JP2010075166A - In-vitro immunization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an in-vitro immunization method by which an antigen-specific antibody having a high IgG content can be produced in high efficiency. <P>SOLUTION: There is provided the two-step in-vitro immunization method including: applying the first immunization process having an antigen sensitization step for sensitizing an immunization cell with a target antigen in vitro and a culture step for culturing an antibody-producing cell in a culture solution containing cytokine, and then applying the second immunization process having an antigen sensitization step for sensitizing the obtained target antigen-specific antibody-producing cell with the same antigen in vitro and a culture step for culturing in a culture solution containing cytokine. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an in vitro immunization method for producing an antigen-specific antibody having a high IgG ratio with high efficiency.

  Antibodies (immunoglobulins) play an important role in the defense mechanism of vertebrate infection, and are produced by B cells among immune cells (lymphocytes). In mammals such as humans, B cells with the ability to make different antibodies present on their cell surfaces antibodies that are said to be tens of millions or more and hundreds of millions or more. A corresponding system is in place. In response to specific B cells whose antibody production function is activated by antigen stimulation, a mechanism that promptly promotes antibody production activation is switched on in the body, and helper T cells and the like act as antigen-specific antibodies. Since the production cells proliferate, it is often possible to obtain antigen-specific antibody-producing cells by immunizing experimental animals with a sufficient amount of antigen. It is difficult to obtain antibodies with high specificity. Therefore, it is necessary to administer an antigen to an animal together with an adjuvant capable of activating immunity, but generally used adjuvants such as FCA (Freund's complete adjuvant) often cause side effects such as arthritis in immunized animals. . Further, in order to induce a sufficient immune response, it is necessary to increase the dose at one time and to administer a large number of times, which requires treatment with great pain to animals, and there is a problem from the viewpoint of animal protection. Therefore, it has been desired to develop a technique capable of producing highly antigen-specific antibodies with high efficiency in in vivo immunization.

As human therapeutic antibodies, human antibodies with low antigenicity to humans are desired, but antigens cannot be directly sensitized to humans. Preparation of a chimeric antibody or a humanized antibody (CDR antibody) from a mouse antibody or the like requires a complicated and advanced technique, and a human antibody from a transgenic mouse has not yet reached a technique capable of dealing with all antigens. In addition, since these techniques are all experimental animals but use mouse living bodies, if the toxicity of the antigen is too high, sufficient immunity cannot be achieved from the viewpoint of animal protection.
On the other hand, in vitro immunization involves inducing immune cells (lymphocytes) to antigens outside the body to induce B cells that secrete antigen-specific antibodies, and then producing hybridomas by cell fusion, and using antigen-specific monoclonal antibodies. This is a production technique, and has the great advantage that it is possible to produce an antibody even with an antigen harmful to an individual, in addition to the advantage that the amount of antigen required is smaller and the immunostimulation period is shorter than in vivo immunization. Therefore, when applied to human lymphocytes, human antibodies can be obtained without going through complicated steps such as chimeric antibodies and humanized antibodies. Antigen-specific antibody induction is also possible for human peripheral blood lymphocytes by performing antigen sensitization in the presence of L-leucyl-L-leucine methyl ester (LLME) (Non-patent Document 1). In addition, a technique (Non-patent Document 2) for increasing induction efficiency in the presence of muramyl dipeptide (MDP), interleukin 2 or interleukin 4 has been developed, and there is a great expectation especially for human monoclonal antibody production.

However, in the case of in vitro immunization, the mechanism that promotes the activation of antibody-producing cells in the body does not work, so it is impossible to obtain a sufficient amount of antibody-producing cells with high antigen specificity. In addition to the high degree of technology, class switching does not work well, and the majority of the antibody classes produced are difficult to handle. For this reason, it is not widely used yet, although its great utility is attracting attention.
Accordingly, there has been a strong demand for the development of a technique that can reliably produce an antigen-specific antibody having a high IgG content with high efficiency in in vitro immunization.
JP-A-4-281799 JP 2004-121237 A E. Lindner-Olsson et al. Animal Cell technology: From Target to Market Kluwer Academic Publisher, 2001, p.171-174 Cytotechnology 31, 131-139, 1999 Feltkamp et al. 1993, Kast et al. 1991 Mizumachi et al. Biosci. Biotechnol. Biochem., 67 (4) 712-719 2003 Takatsu et al. International Immunopharmacology 3 783-800 2003 Krieg et al. Nature 374 546-549 1995

  An object of the present invention is to provide an in vitro immunization method that can reliably produce an antigen-specific antibody having a high IgG content with high efficiency.

