Mutant antibodies, methods for producing mutant antibodies

Abstract

To modify the affinity of antibodies or small molecule antibodies for protein A. [Solution] The 65th amino acid residue and / or the 82ath amino acid residue in Kabat numbering. Either replace the anoacid residues with glycine and asparagine, respectively, or replace the 65th glycine residue with another amino acid.

Description

[Technical Field]

[0001] This invention utilizes the affinity for protein A to purify antibody molecules or small molecule antibodies. This invention relates to a mutant antibody, which is a molecule into which an amino acid substitution mutation has been introduced at a predetermined position, and to a method for producing said mutant antibody. [Background technology]

[0002] Antibodies, which are functional proteins with high specificity and affinity for antigens, are prepared using animal cells. Antibodies expressed in animal cells have an affinity between the Fc region within the molecule and protein A. The antibodies are purified using animal cells. However, the antibody manufacturing process using animal cells has the challenge of being extremely costly. Therefore, in order to prepare more inexpensive antibody drugs, the development of small molecule antibodies that include variable domains of the antibody heavy chain (VH) and light chain (VL), which are involved in binding to the target antigen, is underway.

[0003] Because small molecule antibodies do not have an Fc region, they cannot be purified using protein A, and are usually tagged. The attached molecule is synthesized and purified by immobilized metal affinity chromatography (IMAC), which utilizes the interaction between the tag and the metal ion. For example, when a 6x histidine tag (His tag) is attached to the end of a small molecule antibody... In addition, IMAC, which utilizes interactions with nickel ions and cobalt ions, is used.

[0004] However, concerns have been raised about antibody purification methods using IMAC, including sample heterogeneity due to antibody aggregation and tag degradation, induction of immunogenicity, leakage of metal ions from the column, and impact on pharmacokinetics (Non-patent Literature 1: MAbs 2014, 6 (6), 1551-1559 and Non-patent Literature 2: Theranostics 2014, 4 (7), 708-720).

[0005] Furthermore, in addition to the His tag mentioned above, GST tags, HA tags, FLAG tags, and Myc tags can be used for the purification of small molecule antibodies. However, while the GST tag has high specificity because it utilizes an enzyme substrate reaction and promotes the soluble expression of the target protein, its tag size is 28 kDa. Due to their large size, tag cleavage is required after purification, making the process cumbersome. Furthermore, while HA tags, FLAG tags, and Myc tags enable high purity with short peptide sequences, the use of IgG antibodies as purification ligands results in high costs, which is a challenge. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] MAbs 2014, 6 (6), 1551-1559 [Non-Patent Document 2] Theranostics 2014, 4 (7), 708-720. [Non-Patent Document 3] Proc. Natl. Acad. Sci. USA 2000, 97 (10), 5399-5404 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] By the way, protein A is used in the purification of antibodies that have an Fc region, It has affinity for some of the subclasses belonging to VH3 within the chain variable region (VH region). It is known that (Non-Patent Document 3). However, the VH region having affinity for protein A Research results on the VH region, which does not have affinity for protein A, are insufficient, and the predetermined antibody No technology was known to modify the affinity of body or small molecule antibodies for protein A. .

[0008] Therefore, in view of the above circumstances, the present invention relates to protein A in antibodies or low molecular weight antibodies. We developed a technology that can modify the affinity for a VH region that does not have affinity for protein A. To confer such affinity to, or to the VH region having affinity for protein A A mutant antibody in which affinity for protein A is modified by reducing the mutation, and the mutation The objective is to provide a method for producing antibodies. [Means for solving the problem]

[0009] In order to achieve the above-mentioned objective, the present inventors conducted diligent research and found that certain amino acid residues in the VH region contribute particularly deeply to the affinity for protein A, and that these amino acid residues We discovered that by modifying the base, we can alter the affinity for protein A. This led to the completion of the present invention.

[0010] This invention encompasses the following: (1) The 65th amino acid residue and / or the 82a amino acid residue in Kabat numbering. A mutant antibody having a VH region in which the 65th position is glycine and the 82a position is asparagine, achieved by substituting the acid residues with glycine and asparagine, respectively. (2) Furthermore, the 16th amino acid residue in Kabat numbering is lysine, arginine By substituting with an amino acid selected from the group consisting of lysine, arginine, and glycine, the mutant antibody according to (1), wherein the 16th amino acid is an amino acid selected from the group consisting of lysine, arginine, and glycine. (3) The mutant antibody according to (1), which is scFv, Fv, Fab, Fab', F(ab')2 or VHH containing the VH region. (4) By amino acid substitution, the 19th amino acid residue in Kabat numbering is arginine, the 70th is serine, the 81st is glutamic acid, and the 82bth is serine. The mutant antibody according to (1), characterized in that. (5) An antibody having an affinity for protein A, having a VH region in which the 65th amino acid residue in Kabat numbering is glycine, and having a VH region in which the 65th amino acid residue is substituted with an amino acid residue other than glycine, and having an affinity for protein A. The mutant antibody with reduced affinity compared to that before substitution mutation. (6) The mutant antibody according to (5), which is scFv, Fv, Fab, Fab', F(ab')2 or VHH containing the VH region. (7) The mutant antibody according to (5), wherein the amino acid residue other than glycine is alanine. (8) A method for producing a mutant antibody, comprising the step of purifying the mutant antibody according to any one of (1) to (4) using the affinity for protein A. (9) Further comprising the step of culturing cells expressing the mutant antibody and obtaining the cells or an extract of the cells, and purifying the mutant antibody contained in the cells or the extract using the affinity for protein A. The method for producing a mutant antibody according to (8), characterized in that.

Advantages of the Invention

[0011] According to the present invention, the affinity for protein A in an antibody or a small molecule antibody is modified. This is possible. In other words, the mutant antibody according to the present invention can acquire affinity for protein A through a substitution mutation of a predetermined amino acid residue. This involves reducing affinity for protein A through substitution mutations of specific amino acid residues. It is possible.

[0012] Furthermore, in the method for producing mutant antibodies according to the present invention, mutant antibodies that have acquired affinity for protein A through substitution mutations of predetermined amino acid residues can be purified using their affinity for protein A. [Brief explanation of the drawing]

[0013] [Figure 1] This is a characteristic diagram showing the multiple alignments of the amino acid sequences of VH1 to VH7. [Figure 2] This is a characteristic diagram showing the multiple alignment of VH3, VH(5H) of Ex3 sc1, and VH(OH) of Ex3 sc1. [Figure 3] This is a characteristic diagram schematically illustrating the interaction between glycine at position 65 and protein A in antibody 2A2, which belongs to the VH3 group. [Figure 4] This is a schematic characteristic diagram illustrating the interaction with protein A when the 65th glycine in antibody 2A2, which belongs to VH3, is substituted with asparagine or aspartic acid. [Figure 5] This characteristic diagram shows the results of evaluating the affinity of purified Ex3 sc1-8m(A) and Ex3 sc1-2m(B) to protein A. [Figure 6] This is a characteristic diagram showing the results of cell binding evaluation for TFK-1 cells (A) and T-LAK cells (B). [Figure 7] This is a characteristic diagram showing the results of cancer cell cytotoxicity testing using the MTS assay. [Figure 8]This characteristic diagram shows the results of evaluating the affinity of OKT3 scFv(A), OKT3 scFv-D65G(B), OKT3 scFv-D82aN(C), OKT3 scFv-D65G / D82aN(D), OKT3 scFv-D65A / D82aN(E), and OKT3 scFv-D65N / D82aN(F) with Protein A. [Figure 9] This is an electrophoretic image showing the results of SDS-PAGE analysis of fractions obtained after purification of Ex3 sc1 and Ex3 sc1-8m by Ni2+ affinity chromatography. [Figure 10] This is an electrophoretic image showing the results of SDS-PAGE analysis of fractions obtained after purification of Ex3 sc1-8m and Ex3 sc1-8m-tag(-) by protein A affinity chromatography. [Figure 11] This characteristic diagram shows the chromatogram obtained by gel filtration chromatography of the eluted fraction from Ni2+ affinity chromatography or protein A affinity chromatography. [Figure 12] This is a characteristic diagram showing the results of cell binding evaluation tests performed on Ex3 sc1, Ex3 sc1-8m, and Ex3 sc1-8m-tag(-). [Figure 13] This is a characteristic diagram showing the results of cytotoxic activity tests performed on Ex3 sc1, Ex3 sc1-8m, and Ex3 sc1-8m-tag(-). [Figure 14] This characteristic diagram shows the results of cytotoxic activity tests performed on Ex3 sc1, Ex3 sc1-8m, and Ex3 sc1-8m-tag(-) at concentrations ranging from 0.1 to 10 pM. [Figure 15] This characteristic diagram shows the results of SDS-PAGE expression testing for the wild-type 528 scFv-HL-WT (no mutation introduced), the mutant 528 scFv-HL-6m (with a sextuplet mutation), and the heptuplet mutant (with an additional mutation introduced to the sextuplet mutation). [Figure 16]This characteristic diagram shows the results of evaluating the affinity for protein A of purified wild-type 528 scFv-HL-WT, the mutant 528 scFv-HL-6m with a sextuple mutation, and the heptagonal mutant with an additional mutation introduced to the sextuple mutation. [Modes for carrying out the invention]

