Polypeptide derived from antibody light-chain having her2 decomposition activity, her2-binding polypeptide comprising said polypeptide, methods for producing said polypeptides, and use of said polypeptides

Abstract

The present invention provides a super antibody enzyme that comprises an antibody having a high anti-cancer effect and imparted with a high enzymatic activity. The present invention relates to a polypeptide in which hypervariable regions are derived from an anti-HER2 antibody light-chain, such as those mentioned in (1a) below. (1a) A polypeptide comprising an amino acid sequence that includes a CDR1 region comprising the amino acid sequence represented by SEQ ID NO:1, a CDR2 region comprising the amino acid sequence represented by SEQ ID NO:2 and a CDR3 region comprising the amino acid sequence represented by SEQ ID NO:3, and has a 70th aspartic acid residue in a variable region in accordance with the Kabat numbering scheme, wherein 95th and 96th proline residues in a variable region in accordance with the Kabat numbering scheme are deleted or are substituted by amino acid residues other than proline residues.

Description

Polypeptide derived from antibody light chain having HER2 degrading activity, HER2-binding polypeptide containing said polypeptide, methods for producing them, and uses thereof

[0001] The present invention relates to a polypeptide derived from the light chain of an anti-HER2 antibody having HER2 degrading activity, a HER2-binding polypeptide containing said polypeptide, methods for producing these polypeptides, and cancer therapeutic agents containing these polypeptides.

[0002] An antibody is composed of a heavy chain (H chain) and a light chain (L chain). The heavy chain and the light chain are composed of a variable region (VR: Variable Region) and a constant region (CR: Constant Region), and the variable region has a hypervariable region (CDR: Complimentary Determining Region).

[0003] In recent years, antibodies with enzyme-like activity, i.e., antibody enzymes, have been attracting attention. Because antibody enzymes combine the high molecular recognition ability of antibodies with enzymatic activity, they are expected to be applied in many fields, including medicine, the chemical industry, and the food industry. In particular, antibody enzymes that are highly specific to target molecules and can damage target molecules through their enzymatic activity are expected to become excellent anticancer drugs with few side effects. In particular, human-type antibody enzymes are expected to have few side effects when administered to the human body, so pharmaceutical companies both in Japan and abroad are eagerly awaiting the development of useful human-type antibody enzymes.

[0004] The inventors have conducted various studies on antibody enzymes (see, for example, Patent Documents 1 to 4).

[0005] For clinical use of antibody enzymes as pharmaceuticals, it is important to be able to stably mass-produce antibody enzymes with sufficient activity. However, many antibody enzymes have insufficient enzymatic activity, and when artificially synthesized using intracellular or extracellular expression systems using genetic recombination technology, their performance is unstable and there is a problem of large lot-to-lot variation. Therefore, the present inventors conducted various studies to produce antibody enzymes consisting of antibody light chains with higher enzymatic activity, and reported that deletion or substitution of the proline residue at position 95 in the variable region of the amino acid sequence of the antibody light chain, according to the Kabat classification, causes the light chain to form a catalytic triad conformation (D1 / S27 / H93), resulting in an antibody light chain with higher enzymatic activity (super antibody enzyme) (Patent Document 5).

[0006] Japanese Patent Publication No. 2006-197930 WO 2011 / 102517 WO 2013 / 133253 WO 2015 / 025786 WO 2021 / 015237

[0007] An object of the present invention is to provide a super antibody enzyme in which high enzymatic activity is imparted to an antibody having a high anti-cancer effect.

[0008] Therefore, studies were conducted to confer enzymatic activity to the light chain of an anti-HER2 antibody that has excellent anti-cancer effects against cancers that overexpress HER2, such as breast cancer. However, since this light chain does not contain S27a / H93 as described in Patent Document 5, it was thought that deleting the 95th proline residue in the variable region would not confer enzymatic activity. However, the present inventors discovered that when the 95th and 96th proline residues were simultaneously deleted from the variable region of the anti-HER2 antibody light chain, contrary to expectations, a polypeptide derived from the anti-HER2 antibody light chain having enzymatic activity nearly four times that of the light chain was obtained, and further discovered that the polypeptide could be used to obtain a new HER2-targeted drug having one or more activities selected from antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neutralizing activity in addition to HER2 degradation activity, thereby completing the present invention.

[0009] That is, the present invention provides the following [1] to

[13] : [1] A polypeptide derived from the light chain of an anti-HER2 antibody, wherein the hypervariable region is a polypeptide selected from the group consisting of the following (1a) to (1c): (1a) A polypeptide having a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and having a deletion or an amino acid residue other than a proline residue at positions 95 and 96 of the variable region according to the Kabat classification; (1b) A polypeptide having a CDR1 region consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and having a deletion or an amino acid residue other than a proline residue at positions 95 and 96 of the variable region according to the Kabat classification; (1c) A polypeptide having a CDR1 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and having a deletion or an amino acid residue other than a proline residue at positions 95 and 96 of the variable region according to the Kabat classification. [2] A polypeptide derived from the light chain of an anti-HER2 antibody, whose variable region is a polypeptide selected from the group consisting of the following (2a) to (2c):(2a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence shown in SEQ ID NO: 4, and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues; (2c) a polypeptide which has 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues. [3] The polypeptide according to [1] or [2], further comprising an anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12. [4] The polypeptide according to [3], in which the anti-HER2 antibody light chain hypervariable region according to [1] and the anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12, or the anti-HER2 antibody light chain variable region according to [2] and the anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12 are linked via a linker. [5] The polypeptide according to [4], further to which an Fc region is linked. [6] The polypeptide according to [3], in which a light chain constant region linked to the anti-HER2 antibody hypervariable region according to [1] and a heavy chain CH1 region linked to the heavy chain variable region shown in SEQ ID NO: 12; or a light chain constant region linked to the anti-HER2 antibody variable region according to [2] and a heavy chain CH1 region linked to the heavy chain variable region shown in SEQ ID NO: 12; are associated via a disulfide bond. [7] A polypeptide in which a heavy chain CH2 and a heavy chain CH3 are linked to the CH1 region of the polypeptide according to [6].[8] A method for producing a polypeptide derived from the light chain of an anti-HER2 antibody having enzymatic activity or improved enzymatic activity, wherein the hypervariable region comprises: (1a) a polypeptide comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification; (1b) a polypeptide comprising a CDR1 region consisting of an amino acid sequence in which one or several amino acids have been substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence in which one or several amino acids have been substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification; or (1c) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1. a CDR1 region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:2, and a CDR3 region comprising SEQ ID NO:3, wherein the polynucleotide encoding the polypeptide derived from the light chain of an anti-HER2 antibody is a polypeptide having an aspartic acid at position 70 of the variable region according to the Kabat classification, and wherein the amino acid sequence has proline residues at positions 95 and 96 of the variable region according to the Kabat classification, by modifying the polynucleotide so that the proline residues are deleted or substituted, thereby obtaining a polynucleotide encoding the polypeptide derived from the light chain of an anti-HER2 antibody, wherein the variable region has an amino acid sequence in which the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or have amino acid residues other than proline; and an expression step of expressing the polypeptide derived from the light chain of an anti-HER2 antibody having enzymatic activity in an intracellular or extracellular expression system using an expression vector comprising the polynucleotide encoding the HER2 light chain after the modification step.[9] A method for producing a polypeptide derived from the light chain of an anti-HER2 antibody having enzymatic activity or improved enzymatic activity, comprising: a modification step of modifying a polynucleotide encoding a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region of which is (2a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, added, or deleted in the amino acid sequence set forth in SEQ ID NO: 4 and which has an aspartic acid at position 70 of the variable region according to the Kabat classification; or (2c) a polypeptide which has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, wherein the 95th and 96th amino acid residues of the variable region according to the Kabat classification are proline residues, so that the proline residues are deleted or substituted, thereby obtaining a polynucleotide encoding a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region of which is a polypeptide consisting of an amino acid sequence in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are deleted or have amino acid residues other than proline residues; a production method comprising: an expression step of expressing an enzymatically active polypeptide derived from the light chain of an anti-HER2 antibody in an intracellular or extracellular expression system using an expression vector comprising a polynucleotide encoding the HER2 antibody light chain after the modification step.

