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IgG binding peptide

外国特許コード F130007492
掲載日 2013年7月10日
出願国 アメリカ合衆国
出願番号 31226807
公報番号 20100297606
公報番号 8372950
出願日 平成19年11月2日(2007.11.2)
公報発行日 平成22年11月25日(2010.11.25)
公報発行日 平成25年2月12日(2013.2.12)
国際出願番号 JP2007071754
国際公開番号 WO2008054030
国際出願日 平成19年11月2日(2007.11.2)
国際公開日 平成20年5月8日(2008.5.8)
優先権データ
  • 特願2006-299566 (2006.11.2) JP
  • 2007JP071754 (2007.11.2) WO
発明の名称 (英語) IgG binding peptide
発明の概要(英語) The present invention provides a peptide capable of specifically binding to human IgG.
In particular, the present invention relates to a human IgG binding peptide tag of 11 to 16 amino acids in length, comprising at least an amino acid sequence of the formula I: C-(X)n-W-X-X-X-W-(X)m-C (I) (SEQ ID NO: 17) wherein n and m are each an integer of 1 or more and the sum n+m is 4 or 5, wherein X-X-X in the formula I contains no cysteine residue, and wherein said amino acid sequence satisfies either or both of a) and b): a) (X)n-W in the formula I denotes Za-G-Y-W (SEQ ID NO: 18); and b) W-(X)m in the formula I denotes W-G-L-Zb (SEQ ID NO: 19) wherein Za and Zb are each 0, 1, or more amino acid residues.
従来技術、競合技術の概要(英語) BACKGROUND ART
More than 10 types of antibody are currently marketed in the U.S.A. as medicaments.
Antibody drugs are attracting attention as molecular targeted drugs that can provide the highest certainty, contributing to the rapid enlargement of a field of new medicaments.
Most antibody drugs that are currently under development or in use utilize immunoglobulin G (IgG) class antibodies.
To date, an anti-Fc antibody or Protein A/G that specifically binds to the Fc region of an IgG antibody has often been used for detection or purification of an IgG antibody (Non-patent Documents 1 and 2).
However, an anti-Fc antibody prepared by hybridoma technology or animal immunization has drawbacks such that it is not only expensive, but is also easily denatured upon labeling, for example.
On the other hand, Protein A/G has drawbacks such that it is does not allow one to distinguish between IgGs derived from different organism species and it does not bind to human IgG3.
Protein A/G is generally isolated from bacteria, leading to possible contamination with an endotoxin such as LPS.
This poses a big problem upon the use of a Protein A column for antibody drug purification.
Furthermore, antibody purification using a Protein A column can also lead to a problem such that antibody denaturation takes place due to acid when the antibody is eluded from the column using an acid solution, so that the yield drastically decreases or the quality of the thus purified antibody is lowered.
It is very important to overcome such problems with antibody purification methods using an anti-IgG antibody or Protein A/G so as to develop a novel technique capable of specifically detecting and purifying human IgG.
Many IgG binding peptides that can be used for IgG purification have been reported so far.
Fassina et al., have screened synthetic multimeric peptide libraries and then identified a Protein A peptide mimic that competes with Protein A for interactions with biotin-labeled immunoglobulin (Non-patent Documents 3 and 4).
Also, Ehrlish et al., have isolated an Fc-binding peptide having a Protein A mimic sequence from an M13 phage display library displaying 12-mer or 7-mer linear peptides (Non-patent Document 5).
Krook et al., have also reported identification of a Protein A peptide analog via reaction of a random peptide fUSE5 phage library displaying linear 10-mer peptides with IgG followed by elution with the use of Protein A (Non-patent Document 6).
In 2005, Verdoliva et al., have reported a peptide motif from a cyclic synthetic peptide library, that recognizes regions near the hinge region of an IgG antibody and inhibits competitively the reaction of Fcgamma RIII with IgG Fc (Non-patent Document 7).
Moreover, Suzuki et al., have described peptides having a property of binding to IgG Fc fragments and disclosed a method for detecting IgG using the same (Patent Document 1).
However, it cannot be said that all of these peptide motifs have sufficient IgG binding affinity as tags for purification and/or detection of an IgG antibody.
Meanwhile, in 2000, DeLano et al. have reported polypeptide sequences having binding affinity for the hinge region on a human IgG Fc fragment, such as the Fc-III peptide (DCAWHLGELVWCT; SEQ ID NO: 15), as a result of the use of an M13 phage library displaying cyclic peptides (Non-patent Document 8 and Patent Document 2).
Furthermore, in 2006, Dias et al., have performed engineering so as to further stabilize the circularization of the peptide Fc-III (Non-patent Document 9).
