WO2013125221A1 - Ligand d'affinité pour la purification d'anticorps - Google Patents

Ligand d'affinité pour la purification d'anticorps Download PDF

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Publication number
WO2013125221A1
WO2013125221A1 PCT/JP2013/000961 JP2013000961W WO2013125221A1 WO 2013125221 A1 WO2013125221 A1 WO 2013125221A1 JP 2013000961 W JP2013000961 W JP 2013000961W WO 2013125221 A1 WO2013125221 A1 WO 2013125221A1
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amino acid
seq
sequence shown
sequence
amino
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PCT/JP2013/000961
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English (en)
Japanese (ja)
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靖人 秋山
一道 小澤
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静岡県
一般社団法人ファルマバレープロジェクト支援機構
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Publication of WO2013125221A1 publication Critical patent/WO2013125221A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments

Definitions

  • the present invention relates to a method for identifying an amino acid consensus sequence of a human antibody variable region, a method for screening a polypeptide comprising an amino acid consensus sequence of a human antibody variable region, a molecule that specifically binds to a human antibody variable region, and the screening.
  • the present invention relates to a molecule that specifically binds to a human antibody variable region obtained by the method.
  • Antibodies are widely used in biological research and clinical tests because of their high specific binding ability to specific molecules. In recent years, many attempts have been made to use antibodies themselves as therapeutic agents for treatment, and in particular, development of antibody drugs targeting cancer cells has attracted a great deal of attention.
  • Non-Patent Documents 1 to 3 Protein A is a protein derived from the cell wall of S. aureus and is known to have a strong binding ability to the Fc (constant) region of the antibody molecule. In the constant region, a common structure is conserved across the classes and subclasses of various antibody molecules. Therefore, affinity chromatography using protein A as a ligand purifies various types of antibody molecules with different antigens. Can be used.
  • Protein A is expensive and is known to be immunogenic (see Non-Patent Documents 4 and 5). Protein A shows affinity for the antibody constant region, but does not show affinity for the antibody Fv (variable) region. Therefore, a variable region such as a single chain antibody (scFv antibody) or a diabody antibody is used. It cannot be used for the purification of low-molecular-weight antibodies composed solely of. For this reason, development of an inexpensive and safe ligand that can be used for affinity purification of an antibody having no constant region has been demanded.
  • scFv antibody single chain antibody
  • diabody antibody diabody antibody
  • An object of the present invention is to provide a polypeptide comprising an amino acid consensus sequence of a human antibody variable region, a molecule that specifically binds to an antibody variable region, and a method for screening the molecule.
  • variable region that is an antigen recognition site is considered to be composed of a variety of different sequences for each antibody, and a variable region sequence common to different antibodies was not expected to exist.
  • the present inventors pay attention to amino-produced sequences of variable regions of human antibody proteins from the immune-related database [the international ImMunoGeneTics information system (IMGT)], and identify highly homologous sequences (consensus sequences) for each family. Went. That is, amino acid sequence alignment analysis was performed for each family using amino acid sequence data of human antibody variable regions, and the amino acid sequences shown in SEQ ID NOs: 1 to 24 were found to be consensus amino acid sequences of each family.
  • IMGT international ImMunoGeneTics information system
  • the present invention it is possible to screen a high affinity molecule in an antibody variable region, and to develop a safe and inexpensive ligand that can be used for purification of various antibodies including scFv antibody and diabody antibody. Can do.
  • a molecule that specifically recognizes an antibody variable region obtained by the screening method of the present invention can be used as an affinity ligand for antibody purification.
  • consensus sequences specific for each family identified this time include not only peptide libraries but also ligand screening using phage and other screening methods and affinity ligands for human antibodies such as aptamers. It is the basic target sequence in technology and is very useful.
  • FIG. 2 is a view showing a base sequence encoding the human antibody heavy chain variable region (IGHV1 to 4) consensus sequence of the present invention.
  • FIG. 2 is a view showing a base sequence encoding the human antibody heavy chain variable region (IGHV5-7) consensus sequence of the present invention. It is a figure shown about addition of CDR3 sequence to the human antibody heavy chain variable region consensus sequence of the present invention.
  • the amino acid sequence numbers in the figure follow the Kabat numbering system.
  • MBP Maltose-Binding protein
  • staining MBP fusion human antibody heavy chain variable region consensus sequence of this invention.
  • M represents a molecular weight marker
  • VH1 to 7 represent antibody heavy chain variable region consensus sequences 1 to 7
  • VK1 to 6 represent antibody kappa light chain variable region consensus sequences 1 to 6. It is a figure shown about the screening method of an affinity ligand using the phage display method of this invention. It is a figure shown about the primary screening of the peptide which has a specific binding ability to the human antibody heavy chain variable region family (IGHV3) consensus sequence of this invention. It is a figure which shows the preparation method of the peptide which has a specific binding ability to the human antibody heavy chain variable region family (IGHV3) consensus sequence of this invention.
  • VH heavy chain gene
  • VK and VL light chain gene
  • CDR1, 2 CDR3 is omitted here
  • a method for identifying an amino acid consensus sequence of a human antibody variable region for each VH and VK family and an identified amino acid consensus sequence will be described.
  • the method for identifying the amino acid consensus sequence of the human antibody variable region of the present invention includes (a) human antibody heavy chain variable region family 1 to 7, human antibody ⁇ light chain variable region family 1 to 6, and human antibody ⁇ light chain. A step of aligning amino acid sequences in each of the variable region families 1 to 11; and (b) from the alignment obtained in (a) above, determining the type of amino acid having the highest frequency at each amino acid position in the human antibody variable region.
  • the present invention also relates to a polypeptide comprising the amino acid consensus sequence thus identified, and nucleic acids (DNA and RNA) encoding such a polypeptide.
  • amino acid consensus sequence of the human antibody heavy chain variable region family 1 examples include, for example, (i) the amino acid sequence represented by SEQ ID NO: 1, and (ii) the amino acid at the second position in the amino acid sequence represented by SEQ ID NO: 1, Or M, the amino acid at position 9 is A or P, the amino acid at position 14 is P, L, or T, the amino acid at position 16 is A, S, or T, and The amino acid at position is A or V, the amino acid at position 27 is Y, G or F, the amino acid at position 29 is F or L, and the amino acid at position 30 is T, N, or S, the amino acid at position 31 is G, S, E, or Y, the amino acid at position 32 is Y, L, S, or R, and the amino acid at position 33 is Y, D, S, A , T, or G, and the amino acid at position 34 is M, I, V, or L
  • the amino acid at position 35 is H, N, S, or Q
  • the amino acid at position 38 is R,
  • the amino acid at position 59 is N, G, S, I, or K
  • the amino acid at position 61 is A or S
  • the amino acid at position 63 is K or E
  • the position 64 The amino acid at position 66 is G, E, or D
  • the amino acid at position 67 is R or W
  • the amino acid at position 70 is M, S, or I.
