ ID NO:5, or a variant of SEQ ID NO:6: [L2] SEQ ID NO:11, SEQ ID NO:12, a variant of SEQ ID NO:11, or a variant of SEQ ID NO:12: [L3] SEQ ID NO: 17, SEQ ID NO: 18, a variant of SEQ ID NO: 17, or a variant of SEQ ID NO: 18: [L4] SEQ ID NO: 23, SEQ ID NO: 24, a mutant sequence of SEQ ID NO: 23, or a mutant sequence of SEQ ID NO: 24 (B) [L1] SEQ ID NO:7, SEQ ID NO:8, a variant of SEQ ID NO:7, or a variant of SEQ ID NO:8: [L2] SEQ ID NO: 13, SEQ ID NO: 14, a variant of SEQ ID NO: 13, or a variant of SEQ ID NO: 14: [L3] SEQ ID NO: 19, SEQ ID NO: 20, a variant of SEQ ID NO: 19, or a variant of SEQ ID NO: 20: [L4] SEQ ID NO: 25, SEQ ID NO: 26, a variant of SEQ ID NO: 25, or a variant of SEQ ID NO: 26 (C) [L1] SEQ ID NO:9, SEQ ID NO:10, a variant of SEQ ID NO:9, or a variant of SEQ ID NO:10: [L2] SEQ ID NO: 15, SEQ ID NO: 16, a variant of SEQ ID NO: 15, or a variant of SEQ ID NO: 16: [L3] SEQ ID NO:21, SEQ ID NO:22, a variant of SEQ ID NO:21, or a variant of SEQ ID NO:22: [L4] SEQ ID NO: 27, SEQ ID NO: 28, a variant of SEQ ID NO: 27, or a variant of SEQ ID NO: 28

[0100] Furthermore, although this disclosure is not limited thereto, when further subdividing, the following combinations are preferred examples. (A) (A-1) [L1] SEQ ID NO: 5 or a mutant sequence of SEQ ID NO: 5; [L2] SEQ ID NO: 11 or a mutant sequence of SEQ ID NO: 11; [L3] SEQ ID NO: 17 or a mutant sequence of SEQ ID NO: 17; [L4] SEQ ID NO: 23 or a mutant sequence of SEQ ID NO: 23; (A-2) [L1] SEQ ID NO: 6 or a mutant sequence of SEQ ID NO: 6; [L2] SEQ ID NO: 12 or a mutant sequence of SEQ ID NO: 12; [L3] SEQ ID NO: 18 or a mutant sequence of SEQ ID NO: 18; [L4] SEQ ID NO: 24 or a mutant sequence of SEQ ID NO: 24 (B) (B-1) [L1] SEQ ID NO: 7 or a mutant sequence of SEQ ID NO: 7: [L2] SEQ ID NO: 13 or a mutant sequence of SEQ ID NO: 13: [L3] SEQ ID NO: 19 or a mutant sequence of SEQ ID NO: 19: [L4] or a mutant sequence of SEQ ID NO: 25 or a mutant sequence of SEQ ID NO: 25; (B-2) [L1] SEQ ID NO: 8 or a mutant sequence of SEQ ID NO: 8: [L2] SEQ ID NO: 14 or a mutant sequence of SEQ ID NO: 14: [L3] SEQ ID NO: 20 or a mutant sequence of SEQ ID NO: 20: [L4] SEQ ID NO: 26 or a mutant sequence of SEQ ID NO: 26 (C) (C-1) [L1] SEQ ID NO: 9 or a mutant sequence of SEQ ID NO: 9; [L2] SEQ ID NO: 15 or a mutant sequence of SEQ ID NO: 15; [L3] SEQ ID NO: 21 or a mutant sequence of SEQ ID NO: 21; [L4] SEQ ID NO: 27 or a mutant sequence of SEQ ID NO: 27; (C-2) [L1] SEQ ID NO: 10 or a mutant sequence of SEQ ID NO: 10; [L2] SEQ ID NO: 16 or a mutant sequence of SEQ ID NO: 16; [L3] SEQ ID NO: 22 or a mutant sequence of SEQ ID NO: 22; [L4] SEQ ID NO: 28 or a mutant sequence of SEQ ID NO: 28

[0101] Antigen recognition sequences 1-3 Antigen recognition sequence 1, antigen recognition sequence 2, and antigen recognition sequence 3 may be, in that order, amino acid sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3, which are antibody-derived light chain complementarity determining regions, or may be amino acid sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, which are antibody-derived heavy chain complementarity determining regions. Furthermore, while CDRs are typically derived from animals (and present in animal-derived antibodies), in the present disclosure, antigen recognition sequence 1, antigen recognition sequence 2, and antigen recognition sequence 3 may each be a combination of non-animal-derived amino acid sequences (e.g., artificially created amino acid sequences) having antigen recognition ability corresponding to light chain CDR1, CDR2, and CDR3, or a combination of non-animal-derived amino acid sequences (e.g., artificially created amino acid sequences) having antigen recognition ability corresponding to heavy chain CDR1, CDR2, and CDR3. Thus, antigen recognition sequences 1 to 3 are not particularly limited as long as the three amino acid sequences together are amino acid sequences that can recognize the target antigen, like CDRs 1 to 3 found in conventionally known antibodies.

[0102] Preferred examples of antigen recognition sequences 1 to 3 include, in order, an amino acid sequence encoding light chain CDR1, an amino acid sequence encoding light chain CDR2, and an amino acid sequence encoding light chain CDR3; an amino acid sequence of non-animal origin having antigen recognition ability corresponding to light chain CDR1, an amino acid sequence of non-animal origin having antigen recognition ability corresponding to light chain CDR2, and an amino acid sequence of non-animal origin having antigen recognition ability corresponding to light chain CDR3.

[0103] As described above, the number of amino acid residues in antigen recognition sequence 1 is not limited as long as the three sequences of antigen recognition sequences 1 to 3 together can recognize the target antigen, but the number of amino acid residues in antigen recognition sequence 1 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10. Similarly, the number of amino acid residues in antigen recognition sequence 2 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10. Similarly, the number of amino acid residues in antigen recognition sequence 3 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10.

[0104] In amino acid sequence 1, each sequence (i.e., L1 sequence, antigen recognition sequence 1, L2 sequence, antigen recognition sequence 2, L3 sequence, antigen recognition sequence 3, and L4 sequence, in that order from the N-terminus) is directly or indirectly linked, and when indirectly linked, they are preferably linked via a linker. Linkers are as described below. In amino acid sequence 1, each sequence is preferably directly linked.

[0105] The Protein L-binding polypeptide of the present disclosure is capable of binding to Protein L. In the present disclosure, Protein L is Protein L that can be used by being linked to a support for antibody purification, and supports to which Protein L is linked are commercially available. Therefore, the polypeptide of the present disclosure is not limited as long as it can bind to such Protein L, but more preferably, it is exemplified by a polypeptide that can bind to Protein L with the same or higher efficiency as in Example 5 described below. Whether or not it can bind to Protein L with the same or higher efficiency as in Example 5 can be determined by using a commercially available affinity support for antibody purification, KanCap L (KANEKA KanCap), preferably according to the procedure for "Protein L binding" described in Test Example 3 described below. TM L (50% slurry), a cellulose-based Protein L carrier (manufactured by Kaneka Corporation).

[0106] Amino acid sequence 2 The protein L-binding polypeptide of the present disclosure may further have the following amino acid sequence 2 linked to the N-terminus or C-terminus of amino acid sequence 1. [Amino acid sequence 2] H1 sequence - antigen recognition sequence 4 - H2 sequence - antigen recognition sequence 5 - H3 sequence - antigen recognition sequence 6 - H4 sequence

[0107] H1 sequence The H1 sequence consists of the following (H1-1) or (H1-2): (H1-1) Amino acid sequence represented by SEQ ID NO: 29 (H1-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 29, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 29.

