JP2011072284A - Method for producing antibody and immunogenic composition usable therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunization procedure or the like capable of inducing an efficient immunity against peptides or polypeptides having an amino acid sequence bound to an antibody. <P>SOLUTION: Immunization is performed by using, as the immunogen, two or more peptides or polypeptides bound to the same antibody but having different amino acid sequences so that the antibodies bound to the peptides or polypeptides are induced. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to antibody production methods and immunization methods, as well as immunogenic compositions and immunization products used therefor.

  When inducing immunity with a peptide or polypeptide, the peptide or polypeptide is immunized by binding to what is called a carrier protein (keyhole limpet hemocyanin, thyroglobulin, tetanus toxin, etc.). However, immunity to carrier proteins has increased, and immunity to peptides or polypeptides has not been efficiently induced in many cases (Patent Document 1, Non-Patent Document 1).

  In addition, the method of immunization using chemically synthesized peptides or polypeptides as antigens eliminates the need to isolate and purify naturally-occurring proteins, and does not require the preparation of recombinants. In addition, this is a simple method. However, when peptides or polypeptides themselves are used for immunization, they cannot be incorporated into antigen-presenting cells and in many cases immunity is not established. Furthermore, since the efficiency presented to MHC class II molecules as T cell epitopes is poor, helper T cells that activate immunity could not be efficiently induced (Non-patent Documents 2 and 3).

  Therefore, the synthesized peptide or polypeptide is bound to a substance called a carrier protein by a chemical reaction, and with the help of the carrier protein, it is taken into an antigen-presenting cell or the like and T on an MHC class II molecule on the surface of the antigen-presenting cell. Conventionally, immunization has been performed by a method of presenting cell epitopes (Non-patent Document 4).

  In this case, due to the high immunogenicity of the carrier protein, the immunity induced is occupied by the carrier protein, and the immunity against the peptide or polypeptide is often very low (Patent Document 1, Non-Patent Document). 1). It is also conceivable that immunity against a peptide or polypeptide is suppressed by inducing immunity against a carrier protein. For this reason, development of a method for inducing immunity to a peptide or polypeptide more efficiently has become necessary in the biochemical experiments and the medical / medical fields.

JP-A-8-277297

J.Immunol. 135, 2319 (1985) J.Immunol.164, 625 (2000) By Janet M. Decker, PhD, Immunology Tutorials, Vaccines, [online], last updated: 17 January 2006, University of Arizona (THE UNIVERCITY OF ARIZONA), [Search September 24, 2009], Internet <http://microvet.arizona.edu/courses/mic419/Tutorials/vaccines.html> Infect. Immun. 60, 3947 (1992)

  The present invention relates to an immunization method capable of inducing immunity more efficiently against a peptide or polypeptide, and a method for producing an antibody and an immunogen composition used therefor, in which the above-described problems associated with the immunization method have been solved Etc. is the issue.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the immunization efficiency against a peptide or polypeptide is dramatically improved when immunization is performed with a certain device, and the present invention is completed. It came to.

That is, the gist of the present invention resides in the following (1) to (11).
(1) An antibody comprising inoculating an animal as an immunogen with two or more types of peptides or polypeptides that bind to the same antibody but having different amino acid sequences, and induce an antibody that binds to the peptide or polypeptide Production method.
(2) The method according to (1), wherein the inoculation to the animal is performed simultaneously using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.
(3) The method according to (1), wherein the inoculation to the animal is performed at a fixed time using two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.
(4) In any one of (1) to (3), the two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are peptides selected from the group consisting of an epitope and a mimotope. the method of.

(5) An immunization method characterized in that two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are used as an immunogen.
(6) The method according to (5), wherein the immunization is performed simultaneously using two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.
(7) The method according to (5), wherein the immunization is performed at a predetermined time using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.
(8) The two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are peptides selected from the group consisting of epitopes and mimotopes, the method of.

(9) An immunogenic composition used in the method for producing an antibody according to any one of (1) to (4) or the immunization method according to any one of (5) to (8), wherein An immunogenic composition comprising two or more types of peptides or polypeptides that bind but have different amino acid sequences, simultaneously or individually, as an immunogen.
(10) The immunogenic composition according to (9), wherein the immunogenic composition further contains an adjuvant.
(11) An immunization product comprising an immunogenic composition containing two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences, either simultaneously or individually as an immunogen.

  According to the present invention, a specific antibody against a peptide or polypeptide having an amino acid sequence that binds to an antibody can be raised more efficiently. Thereby, the specific antibody can be efficiently obtained. The immunization method of the present invention is expected to be useful as a vaccine therapy.