  • the amino acid at position 72 is R, E, A or T, the amino acid at position 73 is D or N, and the amino acid at position 74 is T, E, K, M or R;
  • the amino acid at position 76 is I, T, A, or M;
  • the amino acid at position 77 is S or D;
  • the amino acid at position 79 is A or V; and the amino acid at position 84
  • S is R or S
  • the amino acid at position 85 is R or S
  • the amino acid at position 89 is E or D;
  • amino acid consensus sequence of human antibody heavy chain variable region family 2 examples include, for example, (i) the amino acid sequence represented by SEQ ID NO: 2; (ii) the first amino acid is Q in the amino acid sequence represented by SEQ ID NO: 2, Or R, the amino acid at position 2 is I or V, the amino acid at position 5 is K or R, the amino acid at position 10 is T, A, or V, and the amino acid at position 16
  • the amino acid at position 22 is C or R; the amino acid at position 23 is T or A; the amino acid at position 24 is F or V;
  • the amino acid at position is T or N, the amino acid at position 32 is S or A, the amino acid at position 33 is G or R, and the amino acid at position 34 is V, E or M; Yes, the amino acid at position 35 is G, W, R, or C
  • the amino acid at position 36 is V, C, or A, the amino acid at position 37 is G or S, the amino acid at position 50 is L or I, and the amino acid at position 52 is L, The amino
  • amino acid consensus sequence of the human antibody heavy chain variable region family 3 examples include, for example, (i) the amino acid sequence represented by SEQ ID NO: 3; (ii) the first amino acid in the amino acid sequence represented by SEQ ID NO: 3 is Q; E or T, the second amino acid is V or D, the third amino acid is Q or H, the fifth amino acid is V or L, and the sixth amino acid is The amino acid is E or D, the seventh amino acid is S or T, the eighth amino acid is G, R, or A, and the tenth amino acid is G, V, or A
  • the amino acid at position 11 is L or V
  • the amino acid at position 12 is V or I
  • amino acid at position 13 is K, Q, R, or E
  • amino acid at position 16 is G, R or P.
  • the amino acid at position 17 is S or A
  • the amino acid at position 19 is R or K
  • the amino acid at position 20 is L or P
  • the amino acid at position 23 is A or T
  • the amino acid at position 25 is S or C
  • the amino acid at position 26 is G or A
  • the amino acid at position 28 is T or A
  • the amino acid at position 30 is S
  • the amino acid at position 31 is D, S, N, Y, or G
  • the amino acid at position 32 is Y, N, S, A, or H
  • the amino acid at position 33 Is Y, S, E, D, A, V, G, T, W, or P
  • the amino acid at position 34 is M or L
  • the amino acid at position 35 is S, N, H, or D
  • the amino acid at position 37 is I, V, F or A
  • the amino acid at position 38 is R or H
  • the amino acid at position 40 is A or P
  • amino acid consensus sequence of human antibody heavy chain variable region family 4 examples include: (i) the amino acid sequence represented by SEQ ID NO: 4; (ii) the amino acid at the second position in the amino acid sequence represented by SEQ ID NO: 4, Or L, the amino acid at position 6 is E, D, or Q, the amino acid at position 7 is S, W, or L, and the amino acid at position 9 is P, S, or A;
  • the amino acid at position 12 is V or L
  • the amino acid at position 16 is Q, D, E, or G
  • the amino acid at position 23 is T or A
  • the amino acid at position 24 is V ,
  • the amino acid at position 25 is S, T, or Y
  • the amino acid at position 29 is I, V, or F
  • the amino acid at position 30 is S
  • amino acid at position 31 is
  • the amino acid is S or G, and the amino acid at position 32 is G or S; G, S, or D is inserted next to the amino acid at position 32, the amino acid at position 33 is Y or
  • amino acid consensus sequence of human antibody heavy chain variable region family 5 examples include, for example, (i) the amino acid sequence represented by SEQ ID NO: 5; (ii) the amino acid at position 34 in the amino acid sequence represented by SEQ ID NO: 5, Or a sequence in which the amino acid at position 42 is G or R, and the amino acid at position 75 is S or P; (iii) in the amino acid sequence shown in (i) or (ii) above, Preferred examples include amino acid sequences in which one or several amino acids have been deleted, substituted, or added; the sequences shown in any of (i) to (iii).
  • amino acid consensus sequence of human antibody heavy chain variable region family 6 examples include (i) the amino acid sequence represented by SEQ ID NO: 6; (ii) the amino acid sequence represented by SEQ ID NO: 6 lacking one or several amino acids. Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • amino acid consensus sequence of human antibody heavy chain variable region family 7 examples include: (i) the amino acid sequence represented by SEQ ID NO: 7; (ii) the amino acid at position 84 in the amino acid sequence represented by SEQ ID NO: 7, Or a sequence in which the amino acid at position 85 is S or T; (iii) in the amino acid sequence shown in (i) or (ii) above, one or several amino acids are deleted, substituted, or A preferred example is a sequence represented by any one of (i) to (iii) of the added amino acid sequence.
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 1 include (i) the amino acid sequence represented by SEQ ID NO: 8; (ii) the amino acid at the first position in the amino acid sequence represented by SEQ ID NO: 8 is D , A, N, or V, the third amino acid is Q, W, or R, the fourth amino acid is M, or L, and the fifth amino acid is T, or I
  • the amino acid at position 9 is S or F
  • the amino acid at position 10 is T, S, A, L, or F
  • the amino acid at position 11 is L, M, V, or F
  • the amino acid at position 15 is V or T
  • the amino acid at position 20 is T or S
  • the amino acid at position 22 is T, I, or S
  • the amino acid at position 24 is R, Q Or W
  • the amino acid at position 25 is A, V, or M
  • the amino acid at position 27 is Q or E
  • the amino acid at position 28 is G, S, or D
  • the amino acid at position 30
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 2 include (i) the amino acid sequence represented by SEQ ID NO: 9; (ii) the amino acid at the second position in the amino acid sequence represented by SEQ ID NO: 9 is I Or V, the 7th amino acid is T or S, the 11th amino acid is L or S, the 12th amino acid is S or P, and the 17th amino acid is Is Q or E, the amino acid at position 23 is C or F, the amino acid at position 24 is K or R, the amino acid at position 30 is L or V, and position 31
  • the amino acid at position 33 is H, D, or Y, the amino acid at position 33 is D or N, D is inserted after the amino acid at position 33, and the amino acid at position 35 is K, N Or Y, and the amino acid at position 36 is T or N.
