WO2019172401A1 - Polypeptide imitant un anticorps modifié - Google Patents

Polypeptide imitant un anticorps modifié Download PDF

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WO2019172401A1
WO2019172401A1 PCT/JP2019/009244 JP2019009244W WO2019172401A1 WO 2019172401 A1 WO2019172401 A1 WO 2019172401A1 JP 2019009244 W JP2019009244 W JP 2019009244W WO 2019172401 A1 WO2019172401 A1 WO 2019172401A1
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domain
deleted
terminal
antibody
amino acid
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PCT/JP2019/009244
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English (en)
Japanese (ja)
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孝光 宮房
真也 本田
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国立研究開発法人産業技術総合研究所
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Priority to JP2020505124A priority Critical patent/JP7115773B2/ja
Publication of WO2019172401A1 publication Critical patent/WO2019172401A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to polypeptides that mimic denatured antibodies.
  • the present invention relates to a CH3 domain with a C-terminal amino acid deleted, which can specifically bind to a probe that specifically recognizes a denatured antibody.
  • the present invention also relates to a method for measuring a denatured antibody using the polypeptide and a method for screening a compound that specifically interacts with the denatured antibody using the polypeptide.
  • Protein is a group of substances having a function of highly controlling a substance conversion catalyst and signal transmission in a living body. Proteins express these functions by forming specific three-dimensional structures. Once the structure is damaged (denatured), not only is the function lost, but it can be a factor that disturbs the homeostasis of the living body.
  • Antibody molecules that are administered to living bodies as biopharmaceuticals are no exception. Antibodies undergo chemical and physical stresses during the manufacturing process, and part of them change to non-natural three-dimensional structures to form aggregates (in the following description, antibodies having non-natural three-dimensional structures). And their aggregates are collectively referred to as “denatured antibodies”). When a preparation containing this agglomerate in a certain ratio or more is administered, an immune response is induced in the living body, resulting in a decrease in drug efficacy due to the generation of neutralizing antibodies and anaphylactic shock-like symptoms (non-patented). Reference 1).
  • Non-patent document 2 Non-patent document 2
  • Non-Patent Documents 3 to 5 and Patent Documents 1 and 2 A report (Non-patent Documents 3 and 4) in which a molecule that binds to a denatured antibody slightly contained in an antibody sample was obtained as a result of searching for a non-denatured antibody as a target and stress was added. There is a report (Non-Patent Document 5) in which a binding molecule is searched by targeting a denatured antibody and a molecule that binds to the denatured antibody is obtained.
  • Non-Patent Document 5 A report (Non-Patent Document 5) in which a binding molecule is searched by targeting a denatured antibody and a molecule that binds to the denatured antibody is obtained.
  • Proteins are known to be exposed to hydrophobic regions with denaturation. Measurement using a molecule (for example, 1,8-anilinonaphthalenesulfonic acid) that nonspecifically binds to an exposed hydrophobic region is performed for protein structure evaluation (Non-patent Document 6). On the other hand, it is generally understood that the interaction between a denatured protein and a probe is a non-specific phenomenon.
  • a molecule for example, 1,8-anilinonaphthalenesulfonic acid
  • Non-Patent Documents 3 to 5 and Patent Documents 1 and 2 differ from conventional denatured protein recognition probes in that these probes bind specifically to denatured antibodies.
  • the term “specifically binds” as used herein means that it does not bind to a non-denaturing antibody and does not bind to a protein other than the antibody. In order to realize such binding characteristics, it is presumed that the probe strictly recognizes a “specific non-natural three-dimensional structure” that is commonly held by the denatured antibody.
  • Non-Patent Documents 7 and 8 detailed analysis of structural changes caused by stress caused by antibodies has progressed.
  • Non-Patent Documents 7 and 8 we observed a phenomenon in which a region that had a robust three-dimensional structure in a natural three-dimensional structure changed into a flexible structural state due to the collapse of the three-dimensional structure due to stress.
  • the conformation collapses there is no description of the resulting “specific non-natural conformation”.
  • Non-patent Document 9 there is a report that the C-terminal deletion Fc fragment obtained by enzyme digestion reduces IgG multimerization by conformational change. There is no description about "natural type three-dimensional structure”.
  • the denatured antibody produced under stress is a mixture with a natural type antibody, and it is difficult to control the ratio of the denatured antibody in the mixture.
  • Denatured antibodies are aggregates of various association states, and the association state changes with time. In summary, it is very difficult to use a denatured antibody produced by stress as a reference substance.
  • An object of the present invention is to provide a polypeptide that imitates a non-natural three-dimensional structure possessed by a modified antibody and can be used for quantitative evaluation of the modified antibody.
  • An antibody has a specific non-natural three-dimensional structure because a part of the amino acid sequence loses a natural three-dimensional structure due to physical and chemical stress.
  • the present inventor predicted that the “specific non-natural three-dimensional structure” recognized by the probe disclosed in Patent Document 1 exists in the “internal structure exposed by structural change”.
  • the present inventors have conceived of creating a desired non-natural three-dimensional structure by adding a deletion mutation to the amino acid sequence of an antibody.
  • Patent Document 1 specifically binds to the Fc region of an antibody having an unnatural three-dimensional structure. Therefore, the present inventors verified whether the non-natural three-dimensional structure recognized by the probe is present in the CH2 domain or the CH3 domain in the Fc region, and the non-natural three-dimensional structure of the CH3 domain Found to combine.
  • the present invention is based on the above findings and includes the following [1] to [13]: [1] C-terminal truncated CH3 domain described in (i) or (ii) below: (I) a C-terminal deleted CH3 domain consisting of an amino acid sequence in which amino acids are deleted within the range of 5 to 20 residues from the C-terminal side in the amino acid sequence shown in any of SEQ ID NOs: 1 to 7, or , (Ii) 5 to 20 residues from the C-terminal side in the amino acid sequence in which one or several amino acids in the amino acid sequence shown in any of SEQ ID NOs: 1 to 7 are substituted, deleted, inserted and / or added A C-terminal deleted CH3 domain consisting of an amino acid sequence lacking an amino acid within the group and specifically binding to an AF.2A1 polypeptide (SEQ ID NO: 9).