It is a figure which shows the construction scheme of the DNA library for cell-free protein display using LR10A (sequence number 1). It is the photograph of the polyacrylamide gel electrophoresis which shows the coupling | bonding to the bevacizumab (Avastin) of the peptide or polypeptide obtained by the protein display system. It is the photograph of the polyacrylamide gel electrophoresis which shows the binding inhibition to the bevacizumab (Avastin) by the vascular endothelial growth factor (VGEF) of the peptide or polypeptide obtained by the protein display system. It is a figure which shows the reactivity with respect to each antibody of the peptide or polypeptide obtained by the protein display system. It is a figure which shows the antibody titer measurement result (pre-immune result) of the rabbit which immunized the peptide or polypeptide obtained by the protein display system. It is a figure which shows the antibody titer measurement result (the 1st blood collection result) of the rabbit which immunized the peptide or polypeptide obtained by the protein display system. It is a figure which shows the antibody titer measurement result (second blood collection result) of the rabbit which immunized the peptide or polypeptide obtained by the protein display system.

  Below, the typical aspect for implementing this invention is demonstrated concretely, However, This invention is not limited to the following aspects, unless the summary is exceeded.

  In the method for producing an antibody of the present invention, two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are inoculated into humans or animals as an immunogen, and an antibody that binds to the peptide or polypeptide is induced. It has the characteristics to do. The immunization method of the present invention is characterized in that two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are used as an immunogen.

  In the present invention, the peptide or polypeptide used as an immunogen is any peptide having an amino acid sequence that binds to an antibody, that is, having a sequence that can be a B cell epitope and having an ability to elicit immunity. It may have a sequence of

  The probability of becoming a B cell epitope can also be searched by the public web [URL: http://www.imtech.res.in/; Institute of Microbial Technology, Sector 39-A, Chandigarh-160036 (INDIA)]. It is not always necessary that the probability is high. Further, it may be a natural or non-natural sequence containing a B cell epitope obtained by protein display technology based on an existing antibody. This non-natural sequence is a sequence that is recognized by an antibody that recognizes an epitope of an antigen and causes the same reactivity as the antigen, and is hereinafter referred to as “mimotope”. These natural or non-natural sequences containing B cell epitopes are those that "bind to the same antibody but have different amino acid sequences".

  Here, as protein display technology, for example, a method using a mapping molecule between genotype (nucleic acid) and phenotype (protein) (WO98 / 16636, WO2005 / 024018), mRNA display method (FEBS Lett 414 (2): 405-408 (1997)), ribosome display method using cell-free protein synthesis system (Methods, 290, 51-67 (2004)), so-called phage display method (J. Immunol Methods, 316, 67-74. (2006), Blood, 108, 1975-1978 (2006), Proc. Natl. Acad. Sci, USA, 93, 10949-10954 (1996), J. Mol Biol 338 (2 ): 299-310 (2004)). These techniques are known per se, and by using these techniques, a peptide or polypeptide sequence (B cell epitope sequence or mimotope sequence) that reacts with an antibody can be easily identified.

  In addition to the above methods, a bacterial display method (Appl Environ Microbiol 70 (9): 5074-5080 (2004)) for displaying peptides or polypeptides on the cell surface of microorganisms, a peptide or polypeptide on the yeast cell surface. Yeast display method to display (Methods Mol Biol 504: 351-383 (2009)), Mammalian display method to display peptide or polypeptide on the cell surface of animal cells (J Immunol Methods 327 (1-2): 40-52 , (2007)) can also be used.

  These methods are advantageous in that proteins can be displayed on living cells, cells displaying the desired peptide or polypeptide can be isolated by immunological methods (FACS, etc.), and cultured immediately. The size of the peptide or polypeptide library that can be used (type of molecules contained) is as small as 1 / 10,000.

  More specifically, for example, in the method using the mapping molecule, first, a protein (expression) is expressed by translating mRNA encoding a random peptide having puromycin bound to the 3 ′ end using a cell-free translation system. Type) and a corresponding molecule (peptide or polypeptide library) in which mRNA (genotype) encoding the same is linked via puromycin. The antibody is allowed to react with this for a certain period of time, non-reacting mapping molecules are removed, and the process of eluting the mapping molecules bound to the antibodies is repeated several times to select and concentrate the molecules that bind to the antibodies. Further, by analyzing a region encoding a random peptide with respect to the selected / enriched molecule, an epitope or mimotope sequence can be identified.