[10] A cancer therapeutic agent comprising an ingredient selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody according to [1] or [2] above, a HER2-binding polypeptide comprising the polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain.

[11] A pharmaceutical composition for cancer treatment comprising an ingredient selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody according to [1] or [2] above, an anti-HER2 antibody comprising a polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain.

[12] Use of a component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody according to [1] or [2] above, a HER2-binding polypeptide comprising a polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain, for the manufacture of a cancer therapeutic agent.

[13] A component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody according to [1] or [2] above, a HER2-binding polypeptide comprising a polypeptide derived from said light chain, and a functional fragment of an anti-HER2 antibody comprising a polypeptide derived from said light chain, for use in cancer treatment.

[14] A method for treating cancer, comprising administering a component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody according to [1] or [2] above, a HER2-binding polypeptide comprising a polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain.

[0010] The polypeptide derived from the light chain of an anti-HER2 antibody of the present invention has excellent degrading enzyme activity against HER2 peptides, and therefore a component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody, a HER2-binding polypeptide comprising a polypeptide derived from the light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from the light chain is useful as a cancer therapeutic agent that targets HER2 and has one or more activities selected from antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neutralizing activity in addition to HER2 degrading activity.

[0011] 1 shows the base sequence near P95P96 of the trastuzumab light chain gene. 2 shows the results of SDS-PAGE of the trastuzumab light chain. 3 shows the results of SDS-PAGE of the trastuzumab light chain P95ΔP96Δ. 4 shows the results of SDS-PAGE of a polypeptide comprising the scFv trastuzumab light chain variable region P95ΔP96Δ, a linker, and a heavy chain variable region. 5 shows the time-dependent change in the decomposition activity of the trastuzumab light chain, the trastuzumab light chain P95ΔP96Δ, and the scFv trastuzumab light chain P95ΔP96Δ toward the Arg-pNA substrate (R-pNA), a trypsin-like substrate molecule. 6 shows the synthetic design diagrams of the FRET-HER2-1 (a) and FRET-HER2-2 (b) peptides. The cleavage sites of FRET-Her2peptide-1 (579-600) and the structures of its degradation products are shown. The time-dependent changes in degradation activity of trastuzumab light chain (Tmb-L), trastuzumab light chain P95ΔP96Δ (Tmb-L-PΔΔ), scFv trastuzumab light chain (Tmb-scFv), scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFv), and trastuzumab (Trastuzumab) against FRET-HER2 peptide-1 are shown. The amino acid sequence of the variable region of the trastuzumab light chain is shown. The cleavage sites of FRET-Her2peptide-2 (Loop2+Loop3) and the structures of its degradation products are shown.

[0039] Figure 1 shows the time course of degradation activity of trastuzumab light chain (Tmb-L), trastuzumab light chain P95ΔP96Δ (Tmb-L-PΔΔ), scFv trastuzumab light chain (Tmb-scFv), scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFv), and trastuzumab (Trastuzumab) FRET-HER2 peptide-2.

[0040] Figure 1 shows the results of calculation of reaction rate constants by Lineweaver-Burk plot for trastuzumab light chain P95ΔP96Δ and scFv trastuzumab light chain P95ΔP96Δ. The amino acid sequence of #4 polypeptide is shown. 1 shows the results of conformational simulations of the trastuzumab light chain (left) and the trastuzumab light chain P95ΔP96Δ (right).FIG. 1 shows the results of a cytotoxicity test of trastuzumab (Trasutuzumab in the figure), trastuzumab light chain P95ΔP96Δ (L-P95Δ in the figure), scFv trastuzumab light chain P95ΔP96Δ (Sc-FvP95Δ in the figure), and anti-HER2 antibody light chain (Lchain in the figure) against the human breast cancer cell line BT-474. The bar graphs show the results of a cytotoxicity test of trastuzumab (Trastuzumab in the figure), trastuzumab light chain P95ΔP96Δ (L-P95Δ in the figure), scFv trastuzumab light chain P95ΔP96Δ (Sc-FvP95Δ in the figure), and anti-HER2 antibody light chain (Lchain in the figure) against the human breast cancer cell line BT-474. The relative values ​​of the antigen degradation activity test using trastuzumab and its scFv (trastuzumab-scFv, and trastuzumab-scFv-PPΔ in which Pro95 and Pro96 are deleted) are shown.

[0012] In the present invention and the present specification, the term "antibody light chain" refers to an immunoglobulin light chain. The animal species from which the antibody light chain originates is not particularly limited, but is preferably derived from a mammal, and more preferably derived from a human or mouse.

[0013] In the present invention and the present specification, the term "cancer therapeutic drug" refers to a drug that has the activity of killing cancer cells or suppressing or inhibiting their proliferation.

[0014] It is well known in the art that some amino acids among the amino acid residues constituting a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is well known that in addition to artificial modifications, there are mutants in natural proteins that do not significantly change the structure or function of the protein. In this specification, the substitution, addition, or deletion of one or more amino acids in a specific amino acid sequence X is referred to as "mutation."

[0015] Numbering of amino acid sequences according to the Kabat classification can be performed, for example, by analyzing the amino acid sequence of an antibody according to the Kabat numbering system using abYsis software (URL: http: / / www.abysis.org / ) or Discovery Studio (Accelrys Software, San Diego, CA, USA).

[0016] One embodiment of the polypeptide derived from the light chain of an anti-HER2 antibody of the present invention is a polypeptide derived from the light chain of an anti-HER2 antibody, wherein the hypervariable region is a polypeptide selected from the group consisting of the following (1a) to (1c): (1a) A polypeptide having a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and having a deletion or an amino acid residue other than a proline residue at positions 95 and 96 of the variable region according to the Kabat classification; (1b) A polypeptide having a CDR1 region consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and having a deletion or an amino acid residue other than a proline residue at positions 95 and 96 of the variable region according to the Kabat classification; (1c) A polypeptide having a CDR1 region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification, and the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues.

[0017] The light chain variable region (VL region) consists of three hypervariable regions (CDRs: CDR1, CDR2, and CDR3) and four framework regions (FRs) surrounding the three CDRs. In the above embodiment, the CDRs contain specific sequences. The polypeptide (1a) has a CDR1 region consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and is a polypeptide consisting of an amino acid sequence in which the 70th aspartic acid in the variable region according to the Kabat classification is present, and the 95th and 96th proline residues in the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues. Examples of amino acid residues other than proline residues include lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, alanine, glycine, valine, isoleucine, leucine, methionine, and cysteine. Among these, serine and arginine are preferred. It is more preferable that the prolines at positions 95 and 96 are deleted.

[0018] The polypeptide (1b) has a CDR region comprising an amino acid sequence having one or more amino acid substitutions, additions, or deletions in the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region comprising an amino acid sequence having one or more amino acid substitutions, additions, or deletions in the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region having a CDR region having a CDR sequence shown in SEQ ID NO: 3, and the CDR region comprises an amino acid sequence having an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues. Examples of amino acid residues other than proline residues include lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, alanine, glycine, valine, isoleucine, leucine, methionine, and cysteine. Among these, serine or arginine is preferred. Furthermore, it is more preferred that the prolines at positions 95 and 96 are deleted. Examples of substitution, addition, or deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, 2, or 3 include substitution, addition, or deletion of one or two amino acids, preferably substitution, addition, or deletion of one amino acid. Commonly occurring amino acid substitutions include, for example, alanine / serine, valine / isoleucine, aspartic acid / glutamic acid, threonine / serine, alanine / glycine, alanine / threonine, serine / asparagine, alanine / valine, serine / glycine, tyrosine / phenylalanine, alanine / proline, lysine / arginine, aspartic acid / asparagine, leucine / isoleucine, leucine / valine, alanine / glutamic acid, and aspartic acid / glycine.