  • Consisting of the missing amino acid sequence (Ii) ′
  • one or several amino acids in the amino acid sequence represented by any of SEQ ID NOs: 1 to 4 are substituted, deleted, inserted, and / or Alternatively, the added amino acid sequence consists of an amino acid sequence in which amino acids are deleted within the range of 5 to 20 residues from the C-terminal side, and the AF.2A1 polypeptide specifically binds.
  • [5] The C-terminal deleted CH3 domain according to any one of [1] to [3], wherein 5 amino acids are deleted from the C-terminal side.
  • An Fc region fragment comprising the C-terminal deleted CH3 domain according to any one of [1] to [5].
  • An antibody comprising the C-terminal deleted CH3 domain according to any one of [1] to [5].
  • a protein comprising the C-terminal deleted CH3 domain according to any one of [1] to [5].
  • a method for quantitatively measuring a denatured antibody contained in a sample A step of measuring the amount of binding between the AF.2A1 polypeptide (SEQ ID NO: 9) and the denatured antibody contained in the sample, the AF.2A1 polypeptide and the C-terminal deletion according to any one of [1] to [5] A step of measuring the binding amount to the lost CH3 domain, the Fc region fragment according to [6], the antibody according to [7], or the protein according to [8]; And a step of calculating the amount of denatured antibody in the sample from the amount of binding between the C-terminal deleted CH3 domain, the Fc region fragment, the antibody, or a protein and an AF.2A1 polypeptide.
  • [12] A method for screening a compound having affinity for a denatured antibody, A C-terminal deleted CH3 domain according to any one of [1] to [5], an Fc region fragment according to [6], an antibody according to [7], or a protein and a test compound according to [8]
  • a screening method comprising the step of measuring the interaction of.
  • the C-terminal deleted CH3 domain and the antibody or fragment thereof containing the C-terminal deleted domain are homogeneous, stable, and prevent the quantitative evaluation of denatured antibodies.
  • the C-terminal deleted CH3 domain, Fc region fragment, or antibody may be modified with an organic compound or an inorganic compound such as a His tag for use as a reference substance.
  • the CH3 domain lacking the C-terminal amino acid of the present invention is specifically recognized by a probe that specifically binds to a non-natural three-dimensional structure possessed by a denatured antibody.
  • the CH3 domain lacking the C-terminal amino acid of the present invention can be used as a reference substance in quantitative evaluation of denatured antibodies, enabling quantitative evaluation of denatured antibodies, which has been difficult in the past. .
  • FIG. 1 shows a sensorgram showing the binding property between the AF.2A1 polypeptide and the CH2 domain
  • (B) shows a sensorgram showing the binding property between the AF.2A1 polypeptide and the CH3 domain.
  • Fig. 2 (A) is a three-dimensional structure model in which the C-terminal 5 residues are highlighted in black in the Fc region.
  • Fig. 2 (B) is a three-dimensional structure in which the C-terminal 10 residues are highlighted in black in the Fc region.
  • Model, Fig. 2 (C) is a three-dimensional structure model in which the C-terminal 15 residues of the Fc region are highlighted in black, and Fig.
  • FIG. 2 (D) is the C-terminal 20 residues of the Fc region in black. Each of the three-dimensional structure models is shown.
  • FIG. 2 shows a sensorgram showing the binding ratio between AF.2A1 polypeptide and C-terminal deletion variant CH3 domain.
  • (A), (B), (C), (D), (E), (F), and (G) are respectively C-terminal 2, 3, 4, 5, 10, 15, Alternatively, the binding ratio between the CH3 domain with 20 residues deleted and AF.2A1 polypeptide is shown.
  • FIG. 3 (H) shows the binding ratio between the CH3 domain and AF.2A1 polypeptide.
  • FIG. 1 shows the binding ratio between the CH3 domain and AF.2A1 polypeptide.
  • FIG. 5 shows a sensorgram showing the binding affinity between AF.2A1 polypeptide and the CH3 domain with the C-terminal 5 residues deleted, and (B) gave acid stress to AF.2A1 polypeptide.
  • a sensorgram representing the binding affinity of the Fc region is shown.
  • FIG. 5 (A) is a graph showing the storage stability of the binding response to AF.2A1 of the CH3 domain from which the C-terminal 5 residues have been deleted.
  • FIG. 5 is a graph showing the storage stability of the binding response to AF.2A1 of human monoclonal IgG1 that has been non-naturally structured by acid treatment.
  • (A) is an autocorrelation function obtained by evaluating the time change of the particle size of the CH3 domain from which the C-terminal 5 residues are deleted by the dynamic light scattering method.
  • (B) is an autocorrelation function obtained by evaluating the time change of the particle size of human monoclonal IgG1 non-naturally structured by acid treatment by a dynamic light scattering method.
  • a calibration curve is shown with the binding response to AF.2A1 polypeptide on the vertical axis and the concentration of the C-terminal 5-residue deletion domain on the horizontal axis.
  • (A) is a calibration curve prepared using the C-terminal 5-residue deletion domain on day 0 of storage.
  • ⁇ represents a C-terminal 5-residue deletion domain, and ⁇ represents acid stress IgG1 as a measurement sample.
  • (B) is a calibration curve prepared using the C-terminal 5-residue deletion domain on the first day of storage.
  • (C) is a calibration curve prepared using acid stress IgG1 on storage 0 and day 1. ⁇ represents the day 0 of storage, and ⁇ represents the acid stress IgG1 on the first day of storage.