[0014] The present invention will be described in detail below. The mutant antibody according to the present invention acquires a novel affinity for protein A by substituting specific amino acid residues in the heavy chain variable region (VH region) contained in the antibody or small molecule antibody. This refers to a substance, or one in which affinity with protein A has decreased. In other words, a mutation according to the present invention. Antibodies are modified antibodies whose affinity for protein A has been altered through specific substitution mutations.

[0015] The substitution mutations that acquire affinity for protein A are the substitution of the 65th amino acid residue in Kabat numbering with a glycine residue and / or the 82a amino acid residue with an asparagine residue. Either one or both of these substitutions result in Kabat numbering. Antibody having a VH region in which the 65th position is a glycine residue and the 82a position is an asparagine residue. The body or low-molecular-weight antibodies will acquire affinity for protein A.

[0016] Furthermore, the 16th amino acid residue in Kabat numbering is lysine, arginine, and Antibodies or small molecules having a VH region in which the 16th amino acid residue is lysine, arginine, or glycine due to a substitution mutation that replaces the amino acid with one selected from the group consisting of glycine can acquire better affinity for protein A.

[0017] On the other hand, the substitution mutation to reduce affinity with protein A is the substitution of the 65th glycine residue in Kabat numbering with an amino acid other than glycine. This substitution results in a VH region where the 65th amino acid residue in Kabat numbering is an amino acid other than glycine. Antibodies or small-molecule antibodies will have reduced affinity for protein A.

[0018] In this specification, the positions of amino acid residues in the amino acid sequences constituting antibodies and small molecule antibodies are represented by numerical values ​​based on Kabat numbering. Please note that the positions of amino acid residues described herein differ from the numerical values ​​based on the amino acid sequences listed in the sequence listing attached to this book. Furthermore, the Kabat numbering used is that described in *Sequences of proteins of immunological interest*, Elvin A. Kabat et al., NIH publication, no. 91-3242 (National Institutes of Health, 1991, 5th ed.). .

[0019] In this specification, when the term "antibody" is used simply, it is used in its broadest sense, meaning an antibody molecule that exhibits the desired antigen-binding activity, and encompasses various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and small molecule antibodies, as described later. On the other hand, when the term "antibody or small molecule antibody" is used in this specification, "antibody" means an antibody molecule excluding "small molecule antibody" from the definition above.

[0020] A "small molecule antibody" refers to an antibody molecule that contains a portion of a full-length antibody molecule and retains the ability to bind to the antigen to which the full-length antibody molecule would bind. Small molecule antibodies are sometimes also called antibody fragments. Examples of small molecule antibodies are not particularly limited, but include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabolic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and VHH (variable domain of heavy chain of heavy chain antibody). Small molecule antibodies also include multispecific antibodies that are composed of these listed molecules.

[0021] In this invention, the class of antibody is not particularly limited, and there are five major classes of antibodies. It may be any of IgA, IgD, IgE, IgG, and IgM. Also, in the present invention, the antibody The subclass (isotype) is not particularly limited and may be any of the following: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, for example.

[0022] Furthermore, scFv is a single strand formed by linking two variable regions together via linkers or other means as needed. It is a polypeptide. The two variable regions contained in scFv are typically one VH and one VL. However, it may also consist of two VH or two VL domains. Generally, scFv polypeptides contain a linker between the VH and VL domains, thereby forming the paired VH and VL portions necessary for antigen binding. Typically, to form the paired portions between VH and VL within the same molecule, the linker connecting VH and VL is generally a peptide linker with a length of 10 amino acids or more. However, the linker of scFv in the present invention is not limited to such a peptide linker, as long as it does not hinder the formation of scFv.

[0023] Low molecular weight antibodies can be particularly bispecific antibodies. A "bispecific antibody" may be, for example, an antibody in which the heavy chain variable region and the light chain variable region are linked as a single chain (e.g., sc(Fv)2). Alternatively, an antibody-like molecule may be formed by linking scFv (or sc(Fv)2), in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked, to an Fc region (a constant region lacking the CH1 domain). Alternatively, scFv-CH3) may also be used.

[0024] The amino-terminus of each chain that makes up an antibody contains a variable region of approximately 100-110 or more amino acids that are primarily involved in antigen recognition. In the variable region, three loops are located in the heavy and light chains. The domains (i.e., VH and VL) are assembled to form an antigen-binding site. Each loop is also called a complementarity-determining region (hereinafter also referred to as "CDR"). That is, each VH and VL consists of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0025] In the present invention, the variable region of an antibody or small molecule antibody may be of human origin, of non-human mammal origin, or may be modified to contain a mixture of both. Examples of non-human mammals include, but are not limited to, mice, rats, rabbits, goats, sheep, and horses. Similarly, in the present invention, the constant region of an antibody or small molecule antibody may be of human origin, of non-human mammal origin, or may be modified to contain a mixture of both. Examples of non-human mammals include, but are not limited to, mice, rats, rabbits, goats, sheep, and horses. Furthermore, in the present invention, the constant region and variable region of an antibody or small molecule antibody may be derived from different species.

[0026] Here, the 65th amino acid residue in Kabat numbering corresponds to CDR2 in the VH region. It is located at amino acid residue 82a in Kabat numbering, in FR3 in the VH region. It is located there. The VH family consists of seven types, from VH1 to VH7. Table 1 shows examples of antibodies that possess these seven types of VH regions (VH1 to VH7).

[0027] [Table 1]

[0028] Figure 1 shows the multiple alignments of the amino acid sequences of VH1 to VH7 shown in Table 1. Note that in Table 1 and Figure 1, IgM RF 2A2 contains VH (belonging to VH3) whose co-crystal structure with protein A has been elucidated. Furthermore, in the multiple alignments in Figure 1, the positions enclosed in boxes indicate amino acid residues reported to be involved in the interaction between VH3 and protein A, based on the co-crystal structure of VH3, in Graille, M. et al., Proc. Natl. Acad. Sci. USA 2000, 97 (10), 5399-5404. Specifically, the 15th glycine residue, the 17th serine residue, the 19th arginine residue, the 57th lysine residue, the 59th tyrosine residue, the 64th lysine residue, the 65th glycine residue, the 66th arginine residue, the 68th threonine residue, the 70th serine residue, the 81st glutamine residue, and the 82a asparagine residue in the VH region. It has been suggested that the serine residue at position 82b is involved in binding to protein A.

[0029] The amino acid sequence of "VH3(bind)" shown in Figure 1 is designated as Sequence ID No. 1, and the amino acid sequence of "VH1" is shown below. The amino acid sequence of "VH2" was designated as Sequence ID 2, the amino acid sequence of "VH3" as Sequence ID 4, the amino acid sequence of "VH4" as Sequence ID 5, the amino acid sequence of "VH5" as Sequence ID 6, the amino acid sequence of "VH6" as Sequence ID 7, and the amino acid sequence of "VH7" as Sequence ID 8.