[0019] The polypeptide (1c) is a polypeptide having the CDR regions: a CDR1 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1; a CDR2 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2; and a CDR3 region consisting of SEQ ID NO: 3; and the polypeptide has an aspartic acid at position 70 of the variable region according to the Kabat classification, and the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues. Examples of amino acid residues other than proline residues include lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, alanine, glycine, valine, isoleucine, leucine, methionine, cysteine, etc. Among these, serine or arginine is preferred. Furthermore, it is more preferred that the prolines at positions 95 and 96 are deleted. Examples of sequences having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, 2, or 3 include sequences having 95% or more identity, more preferably sequences having 97% or more identity, even more preferably sequences having 98% or more identity, and even more preferably sequences having 99% or more identity.

[0020] The sequence identity of a subject amino acid sequence to a reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula.

[0021] "Sequence identity (%)" = [number of matching amino acids] / [total number of amino acids in the target amino acid sequence] × 100

[0022] Among the polypeptides (1a), (1b), and (1c), the polypeptide (1a) is more preferred. The even more preferred polypeptide (1a) is a polypeptide comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 5, and having an aspartic acid at the 70th position of the variable region according to the Kabat classification.

[0023] Another embodiment of the polypeptide derived from the light chain of an anti-HER2 antibody of the present invention is a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region of which is a polypeptide selected from the group consisting of the following (2a) to (2c): (2a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 4, and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region are deleted or are amino acid residues other than proline residues according to the Kabat classification; (2c) a polypeptide having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region are deleted or are amino acid residues other than proline residues according to the Kabat classification.

[0024] In this embodiment, the entire variable region (including CDRs and frameworks) of the polypeptide derived from the light chain of the anti-HER2 antibody of the present invention contains a specific sequence. Substitutions, additions, deletions, amino acid sequence identity, and deletions or substitutions of amino acid residues with amino acid residues other than proline in the peptides (2a), (2b), and (2c) are the same as those in the peptides (1a), (1b), and (1c) above.

[0025] Among the polypeptides (2a), (2b), and (2c), the polypeptide (2a) is more preferred. The even more preferred polypeptide (2a) is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:6.

[0026] The polypeptide derived from the light chain of the anti-HER2 antibody of the present invention contains the amino acid sequence described above and can be produced by various production methods. However, it is preferred to produce it by the following two embodiments, which include a modification step and an expression step.

[0027] Another aspect of the present invention is a method for producing a polypeptide derived from the light chain of an anti-HER2 antibody having enzymatic activity or improved enzymatic activity, wherein the hypervariable region comprises: (1a) a polypeptide comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3; (1b) a polypeptide comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1 with one or several amino acids substituted, added, or deleted, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2 with one or several amino acids substituted, added, or deleted, and a CDR3 region consisting of SEQ ID NO: 3; or (1c) a polypeptide comprising a CDR1 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3. The method for producing an anti-HER2 antibody light chain comprises: a modification step of modifying a polynucleotide encoding the light chain of an anti-HER2 antibody, the polynucleotide being a polypeptide comprising a CDR2 region consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, wherein the polynucleotide has an amino acid sequence in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are proline residues, by deleting or substituting the proline residues, thereby obtaining a polynucleotide encoding a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region having an amino acid sequence in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are deleted or have amino acid residues other than proline; and an expression step of expressing the enzymatically active polypeptide derived from the light chain of an anti-HER2 antibody using an expression vector comprising the polynucleotide encoding the light chain of the anti-HER2 antibody after the modification step, in an intracellular or extracellular expression system.Another aspect of the present invention is a method for producing a polypeptide derived from the light chain of an anti-HER2 antibody that has enzymatic activity or has improved enzymatic activity, comprising the steps of: modifying a polynucleotide encoding a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region of which is (2a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, added, or deleted in the amino acid sequence set forth in SEQ ID NO: 4 and which has an aspartic acid at position 70 of the variable region according to the Kabat classification; or (2c) a polypeptide which has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are proline residues, by deleting or substituting the proline residues, thereby obtaining a polynucleotide encoding a polypeptide derived from the light chain of an anti-HER2 antibody, the variable region of which is a polypeptide consisting of an amino acid sequence in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are deleted or have amino acid residues other than proline residues; an expression step of expressing a polypeptide derived from the anti-HER2 antibody light chain having enzymatic activity in an intracellular or extracellular expression system using an expression vector containing a polynucleotide encoding the antibody HER2 antibody light chain after the modification step.

[0028] In the modification step, a polynucleotide encoding an antibody light chain, which is a polypeptide whose variable region consists of an amino acid sequence in which the 95th and 96th amino acid residues in the variable region according to the Kabat classification are proline residues, is modified so that the proline residues are deleted or substituted, thereby obtaining a polynucleotide encoding an antibody light chain, which is a polypeptide whose variable region consists of an amino acid sequence in which the 95th and 96th amino acid residues in the variable region according to the Kabat classification are deleted or have amino acid residues other than proline. By performing this step, enzymatic activity can be imparted to an antibody light chain that does not have enzymatic activity, or the enzymatic activity of an antibody light chain that exhibits only low activity can be improved. In other words, this step can also be referred to as a "method for imparting enzymatic activity to an antibody light chain that does not have enzymatic activity" or a "method for improving the enzymatic activity of an antibody light chain." Here, improving the enzymatic activity of an antibody light chain means that the enzymatic activity of the antibody light chain after modification is improved by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more relative to the enzymatic activity of the antibody light chain before modification.

[0029] The method for modifying the polypeptide so as to delete or substitute the proline residues can be carried out by using well-known techniques to easily mutate the proline residues present at positions 95 and 96 of the Kabat classification in the amino acid sequence of the variable region among the amino acid residues constituting the polypeptide. For example, any base in a polynucleotide encoding the polypeptide can be mutated according to known point mutagenesis methods. Furthermore, deletion mutants or substitution mutants can be prepared by designing primers corresponding to any site in a polynucleotide encoding the polypeptide.

[0030] In the expression step, an expression vector containing a polynucleotide encoding the antibody light chain after the modification step is used to express the antibody light chain in an expression system known in the art, such as an intracellular or extracellular expression system. Furthermore, this expression step is appropriately used, independently of the modification step, as a method for producing a HER2-binding polypeptide comprising a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention having HER2 degradation activity, as described below, or a HER2-binding antibody comprising a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention having HER2 degradation activity.

[0031] When a recombinant expression system is used, a method can be employed in which a polynucleotide encoding the antibody light chain of the present invention is incorporated into a recombinant expression vector, which is then introduced into a host capable of expression by a known method, and the polypeptide obtained by translation in the host (transformant) is purified. The recombinant expression vector may or may not be a plasmid, as long as it is capable of introducing the target polynucleotide into the host.

[0032] When introducing a foreign polynucleotide into a host in this manner, it is preferable that the expression vector incorporates a promoter that functions in the host to express the foreign polynucleotide. Methods for purifying recombinantly produced polypeptides vary depending on the host used and the properties of the polypeptide, but the use of tags, etc. makes it relatively easy to purify the target polypeptide.