Therefore, the present inventors examined the conventional in vitro immunization method. As a result, all of the disadvantages that the number of clones is small and the class of antibody produced is IgM are caused by insufficient activation of immunocompetent cells. As a result of intensive research on effective immune system activation methods, we have developed a basic protocol that dramatically improves conventional in vitro immunization methods.
That is, the present invention relates to a basic protocol relating to cell preparation, culture method and optimal immune system stimulating factor. Specifically, immune cells taken out of the body are sensitized and cultured in the presence of cytokines. After that, the obtained antigen-specific antibody-producing cells are obtained, and the sensitization step with the same antigen and the culture step in the presence of cytokines are repeated again to further specifically activate B cells that recognize the target antigen. In addition, it has been found that the production of antigen-specific antibodies particularly in the IgG class is strongly induced. The present invention has been completed with respect to a method for efficiently producing an IgG class antigen-specific monoclonal antibody in vitro from a hybridoma having the antigen-specific antibody-producing cell as a parent.
In addition, as a result of further intensive studies on effective activation methods of the immune system, attention was focused on T cell epitope peptides.
As a T cell epitope, a specific epitope for a specific antigen has heretofore been used to induce and / or enhance a T cell response to the antigen. For example, there are reports on CTL epitopes for tumors (Non-patent Document 3). In either case, a specific antigen-specific T cell epitope peptide is used for the antigen, and the specific helper T cell and / or CTL are in vivo. Induced.
However, even though the ability to activate antibody-producing cells against T cell epitope peptides is expected, it is only activation via helper T cells that recognize T cell epitopes on the antigen. It is not the activation of antibody-producing cells by adding an unrelated T cell epitope. That is, for T cell epitope peptides, based on the antigens from which they are derived, only the ability to activate antibody-producing cells specific to the original antigen has been known.

Contrary to such conventional technical common knowledge, the present inventors dared to test the ability of known T cell epitopes to activate antibodies in in vitro immunization. It has been found that it has a high activation ability for target antigen-specific antibody-producing cells despite addition in vitro. Subsequently, the ability to activate antibody production by a T cell epitope unrelated to the target antigen was also confirmed in normal in vivo immunization.
Based on such knowledge, the present inventors have thought that the common three-dimensional structure formed by three-dimensionally forming various T cell epitopes causes activation of antibody-producing cells. The idea was to design an antibody-producing cell activation peptide based on the three-dimensional structure of the epitope recognition site on the MHC-II side that recognizes. That is, the optimal epitope-like peptide is calculated by calculating the binding energy between each amino acid constituting the MHC-II pocket having a known X-ray crystal structure and each amino acid constituting the T cell epitope-like peptide within the pocket. As a result, it was confirmed that these peptides have the ability to activate antibody-producing cells at the same level or higher than known T cell epitope peptides. That is, the target antigen-specific antibody production using the T cell epitope peptide including the T cell epitope-like peptide consisting of these novel amino acid sequences or the DNA encoding the same as a target antigen-specific antibody-producing cell activator An invention relating to a cell activation method and a method for producing a target antigen-specific monoclonal antibody (IgG) derived from the antibody-producing cell has also been completed, and another application was filed on the same date as the present invention. Further, another application for a T cell epitope-like peptide comprising a novel amino acid sequence has been filed on the same date.
Then, in the in vitro immunization method of the present invention, the present inventors further added the T cell epitope peptides developed simultaneously by the present inventors to a culture medium containing cytokines, thereby further antigen-specific IgG antibodies. It was also found that the induction of is activated, and the present invention about the combined use of the peptides was also completed.