[0030] In this invention, as described above, in order to acquire affinity for protein A, a substitution mutation is introduced into an antibody or small molecule antibody that does not have affinity for protein A, such that the 65th position of the VH region is a glycine residue and the 82a position is an asparagine residue. In addition to mutation, the present invention provides, as described above, an even better affinity for protein A. To achieve this, a substitution mutation is introduced to replace the 16th position in the VH region with a lysine, arginine, or glycine residue. Furthermore, in this invention, additional substitution mutations may be introduced in addition to these substitution mutations. For example, in addition to these substitution mutations, one or more substitution mutations are introduced such that the 15th position in the VH region is a glycine residue, the 17th position is a serine residue, the 19th position is an arginine residue, the 57th position is a lysine residue, the 59th position is a tyrosine residue, the 64th position is a lysine residue, the 66th position is an arginine residue, the 68th position is a threonine residue, the 70th position is a serine residue, the 81st position is a glutamine residue, and the 82b position is a serine residue. It's okay to implement it.

[0031] Furthermore, in this invention, as described above, in order to reduce the affinity for protein A, For antibodies or small molecules that have affinity for tein A, the 65th glycine residue is Substitutional mutations are introduced to result in amino acid residues other than glycine. In addition to these substitutional mutations, one or more substitutional mutations may be introduced in the VH region such that the 15th position is an amino acid residue other than glycine, the 17th position is an amino acid residue other than serine, the 19th position is an amino acid residue other than arginine, the 57th position is an amino acid residue other than lysine, the 59th position is an amino acid residue other than tyrosine, the 64th position is an amino acid residue other than lysine, the 66th position is an amino acid residue other than arginine, the 68th position is an amino acid residue other than threonine, the 70th position is an amino acid residue other than serine, the 81st position is an amino acid residue other than glutamine, the 82a position is an amino acid residue other than asparagine, and the 82b position is an amino acid residue other than serine.

[0032] Here, in order to reduce the affinity for protein A, the 65th glycine residue after substitution is used. In the form in which the 65th glycine residue is replaced with an amino acid residue other than glycine, the substituted amino acid residue is not particularly limited and may be any of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolicine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine. In particular, when the 65th glycine residue is substituted, even if alanine, which has the smallest side chain among the listed amino acids, is used, the affinity for protein A can be reduced.

[0033] Such substitution mutations can be introduced by conventionally known methods. In other words, the mutant antibody according to the present invention can be manufactured by applying genetic recombination technology.

[0034] First, the nucleic acid encoding the antibody or small molecule antibody into which the substitution mutation will be introduced is modified to encode the amino acid sequence in which the target substitution mutation is introduced. More specifically, the nucleic acid is modified so that the codon corresponding to the amino acid residue before the substitution mutation is introduced becomes the codon of the amino acid residue introduced by the substitution mutation. Typically, this involves genetic manipulation or modification to substitute at least one base of the nucleic acid constituting the codon so that it becomes a codon encoding the target amino acid residue. The nucleic acid is subjected to a different treatment. Such modifications can be carried out as appropriate using techniques known to those skilled in the art, such as site-directed mutagenesis and PCR mutation induction.

[0035] Nucleic acids encoding amino acid sequences into which substitution mutations have been introduced are typically held (inserted) into a suitable vector and introduced into host cells. The vector in question must stably hold the inserted nucleic acid. There are no particular restrictions as long as it is done in that way; for example, if E. coli is used as the host, the pBluescript vector (Stratagene) can be used as the cloning vector. In particular, this When a vector is used to produce the mutant antibody according to the invention, it is preferable to use an expression vector. The expression vector is not particularly limited as long as it is a vector that expresses polypeptides in vitro, in E. coli, in cultured cells, or in living organisms. For example, for in vitro expression, the pBEST vector (Promega) is preferred; for E. coli, the pET vector (Invitrogen) is preferred; for cultured cells, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred; and for living organisms, the pME18S vector (Mol Cell Biol. (1988) 8, 466-472) is preferred. .

[0036] There are no particular restrictions on the host cell, and various host cells can be used depending on the purpose. Examples of cells used to express polypeptides include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis, Brevibacillus), fungal cells (e.g., yeast, Aspergillus), and insect cells (e.g., Drosophila S2, Spodoptera SF9). Examples of suitable host cells include animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electro-pulsed poroscopy (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, and microinjection.

[0037] To cause mutated antibodies (antibodies or small molecules) expressed in host cells to be secreted into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, an appropriate secretion signal can be incorporated into the target antibody. If the mutated antibodies (antibodies or small molecules) are secreted into the culture medium, they can be recovered from the culture medium. If the mutated antibodies (antibodies or small molecules) are produced intracellularly, they can be recovered from the resulting lysate after the cells have been collected.

[0038] Conventional methods can be applied to purify the mutant antibodies according to the present invention, including known methods such as ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography.

[0039] In particular, in the case where the aforementioned substitution mutation has resulted in an affinity for protein A In addition, affinity chromatography using protein A is preferred. Furthermore, if the aforementioned substitution mutation reduces affinity for protein A, then the protein Affinity chromatography other than affinity chromatography using In A Fees and other methods described above are preferred. [Examples]

[0040] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0041] The following examples and comparative examples use a small bispecific antibody (hereinafter referred to as Ex3 sc1) targeting human epidermal growth factor receptor (EGFR) and T lymphocyte surface antigen CD3 as a model. For more information on Ex3 sc1, please refer to MAbs, 10(6), 854-863 (2018). The VH(5H) of the anti-EGFR antibody 528 and the VH(OH) of the anti-CD3 antibody OKT3 of Ex3 sc1 belong to the VH1 subclass and VH3 subclass, respectively. Although OH belongs to the VH3 subclass, Ex3 does not have the ability to bind to protein A. .

[0042] [Comparative Example 1] Figure 2 shows the co-crystal structure of VH3 with protein A, and the VH(5H) of Ex3 sc1 and Ex3 The alignment of VH(OH) in sc1 is shown. In Figure 2, the residues involved in the interaction with protein A in VH3, which has affinity for protein A, are enclosed in boxes: G15, S17, R19, K57, Y59, K64, G65, R66, T68, S70, Q80, N82a, and S82b. The amino acid sequence of "VH3 (binding to Protein A)" shown in Figure 2 is shown in Sequence ID No. 9, and the amino acid sequence of Trastuzumab VH The sequence is shown in Sequence ID 10, the amino acid sequence of anti-EGFR VH(5H) is shown in Sequence ID 11, and the amino acid sequence of anti-CD3 VH(OH) is shown in Sequence ID 12.

[0043] We consider that modifying the amino acid sequence of CDR may lead to a decrease in antigen-binding ability. With this in mind, amino acid residues located at FR were chosen as the sites for mutation introduction. Specifically, in this comparative example, the K19R, T70S, E81Q, S82aN, and R82bS mutations at 5H and the D82aN mutation at OH were introduced into Ex3 sc1. The sextuple mutant of Ex3 sc1 with these six mutations introduced is referred to as "Ex3 sc1-6m".

[0044] Site-specific mutagenesis was performed using the Quick Change method for 5H K19R and T70S, and OH D82aN, while mutagenesis was performed using overlap PCR for 5H E81Q, S82aN, and R82bS. Table 2 shows the primers used for targeted mutation introduction, and the primers used for overlap PCR. — is shown in Table 3. In Tables 2 and 3, the locations where mutations were introduced are underlined.

[0045] [Table 2]

[0046] [Table 3]

[0047] Based on the above, the structural gene for Ex3 sc1-6m was obtained. Although details are omitted, the obtained structural gene for Ex3 sc1-6m was inserted into the Brevibacillus expression vector pROXb3 (Protein Express Co., Ltd.). Brevibacillus S5 was transformed using the obtained expression vector. Transformed Brevibacillus S5 was seeded onto MTN agar medium (0.95 L of medium prepared by adding 5% 20% glucose solution after sterilization, consisting of high polypeptone (10 g), yeast extract BSP-B (2 g), bonito extract (5 g), 3MM stock (1% FeSO4·7H2O, 1% MnSO4·7H2O, and 0.1% ZnSO4·7H2O, 1 mL), and 3MM stock, and 3MM stock (1% FeSO4·7H2O, 1% MnSO4·7H2O, and 0.1% ZnSO4·7H2O), and 3MM stock (1 mL), and 3MM stock (1 mL), and 3MM stock (1 mL), 3MM stock (1% FeSO4·7H2O, 1% MnSO4·7H2O, and 1 mL ZnSO4·7H2O)), and incubated at 37°C for 24 hours. The obtained colonies were inoculated into 2 mL of 2SL medium (neomycin: Nm(+), final concentration 50 μg / mL) and incubated at 30°C and 120 rpm for 24 hours. Pre-culture was performed. The 2SL medium was prepared by mixing Phytone Peptone (40g), yeast extract BSP-B (5g), 3MM stock (1mL), and sterile water to 0.9L, and adjusting the pH to 7.2.