  In the phage display method, antibodies are reacted with a peptide or polypeptide library expressed on phage for a certain period of time, non-reacted phage are washed (removed), antibody-binding phage is eluted, and this phage is infected with E. coli. The phage that binds to the antibody is selected and concentrated by repeating the amplification step several times. Furthermore, for an arbitrarily selected phage clone, the epitope or mimotope sequence can be identified by selecting the reactivity to the antibody as an index and analyzing the region encoding the random peptide or polypeptide.

  The antibody that is the basis for identification / analysis of B cell epitopes and mimotopes is not particularly limited, but it is particularly preferable to target pharmaceutical antibodies. Furthermore, specific antibodies against pathogenic microorganisms, viruses, cancer cells, etc. may be targeted. The B cell epitope or mimotope of these antibodies can be expected to be used as a peptide vaccine (Pharmacia Vol. 44, No. 6, 525-527 (2008)).

  The method for preparing the peptide or polypeptide having the epitope or mimotope sequence obtained as described above is not particularly limited, and may be carried out by a conventional method such as chemical synthesis or gene recombination technique. The purification is preferably performed by ordinary chromatography, but is not particularly limited.

The peptide or polypeptide used as the immunogen may be linear or cyclic. Generally, in the case of a linear peptide or polypeptide, it is said that a peptide bond is rotated and a structure is difficult to take. It is thought that the structure is fixed by circularization. It is considered that the antibody induction efficiency is better with the cyclized peptide or polypeptide.
When a plurality of cysteines are present in a peptide or polypeptide, the peptide is oxidized in air and naturally cyclized, and in the reduced state, it is linear.

The size (number of amino acid residues) of the peptide or polypeptide used as the immunogen is usually 25-30, preferably 10-20, more preferably 6-8.
In the present specification, the terms “peptide” and “polypeptide” are generic names of “two or more amino acids bound by peptide bonds” and “usually ten or more amino acids bound”, respectively. Used as

  In immunization, a sequence having a helper T cell epitope is bound to the peptide or polypeptide. The sequence having a helper T cell epitope is a carrier protein used for normal immunization, such as keyhole limpet hemocyanin, thyroglobulin, tetanus toxin, serum albumin, etc. There may be. Alternatively, a sequence that can be a helper T cell epitope searched using a computer may be used.

  Binding of the peptide or polypeptide to the sequence having the helper T cell epitope can be easily performed using an appropriate condensation method such as the cytein-maleimide method, the glutaraldehyde method, the carbodiimide method, or the like.

  The peptide or polypeptide conjugated to the sequence having the helper T cell epitope thus obtained is immunized or inoculated to animals as a composition added with an appropriate carrier, if necessary, or a composition suspended or embedded in an adjuvant. To do. Examples of adjuvants include Freund's complete / incomplete adjuvant, ALUM, liposomes, cholesterol mannan, tapioca and the like. The immunization route includes oral, subcutaneous injection, intramuscular injection, and the like, and can be selected depending on the desired immunization. Examples of animals used for antibody production include mice, rabbits, chickens, goats, horses, cows, sheep, and monkeys.

  In the method of the present invention, two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences, that is, two or more types of peptides or polypeptides selected from epitopes and mimotopes are used as the immunogen.

  In this method, two or more types (plural types) of peptides or polypeptides may be used simultaneously as an immunogen, or each type may be used individually as an immunogen after a certain period of time. In this case, the interval to be used is not particularly limited, and may be usually 30 to 60 days, preferably about 21 to 28 days.

  The combination of peptides or polypeptides to be used is not particularly limited as long as they have different sequences, and any combination selected from the group consisting of an epitope (natural sequence) and a mimotope, a combination of mimotopes, that is, an epitope and a mimotope. Any combination of peptides may be used. The kind to be used is usually two or more kinds, and preferably 3 to 5 kinds of combinations are suitable.

In addition, the ratio of each peptide used for inoculation or immunization is not particularly limited as long as the titer (antibody titer) is increased as compared to the case of using only one kind, and each of the peptides is usually the same amount ( It is appropriate to use the same number of molecules).
Furthermore, the total amount of each peptide used for inoculation or immunization is not particularly limited, and may be set as appropriate based on the antigenicity of the peptide.

  Here, the immunogenic composition of the present invention is used in the above-described method, and two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are simultaneously or individually combined with the immunogen. It has the characteristics in containing. The immunogenic composition may further contain an appropriate carrier or adjuvant as necessary. Usually, an antibody with a higher titer can be raised by including an adjuvant.