  • the amino acid at position 39 is Y, D, S, or N; the amino acid at position 40 is W or C; the amino acid at position 41 is Y, L, or F; and position 42
  • the amino acid at position 44 is K or R, the amino acid at position 48 is S or P, the amino acid at position 50 is Q or R,
  • the amino acid at position 51 is L or R, the amino acid at position 55 is E, L, T, or K, the amino acid at position 56 is V, G, L, or I, and the amino acid at position 58
  • the amino acid is S, N, or Y, the amino acid at position 59 is R or W, the amino acid at position 60 is F, A, or D, and the amino acid at position 69 is G or D
  • a sequence in which the 72nd amino acid is S or A;
  • (iii) the amino acid sequence shown in (i) or (ii) above There are one or a few amino acids are deleted, substituted, or added in the amino acid sequence; of the sequence shown in any one
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 3 include (i) the amino acid sequence represented by SEQ ID NO: 10; (ii) the amino acid at the 4th position in the amino acid sequence represented by SEQ ID NO: 10 is L Or M, the 9th amino acid is A, G, or P, the 13th amino acid is L or V, the 19th amino acid is A or V, and the 24th position.
  • the amino acid at position 28 is R or G, the amino acid at position 28 is S or G, the amino acid at position 32 is S or N, or is deleted, and the amino acid at position 33 is N, or
  • the amino acid at position 35 is A, T, or S; the amino acid at position 43 is Q or L; the amino acid at position 51 is D or G; and position 54
  • the amino acid is N, S, or T, and the 58th position is
  • the amino acid at position 61 is A or D
  • the amino acid at position 68 is S or P
  • the amino acid at position 70 is T or R
  • the amino acid at position 61 is T or R;
  • the amino acid at position 71 is D or E
  • the amino acid at position 78 is S or R
  • the amino acid at position 80 is E or Q
  • the amino acid at position 81 is P or S Any one of (i) to (iii): (iii) an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 4 include (i) the amino acid sequence represented by SEQ ID NO: 11; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 11; Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 5 include (i) an amino acid sequence represented by SEQ ID NO: 12; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 12; Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • Examples of the amino acid consensus sequence of the human antibody kappa light chain variable region family 6 include (i) the amino acid sequence represented by SEQ ID NO: 13; (ii) the first amino acid in the amino acid sequence represented by SEQ ID NO: 13 is E Or D, the second amino acid is I or V, the fourth amino acid is L or M, the ninth amino acid is D or A, and the eleventh amino acid.
  • amino acid at position 16 is K or G
  • amino acid at position 24 is R or Q
  • amino acid at position 27 is Q or E
  • position 28 The amino acid at position 31 is S or N
  • the amino acid at position 32 is S or Y
  • the amino acid at position 34 is H or Y
  • the amino acid at position 43 is S or A
  • the amino acid at position 55 A sequence in which the amino acid is F or I
  • the amino acid at position 73 is L or F
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 1 include (i) the amino acid sequence represented by SEQ ID NO: 14; (ii) the amino acid at the fourth position in the amino acid sequence represented by SEQ ID NO: 14 is L Or V, the amino acid at position 10 is A or V, the amino acid at position 12 is G, E, or A, the amino acid at position 13 is T or A, and position 15
  • the amino acid at position 17 is R or K
  • the amino acid at position 23 is S or T
  • the amino acid at position 28 is N or D
  • the amino acid at position 29 is I or M
  • the amino acid at position 31 is S, N, or A
  • G is inserted after the amino acid at position 31, and the amino acid at position 32 is N Or Y
  • the amino acid at position 33 is Y, A , D, or T
  • the amino acid at position 35 is Y, N, H, or S
  • the amino acid at position 43 is T, or K
  • the amino acid at position 51 is R, S
  • Examples of the amino acid consensus sequence of human antibody ⁇ light chain variable region family 2 include (i) the amino acid sequence represented by SEQ ID NO: 15; (ii) the amino acid at the 8th position in the amino acid sequence represented by SEQ ID NO: 15 is P , A, or R, the ninth amino acid is S or F, the tenth amino acid is A or V, the twelfth amino acid is G or R, and the thirteenth position.
  • the amino acid at position 18 is V or I
  • the amino acid at position 31 is G, S, or D
  • the amino acid at position 33 is N or D
  • the amino acid at position 34 is Y, L, R, or H
  • the amino acid at position 36 is S or F
  • the amino acid at position 40 is Q or K
  • the amino acid at position 41 is H, P, or R
  • the amino acid at position 42 is P, or Is L
  • the amino acid at position 43 is G or S
  • the amino acid at position 44 is K or T
  • the amino acid at position 45 is A or T
  • the amino acid at position 46 is
  • the amino acid at position 47 is K or R
  • the amino acid at position 49 is M or L
  • the amino acid at position 52 is E, D or N
  • the amino acid at position is V or G
  • the amino acid at position 54 is S or N
  • the amino acid at position 55 is K, N or T
  • the amino acid at position 60 is V or I
  • the amino acid at position 61 is P or S
  • Examples of the amino acid consensus sequence of human antibody ⁇ light chain variable region family 3 include (i) the amino acid sequence represented by SEQ ID NO: 16; (ii) the amino acid at the second position in the amino acid sequence represented by SEQ ID NO: 16 is Y Or S, the third amino acid is E, V or G, the fourth amino acid is L or P, the fifth amino acid is T or M, and the seventh position Wherein the amino acid at position 8 is L, H, P, or S, the amino acid at position 9 is S or A, and the amino acid at position 13 is The amino acid at position 14 is L, T, or P, the amino acid at position 15 is G or A, the amino acid at position 16 is Q or K, and The amino acid at position is T, A, or M, and the amino acid at position 18 is A, Is V, the amino acid at position 19 is R or S, the amino acid at position 23 is G, S, or Q, the amino acid at position 25 is N, D, or E; The amino acid at position is N, A, V, K, or S
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 4 include (i) the amino acid sequence represented by SEQ ID NO: 17; (ii) the first amino acid is Q in the amino acid sequence represented by SEQ ID NO: 17; Or L, the amino acid at the 2nd position is P or L, the amino acid at the 7th position is S or P, the amino acid at the 8th position is S or P, and the amino acid at the 13th position Is S or L, the amino acid at position 16 is S or A, the amino acid at position 18 is V or I, the amino acid at position 27 is G or E, and position 30
  • the amino acid at position 32 is I, A, or T, the amino acid at position 34 is A or E, and the amino acid at position 36 is H or Y.