[0048] Next, a 1% dose of the pre-culture solution was inoculated into 2SL medium (neomycin: Nm(+), final concentration 50 μg / mL) to which L-(+)-arginine had been added to achieve a final concentration of 0.1 M. The culture was then performed at 30°C and 120 rpm for 48 hours. The culture medium was centrifuged at 4°C and 5,000xg for 20 minutes to separate it into the culture supernatant and precipitate. The proteins in the culture supernatant were then concentrated by salting out with ammonium sulfate. First, 60% of the culture supernatant was used. Weigh out ammonium sulfate equivalent to the % mass and grind it into particles using a pestle to make it easier to dissolve. The culture supernatant was finely chopped. The culture supernatant was stirred with a stirrer bar in a low-temperature room, taking care not to create bubbles, while ammonium sulfate was added little by little. After stirring for 2 hours, the culture was incubated at 4°C, 5,000xg, for 20 minutes. The precipitate was centrifuged, and approximately 4 mL of 1xPBS was added to dissolve it. Then, to remove any remaining ammonium sulfate, dialysis with 1xPBS was performed three times. The supernatant obtained by centrifugation at 4°C, 15,000xg, for 10 minutes was collected, and a column packed with 1 mL of Ni Sepharose was used. Ni used 2+ Purification was performed by affinity chromatography. SDS-PAGE analysis was performed on the sample. Although the results are not shown in the figure, in all cases, bands were observed near the theoretical molecular weight in the presence of 150 mM and 200 mM imidazole, confirming elution. These eluates were then concentrated by ultrafiltration and subjected to gel filtration chromatography to obtain Ex3 sc1 Ex3 sc1-6m was purified separately.

[0049] In this comparative example, the affinity of purified Ex3 sc1 and Ex3 sc1-6m to protein A was evaluated. In this evaluation, first, 0.1 mL of rProtein A Sepharose Fast Flow was administered to a polyprep container. After packing the chromatography column, the column was equilibrated with 6CV MQ and 50mM Tris-HCl / 200mM NaCl (pH 8.0). Then, 0.2 nmol / 200 μL of the purified sample was applied to the 2CV column. Unbound proteins were eluted by washing with 1x PBS. Subsequently, 0.1 M Gly-HCl (pH 3.0) was added in 2 CVs (3 times) to elute the proteins bound to the column. At that time, the microtube used to collect the eluted sample was pre-filled with 1 M HCl, which is 5% of the elution solution volume. Tris-HCl (pH 9.2) was added to neutralize the eluate.

[0050] Although the results are not shown in the graph, it is known that Ex3 sc1 without the mutation does not bind to protein A, and the majority of Ex3 sc1 applied to the column are flow-through and wash-through. It eluted in the fraction. On the other hand, Ex3 sc1-6m, which had mutations introduced at a total of six locations with the aim of conferring the ability to bind to protein A, also showed elution in the flow-through and wash fractions, and its behavior was almost the same as that of Ex3 sc1.

[0051] Based on the above, the six mutation sites introduced into the Ex3 sc1-6m produced in this comparative example were not sufficient to protect the proteo. It was not possible to improve affinity for in A, and it was thought that further mutation introduction was necessary. .

[0052] [Example 1] The results of Comparative Example 1 revealed that introducing six mutations into the FR region based on the amino acid sequence of VH3, which has affinity for protein A, did not improve affinity for protein A. Therefore, we focused on amino acid residues located in the complementarity-determining region (CDR), which are important for interaction with protein A, and created a cocrystal structure (PDB:1EDD) using the molecular graphic tool PyMOL. The interaction interface between protein A and VH in ) was analyzed.

[0053] Among the residues important for binding to protein A, the amino acid residues located in the CDR are K57, Y59, K64, and G65 (Figures 1 and 2). Of these, Y59 and K64 have the ability to bind to protein A. It is also conserved in Ex3 sc1, which does not contain it. Furthermore, it has been reported that if the 57th amino acid residue is K, R, or T, it can bind to protein A (Crauwels, M. et al., Biotechnol. 2020, 57 (September 2019), 20-28).

[0054] Therefore, we analyzed the interaction interface of the 65th amino acid residue using PyMOL. As a result, in the case of G65, similar to antibody 2A2 in the cocrystal structure, it interferes with binding to protein A. No such interaction was observed (Figure 3). On the other hand, when mutations were introduced to D65 and N65, it was observed that the respective side chains collided with the N43 side chain of protein A (Figure 4). Therefore, in this example, we considered introducing a substitution mutation to the 65th amino acid residue in the VH region located in the CDR. Specifically, the 65th amino acid residue in the VH region located in the CDR is involved in the binding of protein A. To avoid steric hindrance, we decided to introduce the N65G substitution mutation at 5H and the D65G substitution mutation at OH in the Ex3 sc1-6m prepared in Comparative Example 1.

[0055] As a control for evaluating the structural gene and function of Ex3 sc1-6m prepared in Comparative Example 1, the Ex3 sc1 gene was used as a template to perform N65G substitution mutations at 5H and D65G substitution mutations at OH. Specifically, Quick To construct the vector using the change method, the primers shown in Table 4 were designed. The locations where mutations were introduced are underlined.

[0056] [Table 4]

[0057] Using the designed primers, monomutations of N65G and D65G were introduced. The octavalent mutant of Ex3 sc1, in which these two mutations were introduced into Ex3 sc1-6m, is called "Ex3 sc1-8m". Furthermore, the double mutant of Ex3 sc1, in which these two mutations were introduced into Ex3 sc1, is called "Ex3 sc1-2m". The structural genes for Ex3 sc1-8m and Ex3 sc1-2m, constructed in Example 1, were then inserted into the pROXb3 vector, respectively, in the same manner as in Comparative Example 1.

[0058] Expression of both Ex3 sc1-8m and Ex3 sc1-2m was confirmed using the same method as in Comparative Example 1, and they were purified using the same method as in Comparative Example 1. The affinity of the purified Ex3 sc1-8m and Ex3 sc1-2m to protein A was then evaluated using the same method as in Comparative Example 1. The results were... This is shown in Figure 5. In Figure 5, A shows the results for Ex3 sc1-8m, and B shows the results for Ex3 sc1-2m. Figure 5 is a photograph of the gel stained with CBB (Rapid CBB KANTO) after SDS-PAGE. That is the case.

[0059] As shown in Figure 5, when Ex3 sc1-2m was used with mutations only in N65G and D65G, although slight elution was observed in the elution fraction with 0.1M Gly-HCl (pH 3.0), elution mainly occurred through flow-through and washing. This behavior was almost the same as that of Ex3 sc1 prepared in Comparative Example 1. In contrast, when Ex3 sc1-8m, which had mutations introduced at eight locations to improve interaction and avoid steric hindrance, was used, no elution was observed in the flow-through fraction or wash, and all bands derived from Ex3 sc1-8m were confirmed only in the elution fraction. As shown in Comparative Example 1, Ex3 sc1-6m was The fact that Ex3 sc1-8m did not bind to the rotine A column, while it did bind to the protein A column, demonstrates the effectiveness of introducing N65G and D65G mutations to avoid steric hindrance.

[0060] Based on the results of this embodiment, we concluded that affinity for protein A can be improved by combining mutation introduction aimed at enhancing interaction and avoiding steric hindrance.

[0061] [Example 2] Regarding Ex3 sc1-8m, which achieved affinity with protein A in Example 1, "cell binding evaluation" Valency testing and cytotoxic activity testing were performed.