  Here, the carrier is not particularly limited as long as it is pharmacologically or physiologically acceptable, and examples thereof include diphtheria toxin, conjugated albumin, vaccinia virus, and viral shell protein used as a vaccine. .

  Without being bound to any theory anymore, for the following reasons, the use of two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences as immunogens, the induction of particularly efficient immunity Will be possible.

That is, a mimotope is a sequence that is recognized by the same antibody with different amino acid sequences. If the mimotope sequence that reacts with the antibody is in the peptide or polypeptide to be immunized, an antibody having the same antigen-binding ability as the antibody (hereinafter referred to as “similar antibody”) is raised. When it is desired to raise a similar antibody, only this mimotope site is required, but the peptide or polypeptide used for immunization contains other sequences (it is difficult to identify only the mimotope portion). Antibodies are also raised. When immunized with a plurality of peptides or polypeptides of different sequences, including mimotopes, the mimotope moiety stimulates B cells producing similar antibodies in common (because they are structurally common), enhancing the production of similar antibodies. The On the other hand, since sequences other than mimotopes have no commonality, antibodies raised against the respective sequences are not stimulated by the first (single) stimulation, and no further enhancement of production occurs.
Thus, by immunizing different peptides or polypeptides containing mimotope multiple times, only similar antibody-producing B cells can be efficiently stimulated and production of similar antibodies in vivo can be enhanced with high efficiency.

  In the method of the present invention, if booster immunization is performed every 2 to 3 weeks after the initial immunization, immunity is more effectively induced and antiserum having a high titer (antibody titer) is formed.

  In the method for producing an antibody of the present invention, a desired antibody titer (binding to the used immunogen) is confirmed, blood is collected from the immunized animal, and serum is prepared. The obtained antiserum can be used as it is without purification, but after the serum is heat treated to inactivate complement, the immunoglobulin fraction is purified by salting out with ammonium sulfate, ion exchange chromatography, etc. May be. In addition, an antibody that specifically recognizes the peptide or polypeptide used as the immunogen can be obtained by purifying the antibody using a peptide column on which the peptide or polypeptide used as the immunogen is immobilized.

  In addition, antibody-producing cells are collected from the immunized animal and fused with a cultured cell such as a myeloma cell derived from a syngeneic animal by a conventional method to prepare a hybridoma, and an immunoglobulin fraction is prepared from the hybridoma culture solution. A monoclonal antibody can be obtained.

The immunization product of the present invention is characterized by comprising an immunogenic composition containing two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences, either simultaneously or individually as an immunogen. It is what you have.
Such an immunization product is composed of the above-mentioned immunogenic composition, and is provided with the same composition as a reagent kit used for normal animal immunization and a pharmaceutical used for human or animal vaccine therapy.

  The immunization method, immunogenic composition, and immunization product of the present invention can also be used as a peptide vaccine therapy for humans and animals. In that case, T cell epitope sequences and adjuvants that are physiologically and pharmacologically acceptable to humans and animals are used. The peptide or polypeptide serving as an immunogen contains a B cell epitope or mimotope sequence of a specific antibody against a pathogenic microorganism, virus, cancer cell or the like.

  In particular, when an autoantibody against the epitope or mimotope is present in the body during cancer vaccine therapy, an increase in antibody titer may be observed by administration of the peptide alone. In such a case, it is not always necessary to use the T cell epitope bound to the B cell epitope or mimotope as described above.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the scope of the present invention is not limited at all by the following example.
In the following examples, μl represents microliter, ml represents milliliter, M represents mol / liter, mM represents millimol / liter, and μg represents microgram.

Example 1 Selection of Bevacizumab-Binding B Cell Epitope Sequence by Protein Display System Binding to the antibody using the pharmaceutical antibody Bevacizumab [trade name: Avastin, manufactured by Roche] against vascular endothelial growth factor (VEGF) B cell epitope sequence by random peptide display method (Mol Biotechnol. 2009 Feb; 41 (2): 99-105) using a mapping molecule (WO98 / 16636) of genotype (nucleic acid) and phenotype (protein) Acquired as follows.

(1) Construction of DNA library for cell-free protein display As a DNA library for cell-free protein display having a random sequence of 10 or 20 amino acids, DNA encoding SP6 promoter-IRES-random peptide sequence (10 or 20 amino acids) -A DAN fragment (LR10 fragment, LR20 fragment, LCR10 fragment, LCR20 fragment) having a DNA fragment encoding a spacer-Flag tag was prepared by the following method.