  • ITC isothermal titration type calorimetry
  • the present invention provides a CH3 domain in which amino acids are deleted within the range of 5 to 20 residues from the C-terminal side (C-terminal deleted CH3 domain: C-terminal truncated CH3 domain).
  • CH3 domain refers to a region located at the C-terminus of a heavy chain, which is one of the polypeptides forming immunoglobulin G (IgG).
  • IgG immunoglobulin G
  • human IgG there are four types of subclasses IgG1, IgG2, IgG3, and IgG4, and each is identified as a polypeptide having the amino acid sequence set forth in SEQ ID NOs: 1 to 4.
  • the CH3 domain has the property of forming a homodimer by noncovalent bonding. Unless otherwise stated, the CH3 domain refers to a molecule that has formed a homodimer.
  • a similar region is also known as a CH3 domain in IgG derived from mammals other than humans.
  • the CH3 domain of IgG derived from mouse, rat, or rabbit has amino acid sequences described in SEQ ID NOs: 5 to 7, respectively. Is identified as a polypeptide having It is generally accepted that different CH3 domains from different origins retain similar conformations regardless of amino acid sequence homology.
  • RMSD mean square deviation
  • ⁇ -carbon which is one of the indexes representing the similarity of the three-dimensional structure
  • the mean square deviation with respect to the CH3 domain derived from human is the CH3 domain derived from mouse IgG, rat IgG, and rabbit IgG.
  • RMSD of a human IgG-derived CH3 domain and a mouse IgG, rat IgG, or rabbit IgG-derived CH3 domain is shown below.
  • the C-terminal deletion CH3 domain of the present invention retains at least amino acid residues 1 to 87 from the N-terminal side in the amino acid sequence, and further retains 0 to 15 amino acid residues according to the amino acid sequence of the CH3 domain. To do.
  • the residue further retains 15 residues, in other words, the CH3 domain that retains the 1st to 102nd amino acid residues in the amino acid sequence of the CH3 domain. is there.
  • the amino acid when there is no addition / deletion / insertion at the C-terminal of the CH3 domain, the amino acid is deleted in the range of 5 to 20 residues from the C-terminal side.
  • the amino acid Preferably within the range of 5-15 residues (5,6,7,8,9,10,11,12,13,14, or 15 residues) (eg 6-20, 7-20, 6-19) 7-19, etc., and may be in the range of 5-15, more preferably in the range of 5-10 residues (eg, 6-10, 7-10, 6-9, 7-9, etc., 5-5
  • the number of amino acids may be in the range of 10).
  • it is a CH3 domain from which 5 amino acids have been deleted from the C-terminal side.
  • the deletion from the C-terminal side is described in the notation.
  • the amino acid sequence is deleted within the range of 5 to 20 residues from the C-terminal side of the amino acid sequence, thereby imitating the unnatural three-dimensional structure of the denatured antibody in the CH3 domain.
  • amino acids are deleted at less than 5 residues from the C-terminal side, a specific unnatural three-dimensional structure is not formed, and deletion at more than 20 residues is not preferable because it results in a molecule with extremely low structural stability.
  • the amino acid residue at the C-terminus of the CH3 domain and the range of the 20th amino acid residue counted from the amino acid residue ( In other words, it means that the amino acid is deleted at the 88th to 107th amino acid residues in the CH3 domain amino acid sequence or the 430th to 449th amino acid residues in the heavy chain amino acid sequence.
  • the amino acid number of the heavy chain is based on the amino acid sequence described in SEQ ID NO: 8.
  • denatured antibody refers to an antibody whose whole structure or partial structure has been changed by stress due to acid, alkali, heating, freeze-thawing, stirring, or the like. In addition, it refers to the structural state after the whole structure or a part of the structure is changed by applying stress to the denatured antibody, and is called a non-natural three-dimensional structure.
  • the denatured antibody can be an aggregate composed of a plurality of denatured antibodies in which an aggregate is formed starting from a structural change to a non-natural three-dimensional structure.
  • the term “denatured antibody” includes aggregates composed of a plurality of such denatured antibodies.
  • the modified antibody is not particularly limited as long as it contains a CH3 domain having a non-natural three-dimensional structure.
  • immunoglobulin isotypes IgG, IgA, IgD, IgE, IgM, etc.
  • examples of human immunoglobulins include nine classes of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM.
  • animal species from which the denatured antibody is derived and examples include antibodies derived from humans, mice, rats, rabbits, goats, and the like, and antibodies derived from humans are particularly preferred.
  • the denatured antibody is not limited to an antibody derived from such a single animal species, and may be an antibody formed by binding fragments of antibodies derived from different animal species.
  • examples of such antibodies include chimeric antibodies (human antibody Fc region and sequences derived from other animals (for example, mouse antibody, rat antibody, rabbit antibody, goat antibody) (variable region, CDR, etc.) Or a humanized antibody.
  • the antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody. Further, it may be a chimeric protein containing an Fc region.
  • the C-terminal deleted CH3 domain of the present invention mimics the non-natural three-dimensional structure of the denatured antibody in its CH3 domain
  • the CH3 domain of the denatured antibody caused by acid stress Mimics the non-natural three-dimensional structure produced in
  • the non-natural three-dimensional structure refers to a modification of the three-dimensional structure generated in the CH3 domain by acid stress.
  • the acid treatment is not limited, but preferably means exposure under conditions of pH 4.0 or lower, more preferably pH 1.5 to 2.0.
  • immunoglobulin G forms a non-natural structure called Alternative folded state (AFS) that is different from the normal natural structure by treatment with 10 mM Glycine-HCl, 150 mM NaCl, pH 2.0 buffer.
  • AFS Alternative folded state
  • the C-terminal deleted CH3 domain of the present invention mimics the non-natural three-dimensional structure generated in the CH3 domain of a denatured antibody
  • a probe that specifically recognizes the non-natural three-dimensional structure of the denatured antibody has Means binding specifically to the missing CH3 domain.