[0062] <Cell Binding Evaluation Test> In the cell binding evaluation test, EGFR-positive TFK-1 cells 5.0x10 5 5.0x10¹ CD3-positive T-LAK cells 5 Individual cells were used as target cells. The cells were dispensed into Eppendorf tubes, the supernatant was aspirated, and then 20 pmol of [amount missing] was used. Each antibody (Ex3 sc1 or Ex3 sc1-8m) was added, and the samples were left to stand on ice for 30 minutes. The liquid volume was kept the same between samples. Next, two washes were performed using PBS. For washing, 900 μL of PBS was added, followed by centrifugation at 300xg for 5 minutes, after which the supernatant was aspirated and removed. This was done. Next, 100 μL of anti-Ex3 Db rabbit serum, diluted 100-fold, was added as the primary antibody. Then, it was left to stand on ice for 30 minutes. After washing twice with PBS, Alexa Fluor 594 labeled antibody 50 μL of 1 μg rabbit IgG antibody was added, and the mixture was left to stand on ice for 30 minutes. Finally, the mixture was washed twice. After this, 300 μL of PBS is added, and impurities are removed by passing the mixture through a mesh. The fluorescence intensity was measured using a thermometer.

[0063] The results of cell binding evaluation for TFK-1 cells and T-LAK cells are shown in Figure 6. In Figure 6, A The first line shows the results of cell binding evaluation for TFK-1 cells, and the second line shows the results of cell binding evaluation for T-LAK cells. The results are shown. In each graph, the vertical axis represents the number of cells and the horizontal axis represents the fluorescence intensity. A peak shifting to the right indicates stronger binding to the target cells.

[0064] As shown in Figure 6, the histograms of Ex3 sc1 and Ex3sc1-8m show a similar degree of shift regardless of the cell type used. In other words, it was found that even after introducing a total of eight mutations into Ex3 sc1, the same cell-binding ability as before the mutations was maintained.

[0065] <Cytotoxic activity test> In the cytotoxic activity test, cancer cell toxicity was evaluated using the MTS assay. First, the cells were allowed to stand. The supernatant of the cultured TFK-1 cells was aspirated and removed, 1 mL of TrypL Express Enzyme (1X), no phenol red was added, and the flask was incubated in a 37°C, 5% CO2 incubator for about 5 minutes. The flask was then gently removed. The cells are detached by applying a tapping vibration, and then 9 mL of 1xPBS is added and the cells are removed by pipetting. The graft was removed. The cell suspension was centrifuged at 300 xg for 5 minutes. The supernatant was aspirated and removed, and 10 mL of RPMI1640 (containing 10% FBS, PC / SM) was added and resuspended by pipetting. Cell suspension 10 μL of the sample was stained with an equal volume of trypan blue, and the number of cells was measured using a cell counter. Next, TFK-1 cells were placed in a 96-well plate at a rate of 5.0 x 10⁶ per well. 3 Seed so that cells are per 100 μL Therefore, put 1.2x10 in the reservoir 6Cells were separated and suspended in 12 mL of culture medium. Then, 100 μL of the cell suspension was seeded into each well of a 96-well plate. After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was aspirated and removed. Next, 2.0 x 10⁶ T-LAK cells were added per well. 4 Add 50 μL of cells, or 100 μL each of MilliQ water or RPMI1640 medium. Then add T-LAK cells. 50 μL of each sample, with concentrations varied at 5 different points, was added to each well, and the plate was incubated at 37°C and 5% CO2 for 24 hours. After removing the supernatant and washing three times with 1xPBS, 1 mL of MTS reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay) was added to 11 mL of RPMI1640 medium, and 100 μL of the suspension was added to each well. Incubation was performed at 37°C and 5% CO2 for 40 minutes to 1 Incubate for approximately 1 hour, then use a plate reader to measure the absorbance at 490 nm (reference: 655 nm). The absorbance was measured. Wells containing only RPMI1640 were incubated until the absorbance reached approximately 0.8, and the cancer cell toxic activity was calculated using the detected data.

[0066] The MTS reagent contains a tetrazolium salt, which, upon reduction, is converted into a colored formazan product soluble in culture medium, and PES, an electron acceptor. The tetrazolium salt is derived from a dehydrogenase of living cells. Since it is reduced by NADH or NADPH and converted into a colored formazan product with absorption at 490 nm, a proportional relationship exists between the number of viable cells and the amount of formazan product. Using this principle, cancer cytotoxic activity was calculated using the following formula. Cancer cell toxic activity [%] = (1 - (AC)) / (BC) × 100 In the formula, A: the average absorbance of the well to which the sample was added, B: the well to which only the culture medium was added (positive control), and C: the well to which MQ was added (negative control).

[0067] The results of the cancer cell cytotoxicity test using the MTS assay are shown in Figure 7. Both the sc1 and Ex3 sc1-8m antibodies showed an increase in cancer cell cytotoxicity in a concentration-dependent manner. In other words, no difference in cancer cell cytotoxicity was observed before and after the introduction of the eight mutations. This revealed that the eight mutations introduced did not affect the functions induced by Ex3 sc1.

[0068] [Example 3] In this example, using the anti-CD3 antibody OKT3, which is a constituent domain of Ex3 sc1 used in Examples 1 and 2, we investigated amino acid substitution mutations essential for affinity with protein A in the VH(OH) of OKT3. The VH(OH) of OKT3 used in this example belongs to the VH3 subclass, but the protein It is known that it does not have affinity for A.

[0069] The D82aN mutation introduced into OH, as verified in Example 2, forms a hydrogen bond with S33 of protein A. It is important for interaction with protein A in order to form. On the other hand, glycine at position 65 is prote It is known to be an important residue for binding with amino acid A, but is it acceptable for other amino acids besides glycine? There is very little experimentally evaluated knowledge regarding amino acids. Therefore, in this example, OKT3 scFv By introducing mutations into D65 and D82a in the OH region, we identified amino acid residues that are important for binding to protein A in VH belonging to the VH3 subclass.

[0070] In this example, a pRA vector encoding the structural gene of OKT3 scFv was used as a template, and the OH group D65 and We attempted to introduce mutations into D82a. Specifically, we introduced mutations into each monomutant (OKT3 scFv-D65G and OKT3 scFv-D82aN) and into the monomutant with the D82aN mutation, using three different types of A Double mutants with substitutions of the mino acid (OKT3 scFv-D65G / D82aN, OKT3 scFv-D65A / D82aN, and OKT3 scFv-D65N / D82aN) were created. Vector construction was performed for each mutant using the quick change method. Therefore, the primers shown in Table 5 were designed. In Table 5, the sites where mutations were introduced are underlined.

[0071] [Table 5]

[0072] Using the designed primers, expression vectors were constructed with the target monomutations (D65G or D82aN) and double mutants (D65G / D82aN, D65A / D82aN, or D65N / D82aN) introduced into the OH group of OKT3 scFv. Monomutations of N65G and D65G were introduced. The mutant with the introduced mutation is called "OKT3 scFv-D65G," and the mutant with the single mutation D82aN is called This variant is referred to as "OKT3 scFv-D82aN". Furthermore, mutants in which the double mutations D65G / D82aN, D65A / D82aN, or D65N / D82aN are introduced to the OH group of OKT3 scFv are referred to as "OKT3 scFv-D65G / D82aN", "OKT3 scFv-D65A / D82aN", and "OKT3 scFv-D65N / D82aN", respectively.

[0073] Unlike in Example 1, the expression vector constructed in the previous step was used to shape E. coli DH5α The quality was changed. Using the culture supernatant of transformed E. coli DH5α, Ni 2+ Affinity Chromatography Mutants created using Graph were purified. Expression and purification were confirmed for all mutants using the same method as in Comparative Example 1. The OKT3 scFv-D65G, OKT3 scFv-D82aN, OKT3 scFv-D65G / D82aN, and OKT3 scFv-D65A / D82aN mutants that were purified in this example were also purified. And for OKT3 scFv-D65N / D82aN, and OKT3 scFv before mutation introduction (control), Similar to Comparative Example 1 and Example 1, the affinity with Protein A was evaluated. The results are shown in Figure 8. In Figure 8, A shows the results for OKT3 scFv (control), and B shows the results for OKT3 scFv-D65G. The results are shown as follows: C shows the result for OKT3 scFv-D82aN, D shows the result for OKT3 scFv-D65G / D82aN, E shows the result for OKT3 scFv-D65A / D82aN, and F shows the result for OKT3 scFv-D65N / D82aN.