[0033] When a cell-free expression system (cell-free protein synthesis system) is used, it is preferable to add a polynucleotide encoding the antibody light chain of the present invention to a solution containing components necessary for protein translation and synthesis, such as ribosomes and t-RNA, and incubate the solution at an appropriate temperature, thereby purifying the synthesized polypeptide.

[0034] Examples of cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using Escherichia coli S30 extract, and systems using extracts of cellular components obtained from devacuolated plant protoplasts. Generally, eukaryotic cell systems, i.e., systems using wheat germ extract or rabbit reticulocyte extract, are selected for translation of eukaryotic genes. However, any of the above synthesis systems may be selected taking into consideration the origin of the gene to be translated (prokaryote / eukaryote) and the intended use of the protein after synthesis. Various commercially available kits can be used as these synthesis systems.

[0035] Since many of the various virus-derived gene products express their activity after translation through complex biochemical reactions involving intracellular membranes such as the endoplasmic reticulum and Golgi apparatus, the addition of intracellular membrane components (e.g., microsomal membranes) is necessary to reproduce various biochemical reactions in a test tube. A cellular component extract obtained from devacuolated plant protoplasts is preferred because it can be used as a cell-free protein synthesis solution that retains intracellular membrane components and does not require the addition of microsomal membranes.

[0036] As used herein, the term "intracellular membrane component" refers to organelles composed of lipid membranes present in the cytoplasm (i.e., intracellular granules in general, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplasts, and vacuoles). In particular, the endoplasmic reticulum and Golgi apparatus play important roles in post-translational modification of proteins and are essential cellular components for the maturation of membrane proteins and secretory proteins.

[0037] Polypeptides derived from antibody light chains of the present invention can also be isolated from cells or tissues that naturally express the antibody light chains, for example, antibodies or oligonucleotides can be used to identify cells or tissues that naturally express the antibody light chains of the present invention.

[0038] Alternatively, the antibody light chain-derived polypeptide of the present invention can be chemically synthesized. The method of chemical synthesis is not particularly limited, and any method used for chemically synthesizing polypeptides may be used.

[0039] The polypeptide derived from the antibody light chain of the present invention obtained in the expression step can be purified using known antibody purification methods, but the purification method comprising a first purification step, an addition step, and a second purification step described in Patent Document 5 can also be used.

[0040] As shown in the Examples below, the thus obtained polypeptide derived from the light chain of an anti-HER2 antibody of the present invention has nearly four times the HER2-degrading enzymatic activity of the antibody light chain before modification. Furthermore, components selected from the group consisting of polypeptides derived from the light chain of an anti-HER2 antibody of the present invention, HER2-binding polypeptides comprising said polypeptides derived from said light chains, and functional fragments of HER2-binding polypeptides comprising said polypeptides derived from said light chains are useful as cancer therapeutic agents that target HER2 and have, in addition to HER2 degrading activity, one or more activities selected from antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neutralizing activity. As part of a factor analysis of the enzymatic activity of the polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a computer simulation of the conformation of the antibody light chain was performed, and it was found that, instead of the conventional catalytic triad structure (D1 / S27a / H93), S26 / R24 / D70 appears to be a conformation suitable for HER2 hydrolysis. Without intending to be bound by any particular theory, it is speculated that the polypeptide derived from the light chain of the anti-HER2 antibody of the present invention exhibits high enzymatic activity due to the structure of the catalytic triad residues S26 / R24 / D70.

[0041] Another aspect of the present invention is a cancer therapeutic agent containing an ingredient selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a HER2-binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising said polypeptide derived from said light chain. Another aspect of the present invention is a pharmaceutical composition for cancer treatment containing an ingredient selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a HER2-binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising said polypeptide derived from said light chain. Another aspect of the present invention is use of an ingredient selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a HER2-binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising said polypeptide derived from said light chain, for the manufacture of a cancer therapeutic agent. Another aspect of the present invention is a component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a HER2-binding polypeptide comprising a polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain, for use in treating cancer. Another aspect of the present invention is a method for treating cancer, comprising administering a component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention, a HER2-binding polypeptide comprising a polypeptide derived from said light chain, and a functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from said light chain.

[0042] The active ingredient used in the cancer therapeutic drug is one or more selected from the group consisting of polypeptides derived from the light chain of the anti-HER2 antibody of the present invention, HER2-binding polypeptides comprising the polypeptides derived from the light chain, and functional fragments of HER2-binding polypeptides comprising the polypeptides derived from the light chain. Examples of polypeptides derived from the light chain of the anti-HER2 antibody of the present invention include polypeptides selected from (1a), (1b), and (1c) above, polypeptides selected from (2a), (2b), and (2c) above, and the entire light chain including these polypeptides and the light chain constant region.

[0043] The HER2-binding polypeptide comprising a light chain may be an antibody comprising a constant region or an Fc region. The class of the antibody is not particularly limited and includes antibodies having any isotype, such as IgG, IgM, IgA, IgD, or IgE. Considering ease of purification, IgG is preferred. The HER2-binding polypeptide comprising a light chain also includes monoclonal and polyclonal antibodies, as well as HER2-binding antibodies comprising the light chain, such as antibodies that retain the ability to specifically bind to an antigenic determinant and antibody variants and derivatives, such as T-cell receptor fragments. The type of HER2-binding antibody comprising the light chain is not particularly limited, and includes mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, sheep antibodies, camel antibodies, and avian antibodies, as well as genetically engineered antibodies that have been artificially modified to reduce heterologous antigenicity against humans, such as chimeric antibodies and humanized antibodies, as appropriate.

[0044] The functional fragment of a HER2-binding polypeptide comprising a polypeptide derived from the light chain may be a minibody such as an antibody fragment, or a modified antibody. Specific examples of antibody fragments include Fv, Fab, F(ab')2, Fab-Fc, F(ab')2-pFc'scFab, and scFv. Examples of scFv include tandem di-scFv, diabody, tandem tri-scFv, and tri(a)body. More specific examples include VL-CL, VL-VH, VL-VL, and VL-VL-VL. From the viewpoint of increasing the valence of the light chain of the anti-HER2 antibody of the present invention, scFvs such as VL-VL and VL-VL-VL are preferred.

[0045] Specific examples of HER2-binding polypeptides comprising the light chain-derived polypeptides, HER2-binding antibodies comprising the light chain-derived polypeptides, and functional fragments of HER2-binding polypeptides comprising the light chain-derived polypeptides are as follows: (a) A polypeptide comprising an anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12 in addition to a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention (a polypeptide selected from (1a), (1b), and (1c) above, or a polypeptide selected from (2a), (2b), and (2c) above). (b) A polypeptide in which a polypeptide derived from the light chain of an anti-HER2 antibody of the present invention (a polypeptide selected from (1a), (1b), and (1c) above, or a polypeptide selected from (2a), (2b), and (2c) above) and an anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12 are linked via a linker. (c) A polypeptide in which two of the polypeptides of (b) above are linked via a linker. (d) A polypeptide comprising a light chain constant region linked to a polypeptide derived from the light chain of the anti-HER2 antibody of the present invention (a polypeptide selected from (1a), (1b), and (1c) above, or a polypeptide selected from (2a), (2b), and (2c) above), and a heavy chain CH1 region linked to the heavy chain variable region of the anti-HER2 antibody shown in SEQ ID NO: 12. (e) The polypeptide of (d) above, in which the light chain variable region and heavy chain variable region are linked via a linker. (f) A polypeptide further comprising an Fc region linked to the heavy chain CH1 region of (d) or (e). The polypeptide of (c) above, in which a heavy chain CH1 region linked to a heavy chain variable region shown in SEQ ID NO: 12 linked to a polypeptide derived from the light chain of the anti-HER2 antibody of the present invention (a polypeptide selected from (1a), (1b), and (1c), a polypeptide selected from (2a), (2b), and (2c), or the entire light chain including these polypeptides and the light chain constant region) is associated via a disulfide bond. (g) A polypeptide in which a heavy chain CH2 and a heavy chain CH3 are linked to the CH1 region of the polypeptide of (f).