  • the “probe that specifically recognizes a non-natural three-dimensional structure” refers to a probe that specifically recognizes a non-natural three-dimensional structure generated in a denatured antibody, and particularly includes AF.2A1 polypeptide. Can do.
  • the AF.2A1 polypeptide when the C-terminal deleted CH3 domain of the present invention mimics the non-natural conformation that occurs in the CH3 domain of a denatured antibody, the AF.2A1 polypeptide is against the C-terminal deleted CH3 domain. It means to combine stoichiometrically. Specifically, it means that a state in which one molecule of AF.2A1 polypeptide binds to one molecule of C-terminal deleted CH3 domain is retained. In order for the C-terminal deleted CH3 domain to retain such properties, the binding interface with the AF.2A1 polypeptide must be formed equally on all molecules, and the binding interface is not impaired by aggregate formation or the like. Two points are important.
  • the modified antibody obtained by treatment with acid, alkali, heating, freezing and thawing, stirring, etc. with respect to the C-terminal deleted CH3 domain of the present invention is generally difficult to show stoichiometric binding. The reason is that it is first mixed with a natural non-denatured antibody and is estimated to be about several tens of percent of the whole antibody molecule. In addition, denatured antibodies form aggregates, and some antibody molecules are buried within the aggregates and are therefore not supplemented by AF.2A1 polypeptide.
  • the ratio between the non-denatured antibody and the denatured antibody, and the ratio of the molecule denatured within the aggregate among the denatured antibodies varies greatly depending on the conditions of the denaturation treatment, the storage conditions after the denaturation treatment, and the storage period. These properties make it difficult to control the stoichiometric ratio of the binding between the denatured antibody obtained by the denaturation treatment and the denatured antibody recognition probe.
  • AF.2A1 polypeptide is known and disclosed in, for example, pamphlet of International Publication No. 2014/115229. As described above, “AF.2A1 polypeptide” has been reported to have a specific binding activity to an Fc region that has formed a non-natural structure, and the present inventors have also reported that it is within the CH3 domain. It has been found that it has a specific binding activity to a non-natural three-dimensional structure. In the present specification, “AF.2A1 polypeptide” has, in addition to the above-mentioned activity, an activity of binding to an aggregate comprising an antibody or Fc region-containing protein having an unnatural three-dimensional structure. In addition, AF.2A1 polypeptide has binding activity to an antibody having an unnatural three-dimensional structure or an aggregate containing an Fc region-containing protein, but does not bind to a natural human antibody Fc region.
  • AF.2A1 polypeptide can be identified by the amino acid sequence set forth in SEQ ID NO: 9.
  • the AF.2A1 polypeptide can also be specified by a base sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 9.
  • AF.2A1 polypeptide consists of a polypeptide encoded by the base sequence set forth in SEQ ID NO: 10.
  • AF.2A1 polypeptide includes its analogs.
  • An analog of AF.2A1 is composed of a polypeptide similar to AF.2A1 polypeptide, and has the same function as that of AF.2A1 polypeptide (ie, a CH3 domain having a non-natural three-dimensional structure or an antibody containing the CH3 domain) Or a polypeptide having a binding activity to an aggregate formed from a fragment thereof.
  • the analog of AF.2A1 polypeptide is one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 9 as long as the binding activity to the CH3 domain having a non-natural conformation is not impaired.
  • severeal amino acids refers to, for example, 1 to 8, 1 to 6, preferably 1 to 3, more preferably 1 or 2 amino acids.
  • the number of amino acid residues in the polypeptide sequence is not limited as long as it includes the amino acid sequence set forth in SEQ ID NO: 9 and has the above binding activity.
  • Specific examples of analogs of AF.2A1 polypeptide include, but are not limited to, those consisting of the amino acid sequences set forth in SEQ ID NOs: 11 to 33, for example.
  • the antibody from which the C-terminal deleted CH3 domain of the present invention is derived is not particularly limited and includes, for example, immunoglobulin isotypes (IgG, IgA, IgD, IgE, IgM, etc.) and subclasses of those isotypes.
  • immunoglobulin isotypes IgG, IgA, IgD, IgE, IgM, etc.
  • the animal species of the antibody from which the CH3 domain is derived is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, goats, and the like, and antibodies derived from humans are particularly preferable.
  • the C-terminal deleted CH3 domain of the present invention can be specified by the amino acid sequences shown in SEQ ID NOs: 1-7.
  • the C-terminal deleted CH3 domain of the present invention can be identified in one embodiment as follows: (I) a C-terminal deletion CH3 domain (C) consisting of an amino acid sequence in which amino acids are deleted within the range of 5 to 20 residues from the C-terminal side in the amino acid sequence shown in any of SEQ ID NOs: 1 to 7 -terminal truncated CH3 domain), or (Ii) in the amino acid sequence in which one or several amino acids of the amino acid sequences shown in SEQ ID NOs: 1 to 7 are substituted, deleted, inserted, and / or added, 5 from the C-terminal side
  • a C-terminal deleted CH3 domain consisting of an amino acid sequence lacking amino acids within the range of ⁇ 20 residues and specifically binding to AF.2A1 polypeptide.
  • the number is, for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6 More preferably, it is 2 to 3.
  • the C-terminal deletion CH3 domain of the present invention has a C-terminal amino acid deleted in a specific number of residues in the amino acid sequence shown in any of SEQ ID NOs: 1 to 7. “Addition of several amino acids” does not include addition of the amino acid sequence to the C-terminus.
  • the C-terminal deletion CH3 domain of the present invention does not exclude that an additional sequence including a purification tag such as a His tag is added to the N-terminal or C-terminal separately from the amino acid mutation.