[0108] SEQ ID NO: 29 is as follows: QSX e1 X e2 ESX e3 GRLVX e4 PGX e5 X e6 LTLTCTXe7 (X e1 is V or L, and X e2 is K, R or E, and X e3 is G or E, and X e4 is T or M, and X e5 is T or G, and X e6 is P or S, and X e7 is A or V)

[0109] The amino acid sequence represented by SEQ ID NO:29 is not limited thereto, but is preferably exemplified by the amino acid sequence represented by any one of SEQ ID NOs:33 to 38. The amino acid sequences of SEQ ID NOs:33 to 38 and their isoelectric points are shown in Table 1, together with the amino acid sequence represented by SEQ ID NO:29.

[0110] [Table 5]

[0111] As shown in Table 5, the sequence with the lowest isoelectric point among SEQ ID NOs: 33 to 38 is SEQ ID NO: 38 (with an isoelectric point of approximately 4.5). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 29 is preferably 4.5 or higher, more preferably 4.5 to 10, and even more preferably 4.5 to 9, for example.

[0112] In (H1-2), "plural" refers to 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. As in the explanation for (H1-2), preferred examples of mutations in the amino acid sequence of (H1-2) include amino acid substitutions, and the substitutions are as explained above. Preferred examples of the isoelectric point of the amino acid sequence of (H1-2) include 4.5 or more, more preferably 4.5 to 10, and even more preferably 4.5 to 9.

[0113] The number of amino acid residues in the amino acid sequence of (H1-2) is, for example, 20 to 25, preferably 22, 23 or 24, and more preferably 23, for example.

[0114] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (H1-2) include amino acid sequences in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by any one of SEQ ID NOs: 33 to 38, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (H1-2) include the amino acid sequences shown in the following (H1-2-33) to (H1-2-38).

[0115] (H1-2-33) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 33 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 33 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 33").

[0116] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 33 is approximately 8.2, the isoelectric point of the mutant sequence of SEQ ID NO: 33 is preferably 8.2 or higher, more preferably 8.2 to 10, and even more preferably 8.2 to 9, etc.

[0117] (H1-2-34) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 34 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 34 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 34").

[0118] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 34 is approximately 8.2, the isoelectric point of the mutant sequence of SEQ ID NO: 34 is preferably 8.2 or more, more preferably 8.2 to 10, and even more preferably 8.2 to 9, etc.

[0119] (H1-2-35) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 35 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 35 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 35").

[0120] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 35 is approximately 6.1, the isoelectric point of the mutant sequence of SEQ ID NO: 35 is preferably 6.1 or more, more preferably 6.1 to 10, and even more preferably 6.1 to 9, etc.

[0121] (H1-2-36) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 36 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 36 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 36").

[0122] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 36 is approximately 8.2, the isoelectric point of the mutant sequence of SEQ ID NO: 36 is preferably 8.2 or more, more preferably 8.2 to 10, and even more preferably 8.2 to 9, etc.

[0123] (H1-2-37) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 37 and which has been substituted or otherwise modified, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 37 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 37").

[0124] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 37 is approximately 6.1, the isoelectric point of the mutant sequence of SEQ ID NO: 37 is preferably 6.1 or more, more preferably 6.1 to 10, and even more preferably 6.1 to 9, etc.

[0125] (H1-2-38) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 38 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 38 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 38").

[0126] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 38 is approximately 4.5, the isoelectric point of the mutant sequence of SEQ ID NO: 38 is, for example, 4.5 or more, more preferably 4.5 to 10, and even more preferably 4.5 to 9.

[0127] H2 sequence In amino acid sequence 2, the H2 sequence consists of the following (H2-1) or (H2-2). (H2-1) the amino acid sequence represented by SEQ ID NO: 30; (H2-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 30, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 30.

[0128] SEQ ID NO: 30 is as follows: WVRQAPGX f1 GLE (X f1 is K or R)

[0129] The amino acid sequence represented by SEQ ID NO: 30 is not limited thereto, but is preferably exemplified by the amino acid sequence represented by SEQ ID NO: 39 or 40. The amino acid sequences of SEQ ID NOs: 39 and 40 and their isoelectric points are listed in Table 6, along with the amino acid sequence represented by SEQ ID NO: 30.

[0130] [Table 6]

[0131] As shown in Table 6, the sequence with the lowest isoelectric point of SEQ ID NO: 39 (with an isoelectric point of approximately 8.7) is SEQ ID NO: 39 out of SEQ ID NO: 39 and 40. Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 30 is preferably 8.7 or higher, more preferably 8.7 to 11, and even more preferably 8.7 to 10, etc.

[0132] In (H2-2), "plural" refers to 2 to 5, 2 to 4, or 2 or 3. As explained in (L1-2), preferred examples of mutations in the amino acid sequence of (H2-2) include amino acid substitutions, and the substitutions are explained as above. Preferred examples of the isoelectric point of the amino acid sequence of (H2-2) include 8.7 or more, more preferably 8.7 to 11, and even more preferably 8.7 to 10.

[0133] The number of amino acid residues in the amino acid sequence of (H2-2) is, for example, 8 to 15, preferably 10, 11, 12 or 13, and more preferably 11, for example.

[0134] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (H2-2) include amino acid sequences in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 39 or 40, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (H2-2) include the amino acid sequences shown in the following (H2-2-39) and (H2-2-40).

[0135] (H2-2-39) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 39 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 39 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 39").

[0136] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 39 is approximately 8.7, the isoelectric point of the mutant sequence of SEQ ID NO: 39 is preferably 8.7 or higher, more preferably 8.7 to 11, and even more preferably 8.7 to 10, etc.

[0137] (H2-2-40) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 40 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 40 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 40").

[0138] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 40 is approximately 9.6, the isoelectric point of the mutant sequence of SEQ ID NO: 40 is preferably 9.6 or higher, more preferably 9.6 to 11, and even more preferably 9.6 to 10, etc.

[0139] H3 array The H3 sequence consists of the following (H3-1) or (H3-2): (H3-1) the amino acid sequence represented by SEQ ID NO: 31; (H3-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 31, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 31.

[0140] SEQ ID NO: 31 is as follows: YX g1 X g2 WAX g3 GRFTX g4 SX g5 TSTTVDLX g6 X g7 TSX g8 TX g9 X g10 DTATYFC (X g1 is G or A, and X g2 is S or T, and X g3 is K or R, and X g4 is I or V, and Xg5 is R, K or T, and X g6 is K or R, and X g7 is M or I, and X g8 is L or P, and X g9 is T or A, and X g10 is E or S)

[0141] The amino acid sequence represented by SEQ ID NO: 31 is not limited thereto, but is preferably exemplified by any of the amino acid sequences represented by SEQ ID NOs: 41 to 46. The amino acid sequences and their isoelectric points of each of SEQ ID NOs: 41 to 46 are shown in Table 7, along with the amino acid sequence represented by SEQ ID NO: 31.

[0142] [Table 7]

[0143] As shown in Table 7, the sequence with the lowest isoelectric point among SEQ ID NOs: 41 to 46 is SEQ ID NO: 45 (with an isoelectric point of approximately 6.1). Therefore, the isoelectric point of the amino acid sequence represented by SEQ ID NO: 31 is preferably 6.1 or higher, more preferably 6.1 to 11, and even more preferably 6.1 to 10.

[0144] In (H3-2), "plural" refers to 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. As in the explanation for (L1-2), preferred examples of mutations in the amino acid sequence of (H3-2) include amino acid substitutions, and the substitutions are as explained above. Preferred examples of the isoelectric point of the amino acid sequence of (H3-2) include 6.1 or more, more preferably 6.1 to 11, and even more preferably 6.1 to 10.

[0145] The number of amino acid residues in the amino acid sequence of (H3-2) is, for example, 32 to 40, preferably 34, 35 or 36, and more preferably 35, for example.

[0146] Although not limiting the present disclosure, preferred examples of the amino acid sequence of (H3-2) include amino acid sequences in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by any one of SEQ ID NOs: 41 to 46, and which have an isoelectric point equal to or higher than that of the amino acid sequence before the substitution, deletion, insertion, or addition (before the mutation). That is, preferred examples of the amino acid sequence of (H3-2) include the amino acid sequences shown below in (H3-2-41) to (H3-2-46).