[0046] The two or more polypeptides are preferably linked by a covalent bond such as a polypeptide bond. The covalent bond may be a direct polypeptide bond or an indirect polypeptide bond via a linker sequence. The linker sequence typically uses a flexible amino acid, such as an amino acid selected from glycine, alanine, and serine. Alternatively, a linker with greater rigidity may be used. The length of the linker sequence can be easily determined. In many cases, the linker sequence is approximately 12 amino acids or less, for example, 10 amino acids or less, or 2 to 10 amino acids in length. Examples of non-covalent bonds include hydrogen bonds, van der Waals forces, and ionic bonds. The association refers to the quaternary structural bond between polypeptides. In other words, when multiple polypeptides that have formed tertiary structures associate to form a single molecule, the association refers to the bond between the polypeptides that have formed the tertiary structure. In one embodiment of the present invention, a polypeptide comprising a light chain variable region and a light chain constant region is associated with a polypeptide comprising a heavy chain variable region and a heavy chain constant region via disulfide bonds, hydrogen bonds, van der Waals forces, ionic bonds, etc. The polypeptide (g) can be obtained by expressing it in an expression system known in the art, such as an intracellular or extracellular expression system, using an expression vector comprising a polynucleotide encoding a polypeptide in which the polypeptide of the present invention consisting of the amino acid sequence set forth in SEQ ID NO: 6 is linked to the polypeptide set forth in SEQ ID NO: 15. In an intracellular expression system, for example, Chinese hamster ovary cells (CHO cells) can be used as host cells.

[0047] Modified antibodies may also be used, including antibodies conjugated with various molecules such as polyethylene glycol (PEG). Such modified antibodies can be obtained by chemically modifying an antibody. Furthermore, in the present invention, antibodies with modified sugar chains can also be used for the purpose of enhancing cytotoxic activity. Furthermore, in the present invention, HEXABODY technology, which confers complement-dependent cytotoxic activity, can also be applied as appropriate.

[0048] The cancer therapeutic agent of the present invention is effective against cancers that overexpress HER2, and is preferably used to treat, for example, breast cancer, gastric cancer, salivary gland cancer, and other cancers that overexpress HER2.

[0049] The cancer therapeutic drug of the present invention can be administered orally or parenterally to animals, including humans, with cancer, but parenteral administration is preferred. Specific administration methods include mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, and transdermal administration. The cancer therapeutic drug of the present invention can be administered at a dose of preferably 0.01 to 30 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight, and even more preferably 0.1 to 1 mg / kg body weight, in terms of the protein amount of the active ingredient. As for the administration method, after a single administration, administration can also be performed over a certain period of time, for example, at intervals of 2 to 22 days.

[0050] The cancer therapeutic agent of the present invention can be prepared as a cancer therapeutic agent composition by containing, in addition to the active ingredient, a pharmaceutically acceptable carrier, diluent, or excipient (including a combination thereof). Pharmaceutically acceptable carriers and excipients are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The selection of a pharmaceutically acceptable carrier, excipient, or diluent can be easily determined by one skilled in the art in accordance with the route of administration and standard pharmaceutical practice. The cancer therapeutic agent composition of the present invention can further contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0051] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0052] Example 1 (Production of Polypeptide Derived from the Antibody Light Chain of the Present Invention and Enzyme Activity) a. Introduction of Mutations into the Antibody Light Chain (1) The sequence of the trastuzumab light chain near P95P96 is shown in Figure 1 and SEQ ID NO: 7. (2) Primer Sequence and Template DNA Forward primer (red): ACC TTC GGT CAG GGT ACC AAA GTT GA (SEQ ID NO: 8) Reverse primer (blue): GGT GGT GTA GTG CTG CTG GCA GTA G (SEQ ID NO: 9) trastuzumab-L / pET20b(+) 113.5 ng / μL (3) Procedure The template plasmid was diluted to 50 ng / mL with sterile MilliQ. All reagents except KOD-plus were added to a PCR tube, mixed, spun down and collected at the tip of the tube.

[0053]

[0054] A KOD-plus was added, gently tapped, and then spun down. Inverse PCR (iPCR) was performed using a Thermal Cycler. Initial denaturation: 94°C, 2 min Denaturation: 98°C, 10 seconds Annealing & Extension: 71°C, 5 min (5 cycles) 4°C, Hold 10 μL of the iPCR reaction solution was aliquoted and frozen for electrophoresis. 1.6 μL of Dpn I was added to the remaining 40 μL of iPCR reaction solution. The mixture was incubated at 37°C for 1 hour to digest the template DNA. Agarose electrophoresis was performed to confirm Dpn I digestion. To confirm the band position, a template plasmid (100 ng / lane) was used, and 5 μL of the iPCR solution was electrophoresed per lane. The agarose concentration was 1%. Dpn I digestion was confirmed. A self-ligation solution was prepared in a PCR tube according to the table below, and the reaction was carried out at 16°C for 1 hour.

[0055]

[0056] Competent cells, DH5, were thawed in an ice bath until they became sherbet-like. 10 μL of the ligation reaction solution was added and left to stand for 30 min. The cells were immersed in a 42°C water bath for 30 seconds, returned to the ice bath, and left to stand for 2 minutes. 900 μL of SOC medium and the competent cells were added to a round tube, and revival culture was carried out at 37°C and 200 rpm for 1 hour. The revival culture was inoculated onto an LB plate supplemented with 100 μg / mL ampicillin (Amp) and cultured overnight at 37°C. The next day, eight colonies were selected and streaked using the same plate, followed by the preparation of a master plate. Colony PCR was performed using the cells from the Master Plate, and the plasmid was extracted from the cells in which amplification of approximately 660 bp (full-length light chain) was confirmed, and the sequence was confirmed by sequence analysis. Glycerol stocks were then prepared from two colonies, and the plasmid was simultaneously inserted into BL21(DE3)pLysS to prepare cells for expression.

[0057] b. Cultivation, Recovery, and Purification. The transformants were grown in 1 L of Luria-Bertani medium containing 100 μg / mL ampicillin at 37°C to an A600nm of 0.6 and then incubated with 0.01 mM IPTG at 18°C ​​for 20 hours. The cells were harvested by centrifugation (3500 g, 4°C, 10 min) and resuspended in 100 mL of a solution containing 250 mM NaCl and 25 mM Tris-HCl, pH 8.0. The cells were sonicated three times in an ice bath for 2 min each, followed by centrifugation (21,475 g, 4°C, 20 min). The expressed human light chain was collected as the supernatant. The supernatant was first subjected to Ni-NTA column chromatography (Qiagen) equilibrated with 25 mM Tris-HCl, pH 8.0, containing 250 mM NaCl. Elution was performed by increasing the imidazole concentration from 0 and / or 30-300 mM. After completion of Ni-NTA column chromatography, a solution of 50 μM CuCl (1.25 eq of light chain) was added to the eluate, based on a UV / VIS absorbance of 1.0 at A600 nm corresponding to approximately 1 mg / mL (40 μM light chain). This is important for forming a homogeneous (dimeric) structure. The solution containing the light chain and copper ions was then dialyzed against 50 mM Tris-HCl buffer, pH 8.0, for approximately 20 hours. After removing some aggregates by centrifugation (17,800 g, 4°C, 20 min), the solution was concentrated to 2 mg / mL and subjected to cation exchange chromatography using a column of SP-5PW (TOSOH, Japan) with a gradient of NaCl (0.0% to 15.0%) in Tris-HCl (pH 8.0) buffer on a purification device (AKTA system, GE Healthcare Japan, Tokyo) or size exclusion chromatography on an AKTA system (column; HiLoad) using PBS (pH 7.4) buffer containing 137 mM NaCl as the eluent solvent. TM 16 / 60Superdex TMThe solution was subjected to a 200 pg / ml (GE Healthcare) run. The eluate was then collected and dialyzed against 20 mM Tris-HCl / 150 mM NaCl buffer (pH 8.5) for approximately 17 hours. The solution was then concentrated using an Amicon Ultra 10000 (Millipore, USA). EDTA was added to the solution to a concentration of 50 mM, and the solution was incubated at 4°C for 1 hour. It was then dialyzed twice against 2 L of PBS. After confirming complete removal of Cu(II) by UV / VIS spectroscopy, the solution was filtered using a 0.2 μm membrane filter (Merck-Millipore) and stored at 4°C or frozen. Protein concentration was determined by the Bradford method using the Lowry method with a DC protein assay kit (Bio-Rad).