  Using the DNA fragment [LR10A (FIG. 1A)] shown in SEQ ID NO: 1 (chemical synthesis by BEX) and the DNA fragment (3-LR10A) shown in SEQ ID NO: 2 (chemical synthesis by BEX) as a template, KOD polymerase (Toyobo Co., Ltd.) was used to obtain a double-stranded DNA fragment by extension reaction. The extension reaction was performed in the RCR cycle 6 times in the absence of the primer and the reaction composition described in the attached document of the polymerase (FIG. 1B). Using the obtained amplified DNA fragment and SEQ ID NO: 3 (lib5; chemically synthesized at BEX) as a template, a double-stranded DNA fragment that can be transcribed and translated in vitro under the same reaction conditions using KOD polymerase in the same manner. .

  This DNA fragment was separated by 5% polyacrylamide gel electrophoresis, a DNA template having a desired size was excised, and purified by a purification kit (QIAqick Gel Extraction Kit, manufactured by Qiagen). The structure of the obtained DNA fragment (LR10 fragment) is shown in FIG. 1C. The amino acid sequence encoded by this DNA fragment is shown in SEQ ID NO: 7. In the text of this specification, the DNA sequence is capitalized and the amino acid sequence is single letter.

  A DNA library for cell-free protein display having a random sequence of 20 amino acids [DNA fragment encoding SP6 promoter-IRES-random peptide sequence (20 amino acids) -spacer-Flag tag] (LR20 fragment) A DNA fragment (LR20A) shown in SEQ ID NO: 4 (chemical synthesis at BEX) was used instead of the DNA fragment shown in No. 1 and prepared in the same manner as described above. The amino acid sequence encoded by the DNA fragment (LR20 fragment) is shown in SEQ ID NO: 8.

  Cell-free protein display DNA library having a peptide or polypeptide sequence in which C (cysteine) is bonded before and after a random sequence of 10 amino acids [SP6 promoter-IRES-DNA coding for random peptide sequence (10 amino acids) -spacer- Except that the DNA fragment (LCR10 fragment) encoding the Flag tag] (LCR10 fragment) was used instead of the DNA fragment represented by SEQ ID NO: 1 (SEQ ID NO: 5) (chemical synthesis at BEX). Prepared in a similar manner. The amino acid sequence encoded by the DNA fragment (LCR10 fragment) is shown in SEQ ID NO: 9.

  Cell-free protein display DNA library having a peptide or polypeptide sequence in which C is bound before and after a random sequence of 20 amino acids [DNA-spacer-Flag tag encoding SP6 promoter-IRES-random peptide sequence (20 amino acids)] An encoding DNA fragment] (LCR20 fragment) was prepared in the same manner except that the DNA fragment (LCR20A) (chemical synthesis at BEX) shown in SEQ ID NO: 6 was used instead of the DNA fragment shown in SEQ ID NO: 1. The amino acid sequence encoded by the DNA fragment (LCR20 fragment) is shown in SEQ ID NO: 10.

Sequence number 1 (LR10A)
5'CAACAACAACAACAAACAACAACAAAATGGGAPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKGGTGGAAGCGGAGGCGACTAC3 '
[In the sequence, P, Q, and K indicate mixed nucleic acids, and the mixing ratio of each is P = T: C: A: G = 15: 25: 30: 30, Q = T: C: A: G = 30. : 30:30:10, K = T: C: A: G = 50: 0: 0: 50 (the same applies to SEQ ID NOs: 4 to 6). ]

SEQ ID NO: 2 (3-LR10A)
5'TTTCCCGCCGCCCCCCGTCCAGATGAAGAACCGCCCTTGTCATCGTCATCCTTGTAGTCGCCTCCGCTTCCACC3 '

SEQ ID NO: 3 (lib5)
5'GGAAGATCTATTTAGGTGACACTATAGAACAACAACAACAACAAACAACAACAAAATG3 '

SEQ ID NO: 4 (LR20A)
5'CAACAACAACAACAAACAACAACAAAATGGGAPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKGGTGGAAGCGGAGGCGACTAC3 '

SEQ ID NO: 5 (LCR10A)
5'CAACAACAACAACAAACAACAACAAAATGGGACGTPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKCGTGGTGGAAGCGGAGGCGACTAC3 '

Sequence number 6 (LCR20A)
5'CAACAACAACAACAAACAACAACAAAATGGGACGTPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKPQKCGTGGTGGAAGCGGAGGCGACTAC3 '

SEQ ID NO: 7
MGXXXXXXXXXXGGSGGDYKDDDDKGGSSSGRGAAG
[In the sequence, X represents an arbitrary amino acid (the same applies to SEQ ID NOS: 12 to 14 below). ]

