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EP-4737480-A1 - 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

EP4737480A1EP 4737480 A1EP4737480 A1EP 4737480A1EP-4737480-A1

Abstract

The present invention provides an antigenase obtained by imparting high enzymatic activity to an antibody having a high anticancer effect. The present invention relates to a polypeptide including hypervariable regions derived from an anti-HER2 antibody light chain, as described in (1a) or the like: (1a) a polypeptide consisting of an amino acid sequence that includes a CDR1 region consisting of an amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and has an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein proline residues at positions 95 and 96 of a variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues.

Inventors

  • HIFUMI, EMI
  • UDA, TAIZO

Assignees

  • Japan Science and Technology Agency

Dates

Publication Date
20260506
Application Date
20240628

Claims (14)

  1. A polypeptide having HER2 decomposition activity, wherein a hypervariable region is selected from the group consisting of (1a) to (1c): (1a) a polypeptide consisting of an amino acid sequence that includes a CDR1 region consisting of an amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and has an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein proline residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues; (1b) a polypeptide consisting of an amino acid sequence that includes 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 set forth 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 set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and has an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues; and (1c) a polypeptide consisting of an amino acid sequence that includes a CDR1 region consisting of an amino acid sequence having 90% or more identity with 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 with the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and has an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues.
  2. A polypeptide having HER2 decomposition activity, wherein a variable region is selected from the group consisting of (2a) to (2c): (2a) a polypeptide consisting of an amino acid sequence set forth 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 set forth in SEQ ID NO: 4, the amino acid sequence having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues; and (2c) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues.
  3. The polypeptide according to claim 1 or 2, further comprising a variable region of an anti-HER2 antibody heavy chain as set forth in SEQ ID NO: 12.
  4. The polypeptide according to claim 3, wherein the hypervariable region of an anti-HER2 antibody light chain according to claim 1 and the variable region of an anti-HER2 antibody heavy chain as set forth in SEQ ID NO: 12, or the variable region of an anti-HER2 antibody light chain according to claim 2 and the variable region of an anti-HER2 antibody heavy chain as set forth in SEQ ID NO: 12, are linked via a linker.
  5. The polypeptide according to claim 4, further comprising an Fc region linked thereto.
  6. The polypeptide according to claim 3, wherein a light-chain constant region linked to the hypervariable region of an anti-HER2 antibody according to claim 1 and a heavy-chain CH1 region linked to a heavy-chain variable region set forth in SEQ ID NO: 12 are associated through a disulfide bond; or a light-chain constant region linked to the variable region of an anti-HER2 antibody according to claim 2 and a heavy-chain CH1 region linked to a heavy-chain variable region set forth in SEQ ID NO: 12 are associated through a disulfide bond.
  7. A polypeptide comprising a heavy-chain CH2 and a heavy-chain CH3 that are linked to the CH1 region of the polypeptide according to claim 6.
  8. A method for producing a polypeptide derived from an anti-HER2 antibody light chain having enzymatic activity or improved enzymatic activity, the method comprising: a modification step of modifying a polynucleotide encoding an anti-HER2 antibody light chain in which a hypervariable region is (1a) a polypeptide comprising a CDR1 region consisting of an amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme; (1b) a polypeptide comprising 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 set forth 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 set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme; or (1c) a polypeptide comprising a CDR1 region consisting of an amino acid sequence having 90% or more identity with 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 with the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein the polypeptide consists of an amino acid sequence in which amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are proline residues, so that the proline residues are deleted or substituted to give a polynucleotide encoding a polypeptide derived from an anti-HER2 antibody light chain consisting of an amino acid sequence in which amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues; and an expression step of expressing a polypeptide derived from an anti-HER2 antibody light chain having enzymatic activity, by using an expression vector comprising a polynucleotide encoding an anti-HER2 antibody light chain after the modification step, in an intracellular or extracellular expression system.
  9. A method for producing a polypeptide derived from an anti-HER2 antibody light chain having enzymatic activity or improved enzymatic activity, the method comprising: a modification step of modifying a polynucleotide encoding a polypeptide derived from an anti-HER2 antibody light chain in which a variable region is (2a) a polypeptide consisting of an amino acid sequence set forth 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 set forth in SEQ ID NO: 4, the amino acid sequence having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme; or (2c) a polypeptide having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 and having an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein the polypeptide consists of an amino acid sequence in which amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are proline residues, so that the proline residues are deleted or substituted to give a polynucleotide encoding a polypeptide derived from an anti-HER2 antibody light chain, wherein the variable region is a polypeptide consisting of an amino acid sequence in which amino acid residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues; and an expression step of expressing a polypeptide derived from an anti-HER2 antibody light chain having enzymatic activity, by using an expression vector comprising a polynucleotide encoding an anti-HER2 antibody light chain after the modification step, in an intracellular or extracellular expression system.
  10. An anticancer agent comprising a component selected from the group consisting of: the polypeptide derived from an anti-HER2 antibody light chain according to claim 1 or 2; a HER2-binding polypeptide comprising the polypeptide derived from the light chain; and a functional fragment of the HER2-binding polypeptide comprising the polypeptide derived from the light chain.
  11. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a component selected from the group consisting of: the polypeptide derived from an anti-HER2 antibody light chain according to claim 1 or 2; a HER2-binding polypeptide comprising the polypeptide derived from the light chain; and a functional fragment of the HER2-binding polypeptide comprising the polypeptide derived from the light chain.
  12. Use of a component for production of an anticancer agent, the component being selected from the group consisting of: the polypeptide derived from an anti-HER2 antibody light chain according to claim 1 or 2; a HER2-binding polypeptide comprising the polypeptide derived from the light chain; and a functional fragment of the HER2-binding polypeptide comprising the polypeptide derived from the light chain.
  13. A component for use in treatment of cancer, the component being selected from the group consisting of: the polypeptide derived from an anti-HER2 antibody light chain according to claim 1 or 2; a HER2-binding polypeptide comprising the polypeptide derived from the light chain; and a functional fragment of an anti-HER2 antibody comprising the polypeptide derived from the light chain.
  14. A method for treating cancer, the method comprising administering a component selected from the group consisting of: the polypeptide derived from an anti-HER2 antibody light chain according to claim 1 or 2; a HER2-binding polypeptide comprising the polypeptide derived from the light chain; and a functional fragment of the HER2-binding polypeptide comprising the polypeptide derived from the light chain.