That is, the present invention relates to an optimal protocol for in vitro immune stimulation and includes the following inventions.
[1] After subjecting immune cells to an antigen sensitization step for sensitizing a target antigen in vitro and a culture step for culturing antibody-producing cells in a culture solution containing cytokines, The target antigen-specific antibody-producing cells obtained are subjected to a second immunization step in which an antigen sensitization step with the same antigen in vitro and a culture step in a culture solution containing cytokines are performed, Two-step in vitro immunization method.
[2] The two-step in vitro immunization method according to [1], wherein the immune cells are provided with a T cell removal step in advance of the first immunization step.
[3] The two-step in vitro immunization method according to [2], wherein the T cell removing step uses a CD8-specific antibody and a CD49-specific antibody.
[4] The T medium epitope peptide is further contained in the culture medium used in at least one of the antigen sensitization step or the culture step in any of the first immunization step and the second immunization step. The two-step in vitro immunization method according to any one of [1] to [3].
[5] The two-step in vitro immunization method according to [4], wherein the T cell epitope peptide is a peptide represented by any of the following formulas (I) to (VII):
Formula (I):
QYIKANSKFIGITEL
Formula (II):
VTYDNESLLSAHKVE
Formula (III):
RGIFFX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ KEI (where X ₁ is Y or Q, X ₂ is V or T, X ₃ is F, H or K, X ₄ is A or G, X ₅ is A or G, X ₆ is Y or H).
Formula (IV):
FQDN NAX ₇ X ₈ X ₉ X ₁₀ X ₁₁ AVF
(Wherein X ₇ is A or V, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H).
Formula (V):
_{RGIYNAVX 8 X 9 X 10 X 11} AVF
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).
Formula (VI):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AEI
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).
Formula (VII):
NNYX ₁₂ X ₁₃ VX ₁₄ AAX ₁₅ X ₁₆ NN
(In the formula, X ₁₂ is N or G, X ₁₃ is N or G, X ₁₄ is N or G, X ₁₅ is N or G, and X ₁₆ is A or K.) .
[6] The T cell epitope peptide is
In the formula (I), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “QYI” on the N-terminal side and “TEL” on the C-terminal side,
In the formula (II), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “V” on the N-terminal side and “AHKVE” on the C-terminal side,
In the formula (III), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side,
In the formula (IV), a peptide in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “FQDA” on the N-terminal side and “VF” on the C-terminal side,
In the formula (V), a peptide in which one or more amino acids among amino acids consisting of “RGI” on the N-terminal side and “VF” on the C-terminal side are substituted with any other amino acid,
A peptide in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side in the formula (VI),
In the formula (VII), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “NN” on the N-terminal side and “NN” on the C-terminal side,
The in vitro immunization method according to [5] above, wherein
[7] Producing an antigen-specific monoclonal antibody comprising a step of fusing antigen-specific antibody-producing cells obtained by the two-step in vitro immunization method according to any one of [1] to [6] with myeloma cells A method for producing a hybridoma.
[8] A method for producing a target antigen-specific monoclonal antibody, wherein the hybridoma according to [7] is used.

  The in vitro immunization method of the present invention has made it possible to induce antigen-specific IgG producing cells, which has been difficult in the past. By using the hybridoma obtained by the present invention, an antigen-specific IgG monoclonal antibody can be produced efficiently and can be applied to a very wide range of uses.

In the present invention, in addition to the first immunization step in which an in vitro antigen sensitization step and a culture step are provided for immune cells, the sensitization step by the same antigen and the second immunization step in which a culture step is provided again. The third immunization step is further repeated as necessary.
Specific procedures, medium conditions, culture conditions, etc. in the sensitization process and culture process used in the immunization process after the second immunization process are the antigen sensitization process and culture process used in the first immunization process. All the procedures and conditions that can be used in are applicable. All procedures and conditions in all immunization steps may be the same or different. Moreover, in order to detect antibody-producing cell colonies producing antigen-specific antibodies at high concentrations, antigen-specific antibodies may be detected using the culture supernatant.
Subsequently, the obtained antigen-specific antibody-producing cells are cell-fused with myeloma cells or the like by a conventional method, whereby a hybridoma that highly produces an antigen-specific IgG monoclonal antibody can be obtained.

Hereinafter, explanations of terms used in the present invention and specific embodiments of each step will be described, but the present invention is not limited thereto.
In the present invention, “immune cell” refers to a lymphocyte, which may be derived from any tissue as long as it is an antibody-producing cell and contains B cells. For example, peripheral blood, lymph obtained at the time of surgery Those obtained from a node or spleen can be used. The derived biological species may be derived from any mammal, but in order to obtain an antigen-specific monoclonal antibody used for treatment, lymphocytes derived from the same biological species, for example, human-derived lymphocytes are preferred.
In the present invention, it is preferable to provide a T cell removal step in advance prior to in vitro antigen sensitization, but it may be applied to the antigen sensitization step without providing it.
In the T cell removal step of the present invention, CD8 positive T cells (helper T cells) and / or NK cells are preferably removed. In this case, a CD8 specific antibody such as CD8a or CD49 specific antibody such as CD49b is used. To remove. When immunizing human peripheral blood in the presence of L-leucylleucine methyl ester (LLME), CD11c positive cells that suppress LLME action are preferably removed using an anti-CD11c antibody. As a T cell removal method using a CD8-specific antibody, a CD49-specific antibody or the like, a precipitation method or a cell sorting method may be used. It is preferable to magnetically separate by an LD column using beads. (Cell separation by CD8a (Ly-2) microbead (mouse), cell separation by CD49b (DX5) microbead (mouse)-Miltenyi Biotech Co., Ltd.)