  • the amino acid at position 39 is Q or R;
  • the amino acid at position is G or E, the amino acid at position 42 is K or R, the amino acid at position 43 is A, G or S, and the amino acid at position 45 is R or Q;
  • the amino acid at position 47 is L or I;
  • the amino acid at position 50 is L or V;
  • the amino acid at position 51 is E, N or K;
  • the amino acid at position 52 is G Or S
  • the amino acid at position 53 is S or D
  • the amino acid at position 56 is Y or H
  • the amino acid at position 57 is N or S, and the amino acid at position 60 Is S or D
  • the amino acid at position 62 is V or I
  • the amino acid at position 67 is S or M
  • the amino acid at position 74 is D or E
  • position 79 Amino acid is I or F
  • amino acid at position 81 is N or S
  • amino acid consensus sequence of the human antibody ⁇ light chain variable region family 5 examples include (i) the amino acid sequence represented by SEQ ID NO: 18; (ii) the second amino acid in the amino acid sequence represented by SEQ ID NO: 18 is A Or P, the amino acid at position 8 is S, A, T, or P, the amino acid at position 10 is L, S, or H, and the amino acid at position 14 is P or S
  • the amino acid at position 16 is A or E
  • the amino acid at position 18 is A or V
  • the amino acid at position 19 is S or R
  • the amino acid at position 20 is L or F
  • the amino acid at position 23 is T or M
  • the amino acid at position 25 is R, S, or P
  • the amino acid at position 27 is G or D
  • the amino acid at position 28 is I or F
  • the amino acid at position 29 is N, or S
  • the amino acid at position 30 is V or L
  • the amino acid at position 32 is T, S, or D
  • the amino acid at position 33 is Y or F
  • the amino acid at position 44 is S or N
  • the amino acid at position 45 is P or L
  • the amino acid at position 47 is Q or R
  • the amino acid at position 51 is R, S, Or Y
  • the amino acid at position 53 is K, Y, or H
  • the amino acid at position 57 is D, S, or N
  • the amino acid at position 59 is Q, H, or G
  • the amino acid at position 72 is K or N
  • the amino acid at position 76 is A, T or S
  • A is amino acid A or T
  • the amino acid at position 80 is I or L
  • the amino acid at position 82 is L, V, or R
  • the amino acid at position 88 is S or P
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 6 include (i) the amino acid sequence represented by SEQ ID NO: 19; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 19; Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 7 include (i) the amino acid sequence represented by SEQ ID NO: 20; (ii) the second amino acid in the amino acid sequence represented by SEQ ID NO: 20 is A Or T, the amino acid at position 23 is G or A, the amino acid at position 33 is H or Y, the amino acid at position 36 is Y or N, and the amino acid at position 48 Is a sequence in which T is T or A, the amino acid at position 52 is D or S, the amino acid at position 78 is S or L, and the amino acid at position 80 is A or V; A) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in (i) or (ii) above; the sequence shown in any one of (i) to (iii) Can be preferably exemplified .
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 8 include (i) the amino acid sequence represented by SEQ ID NO: 21; (ii) the amino acid at position 63 in the amino acid sequence represented by SEQ ID NO: 21 is R Or a sequence that is C; (iii) above (i) or (ii) Examples of the amino acid sequence shown in (i) to (iii) in which one or several amino acids are deleted, substituted, or added are preferred. .
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 9 include (i) the amino acid sequence represented by SEQ ID NO: 22; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 22; Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 10 include (i) the amino acid sequence shown in SEQ ID NO: 23; (ii) the amino acid at position 28 in the amino acid sequence shown in SEQ ID NO: 23 is N Or a sequence in which the amino acid at position 37 is L or P, the amino acid at position 38 is Q or E, and the amino acid at position 63 is L or F; (iii) The amino acid sequence shown in (i) or (ii) is preferably an amino acid sequence in which one or several amino acids are deleted, substituted, or added; the sequence shown in any one of (i) to (iii) Can be exemplified.
  • Examples of the amino acid consensus sequence of the human antibody ⁇ light chain variable region family 11 include (i) the amino acid sequence represented by SEQ ID NO: 24; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 24; Examples of the amino acid sequence that has been deleted, substituted, or added include the sequence shown in (i) or (ii).
  • the type of family used for each VH and VK is specified, and the frequency of each family used is IGHV3 or IGHV4 for VH and IGKV1 for VK. Is said to be expensive.
  • Herceptin registered trademark, in general
  • an anti-HER2 humanized monoclonal antibody that exhibits an antitumor effect by specifically binding to the HER2 protein which is the gene product of the human oncogene HER2 / neu (c-erbB-2)
  • IGHV3 and IGKV1 are used in the case of the name; trastuzumab)
  • IGHV7 and IGKV1 are used in the case of Avastin (registered trademark, generic name: bevacizumab), which is a humanized monoclonal antibody against anti-vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • IGHV4 and IGKV1 are used, and human monoclonal antibodies against epidermal growth factor receptor are used.
  • a Vectivix registered trademark, generic name: panitumumab
  • each human (humanized) antibody can be used for purification of various human (humanized) antibodies by screening molecules such as peptides that specifically bind to the human antibody variable region matched to the family. Can be obtained universally.
  • the method for screening a molecule that specifically binds to the human antibody variable region of the present invention includes (a) a step of preparing a screening polypeptide comprising a polypeptide comprising the amino acid consensus sequence of the human antibody variable region of the present invention; (B) a screening method comprising: sequentially contacting the polypeptide prepared in step (a) with a test substance and selecting the test substance that binds to the polypeptide;
  • the method for preparing the screening polypeptide containing the amino acid consensus sequence of the present invention in the step (a) is not particularly limited, and is prepared by a general chemical synthesis method according to the amino acid sequence of the screening polypeptide. It corresponds to the amino acid sequence of the above screening polypeptide.
  • the prepared polypeptide for screening is prepared by ion exchange chromatography. It is preferable to purify according to usual methods such as partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method and the like.
  • the screening polypeptide is not particularly limited as long as it contains a polypeptide comprising the amino acid consensus sequence of the human antibody variable region of the present invention, but in order to form the three-dimensional structure of the human antibody variable region, It is preferable that the polypeptide comprising the amino acid sequence of the CDR3 region of the human antibody variable region is added to the C-terminus of the polypeptide comprising the amino acid consensus sequence of the variable region. Or a marker sequence or tag sequence for use in purification.