[0058] SDS-PAGE Analysis: After light chain purification, SDS-PAGE analysis with CBB staining was performed. Figure 2A shows the results of the trastuzumab light chain under reducing and non-reducing conditions. Here, under non-reducing conditions, bands of approximately 46 and 28 kDa corresponding to the dimer and monomer, respectively, were observed. Under reducing conditions, only the 28 kDa band corresponding to the monomer was detected. SDS-PAGE analysis revealed almost no bands other than the light chain monomer and dimer bands, suggesting that the trastuzumab light chain was highly purified. Under non-reducing conditions, the monomer band was faint, but the dimer band was clear, indicating that the dimer was predominant in solution. Figure 2B shows the results of SDS-PAGE for a light chain variant that does not contain Pro95 and Pro96. The results were similar to those obtained with the trastuzumab light chain, except that a faint band around 30 kDa was observed under non-reducing conditions. The results of SDS-PAGE of the purified scFv trastuzumab light chain P95ΔP96Δ are shown in Figure 2C. In this case, a single band around 31 kDa was detected under both reducing and non-reducing conditions. No other bands were detected, indicating that the scFv trastuzumab light chain P95ΔP96Δ was highly purified.

[0059] c. Enzyme Activity Measurement Method (1) Method Using Arg-pNA Substrate: Cleavage of the p-nitroanilide amide bond to the C-terminal aa in the R-pNA substrate (Peptides Institute Inc., Osaka, Japan) was measured at 37°C in a 96-well plate (96-well plate / 353075, Becton-Dickinson, New Jersey, USA) in 100 mM Glycine / 50 mM Tris-HCl buffer containing 0.025% Tween 20 (TGT buffer; pH 7.7). Purified light chain (20 μL) was mixed with 180 μL of the synthetic substrate R-pNA. The final concentrations of light chain and substrate were 10 μM and 200 μM, respectively. Para-nitroaniline formed from the substrate catalyzed by the light chain was detected by measuring absorbance at 405 nm, while 620 nm was used as a reference using a microplate reader (Scan 3.1 for Multiscan FC, Thermo Fisher Scientific, MA, USA). Catalytic activity was estimated from the amount of p-nitroaniline produced.

[0060] The trypsin-like substrate molecule, Arg-pNA substrate (hereinafter referred to as R-pNA), was used to examine catalytic activity. Figure 3 shows the reaction time course of R-pNA cleavage. The three tested samples gradually hydrolyzed the synthetic substrate R-pNA, resulting in the formation of p-nitroaniline (pNA). The degradation reaction was monitored for up to 48 hours. Of the three samples, trastuzumab light chain, trastuzumab light chain P95ΔP96Δ, and scFv trastuzumab light chain P95ΔP96Δ, trastuzumab light chain showed the lowest catalytic activity. On the other hand, trastuzumab light chain P95ΔP96Δ showed the highest activity. ScFv trastuzumab light chain P95ΔP96Δ showed activity intermediate between that of trastuzumab light chain and trastuzumab P95ΔP96Δ. The initial reaction rates were estimated to be 0.13 μM / hr for the trastuzumab light chain, 0.24 μM / hr for the trastuzumab light chain P95ΔP96Δ, and 0.20 μM / hr for the scFv trastuzumab light chain P95ΔP96Δ at a concentration of 4.3 μM for each molecule. The peptidase activity was similar to that of the 22F6 human catalytic antibody (reaction rate; 0.09 μM / hr at a concentration of 4.0 μM for the 22F6 catalytic antibody light chain).

[0061] (2) Dissociation constant (K DThe binding affinity of HER2 to trastuzumab light chain (Tmb-L-wt), trastuzumab light chain P95ΔP96Δ (Tmb-L-PPΔ), scFv trastuzumab light chain (Tmb-scFv(wt)), scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFvPPΔ), and trastuzumab was measured using an Octet N1 measuring instrument (Sartorius AG, Germany) and an Advanced Kinetics method according to the protocol. Recombinant HER2 molecules were bound to an avidin tag (SINO Biochemical; 10004-HCCH-B) using a streptavidin-immobilized biosensor chip (SA-biosensor). The experiment consisted of a five-step sequential assay at room temperature. First, the SA-biosensor was immersed in 250 μL of PBS buffer in a microtube (ClickFit, LightBlock, PP, Black, 0.5 mL, TreffLab, Degersheim, Switzerland) for 30 seconds. Then, the SA-biosensor was immersed in a drop holder containing 4 μL of 100 nM recombinant HER2 molecules for 120 seconds. In the third step, the SA-biosensor (immobilized with recombinant HER2 molecules) was immersed in PBS in a microtube for washing. In the fourth step, the SA-biosensor bound to the recombinant HER2 molecule was immersed in a drop holder containing 4 μL of antibody solution for 120 seconds. In the final (fifth) step, the SA-biosensor bound to the antibody was again immersed in PBS buffer to measure dissociation. Data analysis was performed using Octet N1 software. The dissociation constant (K D Various concentrations of each antibody were prepared to obtain accurate estimates of the dissociation constants (K), association values ​​(k), and dissociation values ​​(k). D ) was calculated as kd / ka. As a result, as shown in Table 3, the dissociation constant (K D The dissociation constant (K D ) was almost equivalent to the scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFvPPΔ).

[0062]

[0063] (3) Method using FRET-HER2 peptide (a) FRET-HER2 peptide was synthesized to examine catalytic activity and conduct kinetic studies. The targeting sequence of the Her2 molecule should contain the epitope region of trastuzumab. The epitope sequence is considered to be aa 563-626. Loop3 (PDB: 1N8Z and 6OGE). 593 KFP DEEGA 600 The sequence of the following strongly interacts with the light chain of trastuzumab (see PDB: 1N8Z and 6OGE). 579 PSGVKPDLSYMPIWKFPDEEGA 600 This region was extracted and synthesized as FRET-Her2 peptide-1 (FIG. 4 a), FRET-Her2peptide-1 (579-600). The cleavage sites of this FRET-Her2peptide-1 (579-600) and the structures of its degradation products are shown in FIG. 5.