  • AF.2A1 polypeptide specifically binds means that AF.2A1 polypeptide specifically binds to the non-natural three-dimensional structure of the denatured antibody in a solution containing the denatured antibody and the non-denatured antibody. Similarly, it specifically refers to binding to the C-terminal deleted CH3 domain in the presence of a non-denatured antibody.
  • the C-terminal deleted CH3 domain of the present invention binds AF.2A1 with a dissociation constant of 10 ⁇ M or less. It binds with a dissociation constant of preferably 1 ⁇ M or less, more preferably 100 ⁇ M or less.
  • the present invention provides a CH3 domain in which amino acids are deleted within the range of 5 to 20 residues from the C-terminal side as a polypeptide that mimics the non-natural three-dimensional structure of a denatured antibody.
  • This C-terminal deleted CH3 domain can be prepared according to a known method.
  • a strategy for designing a polypeptide that mimics such a denatured antibody it is effective to add a mutation to the amino acid sequence. That is, a non-natural three-dimensional structure can be stably realized by adding deletion / insertion / substitution to a part of the amino acid sequence of an antibody. However, when adding such mutations, care must be taken so that the stability of the whole molecule is not impaired.
  • the binding response to a probe is evaluated.
  • ELISA surface plasmon resonance
  • BLI biolayer interferometry
  • SPR has established a method for determining the concentration of binding molecules present in a sample solution without using a standard substance (Calibration-free concentration analysis (CFCA)).
  • CFCA is a powerful technique for determining the concentration of an active substance in a sample containing contaminating molecules (Pascale et al., Anal Biochem. 1997 1997 Jul 1; 249 (2): 165-73.).
  • CFCA By using CFCA, it is possible to evaluate the ratio of polypeptides that mimic denatured antibodies. Specifically, by determining the concentration of the molecule that retains the structure imitating the modified antibody that binds to the probe from CFCA, and dividing by the concentration determined from the absorbance etc., evaluate the ratio of the polypeptide that mimics the modified antibody can do.
  • DLS dynamic light scattering
  • Another embodiment of the present invention provides an Fc region fragment or antibody comprising the C-terminal deleted CH3 domain of the present invention described above. Regions other than the Fc region and the C-terminal deleted CH3 domain that constitutes the antibody can be derived from the same animal species or isotype (including subclasses) from the C-terminal deleted CH3 domain, or from different animal species , May be isotype (including subclass). As used herein, an antibody may be a fragment thereof as long as it contains a CH3 domain.
  • Another aspect of the present invention provides a protein comprising the C-terminal deleted CH3 domain of the present invention described above.
  • the protein is not particularly limited as long as it contains a C-terminal deleted CH3 domain and can specifically bind to AF.2A1 polypeptide.
  • Specific examples of the protein of the present invention include, for example, an enzyme for detection (for example, luciferase, ascariphosphatase, horseradish peroxidase, etc.), tag protein (for example, GFP, MBP, GST, thioredoxin tag, etc.), antibody fragment, etc.
  • an enzyme for detection for example, luciferase, ascariphosphatase, horseradish peroxidase, etc.
  • tag protein for example, GFP, MBP, GST, thioredoxin tag, etc.
  • antibody fragment etc.
  • proteins in which eg, Fab, Fab ′, (Fab ′) 2, scFv, diabody, dsFv, etc
  • the present invention includes a kit for evaluating a modified antibody.
  • the kit for evaluating a denatured antibody of the present invention comprises the above-mentioned C-terminal deleted CH3 domain, or an Fc region fragment, antibody or protein containing the C-terminal deleted CH3 domain.
  • the evaluation kit may contain other reagents and instruments used for evaluating the modified antibody.
  • the evaluation kit of the present invention further comprises an AF.2A1 polypeptide.
  • the present invention provides a method for quantitatively measuring a denatured antibody contained in a sample.
  • the method for measuring a denatured antibody of the present invention comprises a step of measuring the amount of binding between the AF.2A1 polypeptide and the denatured antibody contained in the sample, the AF.2A1 polypeptide, and the C-terminal deleted CH3 domain described above.
  • the step of measuring the binding amount of the Fc region fragment, antibody or protein containing the C-terminal deletion CH3 domain, and the binding amount of the C-terminal deletion CH3 domain, Fc region fragment or antibody and AF.2A1 Calculating the amount of denatured antibody in the sample.
  • the method for measuring the amount of binding between the AF.2A1 polypeptide and the denatured antibody contained in the sample is not particularly limited, and can be performed according to a known method. For example, it can be measured using a surface plasmon resonance method (SPR) using a flow cell to which an AF.2A1 polypeptide is immobilized.
  • SPR surface plasmon resonance method
  • the amount of binding between the AF.2A1 polypeptide and the C-terminal deleted CH3 domain or the Fc region fragment or antibody containing the C-terminal deleted CH3 domain can be measured in the same manner.
  • the amount of denatured antibody To calculate the amount of denatured antibody, create a calibration curve from the measurement results of the amount of binding between AF.2A1 polypeptide and C-terminal deleted CH3 domain or Fc region fragment containing C-terminal deleted CH3 domain or antibody. And can be determined using the calibration curve. That is, the amount of the denatured antibody can be determined by comparing the obtained calibration curve with the measured value of the binding amount of the denatured antibody contained in the AF.2A1 polypeptide and the sample.
  • “quantitatively measuring a denatured antibody” means obtaining a conversion value for a calibration curve using the C-terminal deleted CH3 domain of the present invention as a reference substance.
  • the present invention provides a method for screening a compound having affinity for a denatured antibody.
  • the screening method of the present invention comprises the step of measuring the interaction between the above-mentioned C-terminal deleted CH3 domain or the Fc region fragment or antibody containing the C-terminal deleted CH3 domain and the test compound.
  • the method for measuring the interaction between the C-terminal deleted CH3 domain and the like and the test compound is not particularly limited, and a known method can be used.
  • a known method can be used.