The peptide Fc-III of Delano et al., exhibited its binding affinity with a Kd value of 185 nM for an IgG antibody, but the Kd value exhibited by the improved peptide FcBP-2 of Dias et al. decreased to approximately 2 nM.
It was thus suggested that the peptide FcBP-2 had an extremely strong binding force.
However, the dissociation rate constant, koff, of FcBP-2 was found to be 10-2 sec-1, as a result of analysis of kinetic parameters.
Such dissociation rate is significantly faster than that of general koff (10-3 to 10-5 Sec-1) of an antibody.
This indicates that even if the peptide FcBP-2 is used as a tag for purification or detection of an antibody, the dissociation rate is so fast that the bond between the peptide FcBP-2 and the antibody dissociate rapidly and the bond cannot be retained.
Accordingly, FcBP-2 is inappropriate for use in human IgG purification and/or detection.
Patent Document 3 discloses various human IgG Fc binding peptides.
However, Patent Document 3 does not disclose peptides appropriate for human IgG purification and/or detection, such as peptides capable of sufficiently retaining the bonds with any human IgG subclass and exhibiting no significant binding to IgGs of other organism species.
Patent Document 1: JP Patent Publication (Kokai) No. 2004-187563
Patent Document 2: International Publication WO01/045746 Pamphlet
Patent Document 3: International Publication WO02/086070 Pamphlet
Non-patent Document 1: Ey P. L., et al., Immunochemistry (1978) 15, p. 429-436
Non-patent Document 2: Akerstrom B., et al., J. Immunol., (1985) 135, p. 2589-2592
Non-patent Document 3: Fassina G., et al., J. Mol. Recognit. (1996) 9, p. 564-569
Non-patent Document 4: Fassina G., et al., J. Mol. Recognit. (1998) 11, p. 128-133
Non-patent Document 5: Ehrlich G. K. and Bailon P., J. Mol. Recognit. (1998) 11, p. 121-125
Non-patent Document 6: Krook M., Mosbach K., and Ramstrom O., J. Immunol. Methods (1998) 221, p. 151-157
Non-patent Document 7: Verdoliva A. et al., Chembiochem (2005) 6, p. 1242-1253
Non-patent Document 8: DeLano W. L., et al., Science (2000) 287, p. 1279-1283
Non-patent Document 9: Dias R. L., et al., J. Am. Chem. Soc. (2006) 128, p. 2726-2732

特許請求の範囲(英語) [claim1]
1. A human IgG binding peptide tag that is 11 to 16 amino acids in length, comprising at least an amino acid sequence of formula I:
C-(X)n-W-X-X-X-W-(X)m-C (I) (SEQ ID NO: 17)
wherein n and m are each an integer of 1 or more and the sum n+m is 4 or 5, wherein X-X-X in the formula I contains no cysteine residue, and
wherein said amino acid sequence satisfies either or both of a) and b): a) (X)n-W in the formula I is Za-G-Y-W (SEQ ID NO: 18); and
b) W-(X)m in the formula I is W-G-L-Zb (SEQ ID NO: 19)
wherein Za and Zb are each 0, 1, or more amino acid residues.
[claim2]
2. The peptide tag according to claim 1, wherein said amino acid sequence of the formula I satisfies both a) and b): a) (X)n-W in the formula I is Za-G-Y-W (SEQ ID NO: 18); and
b) W-(X)m in the formula I is W-G-L-Zb (SEQ ID NO: 19)
wherein Za and Zb are each 0, 1, or more amino acid residues.
[claim3]
3. The peptide tag according to claim 1, which consists of any of the amino acid sequences 1) to 11):
1) CGYWRSEWGLC;(SEQ ID NO: 1) 2) CTGFWEREWGLC;(SEQ ID NO: 2) 3) CLYWPRLWGLC;(SEQ ID NO: 3) 4) CTGYWPKAWGLC;(SEQ ID NO: 4) 5) CYWAVRWGLLGC;(SEQ ID NO: 5) 6) CGYWADVWQIHC;(SEQ ID NO: 6) 7) GCGYWRSEWGLCG;(SEQ ID NO: 7) 8) GCTGFWEREWGLCG;(SEQ ID NO: 8) 9) GCTGYWPKAWGLCG;(SEQ ID NO: 9)10) GCGYWRSQWGLCG;(SEQ ID NO: 10)and11) GCTGYWPRAWGLCG(SEQ ID NO: 11).