[0064] The FRET-HER2 peptide (25 μM) was incubated with trastuzumab light chain (Tmb-L), trastuzumab light chain P95ΔP96Δ (Tmb-L-PΔΔ), scFv trastuzumab light chain (Tmb-scFv), scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFv), and trastuzumab (5 μM) in TGT buffer containing 0.02% NaN at 37°C. ex = 320 nm, λ emTransmittance was measured periodically on a chromatograph (F = 405 nm; Thermo Fisher Scientific Oy, Vantaa, Finland). All measurements were performed in duplicate. The results using FRET-HER2 peptide-1 are shown in Figure 6. In this case, the trastuzumab light chain P95ΔP96Δ showed the highest catalytic activity, followed by the scFv trastuzumab light chain P95ΔP96Δ. On the other hand, the trastuzumab light chain showed low activity, and the scFv trastuzumab light chain and trastuzumab showed the lowest activity. The order of activity was similar to that obtained in the Arg-pNA test, except that the trastuzumab light chain P95ΔP96Δ showed the highest activity, followed by the scFv trastuzumab light chain P95ΔP96Δ. The amino acid sequence of the variable region of the trastuzumab light chain is shown in Figure 7 and SEQ ID NO: 4. The amino acid sequence of CDR1 of the trastuzumab light chain is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3. The amino acid sequence of a polypeptide in which P95 and P96 have been deleted from the CDR3 region of the trastuzumab light chain is shown in SEQ ID NO: 5. The amino acid sequence of a polypeptide in which P95 and P96 have been deleted from the variable region of the trastuzumab light chain is shown in SEQ ID NO: 6.

[0065] (4) Method using FRET-HER2 peptide (b) FRET-Her2peptide-2 (Loop2+Loop3) shown in Figure 4b) was synthesized. The cleavage sites of this FRET-Her2peptide-2 (Loop2+Loop3) and the structures of its degradation products are shown in Figure 8. FRET-HER2 peptide-2 (25 μM) was incubated with trastuzumab light chain (Tmb-L), trastuzumab light chain P95ΔP96Δ (Tmb-L-PΔΔ), scFv trastuzumab light chain (Tmb-scFv), scFv trastuzumab light chain P95ΔP96Δ (Tmb-scFv), and trastuzumab (5 μM) in TGT buffer containing 0.02% NaN at 37°C. ex = 320 nm, λ emTransmittance was measured periodically on a chromatograph (F = 405 nm; Thermo Fisher Scientific Oy, Vantaa, Finland). All measurements were performed in duplicate. The results using FRET-HER2 peptide-2 are shown in Figure 9. In this case, the trastuzumab light chain P95ΔP96Δ showed the highest catalytic activity, followed by the scFv trastuzumab light chain P95ΔP96Δ. On the other hand, the trastuzumab light chain showed low activity, and the scFv trastuzumab light chain and trastuzumab showed the lowest activity. The order of activity was similar to that obtained in the Arg-pNA test, except that the trastuzumab light chain P95ΔP96Δ showed the highest activity, followed by the scFv trastuzumab light chain P95ΔP96Δ. The reaction rate constants of the trastuzumab light chain P95ΔP96Δ and the scFv trastuzumab light chain P95ΔP96Δ were calculated by Lineweaver-Burk plot and the results are shown in Figure 10. Both showed high Km values.

[0066] Comparative Example 1 (Changes in enzyme activity due to deletion of P95 in antibody light chain lacking D1 / S27 / H93) P95 was deleted from antibody light chain #4 containing the amino acid sequence of SEQ ID NO: 10 in the same manner as in Example 1, and changes in its enzyme activity were examined. As a result, no improvement in enzyme activity was observed for #4P95Δ. The amino acid sequence of #4 is shown in Figure 11 and SEQ ID NO: 10. The amino acid sequence of the polypeptide obtained by deleting P95 from #4 is shown in SEQ ID NO: 11.

[0067] Example 2 (Conformational Simulation) Using Discovery Studio (Accelrys Software, San Diego, CA, USA), conformational simulations were performed by molecular modeling of #4, #4P95Δ, trastuzumab light chain, trastuzumab light chain P95Δ, and trastuzumab light chain P95ΔP96Δ. As a result, in #4 and #4P95Δ, D1 / S27a / H49 was considered to be a candidate for the catalytic triad residue, but it was not possible to construct the catalytic triad residue. In the case of the trastuzumab light chain, D1 / S26 / H91 was considered to be a candidate for the catalytic triad residue, but it was not possible to construct the catalytic triad residue. On the other hand, quite unexpectedly, the trastuzumab light chain P95ΔP96Δ was able to form a catalytic triad of residues S26 / R24 / D70 ( FIG. 12 ; in the figure, the trastuzumab light chain is on the left and the trastuzumab light chain P95ΔP96Δ is on the right). As a result, it was considered that the polypeptide derived from the light chain of the anti-HER2 antibody of the present invention exhibited nearly four times the enzymatic activity of trastuzumab.

[0068] Example 3 (Cytotoxicity Test of Polypeptides Derived from the Light Chain of an Anti-HER2 Antibody of the Present Invention) BT-474 clone 5 cells were cultured in RPMI-1640 medium (Sigma, R8005, high glucose) supplemented with 10% FBS. When the required number of cells was secured in the logarithmic growth phase, the cells were detached using 0.25% Trypsin-EDTA (Gibco, 25200-072) and cultured at a concentration of 6.4 × 10 4The cells were adjusted to 100 μL / well. The cells were seeded into a 96-well plate at 100 μL / well and cultured at 5% CO and 37°C for 48 hours. The culture medium was removed, and RPMI-1640 medium was added at 50 μL / well. Subsequently, an antibody solution adjusted to 7.06 μM with PBS was added at 50 μL / well, gently mixed, and then cultured at 5% CO and 37°C for 72 hours. 2 μg / mL PI solution (Dojindo, 341-07881) and 2 μg / mL Hoechst 33342 solution (Dojindo, 346-07951) were added, and the cells were incubated at 5% CO and 37°C for 30 minutes, after which morphological observation was performed using a KEYENCE all-in-one microscope (model number BZ-9000). As a result, as shown in Figures 13 and 14, the cytotoxic activity against the human breast cancer cell line BT-474 was shown in the following order: scFv trastuzumab light chain P95ΔP96Δ (in the figures, Sc-FvP95Δ), trastuzumab light chain P95ΔP96Δ (in the figures, L-P95Δ), and trastuzumab (in the figures, Trastuzumab). The anti-HER2 antibody light chain (in the figures, Lchain) showed almost no cytotoxic activity.

[0069] Example 4 (Relative Values ​​in Antigen Decomposition Activity Test of Trastuzumab, Its scFv (Trastuzumab-scFv, and Trastuzumab-scFv-PPΔ in Which Pro95 and Pro96 Are Deleted)) The antigen decomposition activity of Trastuzumab-scFv-PPΔ in Which Pro95 and Pro96 Are Deleted was greatly improved compared to that of Trastuzumab and Trastuzumab-scFv ( FIG. 15 ).

[0070] Example 5 (Synthesis and evaluation of polypeptide (g)) The polypeptide of (g) is expressed in an expression system known in the art, such as an intracellular or extracellular expression system, using an expression vector comprising a polynucleotide encoding a polypeptide in which the polypeptide of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 6 and the polypeptide shown in SEQ ID NO: 15 are linked. Various activities of this polypeptide can be evaluated by comparing it with a control polynucleotide obtained by expressing the polypeptide in an expression system known in the art, such as an intracellular or extracellular expression system, using an expression vector comprising a polynucleotide encoding a polypeptide in which the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 and the polypeptide shown in SEQ ID NO: 15 are linked.