  • a polypeptide containing the amino acid consensus sequence of the human antibody variable region prepared in the step (a) is contacted with a test substance, and a polypeptide containing the amino acid consensus sequence of the human antibody variable region is contacted.
  • a test substance that specifically binds to a peptide
  • test substance is not particularly limited, and specifically, peptides, proteins, nucleic acids, sugars, low molecular compounds, and the like can be preferably exemplified. preferable.
  • the test substance is a peptide
  • the phage display method is a method of screening a peptide having a random amino acid sequence in a phage outer protein (usually about 5 to 12 amino acids) using a phage library displayed as a fusion protein. In accordance with the method described in Smith, G.
  • the molecule that specifically binds to the variable region of the human antibody of the present invention is not particularly limited as long as it is a molecule obtained by the screening method of the present invention, and examples thereof include peptides, proteins, nucleic acids, sugars, and low molecular compounds. However, it is preferably a peptide. Specific examples of such peptides include SEQ ID NOs: 32 to 47 and SEQ ID NOs: 66 to 304.
  • peptides having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the above amino acid sequence are preferably exemplified. be able to.
  • the molecule that specifically binds to the human antibody variable region of the present invention is composed of only the variable region, such as an antibody, in particular, an antibody having no constant region, such as a single-chain antibody (scFv antibody) or a diabody antibody. It is also useful as an affinity ligand for antibody purification. Since the ligand peptide that specifically binds to the human antibody variable region of the present invention is not derived from bacteria, it is safe, unlike protein A derived from bacteria, can be prepared at low cost, and has low immunogenicity. Have advantages.
  • amino acid sequence in which one or several amino acids are deleted, substituted or added examples include, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, particularly 1 or 2. Mention may be made of amino acid sequences in which individual amino acids have been deleted, substituted or added.
  • Aptamer is a single-stranded nucleic acid with a clear three-dimensional structure that can be linked to a target molecule in a manner similar to that of an antibody, and has little immunogenicity (a property that makes an antibody). Development is progressing with representatives such as Macugen (registered trademark), which is a therapeutic agent for age-related macular degeneration, targeting nascent factors. Another application of aptamers that attracts attention is a separating agent for purifying antibodies that utilizes the properties of RNA aptamers that specifically bind to human IgG.
  • the aptamer resin Since elution under acidic conditions is required at the time of purification, the aptamer resin is characterized by being able to elute the antibody in a neutral manner instead of the protein A resin purification method in which inactivation / aggregation of the antibody is pointed out.
  • the amino acid consensus sequence information identified this time particularly RNA encoding a polypeptide comprising an amino acid consensus sequence
  • it can be used as a separation agent for antibody purification that is highly efficient for industrial production. Contributes to the practical application of novel antibody drugs.
  • the DNA encoding the polypeptide comprising the amino acid consensus sequence of the present invention is useful for preparing a polypeptide comprising the amino acid consensus sequence by genetic engineering.
  • Human antibody heavy chain Human antibody heavy chain IGHV1 (31 species): IGHV1-2-02, IGHV1-204, IGHV1-2-03, IGHV1-2-01, IGHV1-8-01, IGHV1-46-01, IGHV1-46-03, IGHV1-46-02, IGHV1- 24-01, IGHV1-69-03, IGHV1-69-13, IGHV1-69-12, IGHV1-69-01, IGHV1-96-07, IGHV1-69-11, IGHV1-69-05, IGHV1-69- 06, IGHV1-69-02, IGHV1-69-08, IGHV1-96-04, IGHV1-69-09, IGHV1-69-10, IGHV1-18-01, IGHV1-18-02, IGHV1-3-01, IGHV1-3-02, IGHV1-58-01, IGHV1-58-02, IGHV -45-01, IGHV1-45-03, IGHV1-45-02.
  • Human antibody heavy chain IGHV2 (24 species): IGHV2-5-04, IGHV2-5-07, IGHV2-5-01, IGHV2-5-03, IGHV2-5-10, IGHV2-5-05, IGHV2-5-06, IGHV2-5-09, IGHV2- 5-02, IGHV2-5-08, IGHV2-70-01, IGHV2-70-13, IGHV2-70-02, IGHV2-70-07, IGHV2-70-08, IGHV2-70-11, IGHV2-70- 03, IGHV2-70-06, IGHV2-70-05, IGHV2-70-04, IGHV2-70-10, IGHV2-70-09, IGHV2-70-12, IGHV2-26-01.
  • Human antibody heavy chain IGHV3 (87 species): IGHV3-11-01, IGHV3-11-03, IGHV3-21-01, IGHV3-21-02, IGHV3-48-01, IGHV3-48-02, IGHV3-48-03, IGHV3-13-01, IGHV3 13-03, IGHV3-13-02, IGHV3-47-01, IGHV3-47-02, IGHV3-9-01, IGHV3-20-01, IGHV3-43-01, IGHV3-43-02, IGHV3-7- 01, IGHV3-7-02, IGHV3-30-3-01, IGHV3-30-3-02, IGHV3-30-17, IGHV3-30-16, IGHV3-30-14, IGHV3-30-11, IGHV3- 30-07, IGHV3-30-04, IGHV3-30-01, IGHV3-30 08, IGHV3-30-09, IGHV3-30-15, IGHV3-30-01, IGHV3-30-08, I
  • Human antibody heavy chain IGHV5 (5 species): IGHV5-51-02, IGHV5-51-04, IGHV5-51-03, IGHV5-51-01, IGHV5-51-05.
  • Human antibody heavy chain IGHV6 (2 types): IGHV6-1-01, IGHV6-1-02.
  • Human antibody heavy chain IGHV7 (3 types): IGHV7-4-1-01, IGHV7-4-1-02, IGHV7-4-1-03.
  • Human antibody kappa light chain Human antibody kappa light chain
  • Human antibody kappa light chain Human antibody kappa light chain
  • Human antibody kappa light chain IGKV1 (28 species): IGKV1-5-01, IGKV1-5-02, IGKV1-5-03, IGKV1-13-02, IGKV1D-13-01, IGKV1-37-01, IGKV1D-37-01, IGKV1-27-01, IGKV1- 33-01, IGKV1D-33-01, IGKV1-39-01, IGKV1D-39-01, IGKV1-17-01, IGKV1-17-02, IGKV1-6-01, IGKV1-16-01, IGKV1-17- 01, IGKV1-12-2-01, IGKV1-12-2-02, IGKV1D-12-01, IGKV1D-12-02, IGKV1D-16-01, IGKV1D-16-
  • IGKV2 Human antibody kappa light chain IGKV2 (13 species): IGKV2-29-02, IGKV2-29-03, IGKV2D-29-02, IGKV2D-29-01, IGKV2-28-01, IGKV2D-28-01, IGKV2-40-01, IGKV2-40-02, IGKV2D- 40-01, IGKV2-24-01, IGKV2D-24-02, IGKV2-30-01, IGKV2D-30-01.