SEQ ID NO: 8
MGXXXXXXXXXXXXXXXXXXXXGGSGGDYKDDDDKGGSSSGRGAAG

SEQ ID NO: 9
circular 10aa
MGCXXXXXXXXXXCGGSGGDYKDDDDKGGSSSGRGAAG

SEQ ID NO: 10
circular 20aa
MGCXXXXXXXXXXXXXXXXXXXXCGGSGGDYKDDDDKGGSSSGRGAAG

(2) Preparation of mRNA for cell-free protein display Each of the four types of DNA fragments prepared in (1) above was used as a DNA library for protein display in the presence of a CAP analog (RiboMax Large RNA Production System- It was transcribed into mRNA using SP6 (manufactured by Promega). Next, puromycin linker (WO2005 / 024018) was ligated to the 3 ′ end of this mRNA using T4 RNA ligase (Takara) according to a conventional method. This ligated molecule was purified by an isopropyl alcohol precipitation method according to a conventional method (hereinafter referred to as “purified ligated product”).

(3) Purification of DNA / RNA hybrid type protein display molecule Each of 25 μg of the purified ligation product is translated by translating 960 μl of wheat germ cell-free translation system (manufactured by Zoigene) at 26 ° C. for 1 hour. A protein-nucleic acid linking molecule (protein display molecule) in which puromycin at the 3 ′ end of mRNA is bound to the C-terminus of a peptide or polypeptide chain (protein), ie, linking a protein and a nucleic acid (mRNA) encoding the protein. Molecules were prepared.
Thereafter, the protein display molecule was purified by Oligo (dT) agarose (manufactured by Takara) using the polyA sequence inside the puromycin linker according to a conventional method.

  Next, using the sequence within the puromycin linker in the protein display molecule as a primer, cDNA was synthesized by a cDNA synthesis kit (Superscript III, manufactured by Invitrogen) according to a conventional method, and a DNA / RNA hybrid protein display molecule ( This is hereinafter referred to as “a hybrid type display molecule”).

  The obtained hybrid type display molecule was diluted 10-fold with PBS buffer solution (0.1 mM EDTA, 0.05% Tween20) containing 0.1% calf serum albumin and tRNA (0.1 mg / ml). Thereafter, 80 μl of anti-FLAG antibody-fixed agarose beads (manufactured by Sigma) were added and reacted overnight at 4 ° C., and the bead fraction was collected according to a standard method. The bead fraction was washed 3 times with 0.2 ml of washing solution (50 mM Tris-HCl pH 7.5, 150 mM KCl, 0.1% Tween20), and then washed with 240 μl of 100 μM 3 × FLAG peptide (manufactured by Sigma). Molecules bound in (1) were eluted according to a conventional method. The eluate was diluted by adding calf serum albumin and tRNA to a final concentration of 0.1% calf serum albumin and tRNA (0.1 mg / ml) to a volume of 0.6 ml.

(4) Affinity separation (concentration and selection of hybrid display molecules)
For the purpose of removing non-specifically adsorbed molecules on the antibody, each of the hybrid type display molecules obtained in (3) above was combined with 10 μl of human G (manufactured by Sigma) protein G-Sepharose beads (manufactured by GE Healthcare). And 30 minutes at room temperature. After removing the bead-adsorbed fraction by a conventional method, 10 μl of the target antibody bevacizumab (Roche) -bound Protein G-Sepharose beads was added to the non-adsorbed fraction and reacted at room temperature for 1 hour.

The bead-bound fraction was collected by a conventional method, washed 3 times with 0.1 ml of washing solution, and the bead-bound molecule was eluted twice with 40 μl of 0.1 M glycine hydrochloride buffer (pH 2.5). The eluate was quickly neutralized with 8 μl of 1M Tris-HCl buffer (pH 8.0). Using a part, the amount of the hybrid type display molecule bound to bevacizumab was measured by SYBR Green method (SYBR Premix Ex Taq ^™ , manufactured by Takara) by quantitative PCR (PRISM7000, manufactured by ABI). Then, it re-amplified by PCR and used for the next concentration / selection step. Subsequent concentration / selection steps were performed in the same manner as described above.

(5) Cloning and Sequence Confirmation of Concentrated Molecules After repeating the above step (4) 6 times, the DNA sequence encoding the protein part of the hybrid display molecule bound to bevacizumab is cloned into a cloning vector kit (pGEM T Easy Vector (manufactured by Promega) was cloned, the base sequence was confirmed according to a conventional method, and the amino acid sequence of the random peptide portion was determined. As a result, B cell epitope sequence candidates that bind to the following five types of bevacizumab were obtained.