Description

Technical Field The present invention relates to a polypeptide derived from an anti-HER2 antibody light chain having HER2 decomposition activity, a HER2-binding polypeptide comprising the polypeptide, a method for producing the same, and an anticancer agent containing the same. Background Art An antibody is composed of a heavy chain (H chain) and a light chain (L chain). The heavy chain and the light chain are each composed of a variable region (VR) and a constant region (CR), the variable region including hypervariable regions (CDRs: complementarity-determining regions). In recent years, an antibody having enzyme-like activity, that is, an abzyme, has attracted considerable attention. Such an abzyme is expected to be applied in many fields, including the medical field, the chemical industry, and the food industry, because it has both high molecular recognition ability of an antibody and enzymatic activity. In particular, an abzyme that has high specificity to a target molecule and is capable of exhibiting cytotoxicity against the target molecule by enzymatic activity is expected to be used as an effective anticancer drug with few side effects. Particularly, since a human abzyme is expected to cause few side effects when administered to the human body, domestic and foreign pharmaceutical companies have been waiting for the development of useful human abzymes. The present inventors have conducted various studies on abzymes (see, for example, Patent Literatures 1 to 4). In order to clinically use an abzyme as a pharmaceutical product, it is important to stably mass-produce an abzyme having sufficient activity. However, many abzymes exhibit insufficient enzymatic activity. When the abzymes are artificially synthesized by an intracellular or extracellular expression system using a genetic recombination technique, there is a problem of unstable performance and significant lot-to-lot variation. Therefore, the present inventors have conducted various studies to produce an abzyme composed of an antibody light chain having higher enzymatic activity and reported that, in an amino acid sequence of an antibody light chain, deletion or substitution of the 95th proline residue in a variable region in accordance with the Kabat numbering scheme enables the light chain to form the steric conformation of catalytic triad residues (D1/S27/H93), thereby yielding an antibody light chain (antigenase) with enhanced enzymatic activity (Patent Literature 5). Citation List Patent Literature Patent Literature 1: JP-A-2006-197930Patent Literature 2: WO 2011/102517Patent Literature 3: WO 2013/133253Patent Literature 4: WO 2015/025786Patent Literature 5: WO 2021/015237 Summary of Invention Technical Problem It is an object of the present invention to provide an antigenase obtained by imparting high enzymatic activity to an antibody having a high anticancer effect. Solution to Problem As a result of studies for imparting enzymatic activity to an anti-HER2 antibody light chain having an excellent anticancer effect against cancers that overexpress HER2, such as breast cancer, deletion of a proline residue at position 95 of the variable region has been considered insufficient to impart enzymatic activity to the light chain, since it lacks S27a/H93 described in Patent Literature 5. However, the present inventors have found that simultaneous deletion of the proline residues at positions 95 and 96 of the variable region of the anti-HER2 antibody light chain unexpectedly yields a polypeptide derived from the anti-HER2 antibody light chain having enzymatic activity close to 4 times that of the light chain, and also that the use of the polypeptide enables the development of a novel HER2-targeted therapeutic agent having, in addition to HER2 decomposition activity, one or more activities selected from the group consisting of antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neutralizing activity. Thus, the present invention has been accomplished. Accordingly, the present invention provides [1] to [14] below. [1] A polypeptide derived from an anti-HER2 antibody light chain, wherein a hypervariable region is selected from the group consisting of (1a) to (1c): (1a) a polypeptide consisting of an amino acid sequence that includes a CDR1 region consisting of an amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region consisting of an amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region consisting of an amino acid sequence set forth in SEQ ID NO: 3, and has an aspartic acid residue at position 70 of a variable region in accordance with the Kabat numbering scheme, wherein proline residues at positions 95 and 96 of the variable region in accordance with the Kabat numbering scheme are deleted or substituted with amino acid residues other than proline residues;(1b) a polypeptide consisting of an amino acid sequence that includes a CDR1 region consisting of an amino acid sequence in which one or several amino aci