[0074] As shown in Figure 8, in OKT3 scFv before mutation introduction, the lysis in the flow-through and wash fractions was Elution was observed. Furthermore, elution was also observed in the flow-through and wash fractions of the single mutants OKT3 scFv-D65G and OKT3 scFv-D82aN, and their behavior was almost identical to that of OKT3 scFv. The results were consistent with those obtained with Ex3 sc1-6m and Ex3 sc1-2m, which were introduced solely to improve interactions or avoid steric hindrance. Taken together, these results suggest that these single mutations are effective. During the introduction process, it became clear that it was not possible to impart affinity for protein A to OKT3 scFv.

[0075] On the other hand, as shown in Figure 8, in the double mutant OKT3 scFv-D65G / D82aN, which incorporates these two mutations, the band intensity in the flow-through and wash fractions decreased, while the band intensity in the emulsion fraction increased. The degree of affinity increased. This result is consistent with the results of Ex3 sc1-8m, which combines mutation introduction aimed at improving interaction and avoiding steric hindrance. It indicates that, in addition to avoiding steric hindrance by the D65G mutation, the formation of hydrogen bonds by the D82aN mutation contributes to the improved affinity for protein A.

[0076] Furthermore, as shown in Figure 8, D65 is the amino acid with the smallest side chain among amino acids with side chains. Nin (side chain; -CH3) and asparagine (side chain; -CH2-CO-NH2) with the polarity of the side chain neutralized. Even after substitution, elution was observed in the Flow Through and Wash fractions, as well as in the Elution fraction. It was not found. This result indicates that in order to acquire affinity with protein A, the 65th position in the VH region is necessary. This indicates that it is essential to have glycine as the amino acid residue and that there is no tolerance for other amino acids. In other words, in antibodies or small molecules that have gained affinity for protein A by having glycine as the 65th amino acid residue in the VH region, By substituting n with other amino acid residues, the affinity for protein A is reduced or lost. It can be done.

[0077] [Example 4] In this example, the c-Myc tag and His tag were removed from the octuplet mutant "Ex3 sc1-8m" of Ex3 sc1 prepared in Example 1, and purification using its affinity for protein A was investigated. In this example, Ex3 sc1-8m-tag(-) was prepared by removing the c-Myc tag and His tag from "Ex3 sc1-8m" prepared in Example 1.

[0078] To remove the c-Myc tag and His tag from pROX3-Ex3 sc1-8m, which was obtained by inserting Ex3 sc1-8m prepared in Example 1 into the pROXb3 vector, an Fw primer containing the restriction enzyme site of NcoI and a Rev primer for adding a stop codon and the restriction enzyme site of HindIII to the C-terminus of Ex3 sc1-8m were designed. The designed primers are shown in Table 6.

[0079]

Table 6

[0080] Ex3 sc1-8m-tag(-) was amplified by PCR using these primer sets and inserted into the pROX3 vector in the same manner as in Example 1. Using the obtained expression vector, Brevibacillus sp. S5 was transformed as in Comparative Example 1, and the expression of Ex3 sc1-6m was confirmed by SDS-PAGE and Western blotting analysis. Subsequently, the transformed Brevibacillus sp. S5 was cultured using a flask, and an attempt was made to purify Ex3 sc1-8m-tag(-) from the culture supernatant in the same manner as in Comparative Example 1. Also, as experimental controls, Ex3 sc1 and Ex3 sc1-8m were similarly prepared. Note that 300 mL each of Ex3 sc1-8m-tag(-) and Ex3 sc1 and 600 mL of Ex3 sc1-8m were cultured.

[0081] For the obtained culture supernatants, the entire amount of Ex3 sc1 and half of Ex3 sc1-8m were purified by Ni affinity chromatography using a column packed with 1 mL of Ni Sepharose, and half of Ex3 sc1-8m and the entire amount of Ex3 sc1-8m-tag(-) were purified by protein A affinity chromatography using a column packed with 1 mL of rProteinA Sepharose.

[0082] 2+ 2+ 2+ Affinity Elution by chromatography was performed by increasing the imidazole concentration in 1xPBS from 0mM, 10mM, 50mM, 150mM, 200mM, 300mM, and 500mM, and was performed at 50CV, 100CV, 20CV, 10CV, 10CV, 10CV, and 10CV respectively. Elution by protein A affinity chromatography was performed as follows: After washing 20 CV with 1x PBS and 5 CV with 0.1 M Glycine buffer (pH 4.0), elution was performed five times in 3 CV increments using 0.1 M Glycine buffer (pH 3.0). The purity of the eluted fraction was determined by SDS-PAGE. After confirming this, the eluate in which the band of the target protein was clearly observed was concentrated by ultrafiltration, and then gel filtration chromatography was performed using a Superdex 200 Increase 10 / 300 GL column. The nomer fraction was separated. Furthermore, SDS-PAGE was performed on the molecular weight peak near the target protein to evaluate whether the target protein had been purified. The fraction containing the target antibody was concentrated by ultrafiltration, filtered, and then used for subsequent evaluation.

[0083] Ni 2+ The fraction obtained was purified by affinity chromatography. Figure 9 shows the results of the SDS-PAGE analysis, including the protein A affinity chromatography. Figure 10 shows the results of SDS-PAGE analysis performed on the fraction obtained after purification using the ionizer.

[0084] As shown in Figure 9, Ni 2+ In affinity chromatography, elution was confirmed for both Ex3 sc1 and Ex3 sc1-8m antibodies in the presence of 150 mM and 200 mM imidazole. As shown in Figure 10, in protein A affinity chromatography, Ex3 Elution was confirmed in the fractions using 0.1M Gly-HCl (pH 3.0) for both the sc1-8m and Ex3 sc1-8m-tag(-) antibodies. The fractions from which elution of each antibody was confirmed were concentrated by ultrafiltration, and then subjected to gel filtration chromatography. The chromatograms from the gel filtration chromatography are shown in Figure 11. Ni 2+ Ex3 sc1 and Ex3 sc1-8m purified by affinity chromatography showed peaks near their theoretical molecular weights. On the other hand, Ex3 sc1-8m and Ex3 sc1-8m-tag(-) purified by protein A affinity chromatography had lower molecular weights than their theoretical molecular weights. The peak shifted towards the high molecular weight side, which was considered an operational issue. In particular, with Ex3 sc1-8m-tag(-), the absence of the artificial tag caused the peak to shift towards the low molecular weight side. When these peaks were examined for purification using SDS-PAGE, a nearly single band was observed near the theoretical molecular weight of each antibody, suggesting that the target antibody had been purified to a high degree of purity.

[0085] Therefore, in each Ex3 sc1, fractions in which a clear band was observed by SDS-PAGE were selected. After concentration by ultrafiltration, the sample was used for subsequent evaluations. In this example, cell binding evaluation tests and cytotoxic activity tests were performed in the same manner as in Example 2.

[0086] The results of the cell binding evaluation test are shown in Figure 12. As shown in Figure 12, the cell binding evaluation results for TFK-1 cells or T-LAK cells of each Ex3 sc1 prepared in this example showed that either cell... Even when using this method, the histogram shifted to a similar extent regardless of the antibody purification method or the presence or absence of tags. Therefore, antibodies with a total of 8 mutations introduced into Ex3 sc1 were tagged with His. Even when purified using affinity with protein A without utilizing other methods, mutations may still be present. It has been revealed that it has similar cell-binding ability to Ex3 sc1 without a tag and Ex3 sc1-8m with a tag. Ta.