  • SPR surface plasmon resonance method
  • a compound that interacts with the C-terminal deleted CH3 domain, etc. by the screening method of the present invention has an affinity for the denatured antibody.
  • Candidate compound Since the C-terminal deleted CH3 domain mimics the non-natural three-dimensional structure of a denatured antibody, a compound that interacts with the C-terminal deleted CH3 domain, etc. by the screening method of the present invention has an affinity for the denatured antibody.
  • the present invention provides a reference reagent (reagent composition) for quantitative determination of a denatured antibody.
  • the reference reagent contains the C-terminal deleted CH3 domain of the present invention or an Fc region fragment or antibody or protein containing the C-terminal deleted CH3 domain as a reference substance.
  • the C-terminal deleted CH3 domain, Fc region fragment or antibody or protein contained is homogeneous, stable, does not cause association / aggregation that interferes with quantitative evaluation of denatured antibodies, and is a probe AF.2A1 By specifically binding to the polypeptide, it functions as a reference substance in quantitative evaluation of the denatured antibody.
  • the C-terminal deleted CH3 domain, Fc region fragment or antibody may be modified for use as a reference substance.
  • Substances used for modification include histidine tags, fluorescent organic compounds such as biotin and fluorescein, stable isotopes, phosphate groups, acyl groups, amide groups, ester groups, epoxy groups, polyethylene glycol (PEG), lipids, sugars
  • fluorescent inorganic compounds such as chains, nucleic acids and quantum dots, and gold colloids.
  • Example 1 Identification of domain recognized by AF.2A1 polypeptide
  • the domain recognized by the AF.2A1 polypeptide was identified by affinity evaluation using biolayer interferometry (BLI method).
  • the BLI method is a method for measuring specific interactions between biopolymers, and is recognized as an excellent method that can stably evaluate samples that can form aggregates. Is.
  • the present inventor uses E. coli as a host for the CH3 domain of human IgG1 consisting of the amino acid sequence shown in SEQ ID NO: 34 having a histidine tag at the N-terminus and the CH3 domain of human IgG1 consisting of the amino acid sequence shown in SEQ ID NO: 35.
  • E. coli prepared using a large expression system and purified using nickel chelate affinity chromatography and size exclusion chromatography. As a result, purified samples containing CH2 domain or CH3 domain were obtained.
  • Acid stress was applied by dialyzing 3 mL of each purified sample diluted to 1 mg / mL against 300 mL of acid-treated buffer (100 mM glycine-hydrochloric acid (pH 2.0)) overnight. Next, add 1.9 mL of neutralization buffer (10 mM citrate-sodium hydroxide buffer (pH 6.0), 150 mM sodium chloride, 0.05% (v / v) Tween 20) to 50 ⁇ g / mL for each 100 L dialysis sample. The sample containing CH2 domain or CH3 domain subjected to acid stress was obtained.
  • neutralization buffer 10 mM citrate-sodium hydroxide buffer (pH 6.0), 150 mM sodium chloride, 0.05% (v / v) Tween 20
  • AF.2A1 polypeptide with biotin added at the N-terminal was immobilized on biosensor SA (Pall Fortebio) having streptavidin on the sensor surface via the streptavidin-biotin interaction.
  • biosensor SA Pall Fortebio
  • the affinity between acid-stressed CH2 or CH3 domain and AF.2A1 polypeptide was measured by BLI.
  • BLI was measured using Octet RED 96 (Pall Fortebio) at a reaction temperature of 30 ° C. The observation results were analyzed with Octet Data Analysis 8.0.
  • the amino acid sequence from the N-terminal side of the CH3 domain of human IgG1 to the 106th and subsequent amino acids is the second residue
  • the 105th and subsequent amino acids (the N-terminal side of the heavy chain amino acid sequence 3 amino acids from the 447th and later (corresponding to the 446th and subsequent amino acids from the N-terminal side of the heavy chain amino acid sequence) 4 residues
  • 103th amino acid (from the heavy chain amino acid sequence N) 5 residues from the 445th amino acid from the end side
  • 93th amino acid (from the heavy chain amino acid sequence) 15 amino acids from the N-terminal side of the sequence (corresponding to the 435th and later) or 20 residues after the 88th amino acid (corresponding to the 430th and later from the heavy chain amino acid sequence) Body (herein
  • the SPR method has been recognized as an excellent method that can measure specific interactions between biopolymers over time and can be quantitatively interpreted. It is recognized as an excellent method for measuring the concentration of molecules.
  • Biacore T200 (GE Health care) was used for SPR measurement.
  • AF.2A1 polypeptide was immobilized on the flow cell of sensor chip CM5 (GE Health care) using an amide coupling reaction.
  • As the running buffer HBS-P (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.05% v / v Surfactant P20) was used, and measurement was performed at a reaction temperature of 25 ° C.
  • the C-terminal deletion variant of the CH3 domain was prepared in the same manner as the CH3 domain.
  • a sample diluted with a running buffer to 0.1 mg / mL based on the absorbance and further diluted to a final concentration of 50 nmol / L was subjected to the experiment.
  • the flow rate was measured at three points of 5, 20, and 100 ⁇ L / min (FIG. 3).
  • Biacore T200 evaluation software version 2.0 was used for analysis of observation results. No binding response was obtained in the 2, 3, or 4 residue deletion mutants, and no binding response was obtained in the 5, 10, 15, or 20 residue deletion mutants.
  • the ratio obtained by dividing the concentration of the obtained binding molecule by the C-terminal deletion variant concentration of the CH3 domain in the sample calculated based on the absorbance is the 5, 10, 15, 20 residue deletion mutant. They were 92%, 84%, 36%, and 36%, respectively (Table 2). In the CH3 domain, the response was below the lower limit of quantification. From the above results, it was shown that the CH3 domain from which 5 to 20 C-terminal amino acid residues were deleted binds to AF.2A1. Further, in this example, a variant in which 5 residues were deleted was most suitable as a molecule that mimics a denatured antibody.