  • Human antibody kappa light chain IGKV3 (12 species): IGKV3-11-01, IGKV3D-11-01, IGKV3-11-02, IGKV3-20-01, IGKV3D-20-01, IGKV3-20-02, IGKV3-7-01, IGKV3-7-03, IGKV3-3 7-02, IGKV3D-7-01, IGKV3-15-01, IGKV3D-15-01.
  • Human antibody kappa light chain IGKV4 (1 type): IGKV4-1.
  • Human antibody kappa light chain IGKV5 (1 type): IGKV5-2.
  • Human antibody kappa light chain IGKV6 (3 types): IGKV6-21, IGKV6D-21, IGKV6D-41.
  • IGLV1 Human antibody ⁇ light chain> Human antibody ⁇ light chain IGLV1 (11 species): IGLV1-47-01, IGLV1-47-02, IGLV1-44-01, IGLV1-36-01, IGLV1-40-02, IGLV1-40-03, IGLV1-40-01, IGLV1-50-01, IGLV1- 51-01, IGLV1-52-02, IGLV1-41-01.
  • IGLV2 Human antibody ⁇ light chain IGLV2 (20 species): IGLV2-8-01, IGLV2-8-03, IGLV2-8-02, IGLV2-11-01, IGLV2-11-02, IGLV2-11-03, IGLV2-14-03, IGLV2-14-04, IGLV2- 14-01, IGLV2-14-02, IGLV2-23-01, IGLV2-23-03, IGLV2-23-02, IGLV2-18-02, IGLV2-18-04, IGLV2-18-01, IGLV2-18- 03, IGLV 2-33-01, IGLV 2-33-02, IGLV 2-33-03.
  • IGLV3 Human antibody ⁇ light chain IGLV3 (18 species): IGLV3-9-01, IGLV3-9-02, IGLV3-12-01, IGLV3-12-02, IGLV3-21-02, IGLV3-21-03, IGLV3-21-01, IGLV3-25-02, IGLV3 25-03, IGLV3-25-01, IGLV3-16-01, IGLV3-10-01, IGLV3-10-02, IGLV3-27-01, IGLV3-22-01, IGLV3-1-01, IGLV3-19- 01, IGLV3-32-01.
  • Human antibody ⁇ light chain IGLV4 (6 species): IGLV4-60-01, IGLV4-60-02, IGLV4-60-03, IGLV4-69-01, IGLV4-69-02, IGLV4-3-01.
  • Human antibody ⁇ light chain IGLV5 (8 species): IGLV5-45-02, IGLV5-45-03, IGLV5-45-01, IGLV5-39-01, IGLV5-39-02, IGLV5-48-01, IGLV5-37-01, IGLV5-52-01.
  • Human antibody ⁇ light chain IGLV6 (1 type): IGLV6-57-01.
  • Human antibody ⁇ light chain IGLV7 (3 types): IGLV7-46-01, IGLV7-46-02, IGLV7-43-01.
  • Human antibody ⁇ light chain IGLV8 (3 types): IGLV8-61-01, IGLV8-61-03, IGLV8-61-02.
  • Human antibody ⁇ light chain IGLV9 (3 types): IGLV9-49-01, IGLV9-49-03, IGLV9-49-02.
  • Human antibody ⁇ light chain IGLV10 (3 types): IGLV10-54-01, IGLV10-54-03, IGLV10-54-02.
  • Human antibody ⁇ light chain IGLV11 (1 type): IGLV11-55-01.
  • Human antibody heavy chain IGHV1 consensus sequence (SEQ ID NO: 1: QVQLVQSGAEVKKPGASVKVSCKASGYTFTYAMHWVRQAPGQGLEWMGWINPNSGNTNYAQKFQGRVTMTRDTTSTAYMELSSLRSTEDTAVYYC)
  • Human antibody heavy chain IGHV2 consensus sequence (SEQ ID NO: 2: QVTLKESGPALVKPTQTLLTTCTFGSFSLSTSTGMGVSWIRQPPPGKALEWLALIDWDDDRYRYSTSLKSRLTISKDDTSKNQVVLTMTNMDPVDTATYYC)
  • Human antibody heavy chain IGHV3 consensus sequence (SEQ ID NO: 3 EVQLVESGGGLVQPGGSLRLSCAASGFTFFSSYAMHWVRQAPGKGLEWVSVISSGSGSTYYADSVKGFRFTISRDNSNKNTLYLQMNSLRAEDTAVYYC Human
  • Human antibody kappa light chain ⁇ Human antibody kappa light chain> Human antibody kappa light chain IGKV1 consensus sequence (SEQ ID NO: 8: DIQMTQSPSSLSASVGDRVTITCRASQGISSYYLAWYQQKPGKAPKLLLIYAASSLQSGVPPSRFSGGSGTDFLTTISSLQPEDFATYYC) Human antibody kappa light chain IGKV2 consensus sequence (SEQ ID NO: 9: DIVMTQTPLSLPPVTPGQPASISSCRSSQSLLHSDGNTTYLDWYLQKPGQSPQLLLIYKVSNRFSGVPDRFSGGSGSDFTLKISRVREAEDVGVYYC) Human antibody kappa light chain IGKV3 consensus sequence (SEQ ID NO: 10: EIVMTQSPATLSLPGEATLSCRASQSVSVSSYLAWYQQKPGQAPRLLIYGASTTRATGIPAFSGSGSGTDFLTTISSLQPEDFAVYYC) Human
  • a nucleotide sequence encoding a human antibody heavy chain consensus sequence added with CDR3 (105-128) was introduced into an expression plasmid (maltose binding protein (MBP) fusion expression).
  • MBP maltose binding protein
  • a human antibody heavy chain consensus sequence expression plasmid was prepared.
  • E. coli transformed with the above human antibody heavy chain consensus sequence expression plasmid was cultured to extract the periplasmic fraction, and purified using amylose resin. Further, purification with nickel beads was performed to prepare an MBP-fused human antibody heavy chain variable region protein (MBP-IGHVDJ-6 ⁇ His).
  • a base sequence in which a pertuzumab kappa light chain CDR3 (105-128) and a histidine tag are added to a base sequence (SEQ ID NO: 48-53) encoding a human antibody kappa light chain consensus sequence (SEQ ID NO: 8-13) is synthesized.