In vitro antigen sensitization in the present invention is performed twice in the first immunization step and the second immunization step or a plurality of times as necessary.
As a sensitization method, first, immune cells from which T cells have been removed (or not removed) are suspended in a culture solution, dispensed into a multiwell plate, and an immunostimulatory substance is added together with a target antigen. And incubate. A well producing an activated antigen-specific antibody in the culture supernatant is selected and cultured in a culture medium in which cytokines such as IL-2 and IL-4, or muramyl dipeptide are further present. In the process, antigen-specific immune cells are grown. Similarly, in the second immunization step, it is preferable to separate the antigen sensitization step and the culture step, but in any one of the multiple immunization steps, an ordinary sensitization technique, IL A technique (such as Patent Documents 1 and 2) may be applied in which antigens are sensitized in the presence of cytokines such as -2, IL-4, and muramyl dipeptide, and simultaneously proliferated.
As an in vitro sensitizing antigen used in the present invention, any target can be targeted, and examples thereof include self antigens, enzymes, sparingly soluble antigens, cells, toxins, bacteria, viruses and the like. . Of course, when producing antibodies for humans, when applied to toxins that are highly toxic to laboratory animals such as mice, the effect of in vitro immunity can be exhibited. In the case of a hapten having a low molecular weight such as a low molecular chemical substance, it is appropriately combined with a carrier protein.
As immunostimulatory substances to be added simultaneously with the antigen, well-known substances are appropriately used. For example, adjuvants such as DNA (ODN) containing CpG motif and muramyl dipeptide (MDP) can be used. Typically, commercially available ODN1826 (Invivogen), MDP (Sigma), or a combination is used.
As a medium for culturing immune cells in the present invention, a normal cell culture medium such as RPMI medium, DMEM medium, Ham-F12 medium, RDF medium, ERDF medium, or a mixed medium thereof can be used. RPMI medium is particularly preferred. 35 to 38 ° C. Preferably, but are cultured at a temperature of 37 ° C., 1% to 10% in the, preferably incubated under about 5% CO ₂ environment. The cell density of immune cells in the medium is in the range of 3 × 10 ^{6 to} 5 × 10 ⁶ cells / ml, preferably 5 × 10 ⁶ cells / ml. The medium can be used as a suspension when collecting immune cells after collection, or as a washing solution when concentrating and separating immune cells from the suspension by centrifugation. It can also be used as a cell culture.
In the present invention, cytokine is added to the medium in the step of culturing the antigen-specific antibody-producing cells. Examples of cytokines used in this process include IL-2, IL-4, IL- 6, cytokines such as IL-10 and IL-21 are appropriately used, and interleukins such as IL-2, IL-4 and IL-21 are particularly preferably used. Specific blending ratios include, for example, IL-2: 1 to 20 ng / ml, IL-4: 1 to 20 ng / ml, preferably IL-2: 5 to 15 ng / ml, IL-4: 2 to 5 ng / ml, IL-10: 5 to 15 ng / ml can be used. Optimal numerical values include, for example, IL-2: 10 ng / ml, IL-4: 2.5 ng / ml, and IL-10: 10 ng / ml.

Hereinafter, the present invention will be described in more detail.
In the present invention, the term “T cell epitope peptide” refers to a T cell epitope peptide derived from a protein different from the target antigen or a T cell epitope-like peptide designed based on the three-dimensional structure of the epitope recognition site on the MHC-II side. (Both are simply referred to as “T cell epitopes”). Typically, 12 to 16 residues, preferably 14 to 15 residues, including T cell epitopes specific to conventionally known proteins, such as CTL epitopes for the proteins or T-helper cell epitopes for the proteins It is a peptide consisting of amino acids and is a T cell epitope unrelated to the target antigen, that is, “T cell epitope peptide other than T cell epitope contained in target antigen” or “T cell epitope peptide not derived from target antigen” .