[0087] The results of the cytotoxic activity test are shown in Figure 13. As shown in Figure 13, the preparations made in this example Results from MTS assays using each Ex3 sc1 showed that all antibodies exhibited an antibody concentration-dependent increase in cancer cell cytotoxicity. Antibodies with a total of 8 mutations introduced into them were tested against the parent of protein A without using His tags, etc. Even when purified using the symmetry method, it was revealed that it possesses similar cancer cell cytotoxic activity to Ex3 sc1 without mutations and Ex3 sc1-8m with tags.

[0088] Furthermore, Ex3 sc1-8m-tag(-) showed slightly higher cancer cell toxicity compared to Ex3 sc1-8m, and a similar trend was confirmed when re-evaluated in the concentration range of 0.1 to 10 pM (Figure 14). This result suggests that cancer cell toxicity increases when the terminal artificial tags of HL-type Ex3 Db and Ex3 sc2 are removed. This is consistent with a previous report (Asano, R. et al., FEBS J. 2010, 277 (2), 477-487). As mentioned above, it has been confirmed that the presence or absence of a tag does not affect cell binding ability. Therefore, the reason why cancer cytotoxic activity was enhanced in the tagless configuration is unclear. However, it is possible that it affected intracellular signaling efficiency and cytokine production efficiency, and consequently influenced cancer cytotoxic activity.

[0089] [Example 5] In this embodiment, the affinity for protein A due to the D65G mutation and / or D82aN mutation described above In addition to its effectiveness in improving sexual performance, other substitution mutations further enhance its affinity for protein A. We considered making it happen. In this example, similar to the Ex3 sc1 used in Examples 1-4, cancer Ex3 ta6 is a small-molecule bispecific antibody that targets EGFR on the cell surface and CD3 on the T cell surface. This was used (Asano, R. et al., MAbs 2018, 10 (6), 854-863). The difference between the two is the order in which the constituent domains are linked; Ex3 sc1 has OL-5H-5L-OH from the N-terminus, while Ex3 ta6 has OL-5H-5L-OH from the N-terminus. It is structured as OH-OL-5H-5L. Although the four constituent domains each have the same amino acid sequence, it is thought that they have different three-dimensional structures due to the difference in their linkage order.

[0090] In this example, as another substitution mutation, the amino acid to be substituted is alanine at the 16th position from the N-terminus. The goal was to use alanine as a residue and substituted it with lysine, arginine, and glycine.

[0091] [Construction of Vector pRA1 / 528 scFv-HL-WT] Using pRA1 / Ex3 ta6, which is formed by inserting Ex3 ta6 into vector pRA1, as a template, the following are shown in Table 7 below. The structural gene fragment of 528 scFv-HL-WT was amplified by PCR using a lymer.

[0092] [Table 7]

[0093] Amplification of the target DNA was confirmed by electrophoresis using 5 μL of the reaction solution after PCR, and the PCR product was purified using the FastGene gel / PCR purification kit with the remaining reaction solution. The purified DNA fragment and pRA1 / EgA1 were digested with NocI and SacII. After electrophoresis using the entire amount of the treated reaction solution, the gel was excised and the target DNA was extracted using the FastGene gel / PCR purification kit. The extracted 528 scFv-HL-WT structural gene fragment was mixed to a total volume of 5 μL at a molar ratio of more than 4 times that of the pRA1 vector fragment. Then, 5 μL of Solution I of DNA Ligation kit Ver.2.1 was added and incubated at 16°C for 1 hour. The ligation reaction was performed by [method]. 50 μL of E. coli DH5α was transformed using the entire reaction solution after ligation. Shaking culture was performed for 20 minutes, and the cells were incubated overnight at 37°C on LB agar (Amp(+); fc 100 μg / mL). Colonies that grew on the plate were picked with a toothpick and added to a test tube containing 3 mL of LB medium (Amp(+); fc 100 μg / mL). Shaking was performed at 37°C and 140 rpm for 18 hours. The plasmids were cultured and extracted using the FastGene plasmid mini-kit. For elution, 50 μL of MQ heated to 70°C was used. The extracted plasmids were also subjected to Europh. The vector was constructed using the DNA sequencing service of InGenomics Inc. I confirmed that.

[0094] [Construction of the mutant vector pRA1 / 528 scFv-HL-6m] To construct the hexavalent mutant 528 scFv-HL-6m, the vector pRA1 / Ex3 ta6-8m, in which the mutations K19R, N65G, T70S, E81Q, D82aN, and R82bS were introduced into 528 VH, was used as a template. The structural gene fragment of 528 scFv-HL-6m was amplified using the primers shown in Table 7 above, in the same manner as described above. Furthermore, a pRA1 vector with the structural gene fragment of 528 scFv-HL-6m inserted was constructed in the same manner as described above.

[0095] [Construction of the mutant vector pRA1 / 528 scFv-HL-7m] Using the pRA1 / 528 scFv-HL-6m constructed above as a template, primers were designed to replace the 16th alanine from the N-terminus with lysine, arginine, and glycine using the Quick Change method (Table 8). In the primer sequences shown in Table 8, the mutation sites are underlined.

[0096] [Table 8]

[0097] After PCR using the above primer set, 1 μL of DpnI is added to the reaction mixture and incubated at 37°C for 2 hours to digest the methylated DNA without mutations, and then incubated at 80°C for 15 minutes. DpnI was inactivated by vating. Using 5 μL of the resulting reaction solution, E. coli DH5α was metabolized. The medium was converted. Shaking culture was performed for 30 minutes, and the cells were incubated overnight at 37°C on LB agar medium (Amp(+); fc 100 μg / mL). Colonies that grew on the plate were picked with a toothpick and added to a test tube containing 3 mL of LB medium (Amp(+); fc 100 μg / mL). Shaking culture was performed at 37°C at 140 rpm for 18 hours, and FastGene was used. Plasmid extraction was performed using a plasmid mini-kit. For elution, 50 μL of MQ warmed to 70°C was used. The extracted plasmids were analyzed using the DNA sequencing service of Eurofins Genomics, Inc. to confirm the introduction of mutations.

[0098] A vector into which the seven-fold mutation A16K was introduced into 528 scFv-HL-6m was designated pRA1 / 528 scFv-HL-7m (A16K), a vector into which the seven-fold mutation A16R was introduced was designated pRA1 / 528 scFv-HL-7m (A16R), and a vector into which the seven-fold mutation A16G was introduced was designated pRA1 / 528 scFv-7m (A16G).

[0099] [Preparation of 528 scFv-HL-WT, 6m, and 7m] 0.5 μL each of the vectors pRA1 / 528 scFv-HL-WT, pRA1 / 528 scFv-HL-6m, pRA1 / 528 scFv-HL-7m (A16K), pRA1 / 528 scFv-HL-7m (A16R), and pRA1 / 528 scFv-7m (A16G) constructed above was used to transform 2.5 μL of E. coli BL21 (DE3), and the cells were incubated overnight at 28°C on LB agar (Amp(+); fc 100 μg / mL). The obtained colonies were inoculated into 3 mL of LB medium (Amp(+); fc 100 μg / mL). The cells were then incubated with shaking at 28°C and 170 rpm for 20 hours. 100 mL of auto-induction medium was then added to the OD. 600 =0.0 The pre-culture solution was added to a ratio of 3, and the culture was incubated in a 500 mL baffled flask at 20°C and 170 rpm for 40 hours. After centrifugation of the culture solution at 4°C and 4,800 x g for 20 minutes, the resulting culture supernatant was passed sequentially through membrane filters with pore sizes of 5.0 μm, 3.0 μm, and 1.0 μm, and then filtered using a column packed with 1 mL of Ni Sepharose. 2+ Purification was performed by affinity chromatography. Elution was carried out by increasing the imidazole concentration in PBS to 1 mM, 10 mM, 50 mM, 300 mM, 300 mM, and 1000 mM, each time at 5 CV.

[0100] The results of SDS-PAGE are shown in Figure 15. In the wild type without the introduced mutation ("WT" in Figure 15), the value was 29.5 kDa; in the mutant with the introduced sextapulse ("6M" in Figure 15), the value was 29.4 kDa; in the heptapse mutant with the introduced A16K in addition to the sextapulse ("A16K" in Figure 15), the value was 29.5 kDa; and in the mutant with the introduced A16R in addition to the sextapulse... In the heptapse mutant (labeled "A16R" in Figure 15), a band is observed at 29.5 kDa, and in the heptapse mutant created by introducing A16G into the sexapse mutant (labeled "A16G" in Figure 15), a band is observed at 29.4 kDa. As shown in Figure 15. The target band was clearly observed in the fraction using 300 mM imidazole.