M074 # D12: WLEMHWPAHS (SEQ ID NO: 11)
M075 # H11: CGGRFYDIMNHC (SEQ ID NO: 12)
M075 # C03: CGGRWQTIRSFC (SEQ ID NO: 13)
M075 # A10: RDLRHCESSWHKLVDFYCYT (SEQ ID NO: 14)
M074 # F02: KLEMHFPSHVISVADGWSLF (SEQ ID NO: 15)

(6) Confirmation of binding of the obtained amino acid sequence to bevacizumab The peptide or polypeptide comprising the amino acid sequence obtained in (5) above is a B cell epitope sequence that binds to bevacizumab. The labeling method (Biotechnol Lett. 2007 Jul; 29 (7): 1065-1073) was synthesized as follows as a C-terminal fluorescent label and confirmed by determining its binding by a pull-down assay using bevacizumab.

  First, using the plasmid having the sequence cloned in (5) and lib5 and 3-LR10A described in (1), the DNA portion whose sequence was confirmed was amplified by PCR according to the method described in (1) And purified. From the obtained purified DNA fragment, mRNA was prepared according to a conventional method using RiboMax large RNA Production System-SP6 (manufactured by Promega). Next, using a wheat germ extract protein synthesis system (manufactured by Zoigene), in the presence of 20 μM Cy3-dC Puro [synthesized according to the method described in Biotechnol Lett. 2007 Jul; 29 (7): 1065-1073] The mRNA prepared in (1) was added and reacted at 26 ° C. for 2 hours.

  To the reaction solution, 10 μl of bevacizumab (Roche) -bound Protein G-Sepharose beads was added and reacted at room temperature for 1 hour. As a negative control, the reaction was carried out in the same manner as 10 μl of human IgG (manufactured by Sigma) -conjugated Protein G-Sepharose beads (manufactured by GE Healthcare). The bead-bound fraction was collected by a conventional method, washed 3 times with a 0.1 ml washing solution, and tricine-polyacylamide gel electrophoresis sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 1% 2-mercaptoethanol, 6% glycerol) and reacted at 90 ° C. for 5 minutes. The bound peptide was separated by 16.5% Tricine-polyacrylamide gel electrophoresis and confirmed with a fluorescence image analyzer (Molecular Imager FX, Bio-Rad). The result is shown in FIG.

  In FIG. 2, lane 2 shows the protein adsorbed on bevacizumab (Avastin) -bound beads, and lane 3 shows the protein adsorbed on human IgG (h-IgG) -bound beads as a control. Lane 1 shows the result of applying 1/1 amount of the protein used for each adsorption as it is. As shown in FIG. 2, it was found that any peptide or polypeptide consisting of the amino acid sequence obtained in (5) above bound to bevacizumab (Avastin). The amino acid sequences of these peptides or polypeptides were different from each other and different from the original B cell epitope sequence of bevacizumab.

(7) Confirmation of bevacizumab binding inhibition by vascular endothelial growth factor Peptide or polypeptide [M074 # D12 (SEQ ID NO: 11), M075 # H11 (SEQ ID NO: 12), M075 confirmed binding to bevacizumab in (6) above] # C03 (SEQ ID NO: 13), M075 # A10 (SEQ ID NO: 14), M074 # F02 (SEQ ID NO: 15)] and whether or not the binding to bevacizumab is inhibited by vascular endothelial growth factor (VEGF) . The measurement method was as described in (6), and was performed by adding 12.5 μl of 200 μg / ml VEGF in a measurement system using bevacizumab-bound beads. The result is shown in FIG.

  In FIG. 3, lane 2 shows the protein adsorbed on the bevacizumab (Avastin) -bound beads, and lane 3 shows the protein adsorbed on the bevacizumab-bound beads when VGEF is added. Lane 1 shows the result obtained by directly applying 1/1 amount of the protein used in each adsorption experiment. As shown in FIG. 3, it was found that the binding of bevacizumab to the peptide or polypeptide whose binding to bevacizumab was confirmed in (6) was inhibited by VEGF.

(8) Binding specificity to bevacizumab Peptide or polypeptide whose binding to bevacizumab was confirmed in (6) above [M074 # D12 (SEQ ID NO: 11), M075 # H11 (SEQ ID NO: 12), M075 # C03 (sequence) No. 13), M075 # A10 (SEQ ID NO: 14), M074 # F02 (SEQ ID NO: 15)] were chemically synthesized using the solid phase method at Toray Research Center (Kamakura) as a C-terminal biotin label, and HPLC Purified.