The T cell epitope peptide used in the present invention may be any T cell epitope peptide other than the T cell epitope derived from the target antigen, and in particular, the amino acid sequence represented by the following formulas (I) to (VII) A T cell epitope peptide consisting of
Formula (I):
QYIKANSKFIGITEL
Formula (II):
VTYDNESLLSAHKVE
Formula (III):
RGIFFX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ KEI
(In the formula, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ and X ₆ may be the same or different arbitrary amino acid residues, but X ₁ is Y or Q; _It is particularly preferred that ₂ is V or T, X ₃ is F, H or K, X ₄ is A or G, X ₅ is A or G, and X ₆ is Y or H.
Formula (IV):
FQDN NAX ₇ X ₈ X ₉ X ₁₀ X ₁₁ AVF
(In the formula, X ₇ , X ₈ , X ₉ , X ₁₀ and X ₁₁ may be the same or different arbitrary amino acid residues, but X ₇ is A or V, and X ₈ is G, A Or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is particularly preferably N or H).
Formula (V):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AVF
(In the formula, X ₈ , X ₉ , X ₁₀ and X ₁₁ may be the same or different arbitrary amino acid residues, but X ₈ to X ₁₁ are the same as those in formula (IV), that is, X ₈ Is particularly preferably G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H).
Formula (VI):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AEI
(In the formula, X ₈ , X ₉ , X ₁₀ and X ₁₁ may be the same or different arbitrary amino acid residues, but X ₈ to X ₁₁ are the same as those in formula (IV), that is, X ₈ Is particularly preferably G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H).
Formula (VII):
NNYX ₁₂ X ₁₃ VX ₁₄ AAX ₁₅ X ₁₆ NN
(In the formula, X ₁₂ , X ₁₃ , X ₁₄ , X ₁₅ and X ₁₆ may be the same or different arbitrary amino acid residues, but X ₁₂ is N or G, and X ₁₃ is N Or G, X ₁₄ is N or G, X ₁₅ is N or G, and X ₁₆ is A or K.)

Here, the T cell epitope peptide used in the present invention includes not only a sequence included as a partial sequence of an actual antigen which is a known T cell epitope peptide, but also molecular dynamics analysis software Cerius 2 (Accelrys). For example, novel T cell epitope-like peptides such as formula (III) to formula (VII), which are composed of amino acid sequences designed based on the calculation using, are included.
Such a T cell epitope-like peptide is specifically designed by the following methods (1) to (3).

(1) Based on the crystal structure of MHCII, using the molecular dynamics analysis software Cerius 2 (Accelrys), the binding energy between MHCII and a 14-residue peptide of a T cell epitope whose known crystal structure is known To estimate a peptide sequence that is predicted to have high binding ability.
(2) Adding a correction from the sequence information having the ability to induce the antibody-producing ability of the sequence and other known experiments, further narrowing down the sequence and synthesizing a peptide with a higher binding ability, The ability to induce antibody production is measured. Actually, the antibody-producing cell activation ability is often superior to the original T cell epitope peptide.
(3) In the amino acid sequence of a peptide that is estimated to have a high binding ability, RGI on the N-terminal side and EI on the C-terminal side are not amino acids at positions recognized by MHC-II as T cell epitopes. Even if it is substituted with one or all amino acid residues among the residues, preferably any other amino acid residues within the range of 1 or 2, the antibody-producing cell activation function is retained.

Although a specific embodiment is shown below, since this technique is versatile, the combination of MHC-II and a T cell epitope is not restricted to this.
As MHC-II for presenting T cell epitopes, IA ^d , which is an MHC-II of Balb / c mouse, in which antibody production is efficiently performed, is used, and among known T cell epitopes, this is combined with IA ^d. Ovalbumin's "Peptide 323-339" was selected, whose body crystal structure was already X-ray analyzed. Isolate and purify the complex of bound MHC-II and T cell epitope, perform X-ray analysis after crystallization, calculate binding energy, and determine the amino acid sequence that is estimated to have high binding ability according to the following steps Just design.
Hereinafter, the calculation method (1) will be described in detail.
(A) A complex of MHC-II and a T cell epitope is isolated and purified, crystallized, the crystal structure is subjected to X-ray analysis, and the binding energy is calculated. Here, the IA ^d of mouse MHC-II, which has already been analyzed for the crystal structure of the complex, and the numerical value of Peptide 323-339 derived from Ovalbumin (PDB, 1IAO, 3D structure of MHC class II + OVApeptide ( 1IAO) CA Scott et al Immunity, Vol. 8, 319-329, 1998), and the case of using the numerical value will be described as a typical example.