  • a nucleotide sequence encoding a human antibody kappa light chain consensus sequence added with CDR3 (105-128) was introduced into an expression plasmid (maltose binding protein (MBP) fusion expression), and SEQ ID NOs: 60-65.
  • a human antibody kappa light chain consensus sequence expression plasmid having a base sequence was prepared (FIG. 7).
  • MBP-IGKVDJ-6 ⁇ His MBP fusion human antibody light chain variable region protein
  • SEQ ID NOs: 54 to 59 The prepared MBP fusion human antibody heavy chain variable region protein (MBP-IGHVDJ-6 ⁇ His) and MBP fusion human antibody light chain variable region protein (MBP-IGKVDJ-6 ⁇ His) were subjected to polyacrylamide gel electrophoresis and coomassie brilliant. The results of staining with blue (CBB) are shown in FIGS.
  • a base sequence encoding peptide 3 (SEQ ID NO: 32) was introduced into an expression plasmid (a fusion expression vector with glutathione S transferase (GST)) to prepare a peptide 3 expression plasmid.
  • Escherichia coli transformed with the peptide 3 expression plasmid was cultured to extract a solubilized fraction, and purified with nickel beads to prepare GST fusion peptide 3.
  • the MBP-fused IGHVDJ3 protein prepared in Example 2 was immobilized on a microplate, and ELISA was performed using the GST-fused peptide 3, mouse anti-GST monoclonal antibody, and HRP-labeled sheep anti-mouse IgG monoclonal antibody. And the binding of IGHV3 consensus sequence. From the results shown in FIG. 11, it was revealed that peptide 3 specifically binds to the IGHV3 consensus sequence.
  • FIG. 12A shows the results of SPR analysis of the affinity between the code 4 peptide (SEQ ID NO: 69) and MBP-VH3 (MBP-IGHVDJ3-6 ⁇ His) or control MBP.
  • FIG. 12B shows the results of SPR analysis of the affinity between the code 15 peptide and MBP-VK3 (MBP-IGKVDJ3-6 ⁇ His) or control MBP.
  • Monoclonal phages were prepared for 198 clones other than the arbitrarily selected clones among the eluted phage plaques after four rounds of panning in the primary screening of affinity ligand using the above phage display, and a total of 198 peptides displayed by the phages
  • the amino acid sequence of (SEQ ID NO: 107-304) was identified. These peptides may also be peptides that recognize human antibody heavy chain consensus sequences and human antibody kappa light chain consensus sequences.
  • a target protein of 100 ng per well was placed in a maxisorp immunoplate (Nalge Nunc International), allowed to stand at 37 ° C. for 1 hour, and then the target protein was solidified on the plate by incubation at 4 ° C. over night. Washing of each well was repeated 3 times with PBST (PBS-0.05% Tween 20), 200 ul of 3% BSA (Bovine Serum Albumin) solution was added to each well and blocked at 37 ° C. for 2 hours. The culture supernatant of E. coli containing phages displaying each peptide was diluted 10-fold with 0.5% BSA / PBS, dispensed 100 ul per well, and allowed to stand at room temperature for 1 hour.
  • PBST PBS-0.05% Tween 20
  • BSA Bovine Serum Albumin
  • a rabbit anti-fd bacteriophage antibody (# B7786: Sigma-Aldrich) diluted 2000-fold was dispensed into each well as 100 ul and allowed to stand at room temperature for 1 hour.
  • 100 ul of HRP-labeled anti-rabbit IgG antibody (# NA9340: GE Healthcare) diluted 5000 times as a secondary antibody was dispensed into each well and allowed to stand at room temperature for 1 hour.
  • FIG. 13 shows the results of secondary screening of peptides that recognize the IGHV consensus sequence by the phage ELISA method.
  • buffer buffer only (no target protein)
  • MBP Maltose-Binding-Protein
  • VH MBP fusion IGHV3 consensus sequence target protein (VH3)
  • VH4 and VH6 the target protein mix including VH4 and VH6 are shown respectively.
  • Buffer represents a negative control without target protein solidification
  • the MBP value refers to the value of non-specific binding (background) in the MBP fusion VH protein. In all 19 specimen samples, there was no peptide that recognized VH-specific binding (FIG. 13).
  • FIG. 14 shows the results of secondary screening of peptides recognizing IGKV3, 6 consensus sequences by the phage ELISA method.
  • MBP Maltose-Binding Protein
  • VK3 IGKV3 consensus sequence target protein
  • VK6 MBP fusion IGKV6 consensus sequence target protein.
  • the MBP value refers to the value of non-specific binding in the MBP fusion VK6 protein. Seven of the 179 peptide sequences were confirmed to be peptides that specifically bind to VK3 (FIG. 14).
  • the Herceptin family is generally identified as IGHV3 and IGKV1, but as a result of screening by the phage ELASA method of this Example, higher activity against Herceptin was observed in the IGKV3 peptide.
  • analysis was advanced based on the results in the following examples. As described above, two types of peptides were identified in the primary screening by SPR analysis, and seven types of peptides were identified in the secondary screening by the phage ELISA method (FIG. 15).
  • the purification / recovery rate of Herceptin scFv antibody was analyzed by immunoprecipitation method in which a single-chain antibody was prepared and the single-difference antibody was reacted with the nine types of peptide-bound magnetic beads, and the antibody purification ability of each peptide was evaluated. .
  • the method is shown below.
  • FIG. 100 ul of magnetic beads (Dynabeads M-280 Streptavidin: Invitrogen) were washed with TBST containing 0.5% Tween 20, and then blocked with 3% BSA-added TBST for 1 hour at room temperature. A beaded column was set on a magnetic stand, the supernatant was removed, and the nine peptides biotinylated were adjusted to 100 ⁇ g / mL with TBST, and then 100 ⁇ L was added to the column. The reaction was performed at room temperature for 1 hour with stirring every 10-15 minutes to bind each of the nine peptides to the beads.
  • the beads were washed with TBST and PBS, and a His-tagged scFv antibody (self-prepared) prepared to 100 ⁇ g / mL with PBS was applied to the test peptide-bound beads. 100 ⁇ L was added and an adsorption reaction was performed at 4 ° C. for 16 to 18 hours. After completion of the reaction, the supernatant was removed with a magnetic stand, the beads were collected, 100 ⁇ L of Laemmli solution was added, and the bound peptide was peeled from the beads by heat treatment at 99 ° C. for 10 minutes.
  • the beads were collected on a magnetic stand, the supernatant was collected, reduced with 2-mercaptoethanol (2-ME), and used as a sample for SDS-PAGE.