[0147] (H3-2-41) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 41 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 41 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 41").

[0148] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 41 is approximately 8.1, the isoelectric point of the mutant sequence of SEQ ID NO: 41 is, for example, 8.1 or more, more preferably 8.1 to 11, and even more preferably 8.1 to 10.

[0149] (H3-2-42) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 42 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 42 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 42").

[0150] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 42 is approximately 8.2, the isoelectric point of the mutant sequence of SEQ ID NO: 42 is, for example, 8.2 or more, more preferably 8.2 to 11, and even more preferably 6.1 to 10.

[0151] (H3-2-43) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 43 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 43 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 43").

[0152] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 43 is approximately 8.1, the isoelectric point of the mutant sequence of SEQ ID NO: 43 is, for example, 8.1 or more, more preferably 8.1 to 11, and even more preferably 8.1 to 10.

[0153] (H3-2-44) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 44 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 44 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 44").

[0154] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 44 is approximately 9, the isoelectric point of the mutant sequence of SEQ ID NO: 44 is, for example, 9 or more, more preferably 9 to 11, and even more preferably 9 to 10, etc.

[0155] (H3-2-45) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 45 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 45 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 45").

[0156] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 45 is approximately 6.1, the isoelectric point of the mutant sequence of SEQ ID NO: 45 is, for example, 6.1 or more, more preferably 6.1 to 11, and even more preferably 6.1 to 10.

[0157] (H3-2-46) An amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 46 and which has been substituted or otherwise modified as described above, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 46 (hereinafter, this sequence may be referred to as the "mutated sequence of SEQ ID NO: 46").

[0158] Since the isoelectric point of the amino acid sequence represented by SEQ ID NO: 46 is approximately 8.1, the isoelectric point of the mutant sequence of SEQ ID NO: 46 is, for example, 8.1 or more, more preferably 8.1 to 11, and even more preferably 8.1 to 10.

[0159] H4 sequence The H4 sequence consists of the following (H4-1) or (H4-2): (H4-1) the amino acid sequence represented by SEQ ID NO: 32; (H4-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 32, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 32.

[0160] SEQ ID NO: 32 is as follows:

[0161] [Table 8]

[0162] In (H4-2), "plural" is exemplified by 2 to 5, 2 to 4, or 2 or 3. As explained in (L1-2), preferred examples of mutations in the amino acid sequence of (H4-2) include amino acid substitutions, and the substitutions are explained as above.

[0163] An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 32 and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 32 may be referred to hereinafter as a "mutant sequence of SEQ ID NO: 32" or "(H4-2-32)." The isoelectric point of the mutant sequence of SEQ ID NO: 32 is preferably 5.5 or higher, more preferably 5.5 to 8, and even more preferably 5.5 to 7. The number of amino acid residues in the amino acid sequence of (H4-2) is preferably 8 to 15, preferably 10, 11, 12, or 13, and even more preferably 11.

[0164] In the present disclosure, the H1, H2, H3, and H4 sequences are not limited as described above. Although not limiting the present disclosure, the isoelectric point of an amino acid sequence consisting of only the H1, H2, H3, and H4 sequences, in which these four sequences are directly linked, is preferably 8 or higher, more preferably 8 to 11, and particularly preferably 8 to 10. The isoelectric point is determined in the same manner as described above. The isoelectric points of the amino acid sequences consisting of only the H1, H2, H3, and H4 sequences used in the test examples described below are listed in the "VH frame" in Table 9.

[0165] Furthermore, although not limiting the present disclosure, when the H1 to H4 sequences and the L1 to L4 sequences are combined, the isoelectric point of the amino acid sequence consisting only of the H1 to H4 sequences and the L1 to L4 sequences, in which these eight sequences are more preferably directly linked, is preferably 5 or more, more preferably 5 to 11, and particularly preferably 5 to 10. The isoelectric point is determined in the same manner as described above. The isoelectric points of the amino acid sequences consisting only of the H1 to H4 sequences and the L1 to L4 sequences used in the test examples described below are shown in Table 9 under "FR1-4 without linker."

[0166] The total number of amino acid residues in the H1, H2, H3, and H4 sequences is 75 to 90, preferably 78 to 85, and more preferably 80, for example.

[0167] Each of the sequences H1, H2, H3, and H4 is usually selected from the aforementioned sequences and used in combination, and although there is no limitation thereto, preferred examples include combinations selected from the following (D) to (F). (D) [H1] SEQ ID NO: 33, SEQ ID NO: 34, a variant of SEQ ID NO: 33, or a variant of SEQ ID NO: 34: [H2] SEQ ID NO: 39, SEQ ID NO: 40, a variant of SEQ ID NO: 39, or a variant of SEQ ID NO: 40: [H3] SEQ ID NO: 41, SEQ ID NO: 42, a variant of SEQ ID NO: 41, or a variant of SEQ ID NO: 42: [H4] SEQ ID NO: 32 or a variant of SEQ ID NO: 32 (E) [H1] SEQ ID NO: 35, SEQ ID NO: 36, a variant of SEQ ID NO: 35, or a variant of SEQ ID NO: 36: [H2] SEQ ID NO: 39 or a variant of SEQ ID NO: 39: [H3] SEQ ID NO: 43, SEQ ID NO: 44, a variant of SEQ ID NO: 43, or a variant of SEQ ID NO: 44: [H4] SEQ ID NO: 32 or a variant of SEQ ID NO: 32 (F) [H1] SEQ ID NO: 37, SEQ ID NO: 38, a variant of SEQ ID NO: 37, or a variant of SEQ ID NO: 38: [H2] SEQ ID NO: 39 or a variant of SEQ ID NO: 39: [H3] SEQ ID NO: 45, SEQ ID NO: 46, a variant of SEQ ID NO: 45, or a variant of SEQ ID NO: 46: [H4] SEQ ID NO: 32 or a variant of SEQ ID NO: 32

[0168] Furthermore, although this disclosure is not limited thereto, when further subdividing, the following combinations are preferred examples. (D) (D-1) [H1] SEQ ID NO: 33 or a mutant sequence of SEQ ID NO: 33: [H2] SEQ ID NO: 39 or a mutant sequence of SEQ ID NO: 39: [H3] SEQ ID NO: 41 or a mutant sequence of SEQ ID NO: 41: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32; (D-2) [H1] SEQ ID NO: 34 or a mutant sequence of SEQ ID NO: 34: [H2] SEQ ID NO: 40 or a mutant sequence of SEQ ID NO: 40: [H3] SEQ ID NO: 42 or a mutant sequence of SEQ ID NO: 42: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32 (E) (E-1) [H1] SEQ ID NO: 35 or a mutant sequence of SEQ ID NO: 35: [H2] SEQ ID NO: 39 or a mutant sequence of SEQ ID NO: 39: [H3] SEQ ID NO: 43 or a mutant sequence of SEQ ID NO: 43: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32; (E-2) [H1] SEQ ID NO: 36 or a mutant sequence of SEQ ID NO: 36: [H2] SEQ ID NO: 39 or a mutant sequence of SEQ ID NO: 39: [H3] SEQ ID NO: 44 or a mutant sequence of SEQ ID NO: 44: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32 (F) (F-1) [H1] SEQ ID NO: 37 or a mutant sequence of SEQ ID NO: 37: [H2] SEQ ID NO: 39 or a mutant sequence of SEQ ID NO: 39: [H3] SEQ ID NO: 45 or a mutant sequence of SEQ ID NO: 45: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32; (F-2) [H1] SEQ ID NO: 38 or a mutant sequence of SEQ ID NO: 38: [H2] SEQ ID NO: 39 or a mutant sequence of SEQ ID NO: 39: [H3] SEQ ID NO: 46 or a mutant sequence of SEQ ID NO: 46: [H4] SEQ ID NO: 32 or a mutant sequence of SEQ ID NO: 32

[0169] Antigen recognition sequences 4-6 Antigen recognition sequence 4, antigen recognition sequence 5, and antigen recognition sequence 6 may be, in order, amino acid sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3, which are antibody-derived light chain complementarity determining regions, respectively; or may be amino acid sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, which are antibody-derived heavy chain complementarity determining regions. As described above for antigen recognition sequences 1 to 3, antigen recognition sequence 4, antigen recognition sequence 5, and antigen recognition sequence 6 may each be a combination of non-animal-derived amino acid sequences (e.g., artificially produced amino acid sequences) having antigen recognition ability corresponding to light chain CDR1, CDR2, and CDR3, or a combination of non-animal-derived amino acid sequences (e.g., artificially produced amino acid sequences) having antigen recognition ability corresponding to heavy chain CDR1, CDR2, and CDR3. Thus, antigen recognition sequences 1 to 3 are not particularly limited as long as the three amino acid sequences, as a set, can recognize the target antigen, like CDR1 to CDR3 found in conventionally known antibodies.