In order to measure the binding specificity, as other antibodies, Humira (Abbott), Xolair (Roche), Erbitux (Merck), Actemura (Roche), ReoPro (Lily), human Using IgG (manufactured by Sigma), the antibody was adsorbed on a microtiter plate, and the binding between each antibody and the peptide or polypeptide synthesized above was performed using streptavidin HRP (horseradish peroxidase) according to the ELISA standard method. Binding was detected. The result is shown in FIG.
As is clear from FIG. 4, it was confirmed that all of the obtained B cell epitope sequences reacted specifically with bevacizumab (Avastin).

Example 2 Antibody Initiation Test with Bevacizumab Binding Peptide (B Cell Epitope Sequence) Selected by Protein Display System Among the peptides or polypeptides confirmed to be bevacizumab obtained in Example 1, M74_D12 (SEQ ID NO: 11) ) And M74_F02 (SEQ ID NO: 15) were selected and combined with a protein KLH (keyhole limpet hemocyanin) having a T cell epitope. This was immunized to rabbits, and the increase in antibody titer was measured as follows.

  First, in order to bind M74_D12 (SEQ ID NO: 11) and M74_F02 (SEQ ID NO: 15) to a T cell epitope peptide, Cys- (PEG) 3-GWLEMHWPAHS (SEQ ID NO: 16) and Cys- (PEG) 3-GKLEMHFPSHVISVADGWSLF (SEQ ID NO: 17) was chemically synthesized and fused with KLH by the cysteine maleimide method at the Cys moiety according to a conventional method. The same amount (number of molecules) mixture of these peptides was immunized 0.4 mg for the first time and 0.2 mg for the second time per rabbit.

  Two weeks after the first sensitization, the first blood was collected and the antibody titer was measured. Further, a second sensitization was performed on the same day, and a second blood was collected two weeks later, and an antibody titer against VEGF, which is an antigen of bevacizumab, was measured according to a conventional method. As controls, antibody titers against TNFα (tumor necrosis factor α), EGFR (endothelial cell factor receptor), GST (glutathione S transferase), KLH (keyhole limpet hemocyanin), PBS (phosphate physiological saline buffer) are also included. It measured similarly. The results are shown in FIGS. 5 to 7 show the pre-immunization result, the first vote result, and the second vote result, respectively.

  As is apparent from FIGS. 5, 6, and 7, by combining and immunizing M74_D12 and M74_F02 among the peptides or polypeptides confirmed to be bound to bevacizumab obtained in Example 1, the antigen of bevacizumab It was confirmed that the antibody titer against VEGF can be increased efficiently.

  We believe that the production of vaccines and antibodies using synthetic peptides without using recombinant proteins will become more and more useful in the future in terms of cost and safety. However, it has been difficult in the past to efficiently elicit a desired antibody from a peptide. By using the present invention, since a desired antibody can be induced more easily, it is possible to easily produce an inexpensive and highly safe vaccine and antibody using a synthetic peptide. Thus, the present invention can be particularly suitably used in the life science field, the pharmaceutical / pharmaceutical field, and the like.

Claims (11)

  A method for producing an antibody, comprising inoculating an animal as an immunogen with two or more types of peptides or polypeptides that bind to the same antibody but having different amino acid sequences, and induce an antibody that binds to the peptide or polypeptide.   The method according to claim 1, wherein the inoculation to the animal is performed simultaneously using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.   The method according to claim 1, wherein the immunization to the animal is performed at a predetermined time using two or more kinds of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.   The method according to any one of claims 1 to 3, wherein the two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are peptides selected from the group consisting of an epitope and a mimotope.   An immunization method comprising using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences as an immunogen.   6. The method according to claim 5, wherein the immunization is performed simultaneously using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.   The method according to claim 5, wherein the immunization is performed at a predetermined time using two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences.   The method according to any one of claims 5 to 7, wherein the two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences are peptides selected from the group consisting of an epitope and a mimotope.   An immunogenic composition used in the method for producing an antibody according to any one of claims 1 to 4 or the immunization method according to any one of claims 5 to 8, wherein the composition binds to the same antibody. An immunogenic composition comprising two or more types of peptides or polypeptides having different amino acid sequences as an immunogen, simultaneously or separately.   The immunogenic composition of claim 9, wherein the immunogenic composition further comprises an adjuvant.   A product for immunization comprising an immunogenic composition containing two or more types of peptides or polypeptides that bind to the same antibody but have different amino acid sequences, either simultaneously or individually, as an immunogen.