  • Example 3 Evaluation of the affinity of a 5-residue deleted CH3 domain for AF.2A1 polypeptide
  • the affinity of 5-residue deleted CH3 domain and AF.2A1 polypeptide was evaluated by SPR method.
  • Biacore T200 GE Health care
  • a 5-residue-deleted CH3 domain was immobilized on a flow cell of a sensor chip CM5 (GE Health care) using an amide coupling method.
  • a sensor chip was prepared by adjusting an Fc region subjected to acid stress under the same conditions as in the acid treatment described in Example 1 and immobilizing the acid stress Fc region.
  • HBS-P 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.05% v / v Surfactant P20
  • measurement was performed at a reaction temperature of 25 ° C.
  • the analyte sample was prepared by dissolving AF.2A1 polypeptide in a running buffer solution and preparing it from 200 nmol / L to 12.5 nmol / L in a dilution series with a common ratio of 2 (FIG. 4).
  • the dissociation constant was calculated using Biacore T200 evaluation software version 2.0.
  • the binding kinetic parameters (dissociation constant, binding rate constant, dissociation rate constant) in the 5-residue-deleted CH3 domain and the acid stress Fc region were comparable. It was confirmed that the 5-residue-deleted CH3 domain retained the same non-natural three-dimensional structure as the Fc region to which acid stress was added (Table 3).
  • a 5-residue deleted CH3 domain was prepared in the same manner as described in Example 2, and diluted with HBS-P (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.05% v / v Surfactant P20) was adjusted to 1 mg / mL.
  • Acid stress IgG1 was subjected to acid stress by adding 100 mL of acid-treated buffer (100 mM glycine-hydrochloric acid (pH 2.0)) to 1 mL of commercially available monoclonal IgG1 antibody (10 mg / mL) and dialyzing overnight. .
  • Biacore T200 (GE Health care) was used for SPR measurement.
  • AF.2A1 polypeptide was immobilized on the flow cell of sensor chip CM5 (GE Health care) using the amide coupling method.
  • As the running buffer HBS-P was used, and measurement was performed at a reaction temperature of 25 ° C.
  • the analyte sample was prepared by dissolving a 5-residue-deleted CH3 domain stored in 0, 1, 3, or 5 days or acid stress IgG1 in a running buffer to 1 ⁇ g / mL (FIG. 5). .
  • the binding response was calculated using Biacore T200 evaluation software version 2.0.
  • the binding of the 5-residue-deleted CH3 domain to the AF.2A1 polypeptide was not significantly changed in the 5 days after storage.
  • acid stress IgG a significant decrease in binding was observed between the 0-day storage sample and the 1-day storage sample.
  • the 5-residue deleted CH3 domain was shown to be more conserved in the binding response to AF.2A1 polypeptide compared to acid stressed IgG1.
  • Example 5 Stability of molecular size of 5-residue-deleted CH3 domain, comparison with acid stress IgG1
  • the 5-residue-deleted CH3 domain and acid stress IgG1 were stored under low temperature conditions (4 ° C.), and the change in molecular size over time was evaluated by DLS. It is recognized that DLS is an excellent method capable of measuring molecules with sizes from protein monomers (1-10 nm) to aggregates ( ⁇ 1000 nm).
  • a 5-residue-deleted CH3 domain and acid stress IgG1 were prepared in the same manner as described in Example 4. ZetaSizer Nano S (Malvern) was used for the measurement of DLS. Measurement was performed at a measurement temperature of 30 ° C. The 5-residue-deleted CH3 domain and acid stress IgG1 were subjected to the experiment with 1 mg / mL. For the analysis of the analysis results, an autocorrelation function was calculated using ZetaSizer softwear version 7.02 (FIG. 6). As a result, no significant change was observed in the molecular size of the 5-residue-deleted CH3 domain over 5 days after storage. On the other hand, acid stress IgG1 was observed to increase significantly in molecular size in samples stored for 1 day. The 5-residue-deleted CH3 domain was shown to have higher storage stability in molecular size compared to acid stress IgG1.
  • the average particle size of acid stress IgG1 stored for 1, 3 and 5 days was calculated to be 2.1, 4.4 and 4.4 ⁇ m, respectively.
  • the reason why the binding response to the AF.2A1 polypeptide was remarkably decreased is considered to be because the binding to the ligand on the sensor chip was inhibited by steric hindrance.
  • Example 6 Quantification of denatured antibody using 5-residue-deleted CH3 domain as a reference substance
  • a calibration curve is prepared by using a 5-residue-deleted CH3 domain or acid stress IgG1 as a reference substance, taking the reference substance concentration on the horizontal axis, and the binding response to the AF.2A1 polypeptide on the vertical axis.
  • An example of quantifying the denatured antibody concentration is shown below.
  • AF.2A1 polypeptide was immobilized on the flow cell of sensor chip CM5 (GE Health care) using the amide coupling method.
  • HBS-P 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.05% v / v Surfactant P20
  • measurement was performed at a reaction temperature of 25 ° C.
  • a 5-residue-deleted CH3 domain and acid stress IgG1 were prepared in the same manner as described in Example 4. Samples stored for 0 day and 1 day were used for the experiment.
  • the analyte sample for the calibration curve should be prepared by dissolving 5-residue-deleted CH3 domain or acid stress IgG1 in running buffer to 1, 0.3, 0.1, 0.03, 0.01 ⁇ g / mL.
  • the binding response was calculated using Biacore T200 evaluation software version ⁇ 2.0 (Fig. 7).
  • Fig. 7 (A) shows the calibration curve created using the measurement results of the 5-residue-deleted CH3 domain stored on day 0.
  • the calibration curve generated using the measurement results of the 5-residue-deleted CH3 domain stored on day 1 As shown in FIG. Similarly, a calibration curve created using the measurement results of acid stress IgG1 (0-day storage and 1-day storage) is shown in FIG.