[0170] In the present disclosure, when the aforementioned antigen recognition sequences 1 to 3 are, in order, amino acid sequences encoding light chain CDR1 to 3 or amino acid sequences of non-animal origin having antigen recognition ability corresponding to light chain CDR1, CDR2, and CDR3, the antigen recognition sequences 4 to 6 are, in order, amino acid sequences encoding heavy chain CDR1 to 3 or amino acid sequences of non-animal origin having antigen recognition ability corresponding to heavy chain CDR1, CDR2, and CDR3. Furthermore, when the aforementioned antigen recognition sequences 1 to 3 are, in order, amino acid sequences encoding heavy chain CDR1 to 3 or amino acid sequences of non-animal origin having antigen recognition ability corresponding to heavy chain CDR1, CDR2, and CDR3, the antigen recognition sequences 4 to 6 are, in order, amino acid sequences encoding light chain CDR1 to 3 or amino acid sequences of non-animal origin having antigen recognition ability corresponding to light chain CDR1, CDR2, and CDR3.

[0171] As described above, the number of amino acid residues in antigen recognition sequence 4 is not limited as long as the three sequences of antigen recognition sequences 4 to 6 together can recognize the target antigen, but the number of amino acid residues in antigen recognition sequence 4 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10. Similarly, the number of amino acid residues in antigen recognition sequence 5 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10. Similarly, the number of amino acid residues in antigen recognition sequence 6 is, for example, 2 to 30, and preferably 3 to 25, 3 to 20, 3 to 15, or 3 to 10.

[0172] As described above, the origin of antigen recognition sequences 1 to 6 is not limited and may be animal- or non-animal-derived. Therefore, the sequences may be naturally occurring sequences or artificial sequences produced according to procedures known in the field of genetic engineering, etc. Examples of animal origins include, but are not limited to, rabbit antibody, mouse antibody, rat antibody, human antibody, guinea pig antibody, chicken antibody, camel antibody, and shark antibody. Furthermore, the origins of antigen recognition sequences 1 to 6 may be the same or different. Typically, antigen recognition sequences 1 to 3 are derived from the same source, and antigen recognition sequences 4 to 6 are derived from the same source. Preferably, antigen recognition sequences 1 to 6 are derived from the same source. A preferred example is a rabbit antibody. Antigen recognition sequences 1 to 3 and / or antigen recognition sequences 4 to 6 are present in an appropriate set, thereby enabling specific recognition of the target antigen.

[0173] Various antibodies have been commercially available to date, including mouse antibodies (particularly mouse monoclonal antibodies) and rabbit antibodies (particularly rabbit polyclonal antibodies). Rabbit antibodies are said to have higher thermal stability than mouse antibodies, and therefore may be easier to use. However, polyclonal antibodies contain nonspecific antibodies compared to monoclonal antibodies, and rabbit polyclonal antibodies, which are primarily used as rabbit antibodies, have the problem of low antigen detection accuracy. Furthermore, rabbit monoclonal antibodies are considered more difficult to produce than mouse monoclonal antibodies, and no rabbit monoclonal antibodies capable of binding to Protein L have been known to date. In recent years, not only have animal-derived light chain CDRs 1-3 and / or heavy chain CDRs 1-3 been used as antigen-recognition sequences, but technologies using artificially designed antigen-recognition sequences have also progressed. In the present disclosure, any amino acid sequence with antigen-recognition ability can be used as antigen-recognition sequences 1-3 and even 4-6 using recombinant technologies such as grafting. Thus, according to the present disclosure, any antigen recognition sequence can be introduced as antigen recognition sequences 1 to 3, or even antigen recognition sequences 4 to 6. Furthermore, as described below, the polypeptide can be used as an antibody itself or a part thereof. Therefore, by utilizing the protein L binding ability due to the L1 to L4 sequences, the polypeptide of the present disclosure, and ultimately the antibody, can be easily purified using a protein L-carrying solid phase, etc. Therefore, according to the present disclosure, it is possible to easily provide antibodies having the desired antigen specificity of rabbit antibodies, etc., which have traditionally been considered difficult to purify, and further, because a specific antigen recognition sequence can be introduced in this way, it is possible to provide antibodies with improved antigen detection accuracy compared to polyclonal antibodies.

[0174] In amino acid sequence 2, each sequence (i.e., H1 sequence, antigen recognition sequence 4, H2 sequence, antigen recognition sequence 5, H3 sequence, antigen recognition sequence 6, and H4 sequence, in that order from the N-terminus) is linked directly or indirectly, and when linked indirectly, they are preferably linked via a linker. Linkers are as described below. In amino acid sequence 2, each sequence is preferably linked directly.

[0175] As described above, a polypeptide having the L1, L2, L3, and L4 sequences can bind to Protein L. Therefore, by linking amino acid sequence 2 to amino acid sequence 1, a polypeptide having all of the antigen recognition sequences 1 to 6 can be easily purified and detected using a Protein L-carrying solid phase or the like.

[0176] Amino acid sequence 2 can be linked to the N-terminus or C-terminus of amino acid sequence 1, and is preferably linked to the N-terminus of amino acid sequence 1. Amino acid sequence 1 and amino acid sequence 2 are linked directly or indirectly, preferably indirectly. When linked indirectly, they are preferably linked via a linker. The linker is not limited as long as it does not impair the effects of the present disclosure, and examples thereof include a GS linker, a linker consisting only of glycine (G), a linker consisting only of serine (S), and an EAAAK linker. The length of the linker is not particularly limited, and examples thereof include the number of amino acid residues per linker being preferably 2 to 30, more preferably 3 to 25 or 4 to 20, etc. A preferred example of a GS linker is (GS), where n is an integer of 1 to 6, preferably 2 to 5, and more preferably 3 or 4, etc. An example of the EAAAK linker is (EAAAK)n, where n is an integer of 1 to 6, preferably 2 to 5, and more preferably 3 or 4 (EAAAK is shown in SEQ ID NO: 60). One type of linker may be used alone, or two or more types may be used in combination.

[0177] When amino acid sequence 1 is linked to amino acid sequence 2, the following combinations are exemplified as preferred embodiments of the L1 to L4 sequences constituting amino acid sequence 1 and the H1 to H4 sequences constituting amino acid sequence 2. A combination of (A) and (D) (more preferably a combination of (A-1) and (D-1), or a combination of (A-2) and (D-2)). A combination of (B) and (E) (more preferably a combination of (B-1) and (E-1), or a combination of (B-2) and (E-2)). A combination of (C) and (F) (more preferably a combination of (C-1) and (F-1), or a combination of (C-2) and (F-2)).

[0178] The polypeptide of the present disclosure may be linked with an amino acid sequence encoding any other substance other than amino acid sequence 2, as long as the effect of the present disclosure is not impaired. Examples of such substances include various proteins (e.g., lactoferrin), restriction enzyme sites, various tags (His tag, etc.), etc. One type of other substance may be linked alone, or two or more types may be linked together.