  • the recovered scFv antibody was subjected to electrophoresis using a 12% minigel (Bio-RAD) under the conditions of 25 mA / gel / 40 min, and then transferred to a PVDF membrane under conditions of 40 V-60 minutes and 90 V-2 hours.
  • the transferred PVDF membrane was washed with TBST, and then blocked with TBST containing 1% skim milk at 4 ° C. for 16-18 hours.
  • an HRP-conjugated anti-His tag antibody (Anti-His C-term antibody, Invitrogen) diluted 5000 times with TBST containing 1% skim milk was reacted with the transferred PVDF membrane at room temperature for 1 hour. After completion of the reaction, the PVDF membrane was washed with TBST, and the fluorescence developed with an Amersham ECL kit was measured with a Lumino Image Analyzer (Image Quant LAS 4000: GE Healthcare), and then the Image Quant TL (GE Healthcare). ) was used to quantify the fluorescence intensity.
  • the Herceptin scFv antibody collected by each peptide magnetic bead was subjected to SDS-PAGE electrophoresis, and then the signal intensity of each band was semi-quantified by image analysis (FIG. 17).
  • 1) -7) is a peptide recognizing the IGKV3 consensus sequence identified by the secondary screening
  • 8) is a peptide recognizing the IGKV3 consensus sequence identified by the primary screening
  • 9) is the primary screening.
  • Is a peptide that recognizes the IGHV3 consensus sequence identified by.
  • code 166 peptide code 166 peptide
  • 9 code 4 peptide
  • FIG. 19 shows a graph of the peptide Herceptin scFv antibody recovery rate relative to the control in the order of reaction of each peptide magnetic bead.
  • 20 ⁇ g or 10 ⁇ g of Herceptin scFv antibody protein-SDS-PAGE electrophoresis band was defined as 100%, and the fluorescence intensity of the antibody protein recovered by the first and second reactions was determined. Due to the semi-quantitative analysis method, each total value exceeded 100%.
  • the leftmost graph of Beads Only shows the amount of non-specific (background) binding when magnetic beads not bound with a peptide are used.
  • VH3-4 peptide magnetic beads that bind to the VH of the Herceptin scFv antibody were used, and the supernatant containing the antibody that did not bind to the beads in the first reaction was added to the VL of the antibody. It was made to react with a bead (FIG. 19 center graph). In addition, the antibody recovery rate when each reaction order was reversed was also analyzed (FIG. 19, right graph). As a result, regardless of whether the concentration of the antibody to be reacted is 20 ⁇ g or 10 ⁇ g, the combination of reacting with VK-6 peptide magnetic beads first and then reacting unrecovered antibody with VH3-4 peptide magnetic beads (FIG. 19, right) It was revealed that the graph) had a higher recovery rate.
  • FIG. 20 shows the results of the peptide Herceptin scFv antibody recovery rate by immunoprecipitation using the above five kinds of single amino acid mutant peptide magnetic beads.
  • the recovery was successful in recovering 84% of the highest value of the peptide magnetic beads (code 245 peptide) in which the seventh amino acid was mutated to alanine, whereas the original peptide magnetic beads without the mutation (code 4 peptide). It was 23%. Since the recovery rate is drastically decreased in the mutations of amino acids 8 and 9, amino acids 8 and 9 of the code 4 peptide may be strongly involved in the binding of the Herceptin scFv antibody to the VH chain. Sex was suggested.
  • the amount of recovered antibody was quantified using His-Tag® Protein® ELISA (FIG. 21). Unlike the result of semi-quantitative analysis of band concentration by SDS-PAGE (FIG. 17), the highest recovery rate (80%) was obtained with the code 161 peptide.
  • the present invention can be suitably used in the field of antibody purification.

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Abstract

L'objectif de l'invention est de fournir des molécules qui se lient spécifiquement à une région variable d'un anticorps et un procédé pour le criblage des molécules par : i) la mise en œuvre d'un alignement pour les séquences d'acides aminés dans chaque famille parmi les familles 1-7 à région variable de chaîne lourde d'anticorps humain, les familles 1-6 à région variable de chaîne légère k d'anticorps humain et les familles 1-11 à région variable de chaîne légère λ d'anticorps humain, et l'identification de la séquence consensus d'acides aminés de la région variable d'anticorps humain ; ii) la préparation d'un polypeptide comprenant la séquence consensus d'acides aminés de région variable d'anticorps humain identifiée et l'utilisation du polypeptide pour cribler pour des molécules qui se lient spécifiquement avec la région variable de l'anticorps.
PCT/JP2013/000961 2012-02-21 2013-02-20 Ligand d'affinité pour la purification d'anticorps WO2013125221A1 (fr)

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JP2022017562A (ja) * 2017-01-30 2022-01-25 国立研究開発法人国立循環器病研究センター 血管内皮系細胞に特異的に結合するペプチドの使用、及びペプチド
JP2022527351A (ja) * 2019-04-02 2022-06-01 イーライ リリー アンド カンパニー 結合ペプチドを選択し検出するための方法

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Publication number Priority date Publication date Assignee Title
WO2018139665A1 (fr) * 2017-01-30 2018-08-02 国立研究開発法人国立循環器病研究センター Utilisation d'un peptide se liant de manière unique à des cellules endothéliales vasculaires, et peptide
JP2018121760A (ja) * 2017-01-30 2018-08-09 国立研究開発法人国立循環器病研究センター 血管内皮系細胞に特異的に結合するペプチドの使用、及びペプチド
JP2022017562A (ja) * 2017-01-30 2022-01-25 国立研究開発法人国立循環器病研究センター 血管内皮系細胞に特異的に結合するペプチドの使用、及びペプチド
JP7017726B2 (ja) 2017-01-30 2022-02-09 国立研究開発法人国立循環器病研究センター 血管内皮系細胞に特異的に結合するペプチドの使用、及びペプチド
US11253626B2 (en) 2017-01-30 2022-02-22 National Cerebral And Cardiovascular Center Use for peptide uniquely binding to vascular endothelial cells, and peptide
JP7320796B2 (ja) 2017-01-30 2023-08-04 国立研究開発法人国立循環器病研究センター 血管内皮系細胞に特異的に結合するペプチドの使用、及びペプチド
WO2019238686A1 (fr) * 2018-06-13 2019-12-19 Aziende Chimiche Riunite Angelini Francesco - A.C.R.A.F. S.P.A. Peptides ayant une activité inhibitrice sur le récepteur muscarinique m3
JP2022527351A (ja) * 2019-04-02 2022-06-01 イーライ リリー アンド カンパニー 結合ペプチドを選択し検出するための方法

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