  • the concentration of the denatured antibody contained in the sample is quantified as follows. Assume that the binding response of an unknown sample measured using the sensor chip is 200 RU. When this measurement value is applied to a calibration curve created using the measurement results of the 5-residue-deleted CH3 domain stored on day 0, the denatured antibody concentration in the sample is 0.205 ⁇ g / mL in terms of 5-residue-deleted CH3 domain. Is required.
  • the concentration of the denatured antibody in the sample is calculated to be 0.172 ⁇ g / mL in terms of 5-residue-deleted CH3 domain.
  • the concentration of denatured antibody in the sample was It is calculated as 0.910 ⁇ g / mL in terms of stress IgG1.
  • the measurement result of acid stress IgG1 stored for 1 day when used, the measurement result does not fall within the calibration curve range, and the extrapolated value is 8.18 ⁇ g / mL.
  • the measurement results of unknown samples can be treated as highly reproducible quantitative values compared to when acid-treated IgG1 is also used as a reference substance. It becomes possible.
  • Example 7 Affinity evaluation of 5-residue deleted CH3 domain and AF.2A1 polypeptide
  • the affinity of 5-residue deleted CH3 domain and AF.2A1 polypeptide was evaluated by isothermal titration calorimetry (ITC) method. Nano ITC (TA Instruments) was used for the measurement of ITC.
  • the solvent substitution of 1 mL of 5-residue-deleted CH3 domain was performed by overnight dialysis using 100 mL of HBS (10 mM HEPES-NaOH pH 7.4, 150 mM NaCl) as an external solution. Thereafter, the concentration was adjusted to 200 ⁇ mol / L using HBS as an external solution.
  • the AF.2A1 polypeptide was dissolved to 30 ⁇ mol / L using HBS as an external solution.
  • the 5-residue-deleted CH3 domain filled in the syringe was dropped stepwise on the AF.2A1 polypeptide filled in the cell, and the heat of reaction accompanying the binding was measured.
  • the reaction temperature was measured at 25 ° C.
  • the heat of dilution of the 5-residue deleted CH3 domain was measured by measurement using a cell filled with HBS instead of AF.2A1 polypeptide.
  • the heat of reaction associated with the binding minus the heat of dilution was used for the analysis.
  • the CH3 domain provided by the present invention can be used for quantitative evaluation of denatured antibodies in the quality control technology of biopharmaceuticals, particularly antibody pharmaceuticals. Specifically, it is assumed that it is used as a reference substance in a denatured antibody detection device (for example, affinity-based detection using denatured antibody recognition probe AF.2A1 developed by AIST Biomedical RI Molecular Cell Breeding RG Application to equipment).
  • a denatured antibody detection device for example, affinity-based detection using denatured antibody recognition probe AF.2A1 developed by AIST Biomedical RI Molecular Cell Breeding RG Application to equipment.
  • the CH3 domain provided by the present invention can be used in a screening method for obtaining a compound having a recognition mechanism similar to AF.2A1 (for example, a low molecular compound or an oligonucleic acid).
  • Sequence number 9 Chemical synthesis sequence number 10: Chemical synthesis sequence number 11: Chemical synthesis sequence number 12: Chemical synthesis sequence number 13: Chemical synthesis sequence number 14: Chemical synthesis sequence number 15: Chemical synthesis sequence number 16: Chemical synthesis sequence number 17: Chemical synthesis sequence number 18: Chemical synthesis sequence number 19: Chemical synthesis sequence number 20: Chemical synthesis sequence number 21: Chemical synthesis sequence number 22: Chemical synthesis sequence number 23: Chemical synthesis sequence number 24: Chemical synthesis sequence number 25: Chemical synthesis sequence number 26: Chemical synthesis sequence number 27: Chemical synthesis sequence number 28: Chemical synthesis sequence number 29: Chemical synthesis sequence number 30: Chemical synthesis sequence number 31: Chemical synthesis sequence number 32: Chemical synthesis sequence number 33: Chemical synthesis SEQ ID NO: 34: Chemical synthesis SEQ ID NO: 35: Chemical synthesis

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Abstract

L'invention fournit une molécule qui ressemble à une structure tridimensionnelle non naturelle appartenant à un anticorps modifié, et qui peut être mise en œuvre dans l'évaluation quantitative d'un anticorps modifié. De manière concrète, l'invention fournit un domaine CH3 à délétion C-terminale défini par (i) ou (ii). (i) : un domaine CH3 à délétion C-terminale qui est constitué d'une séquence d'acides aminés présentant une délétion des acides aminés à l'intérieur d'une plage de 5 à 20 résidus depuis un côté terminaison C, dans une séquence d'acides aminés représentée par dans l'une des séquences de SEQ ID NOS : 1 à 7. (ii) : un domaine CH3 à délétion C-terminale qui est constitué d'une séquence d'acides aminés présentant une délétion des acides aminés à l'intérieur d'une plage de 5 à 20 résidus depuis un côté terminaison C, et auquel est lié de manière spécifique un polypeptide AF.2A1, dans une séquence d'acides aminés représentée par dans l'une des séquences de SEQ ID NOS : 1 à 7.
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WO2021193870A1 (fr) * 2020-03-26 2021-09-30 デンカ株式会社 Réactif contenant un anticorps ayant une délétion partielle de région fc

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WO2014115229A1 (fr) * 2013-01-28 2014-07-31 独立行政法人産業技術総合研究所 Peptide liant les anticorps

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2021193870A1 (fr) * 2020-03-26 2021-09-30 デンカ株式会社 Réactif contenant un anticorps ayant une délétion partielle de région fc
EP4130032A4 (fr) * 2020-03-26 2024-03-13 Denka Company Ltd Réactif contenant un anticorps ayant une délétion partielle de région fc

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