[0179] The polypeptides of the present disclosure can be produced according to procedures known in the field of genetic engineering, etc. While not limiting the present disclosure, for example, they can be produced by constructing a polynucleotide having a nucleotide sequence encoding amino acid sequence 1, or by constructing a polynucleotide in which the nucleotide sequence encoding amino acid sequence 1 is linked to the nucleotide sequence encoding amino acid sequence 2, transforming the polynucleotide into a host cell using a vector, and culturing and purifying the cell. In this case, a nucleotide sequence encoding any substance, such as a restriction enzyme site, a tag, or a protein, may be introduced into the nucleotide as needed. Because the polypeptides of the present disclosure have protein L binding properties, they can be easily purified using an antibody purification column to which protein L is immobilized.

[0180] Thus, since the Protein L-binding polypeptide of the present disclosure comprises antigen recognition sequences 1 to 3 or comprises antigen recognition sequences 1 to 3 and antigen recognition sequences 4 to 6, the polypeptide can be used as an antibody itself or as a part of an antibody. Therefore, the present disclosure can also be said to provide an antibody comprising a Protein L-binding polypeptide. Examples of antibodies comprising the polypeptide include scFv, Fab, F(ab)2', scFv-Fc, scAb (single-chain antibody), sdAb (single-domain antibody), VHH, and full-length antibodies. Furthermore, according to the present disclosure, even if an antibody does not originally bind to Protein L, a desired antibody capable of binding to Protein L can be easily produced by replacing the amino acid sequences corresponding to the four light-chain framework regions with the L1 to L4 sequences. Various antibodies can be produced using the polypeptide of the present disclosure according to procedures known in the field of genetic engineering, etc. By utilizing the Protein L-binding ability of the polypeptide of the present disclosure, the antibody can be easily linked to Protein L (for example, to a solid phase such as a column or plate carrying Protein L). The solid phase is not limited as long as it is a solid phase known in the art, which allows for convenient purification, detection, etc. of antibodies having a desired antigen recognition sequence.

[0181] Furthermore, as shown in the Examples below, it was confirmed that the polypeptide of the present disclosure can maintain antigen binding based on the antigen recognition sequence. This indicates that the polypeptide of the present disclosure is particularly useful in antibody purification and detection using a solid phase bound to Protein L, as well as antigen detection using an antigen-antibody reaction.

[0182] As mentioned above, it is known that Protein L can be used as a means for purifying, detecting, and the like antibodies. This means utilizes the specific binding between Protein L and antibodies. Protein L is said to be the first protein isolated from Peptostreptococcus magnus. However, not all antibodies can bind to Protein L. For example, the rabbit-derived single-chain antibody C1R (Comparative Example 1) shown in Test Example 1 below does not bind to Protein L and therefore cannot be purified using a column to which Protein L is bound. According to the present disclosure, as mentioned above, by utilizing the Protein L-binding ability of the polypeptide, polypeptides of the present disclosure having a desired antigen recognition sequence, and therefore antibodies, can be simply and specifically purified and detected using Protein L (e.g., a Protein L-supported solid phase). In the present disclosure, detection also includes the meaning of measurement. Thus, the polypeptides of the present disclosure are useful in immunoassays. Furthermore, as described above, according to the present disclosure, a desired antigen recognition sequence can be arbitrarily selected and used, and therefore the polypeptides of the present disclosure are also useful for suppressing non-specific binding of antibodies (for example, suppressing non-specific binding in immunoassays that is likely to occur when polyclonal antibodies, etc. are used). [Example]

[0183] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0184] Test Example 1 <Test Procedure> Preparation of single-chain antibody (scFv) comprising protein L-binding polypeptide (Examples 1 to 3 and Comparative Example 1) Comparison Example 1 An anti-human CRP rabbit single-chain antibody (C1R) having the amino acid sequence (SEQ ID NO: 47) shown in Figure 1 was produced (Comparative Example 1). C1R is an scFv that can specifically recognize human CRP but does not bind to Protein L. C1R comprises a heavy chain variable region (VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4) and a light chain variable region (VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4) linked via a linker.

[0185] Example 1 In Comparative Example 1 (the C1R), the sequences of the four light chain framework regions VLFR1, VLFR2, VLFR3, and VLFR4 were changed to L1 sequence (SEQ ID NO: 5), L2 sequence (SEQ ID NO: 11), L3 sequence (SEQ ID NO: 17), and L4 sequence (SEQ ID NO: 23), respectively, to prepare a single-chain antibody C1R / VLC2R (Example 1) having the amino acid sequence (SEQ ID NO: 48) shown in Figure 2. That is, Example 1 comprises [amino acid sequence 1], specifically [L1 sequence (SEQ ID NO: 5)-antigen recognition sequence 1-L2 sequence (SEQ ID NO: 11)-antigen recognition sequence 2-L3 sequence (SEQ ID NO: 17)-antigen recognition sequence 3-L4 sequence (SEQ ID NO: 23)].

[0186] Example 2 In Comparative Example 1 (the C1R), the sequences of the four light chain framework regions VLFR1 to 4 were changed in the same manner as in Example 1, and the sequences of the four heavy chain framework regions VHFR1, VHFR2, VHFR3, and VHFR4 were also changed to H1 sequence (sequence number 33), H2 sequence (sequence number 39), H3 sequence (sequence number 41), and H4 sequence (sequence number 32), respectively, to produce a single-chain antibody C1R / C2R (Example 2) having the amino acid sequence shown in Figure 3 (sequence number 49). That is, in Example 2, [amino acid sequence 2] is further linked to the N-terminus of [amino acid sequence 1], and specifically comprises [H1 sequence (SEQ ID NO: 33)-antigen recognition sequence 4-H2 sequence (SEQ ID NO: 39)-antigen recognition sequence 5-H3 sequence (SEQ ID NO: 41)-antigen recognition sequence 6-H4 sequence (SEQ ID NO: 32)] (amino acid sequence 2) and [L1 sequence (SEQ ID NO: 5)-antigen recognition sequence 1-L2 sequence (SEQ ID NO: 11)-antigen recognition sequence 2-L3 sequence (SEQ ID NO: 17)-antigen recognition sequence 3-L4 sequence (SEQ ID NO: 23)] (amino acid sequence 1).

[0187] Example 3 In Example 2 (the C1R / C2R), the sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 (VH CDR1 to 3 and VL CDR1 to 3 in the figure) were changed to the amino acid sequences encoding heavy chain CDR1 to 3 and light chain CDR1 to 3 of another antibody, respectively, to prepare a single-chain antibody C2R (Example 3) having the amino acid sequence shown in Figure 4 (SEQ ID NO: 50). That is, Example 3 is an antibody in which only antigen recognition sequences 1 to 6 have been changed in [amino acid sequence 1] and [amino acid sequence 2] of Example 2.

[0188] Specifically, the single chain antibodies of Examples 1 to 3 and Comparative Example 1 were produced using recombinant Escherichia coli according to the following procedure. · Cultivation and purification 1-1) 1 μL of each scFv expression vector was added to 50 μL of C43 competent cells and cooled on ice for 15 minutes. 1-2) The mixture was incubated at 42°C for 45 seconds to transform the E. coli, and then cooled on ice again. 1-3) The transformed E. coli was inoculated onto an LB agar plate containing Amp (final concentration 50 μg / mL) and cultured overnight at 37°C. 1-4) 10 mL of sterilized 2xYT medium and Amp (final concentration 50 μg / mL) were added to a 50 mL centrifuge tube, and the colony on the agar plate was inoculated and cultured overnight at 30°C and 200 rpm. 1-5) 50 mL of Overnight Express medium and Amp (final concentration 50 μg / mL) were added to a sterilized 500 mL Erlenmeyer flask equipped with a baffle. 1-6) OD of the preculture solution obtained in 4) 600 = 0.1, and cultured at 30°C, 200 rpm for 24 hours. 1-7) The culture medium was transferred to a centrifuge tube and centrifuged at 13,400 xg at 4°C for 20 minutes to separate the culture supernatant from the bacterial cells.