[0071] [Sequence Listing] SEQ ID NO: 1: RASQDVNTAVA (CDR1 region) SEQ ID NO: 2: SASFLYS (CDR2 region) SEQ ID NO: 3: QQHYTTPPT (CDR3 region) SEQ ID NO: 4: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5: QQHYTTT (CDR3 region; ΔP95,96) SEQ ID NO: 6: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence in which Pro95 and Pro06 of the light chain of HER2 antibody have been deleted) SEQ ID NO: 7: CCTACTACTGCCAGCAGCACTACACCACCCCGCCGACCTTCGGTCAGGGTACCAAAGTTGAAATC SEQ ID NO: 8: ACC TTC GGT CAG GGT ACC AAA GTT GA (Forward primer) SEQ ID NO: 9: GGT GGT GTA GTG CTG CTG GCA GTA G (Reverse primer) SEQ ID NO: 10:DVVMTQTPLSLSVTPGEPASISCRSTQSLLDSDGVNPSFDWYVQKPGQSPQLLIHRGFYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (amino acid sequence of #4) SEQ ID NO: 11: DVVMTQTPLSLSVTPGEPASISCRSTQSLLDSDGVNPSFDWYVQKPGQSPQLLIHRGFYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQRIEFLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (amino acid sequence of polypeptide obtained by deleting P95 from #4) SEQ ID NO: 12: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS (heavy chain variable region) SEQ ID NO: 13: scFv sequence-1 (clone in which the antibody HER2 antibody light chain variable region and the antibody HER2 antibody heavy chain variable region are linked with a linker) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGSGGGGGSGGGGSGGGGSKLEVQL VESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSLE SEQ ID NO: 14: scFv sequence-2(A clone in which Pro95 and Pro96 according to the Kabat classification of the HER2 antibody light chain variable region have been deleted and the HER2 antibody heavy chain variable region are linked with a linker) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTTFGQGTKVEIKRGSGGGGSGGGGSGGGGSKLEVQLV ESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSL ... EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A polypeptide having a hypervariable region selected from the group consisting of the following (1a) to (1c), and having HER2 degradation activity: (1a) a polypeptide having a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and consisting of an amino acid sequence in which the 70th aspartic acid in the variable region is deleted or the 95th and 96th proline residues in the variable region are deleted or are amino acid residues other than proline residues in the Kabat classification; (1b) a polypeptide having a CDR1 region consisting of an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and consisting of an amino acid sequence in which the 70th aspartic acid in the variable region is deleted or the 95th and 96th amino acid residues in the variable region are deleted or are amino acid residues other than proline residues in the Kabat classification; (1c) A polypeptide having a CDR1 region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at the 70th position of the variable region according to the Kabat classification, and having an amino acid sequence in which the 95th and 96th amino acid residues of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues.

2. A polypeptide having HER2 degradation activity, the variable region of which is selected from the group consisting of the following (2a) to (2c): (2a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, added or deleted in the amino acid sequence shown in SEQ ID NO: 4, and which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region are deleted or are amino acid residues other than proline residues according to the Kabat classification; (2c) a polypeptide having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, which has an aspartic acid at position 70 of the variable region according to the Kabat classification, and in which the amino acid residues at positions 95 and 96 of the variable region are deleted or are amino acid residues other than proline residues according to the Kabat classification.

3. The polypeptide according to claim 1 or 2, further comprising an anti-HER2 antibody heavy chain variable region as shown in SEQ ID NO:

12.

4. The polypeptide according to claim 3, in which the anti-HER2 antibody light chain hypervariable region according to claim 1 and the anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12, or the anti-HER2 antibody light chain variable region according to claim 2 and the anti-HER2 antibody heavy chain variable region shown in SEQ ID NO: 12 are linked by a linker.

5. The polypeptide according to claim 4, further comprising an Fc region linked thereto.

6. The polypeptide according to claim 3, in which a light chain constant region linked to the hypervariable region of the anti-HER2 antibody according to claim 1 and a heavy chain CH1 region linked to the heavy chain variable region shown in SEQ ID NO: 12; or a light chain constant region linked to the variable region of the anti-HER2 antibody according to claim 2 and a heavy chain CH1 region linked to the heavy chain variable region shown in SEQ ID NO: 12; are associated via a disulfide bond.

7. A polypeptide comprising the CH1 region of the polypeptide according to claim 6 linked to heavy chains CH2 and CH3.

8. A method for producing a polypeptide derived from an anti-HER2 antibody light chain having enzymatic activity or improved enzymatic activity, comprising: a hypervariable region comprising: (1a) a polypeptide comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification; (1b) a polypeptide comprising a CDR1 region consisting of an amino acid sequence in which one or several amino acids have been substituted, added or deleted from the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence in which one or several amino acids have been substituted, added or deleted from the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region consisting of SEQ ID NO: 3, and having an aspartic acid at position 70 of the variable region according to the Kabat classification; or (1c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

1. a polynucleotide encoding an anti-HER2 antibody light chain comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:2, a CDR1 region consisting of the sequence SEQ ID NO:3, a CDR2 region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:2, and a CDR3 region consisting of SEQ ID NO:3, said polynucleotide encoding an anti-HER2 antibody light chain comprising an amino acid sequence having an aspartic acid at position 70 of the variable region according to the Kabat classification, and wherein the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are proline residues, said polynucleotide being modified so as to delete or substitute the proline residues to obtain a polynucleotide encoding a polypeptide derived from the anti-HER2 antibody light chain, said variable region comprising an amino acid sequence in which the amino acid residues at positions 95 and 96 of the variable region according to the Kabat classification are deleted or are amino acid residues other than proline residues; and an expression step of expressing a polypeptide derived from the anti-HER2 antibody light chain having enzymatic activity by an intracellular or extracellular expression system using an expression vector comprising a polynucleotide encoding the HER2 antibody light chain after the modification step.

9. A method for producing a polypeptide derived from an anti-HER2 antibody light chain having an enzymatic activity or an improved enzymatic activity, comprising the steps of: modifying a polynucleotide encoding a polypeptide derived from an anti-HER2 antibody light chain, the variable region of which is (2a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4; (2b) a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence shown in SEQ ID NO: 4 and which has an aspartic acid at position 70 in the variable region according to the Kabat classification; or (2c) a polypeptide which has 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and which has an aspartic acid at position 70 in the variable region according to the Kabat classification, and in which the 95th and 96th amino acid residues in the variable region are proline residues according to the Kabat classification, so that the proline residues are deleted or substituted to obtain a polynucleotide encoding a polypeptide derived from an anti-HER2 antibody light chain, the variable region of which is a polypeptide consisting of an amino acid sequence in which the 95th and 96th amino acid residues in the variable region are deleted or have amino acid residues other than proline residues according to the Kabat classification; an expression step of expressing a polypeptide derived from the anti-HER2 antibody light chain having enzymatic activity, in an intracellular or extracellular expression system, using an expression vector containing a polynucleotide encoding the antibody HER2 antibody light chain after the modification step.

10. A cancer therapeutic agent comprising an ingredient selected from the group consisting of a polypeptide derived from the light chain of the anti-HER2 antibody according to claim 1 or 2, a HER2 binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of a HER2 binding polypeptide comprising said polypeptide derived from said light chain.

11. A pharmaceutical composition for cancer treatment comprising an ingredient selected from the group consisting of a polypeptide derived from the light chain of the anti-HER2 antibody according to claim 1 or 2, a HER2-binding polypeptide comprising said light chain-derived polypeptide, and a functional fragment of a HER2-binding polypeptide comprising said light chain-derived polypeptide.

12. Use of a component selected from the group consisting of a polypeptide derived from the light chain of the anti-HER2 antibody described in claim 1 or 2, a HER2 binding polypeptide comprising said light chain derived polypeptide, and a functional fragment of a HER2 binding polypeptide comprising said light chain derived polypeptide, for the manufacture of a cancer therapeutic agent.

13. A component selected from the group consisting of a polypeptide derived from the light chain of an anti-HER2 antibody described in claim 1 or 2, a HER2-binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of an anti-HER2 antibody comprising said polypeptide derived from said light chain, for use in the treatment of cancer.

14. A method for treating cancer, comprising administering a component selected from the group consisting of a polypeptide derived from the light chain of the anti-HER2 antibody described in claim 1 or 2, a HER2 binding polypeptide comprising said polypeptide derived from said light chain, and a functional fragment of a HER2 binding polypeptide comprising said polypeptide derived from said light chain.

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