[0101] Next, the eluate from the fraction is concentrated by ultrafiltration, and Superdex 200 Increase 10 / 300 GL Purification was performed by gel filtration chromatography using Lamb, and absorbance at 280 nm was observed. The fractions were subjected to SDS-PAGE to evaluate whether the target protein had been purified (not shown). The fractions containing the target antibody were filtered and sterilized, and used for subsequent evaluation.

[0102] [Evaluation of binding to Protein A column] 0.1 mL of rProtein A Sepharose Fast Flow is applied to a polyprep chromatography column. After packing, the column was equilibrated using 6 CV MQ and 50 mM Tris-HCl / 200 mM NaCl (pH 8.0). 200 μL of the purified sample (5.0 μM) was applied, and a total of two washes were performed using 2 CV PBS. This allowed for the elution of proteins not bound to the column. Subsequently, 2 CVs of 0.1 M Gly-HCl (pH 3.0) were added three times, followed by 2 CVs of IgG Elution Buffer once, to elute the proteins bound to the column. At that time, 5% of the eluate volume of 1 M Tris-HCl (pH 9.2) was added to the microcentrifuge tube used to collect the eluted sample in advance to neutralize the eluate.

[0103] The results are shown in Figure 16. In Figure 16, the results for the wild type without the introduced mutation are labeled "WT", the results for the mutant with the introduced sextuple mutation are labeled "6m", the results for the heptative mutant with the introduced A16K in addition to the sextuple mutation are labeled "7M(A16K)", and the results for the heptative mutant with the introduced A16R in addition to the sextuple mutation are labeled "7M(A16K)". The result was designated "7M(A16R)," and the result for the heptextrous mutant, in which A16G was introduced into the hexatextrous mutant, was designated "7M(A16G)." As can be seen from Figure 16, when A16K, A16R, or A16G was further introduced into the hexatextrous mutant... The seven-fold mutation showed improved affinity for protein A compared to the wild type and the six-fold mutation. Understood. Based on the results of this example, the parentage of protein A due to the D65G mutation and / or D82aN mutation is The effect of improving compatibility was shown to be further enhanced by A16K, A16R, or A16G.

[0104] SEQUENCE LISTING <110> Tokyo University of Agriculture and Technology ProteinExpress Co., Ltd. <120> A mutant antibody and a method for producing a mutant antibody <130> P25-0807 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Phe Tyr Asp Pro Thr Ala Pro Asn Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 124 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln Ile Trp Pro Gly Asp Ser Asp Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 129 <212> PRT <213> Homo sapiens <400> 3 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Asn Arg Phe Gln Tyr Cys Ser Ser Thr Thr Cys Tyr Thr 100 105 110 Leu Leu Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 4 <211> 119 <212> PRT <213> Homo sapiens <400> 4 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115 <210> 5 <211> 119 <212> PRT <213> Homo sapiens <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30 Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> Homo sapiens <400> 6 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Phe Tyr Pro Gly Asp Ser Ser Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Asn Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 7 <211> 127 <212> PRT <213> Homo sapiens <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Gly Met Ile Phe Asn Trp Pro Leu Gly Gly Trp 100 105 110 Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 124 <212> PRT <213> Homo sapiens <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Met Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Arg Gln Trp Leu Val Ala Arg Ile Thr Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 121 <212> PRT <213> Homo sapiens <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Lys Phe Tyr Asp Pro Thr Ala Pro Asn Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 120 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 118 <212> PRT <213> Homo sapiens <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Trp Pro Gly Ser Gly Gly Thr Asn Tyr Ala Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Gly Pro Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 12 <211> 119 <212> PRT <213> Homo sapiens <400> 12 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115 <210> 13 <211> 28 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 13 ggcgcgtccg tgcgtgtatc ctgcaaag 28 <210> 14 <211> 28 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 14 ctttgcagga tacacgcacg gacgcgcc 28 <210> 15 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 15 cgcgtgacca tgtcccgtga tacttccatc tcc 33 <210> 16 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 16 ggagatggaa gtatcacggg acatggtcac gcg 33 <210> 17 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 17 gttctgcaa atgaactccc tgcgcccgg 29 <210> 18 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 18 ccgggcgcag ggagttcatt tgcagaaac 29 <210> 19 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 19 taatacgact cactataggg 20 <210> 20 <211> 48 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 20 ggaacgcagg ctgttcagct gcatgtaagc ggtggagatg gaagtatc 48 <210> 21 <211> 32 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 21 cagctgaaca gcctgcgttc cgatgacacc gc 32 <210> 22 <211> 19 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 22 gctagttatt gctcagcgg 19 <210> 23 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 23 agaaaaattt aaaggccgcg tgaccatga 29 <210> 24 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 24 tcatggtcac gcggccttta aatttttct 29 <210> 25 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 25 ccagaaagta aaaggccgct tcaccatct 29 <210> 26 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 26 agatggtgaa gcggcctttt actttctgg 29 <210> 27 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 27 tcagaaagtg aaaggccgct ttaccatta 29 <210> 28 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 28 taatggtaaa gcggcctttc actttctga 29 <210> 29 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 29 tcagaaagtg aaagctcgct ttaccatta 29 <210> 30 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 30 taatggtaaa gcgagctttc actttctga 29 <210> 31 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 31 tcagaaagtg aaaaaccgct ttaccatta 29 <210> 32 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 32 taatggtaaa gcggtttttc actttctga 29 <210> 33 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 33 gtttctgcag atgaacagcc tgcgcccgg 29 <210> 34 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 34 ccgggcgcag gctgttcatc tgcagaaac 29 <210> 35 <211> 27 <212> DNA <213> Artificial <220> <223> Synthetic DNA <220> <221> misc_feature <222> (1)..(3) <223> n is a, c, g, or t <400> 35 nnnccatggc tttcgctgca gatattc 27 <210> 36 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic DNA <220> <221> misc_feature <222> (1)..(3) <223> n is a, c, g, or t <400> 36 nnnaagcttt taggagctaa ccgtgacagg c 31 <210> 37 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic DNA <220> <221> misc_feature <222> (1)..(3) <223> n is a, c, g, or t <400> 37 nnnccatggc ccaggtgcaa ctggtacaat c 31 <210> 38 <211> 22 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 38 cagccgcggc tttgatttct ac 22 <210> 39 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 39 gaaaaaaccg ggccgctccg tgcgtgtat 29 <210> 40 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 40 atacacgcac ggagcggccc ggttttttc 29 <210> 41 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 41 gaaaaaaccg ggcaaatccg tgcgtgtat 29 <210> 42 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 42 atacacgcac ggatttgccc ggttttttc 29 <210> 43 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 43 gaaaaaaccg ggcggctccg tgcgtgtat 29 <210> 44 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 44 atacacgcac ggagccgccc ggttttttc 29

Claims

1. A polypeptide comprising a VH region containing an amino acid sequence in the amino acid sequence shown in Sequence ID No. 12, in which the 66th aspartic acid residue, corresponding to the 65th amino acid residue in Kabat numbering, is replaced with glycine, and the 84th aspartic acid residue, corresponding to the 82a amino acid residue in Kabat numbering, is replaced with asparagine.

2. A low-molecular-weight antibody comprising the polypeptide described in claim 1.

3. A bispecific antibody comprising the polypeptide described in claim 1 or the low molecular weight antibody described in claim 2.

4. A method for producing a low molecular weight antibody or a bispecific antibody, comprising a culture step of culturing cells that express the low molecular weight antibody described in claim 2 or the bispecific antibody described in claim 3.

5. The manufacturing method according to claim 4, comprising an extraction step for obtaining an extract of cells obtained after the culture step.

6. The manufacturing method according to claim 4 or 5, further comprising a purification step of purifying the low molecular weight antibody or bispecific antibody from the culture supernatant obtained after the culture step or from the cell extract obtained after the extraction step, utilizing its affinity for protein A.

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