[0189] · Purification of single-chain antibodies expressed in culture supernatant 2-1) A 1 mL Ni-NTA column was loaded with binding buffer (20 mM imidazole, 2xPBS pH 8.0) and equilibrated. 2-2) To the culture supernatant obtained in 1-7) above, a 200 mM imidazole and 10% Tween 20 solution was added so that the imidazole concentration was 20 mM and the Tween 20 concentration was 1%. 2-3) The solution obtained in 2-2) above was supplied to the column in 2-1) above. 2-4) The column was washed with 5 CV of binding buffer three times. 2-5) 1 mL of elution buffer (250 mM imidazole, 2×PBS pH 8.0) was applied to the column five times and collected as fractions 1-5. 2-6) The fractions that showed absorbance at 280 nm were dialyzed overnight at 4°C against 1x PBS to obtain single-chain antibodies.

[0190] ·Antigen binding The antigen binding properties of the single chain antibodies of Examples 1 to 3 and Comparative Example 1 were evaluated according to the following procedure. 1) Add 100μL of 10μg / mL rCRP to Nunc Maxisorp TM (Thermo Fisher Scientific) was added to each well and incubated overnight at 4°C. 2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. 3) After washing five times with PBST, 100 μL of 2-0 μg / mL scFv was added to each well and incubated at 25°C for 1 hour. 4) After washing five times with PBST, 100 μL of 10,000 diluted Mouse anti-6×His IgG (9C11) POD Conjugated was added to each well and incubated at 25°C for 1 hour. 5) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for up to 15 minutes. 6) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured using a microplate reader.

[0191] 3) Protein L binding The binding ability of the single-chain antibodies of Examples 1 to 3 and Comparative Example 1 to Protein L was evaluated according to the following procedure. 1) Add 100μL of 10μg / mL Protein L to Nunc Maxisorp TM (Thermo Fisher Scientific) was added to each well and incubated overnight at 4°C. 2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. 3) After washing five times with PBST, 100 μL of 10-10 μg / mL biotinylated scFv was added to each well and incubated at 25°C for 1 hour. 4) After washing five times with PBST, 100 μL of 10,000 diluted Mouse anti-6×His IgG (9C11) POD Conjugated was added to each well and incubated at 25°C for 1 hour. 5) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for up to 15 minutes. 6) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured using a microplate reader.

[0192] As a positive control, human IgG (a full-length antibody known to have binding properties to protein L) was used.

[0193] <Result> The results are shown in Figures 5 to 7. Comparative Example 1 (C1R) is a single-chain antibody that can specifically recognize human CRP but does not have binding affinity to Protein L. As shown in Figure 5, antigen-binding affinity to human CRP was observed in Comparative Example 1. On the other hand, as shown in Figure 6, no band was observed in Comparative Example 1 in protein purification using a Protein L column (lane 4, Elution in Figure 6), confirming that Comparative Example 1 does not bind to Protein L.

[0194] In contrast, in Example 1 (C1R / VLC2R), antigen-binding ability to human CRP was confirmed as shown in FIG. 5 , and further, bands were confirmed in protein purification using a Protein L column as shown in FIG. 6 (lane 4, Elution in FIG. 6 ). Example 1 is a single-chain antibody in which the four light chain framework regions (VLFR1-4) present in Comparative Example 1 (C1R) were replaced with the L1-L4 sequences as described above. This demonstrates that the L1-L4 sequences are sequences that have binding ability to Protein L, and that the presence of the L1-L4 sequences enabled the single-chain antibody of Example 1 to bind to Protein L. Furthermore, even when the L1-L4 sequences were replaced in this way, the human CRP antigen-binding ability derived from C1R was maintained, demonstrating that the L1-L4 sequences are polypeptides that can bind to Protein L while maintaining the antigen-binding ability of the antibody.

[0195] When Example 2 (C1R / C2R) was used, antigen-binding ability to human CRP was confirmed, and a band was confirmed in protein purification using a Protein L column (Figures 5 and 7). Example 2 is a single-chain antibody in which not only were the four light chain framework regions present in C1R replaced with the L1 to L4 sequences, but also the four heavy chain framework regions (VHFR1 to VHFR4) were replaced with H1 to H4 sequences as described above. This demonstrates that binding to Protein L is possible even when H1 to H4 sequences are further linked to the L1 to L4 sequences. It also demonstrates that the H1 to H4 sequences do not interfere with binding to Protein L by the L1 to L4 sequences.

[0196] Furthermore, as shown in the table at the bottom of Figure 7, the antibody recovery rate in Example 1 was 93%, and the antibody recovery rate in Example 2 was 84%, demonstrating that Example 1 had higher Protein L binding ability than Example 2. As mentioned above, Example 1 is a single-chain antibody in which the L1 to L4 sequences were grafted to Comparative Example 1, and Example 2 is a single-chain antibody in which not only the L1 to L4 sequences but also the H1 to H4 sequences were grafted to Comparative Example 1. Furthermore, although not shown in the results, when only the H1 to H4 sequences were grafted to Comparative Example 1, no Protein L binding ability was observed. From this, it was determined that the L1 to L4 sequences were mainly responsible for Protein L binding ability.

[0197] Furthermore, in Example 3 (Example 3), in which the heavy chain CDR1-3 and light chain CDR1-3 in Example 2 were replaced with other heavy chain CDR1-3 and light chain CDR1-3 (human CRP (rCRP) is not the target antigen), no antigen-binding ability to human CRP was observed, and bands were confirmed in protein purification using a Protein L column (FIGS. 5 and 7). From this, it was understood that even when the heavy chain CDR1-3 and light chain CDR1-3 are changed, i.e., even when the antigen-recognition sequences are different, binding to Protein L is possible as long as the L1-L4 sequences are present. Although not shown in the results, Example 3 was observed to have specific antigen-binding ability to the original target antigen, C2R.

[0198] These findings demonstrate that by linking an antigen recognition sequence such as a CDR to the L1 to L4 sequences, the antigen recognition sequence, and in turn an antibody having the antigen recognition sequence, can be easily linked to Protein L. Furthermore, by replacing the amino acid sequences corresponding to the four light chain framework regions of an antibody that is not originally capable of binding to Protein L with the L1 to L4 sequences, it is possible to easily prepare a desired antibody capable of binding to Protein L, and it is also possible to perform affinity purification of the antibody using a column to which Protein L is bound, for example.

[0199] Test Example 2 <Test Procedure> Example 4 In Example 3 (C2R) shown in Test Example 1, the L1 to L4 sequences and H1 to H4 sequences were changed to SEQ ID NO: 6 (L1 sequence), SEQ ID NO: 12 (L2 sequence), SEQ ID NO: 18 (L3 sequence), SEQ ID NO: 24 (L4 sequence), SEQ ID NO: 34 (H1 sequence), SEQ ID NO: 40 (H2 sequence), SEQ ID NO: 42 (H3 sequence), and SEQ ID NO: 32 (H4 sequence), respectively, as shown in Example 4 (C2RrepCKV1, SEQ ID NO: 51) in FIG. 8. A single-chain antibody was produced in the same manner as in Example 3. The single-chain antibody produced in this manner was named C2RrepCKV1 (Example 4). That is, Example 4 differs from Example 3 in the sequences of L1 to L4 constituting [amino acid sequence 1] and the sequences of H1 to H3 constituting [amino acid sequence 2]. Specifically, Example 4 has sequences in which eight lysines (K) present in the L1 to L4 sequences and H1 to H3 sequences used in Example 3 have been replaced with arginines (R), and one cysteine ​​(C) has been replaced with serine (S), as shown by the squares in Figure 8.

[0200] Comparative Example 2 C2RrepCKV2 was prepared as Comparative Example 2. Comparative Example 2 (C2RrepCKV2, SEQ ID NO: 52) differs from Example 3 (C2R) in the type of amino acid indicated by the square in Figure 8. For example, the isoelectric point of the L1 sequence in Example 3 is 4.14, whereas the isoelectric point of the sequence used in Comparative Example 2 instead of the L1 sequence is low at 3.57; and the isoelectric point of the L3 sequence in Example 3 is 4.14, whereas the isoelectric point of the sequence used in Comparative Example 2 instead of the L3 sequence is low at 3.77.