[claim4]
3. The peptide tag according to
-- 1) CGYWRSEWGLC; (SEQ ID NO: 1)
--
-- 2) CTGFWEREWGLC; (SEQ ID NO: 2)
--
-- 3) CLYWPRLWGLC; (SEQ ID NO: 3)
--
-- 4) CTGYWPKAWGLC; (SEQ ID NO: 4)
--
-- 5) CYWAVRWGLLGC; (SEQ ID NO: 5)
--
-- 6) CGYWADVWQIHC; (SEQ ID NO: 6)
--
-- 7) GCGYWRSEWGLCG; (SEQ ID NO: 7)
--
-- 8) GCTGFWEREWGLCG; (SEQ ID NO: 8)
--
-- 9) GCTGYWPKAWGLCG; (SEQ ID NO: 9)
--
-- 10) GCGYWRSQWGLCG; (SEQ ID NO: 10)
-- and
--
-- 11) GCTGYWPRAWGLCG (SEQ ID NO: 11).
[claim5]
4. The peptide tag according to any claim 1, wherein a disulfide bond is formed between two cysteine residues in formula I.
[claim6]
5. The peptide tag according to claim 1, which binds to acid-denatured human IgG.
[claim7]
6. A human IgG binding peptide tag that is 11 to 16 amino acids in length, comprising at least an amino acid sequence of formula I:
-- C-(X)n-W-X-X-X-W-(X)m-C (I) (SEQ ID NO: 17)
[claim8]
7. A fusion protein of the following formula: P1-C-(X)n-W-X-X-X-W-(X)m-C-P2
wherein
C-(X)n-W-X-X-X-W-(X)m-C(I)(SEQ ID NO: 17)
is a human IgG binding peptide tag that is 11 to 16 amino acids in length,
wherein n and m are each an integer of 1 or more and the sum n+m is 4 or 5, wherein X-X-X in the formula contains no cysteine residue, and
wherein said human IgG binding peptide tag satisfies either or both of a) and b): a) (X)n-W in the formula is Za-G-Y-W (SEQ ID NO: 18); and
b) W-(X)m in the formula is W-G-L-Zb (SEQ ID NO: 19)
wherein Za and Zb are each 0, 1, or more amino acid residues,
wherein either of P1 and P2 is present, but P1 and P2 are not both present at the same time,
wherein P1, when present, is bonded to the N-terminus of the human IgG binding peptide tag C-(X)n-W-X-X-X-W-(X)m-C (SEQ ID NO:17),
wherein P2, when present, is bonded to the C-terminus of the human IgG binding peptide tag C-(X)n-W-X-X-X-W-(X)m-C (SEQ ID NO:17),
and wherein P1 and P2 each represents a protein.
[claim9]
7. A fusion protein of the following formula:
-- C-(X)n-W-X-X-X-W-(X)m-C (I) (SEQ ID NO: 17)
[claim10]
8. A solid phase support, on which the peptide tag according to claim 1 is immobilized.
[claim11]
9. A method for determining the presence of human IgG or an Fc region-containing fragment thereof in a sample, comprising the steps a) to c): a) contacting a sample with acid to produce an acid-treated sample;
b) contacting the acid-treated sample with the peptide tag according to claim 1; and
c) measuring a binding affinity produced in the step b) between the peptide tag and human IgG or an Fc region-containing fragment thereof, wherein the binding affinity having a dissociation constant of 0.1 nM to 50 nM indicates the presence of human IgG or an Fc region-containing fragment thereof in a sample.
[claim12]
10. The method according to claim 9, wherein the binding affinity is measured by surface plasmon resonance analysis.
[claim13]
11. A method for purifying human IgG or an Fc region-containing fragment thereof in a sample, comprising the steps a) and b): a) contacting the peptide tag according to claim 1 with an acid-treated sample containing human IgG or an Fc region-containing fragment thereof, thereby binding human IgG or an Fc region-containing fragment thereof in the sample to the peptide tag; and
b) separating the human IgG or the Fc region-containing fragment thereof, which is bound to the peptide tag in the step a), from the sample.
[claim14]
12. A method for removing an acid-denatured human IgG or Fc region-containing fragment thereof from a sample, comprising the steps a) and b): a) contacting the peptide tag according to claim 1 with a sample containing acid-denatured human IgG or an Fc region-containing fragment thereof; and
b) removing human IgG or an Fc region-containing fragment thereof bound to the peptide tag prepared by the step a), from the sample.
[claim15]
13. A method for purifying a protein, comprising the steps a) to c): a) producing a fusion protein according to claim 7, and then preparing a sample containing the fusion protein;
b) contacting the sample prepared by step a) with acid-treated human IgG or an Fc region-containing fragment thereof, thereby binding the fusion protein to the human IgG or Fc region-containing fragment thereof; and
c) separating the fusion protein, which is bound to the human IgG or Fc region-containing fragment thereof in the step b), from the sample.
  • 発明者/出願人(英語)
  • ITO YUJI
  • KAGOSHIMA UNIVERSITY
国際特許分類(IPC)
米国特許分類/主・副
  • 530/327
  • 530/326
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