[0201] Comparative Example 3 C2RrepCKV3 was prepared as Comparative Example 3. C2RrepCKV3 (Comparative Example 3, SEQ ID NO: 53) differs from Example 3 (C2R) in the type of amino acid indicated by the square in Figure 8. For example, the isoelectric point of the L1 sequence in Example 3 is 4.14, whereas the isoelectric point of the sequence used in Comparative Example 3 in place of the L1 sequence is low at 3.57; and the isoelectric point of the L3 sequence in Example 3 is 4.14, whereas the isoelectric point of the sequence used in Comparative Example 3 in place of the L3 sequence is low at 3.77.

[0202] Protein L binding The binding ability of each scFv of Example 4 and Comparative Examples 2 to 3 to Protein L was evaluated in the same manner as in Test Example 1, except that in step 4) shown in Test Example 1, the wells were washed five times with PBST and 50,000 diluted SA-HRP was added to each well.

[0203] <Result> The results are shown in Figure 9. As shown in Figure 9, binding to Protein L was observed in Example 4. Furthermore, although not shown in the results, specific antigen binding to the target antigen C2R was also observed in Example 4. In contrast, binding to Protein L was not observed in Comparative Examples 2 and 3.

[0204] In Example 4, the isoelectric points of the L2 and L4 sequences were higher than those of the L2 and L4 sequences of Example 3, and the isoelectric points of the L1 and L3 sequences were the same as those of the L1 and L3 sequences of Example 3 (Table 9). Meanwhile, in Comparative Example 2, the sequences used in place of the L1 to L4 sequences of Example 3 were lower than those of the L1 to L4 sequences of Example 3; for example, the isoelectric point of the sequence used in place of the L1 sequence was 3.57, and the isoelectric point of the sequence used in place of the L3 sequence was 3.77. Similarly, in Comparative Example 3, the isoelectric points of the sequences used in place of at least the L1, L3, and L4 sequences were lower than those of the L1, L3, and L4 sequences of Example 3; for example, the isoelectric point of the sequence used in place of the L1 sequence was 3.57, and the isoelectric point of the sequence used in place of the L3 sequence was 3.77. The amino acid sequences L1 to L4 used in Example 4 were sequences in which amino acids had been substituted so that the isoelectric points were equal to or higher than those of the sequences L1 to L4 used in Example 3. Therefore, it was understood that the desired protein L binding properties could be observed even when the amino acid sequences L1 to L4, which have protein L binding properties, were mutated so that the isoelectric points were equal to or higher.

[0205] [Table 9]

[0206] The isoelectric point was determined using the Expasy protparam tool (https: / / web.expasy.org / protparam / ). This tool calculates the isoelectric point by inputting the amino acid sequence for which the isoelectric point is to be calculated into a specified screen displayed on the screen. The isoelectric point was determined by inputting the amino acid sequence according to the procedure of the tool (Gasteiger E., et al., Protein Identification and Analysis Tools on the Expasy Server, in John M. Walker (ed), The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607; https: / / web.expasy.org / docs / expasy_tools05.pdf).

[0207] Test Example 3 <Test Procedure> Single-chain antibodies (Examples 5 to 8) with antigen specificity different from that of Test Examples 1 and 2 were prepared, and their antigen binding and protein L binding properties were evaluated. The prepared single-chain antibodies had the H1 to L4 sequences, heavy chain (VH) CDRs 1 to 3, L1 to L4 sequences, and light chain (VL) CDRs 1 to 3 sequences shown in Figures 10 and 11, respectively; that is, amino acid sequence 1 was located C-terminally of amino acid sequence 2. These antibodies were named NP-B R4-7 (Example 5), NP-B R4-41 (Example 6), KYN R2-42 (Example 7), and KYN R2-75 (Example 8), respectively. Examples 5 and 6 are antibodies against NP-B as an antigen, and Examples 7 and 8 are antibodies against kynureninase (KYN) as an antigen. These single-chain antibodies were prepared in the same manner as described above.

[0208] · Protein L binding The binding ability of each scFv to Protein L was evaluated according to the following procedure. TM This was carried out according to the instructions for use of L. 1) KanCap L (KANEKA KanCap), a commercially available affinity support for antibody purification TM 50 μL of Protein L (50% slurry, cellulose-based Protein L carrier, manufactured by Kaneka Corporation) was placed in an Eppendorf tube and suspended in 500 μL of 1×PBS. 2) The mixture was centrifuged at 1000 g for 10 minutes and the supernatant was removed. This washing procedure was repeated twice. 3) 100 μL of the purified scFv solution obtained by dialysis in the same manner as in 2-6) above was added to each well and incubated at 25° C. for 1 hour. 4) After centrifugation at 1000 g × 10 min, 20 μL of the supernatant was recovered (supernatant in Figure 12), and the remaining supernatant was removed. 5) The precipitate obtained by centrifugation in 4) above was suspended in 200 μL of 1×PBS, centrifuged, and the supernatant was removed. This washing process was repeated twice. 6) 200 μL of 50 mM citrate buffer (pH 2.5) was added, and the mixture was centrifuged at 1000 g for 10 minutes (elution). 7) 100 μL of the eluate was collected (eluate collection), and the remainder was removed. 8) 10 μL of 1 M Tris-HCl (pH 9.0) was added to 100 μL of the eluate to prepare an eluted sample (elution in FIG. 12). 9) The purified scFv solution (original scFV) used in 3), the supernatant obtained in 4), and the elution obtained in 8) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0209] <Result> The results are shown in Figure 12. As shown in Figure 12, binding to Protein L was observed in all of Examples 5 to 7. In particular, the band observed in the supernatant was fainter than the band observed in the original scFV, indicating that all of Examples 5 to 7 had binding to Protein L. Furthermore, although not shown in the results, binding to Protein L was also observed in Example 8, and both single-chain antibodies also exhibited specific antigen binding to the target antigen NP-B in Examples 5 and 6, and to the target antigen kynureninase in Examples 7 and 8.

[0210] As described above, it can be determined that the L1 to L4 sequences are primarily responsible for Protein L binding. The L1 to L4 sequences were further examined based on Test Examples 1 to 3. As a result, it was found that the amino acid sequences constituting the L1 sequence in Examples 1 to 8 were all similar, and that the isoelectric point of the L1 sequence was approximately 3.6 or higher, as shown in Table 9. Similarly, it was found that the amino acid sequences constituting each of the L2 to L4 sequences in Examples 1 to 8 were also similar, and that the isoelectric point of the L2 sequence was approximately 9.5 or higher, the isoelectric point of the L3 sequence was approximately 4 or higher, and the isoelectric point of the L4 sequence was approximately 4.5 or higher, as shown in Table 9. From these findings, it was determined that the L1 to L4 sequences with these isoelectric points were more preferable in terms of Protein L binding.

[0211] Test Example 4 <Test Procedure> Preparation of lactoferrin-fused single-chain antibodies (Examples 9 to 11) · Example 9 An amino acid sequence encoding lactoferrin was linked via an SG linker to the C-terminus of Example 3 (single-chain antibody C2R) used in Test Example 1, and a HiG-tag was also linked to the C-terminus of the amino acid sequence encoding lactoferrin. The lactoferrin-fused single-chain antibody obtained in this manner was designated scFv(C2R)-LF (Example 9).

[0212] · Example 10 A lactoferrin-fused single-chain antibody, scFv(C1R / C2R)-LF (Example 10), was prepared in the same manner as in Example 9, except that Example 2 (single-chain antibody C1R / C2R) used in Test Example 1 was used. Lactoferrin and a Hig-tag were linked via an SG linker.

[0213] Example 11 In Example 3 (single-chain antibody C2R) used in Test Example 1, heavy chain CDRs 1-3 and light chain CDRs 1-3 were replaced with those of another antibody (anti-B1R antibody), and a Hig-tag, an amino acid sequence encoding lactoferrin, was linked to the C-terminus via an SG linker, as in Example 9 (FIG. 13). The underlined portions in FIG. 13 correspond to the H1-H4 and L1-L4 sequences constituting Example 3 (C2R), in order from the N-terminus. The lactoferrin-fused single-chain antibody obtained in this manner was designated scFv(B1R / C2R)-LF (Example 11).

[0214] Example 12 In Example 3 (single-chain antibody C2R) used in Test Example 1, the H1 to H4 sequences and heavy chain CDRs 1 to 3 were replaced with those of an hCG VHH antibody, and a Hig-tag, an amino acid sequence encoding lactoferrin, was linked to the C-terminus via an SG linker, as in Example 9 (FIG. 14). In FIG. 14, the amino acid sequence encoding the hCG VHH antibody is located N-terminally from the first SG linker, and the underlined portions correspond to the L1 to L4 sequences constituting Example 3 (C2R) from the N-terminus. The lactoferrin-fused single-chain antibody obtained in this manner was designated hCG VHH-C2R(VL)-LFF (Example 12). That is, in Example 12, amino acid sequence 1 was linked to the C-terminus of a known VHH antibody, and lactoferrin was further linked to the C-terminus of that.

[0215] The linkages of lactoferrin and the like in Examples 9 to 12 were prepared by transforming Escherichia coli with an expression vector in which each base sequence had been linked, followed by culturing and purifying, in accordance with the recombination procedure described above.

[0216] Protein L binding The binding ability of each scFv to Protein L was evaluated in the same manner as in Test Example 3. For Example 9, the binding ability to Ni was evaluated separately using a nickel-binding column according to conventional procedures.

[0217] <Result> The results are shown in Figure 15. As shown in lanes 4, 6, 8, and 10 in Figure 15, all of the single chain antibodies of Examples 9 to 12 were found to be able to bind to Protein L, even when linked to lactoferrin.

[0218] A widely known technique utilizes the strong binding of histidine to nickel ions to purify and detect proteins by linking a His-tag to the C-terminus of the amino acid sequence encoding the protein and then using a nickel-bound column. For this reason, in this test example, we attempted to purify the single-chain antibody using a nickel-bound column. However, as shown in lanes 1 and 2 of Figure 15, the single-chain antibody was not detected when using a nickel-bound column. This is thought to be because, although Example 9 has a His-tag at its C-terminus, the presence of lactoferrin, which has a large molecular weight, prevents the His-tag from appearing on the antibody surface to an extent that would allow binding to the nickel-bound column. Therefore, as described above, the antibodies shown in Examples 9 to 11, which contain both amino acid sequence 1 and amino acid sequence 2, and in Example 12, in which amino acid sequence 1 is linked to a VHH antibody, were efficiently purified using a protein L-bound column.

[0219] These findings demonstrate that by using a protein L-binding polypeptide having the L1 to L4 sequences, an antibody can be bound to protein L even when a protein such as lactoferrin is further linked to the antibody.

[0220] For the above reasons, the purification efficiency of lactoferrin-linked antibodies has been very low in the past.On the other hand, since lactoferrin has good adsorption properties to nitrocellulose membranes, a method is known in which lactoferrin is linked to an antibody, and the antibody is indirectly linked to nitrocellulose via the lactoferrin.However, although this indirect binding requires a lactoferrin-linked antibody, as mentioned above, the purification efficiency of lactoferrin-linked antibodies is very low.According to the present disclosure, as mentioned above, the purification efficiency of lactoferrin-linked antibodies can be improved by using a protein L-binding polypeptide.In addition, by applying the antibody having the polypeptide of the present disclosure and linked to lactoferrin to a nitrocellulose membrane in the same manner as in the past, the antibody can be easily indirectly linked to the nitrocellulose membrane via lactoferrin.In this way, according to the present disclosure, antibody purification, which has been difficult in the past, can be easily carried out.

Claims

1. A Protein L binding polypeptide comprising the following amino acid sequence: [Amino acid sequence 1] L1 sequence - antigen recognition sequence 1 - L2 sequence - antigen recognition sequence 2 - L3 sequence - antigen recognition sequence 3 - L4 sequence where: The L1 sequence consists of the following (L1-1) or (L1-2): (L1-1) the amino acid sequence represented by SEQ ID NO: 1; (L1-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 1; The L2 sequence consists of the following (L2-1) or (L2-2): (L2-1) the amino acid sequence represented by SEQ ID NO: 2; (L2-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, and the isoelectric point of the amino acid sequence represented by SEQ ID NO: 2 is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO: 2; The L3 sequence consists of the following (L3-1) or (L3-2): (L3-1) the amino acid sequence represented by SEQ ID NO: 3; (L3-2) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 3; The L4 sequence consists of the following (L4-1) or (L4-2): (L4-1) the amino acid sequence represented by SEQ ID NO: 4; (L4-2) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO:

4.

2. the amino acid sequence described in (L1-1) is an amino acid sequence represented by any one of SEQ ID NOs: 5 to 10, the amino acid sequence described in (L2-1) is an amino acid sequence represented by any one of SEQ ID NOs: 11 to 16, the amino acid sequence described in (L3-1) is an amino acid sequence represented by any one of SEQ ID NOs: 17 to 22, and / or The amino acid sequence described in (L4-1) is an amino acid sequence represented by any one of SEQ ID NOs: 23 to 28. The protein L-binding polypeptide according to claim 1.

3. The antigen recognition sequences 1, 2, and 3 are, in order, an amino acid sequence encoding a light chain CDR1, an amino acid sequence encoding a light chain CDR2, and an amino acid sequence encoding a light chain CDR3; an amino acid sequence encoding a heavy chain CDR1, an amino acid sequence encoding a heavy chain CDR2, and an amino acid sequence encoding a heavy chain CDR3; The protein L-binding polypeptide according to claim 1.

4. The protein L-binding polypeptide according to claim 1, further comprising the following amino acid sequence 2 on the N-terminal or C-terminal side of the amino acid sequence 1: [Amino acid sequence 2] H1 sequence - antigen recognition sequence 4 - H2 sequence - antigen recognition sequence 5 - H3 sequence - antigen recognition sequence 6 - H4 sequence where: The H1 sequence consists of the following (H1-1) or (H1-2): (H1-1) the amino acid sequence represented by SEQ ID NO: 29; (H1-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 29, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 29; The H2 sequence consists of the following (H2-1) or (H2-2): (H2-1) the amino acid sequence represented by SEQ ID NO: 30; (H2-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 30, and the isoelectric point is equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 30; The H3 sequence consists of the following (H3-1) or (H3-2): (H3-1) the amino acid sequence represented by SEQ ID NO: 31; (H3-2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 31, and which has an isoelectric point equal to or higher than that of the amino acid sequence represented by SEQ ID NO: 31; The H4 sequence consists of the following (H4-1) or (H4-2): (H4-1) the amino acid sequence represented by SEQ ID NO: 32; (H4-2) An amino acid sequence in which one or more amino acids have been substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 32, and the isoelectric point is equal to or higher than the isoelectric point of the amino acid sequence represented by SEQ ID NO:

32.

5. The amino acid sequence described in (H1-1) is an amino acid sequence represented by any one of SEQ ID NOs: 33 to 38, the amino acid sequence described in (H2-1) is the amino acid sequence represented by SEQ ID NO: 39 or 40, and / or The amino acid sequence described in (H3-1) above is an amino acid sequence represented by any one of SEQ ID NOs: 41 to 46. The protein L-binding polypeptide according to claim 4.

6. The protein L-binding polypeptide according to claim 4, wherein the antigen recognition sequences 1, 2, and 3 are, respectively, an amino acid sequence encoding a light chain CDR1, an amino acid sequence encoding a light chain CDR2, and an amino acid sequence encoding a light chain CDR3, and the antigen recognition sequences 4, 5, and 6 are, respectively, an amino acid sequence encoding a heavy chain CDR1, an amino acid sequence encoding a heavy chain CDR2, and an amino acid sequence encoding a heavy chain CDR3.

7. An antibody comprising the Protein L-binding polypeptide according to any one of claims 1 to 6.

8. scFv, Fab, F(ab) ２ 8. The antibody of claim 7, used as an scFv-Fc, scAb, sdAb or full-length antibody.

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