(B) Using AutoLudi capable of automatic calculation on the analysis software Cerius 2 of Accelrys, docking simulation was performed on a large number of candidates. In AutoLudi, binding was assessed using the Ludi score. The Ludi score provides a score that correlates with the dissociation constant Ki in the ligand-receptor complex.
The correlation equation between the dissociation constant Ki and the Ludi score is Ludi score = 100 logKi, and the Ludi score represents the strength of binding. By calculating the free energy of binding, the Ludi score is determined. Free energy of bond: ΔG is expressed by the following equation. By searching for amino acid residues that have a large Ludi score, amino acid sequences with high binding are obtained.

(C) Each of the residues from the 1st to the 14th residues, using the binding energy when all 14 amino acids of the peptide of the T cell epitope (Ovalbumin-derived “peptide 323-339”) are exchanged for glycine as a reference value Calculate the interaction energy with MHC-II (IA ^d ) when each position is replaced with another amino acid. At that time, since the conformers with different rotation angles are also calculated, the interaction energy replaced with 94 components at each position is calculated. Replace each position of positions 1-14 of T cell epitope peptide with 94 components, calculate interaction energy for all residues, and first select amino acid residues higher than glycine as the first candidate And tabulate. (Table 1)

(D) Next, examine the interaction status of candidate residues with MHC-II (IA ^d ) (hydrogen bonding, space filling, aromatic interaction, etc.), and investigate the change in interaction from the original residue. While checking, select promising ones and narrow down the candidates for each amino acid residue.

(E) A peptide having all amino acid sequences in which the amino acid residues hit in (d) are combined with the amino acid residues of the original T cell epitope peptide is prepared using a link library, and the binding energy is calculated. , Perform scoring, and select them in descending order of binding energy. (Table 2)
Each peptide shown in Table 2 can be represented by the following formula (III).
Formula (III):
RGIFFX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ KEI
(Wherein X ₁ is Y or Q, X ₂ is V or T, X ₃ is F, H or K, X ₄ is A or G, X ₅ is A or G, X ₆ is Y or H.)

(F) A similar technique was applied to known T cell epitope peptides derived from Mycobacterium tuberculosis proteins in order to show that the technique used to produce T cell epitope-like peptides in the present invention is versatile.
That is, the peptide of the formula (IV) was prepared based on the known T cell epitope peptide “FQDAYNAAGGHNAVF” derived from the sequence in the M. tuberculosis protein by the same procedure as the preparation of the formula (III).
In the peptide represented by the following formula (IV), or in the formula (IV), one or more amino acids among amino acids consisting of “FQDA” on the N-terminal side and “VF” on the C-terminal side are any other amino acids. A substituted peptide that has the ability to activate antibody-producing cells;
Formula (IV):
FQDN NAX ₇ X ₈ X ₉ X ₁₀ X ₁₁ AVF
(Wherein X ₇ is A or V, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H).

(G) In the formula (III), the N-terminal RGI (R1 to R3 in Table 3) and the C-terminal EI (R13 to R14 in Table 3) are the positions recognized by MHC-II as T cell epitopes. Since these are not amino acids, one or more all of these amino acid residues, preferably one amino acid residue at either or both of the N-terminal side and the C-terminal side, is any other amino acid. Even when substituted with a residue, the antibody-producing cell activation function is retained. That is, in the peptide of the present invention, in the formula (III), one or more amino acids among the amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side are substituted with any other amino acid. Peptides that have the ability to activate antibody-producing cells are also included.
The point that R12 in Table 2 is "K" having a positive charge is very characteristic. On the other hand, since R4, R5 and R8 are “F”, it is considered that the space filling effect due to the size of the amino acid is simply reflected. From the conventional experimental knowledge that the tendency is high, it is conceivable that Y is applied to R4, N or G is applied to R5, and A, N, G or H is applied to R8. For the same reason, R6, R11, and R12 may be assigned A to R6, N or H to R11, and A to R12.

(H) Based on the above consideration, peptides of the following formulas (V) and (VI) were prepared. Formula (VI) is an array in which “VF” on the C-terminal side of Formula (V) is changed to “EI”.
In the peptide represented by the following formula (V), or in the formula (V), one or more amino acids among the amino acids consisting of “RGI” on the N-terminal side and “VF” on the C-terminal side are any other amino acids. A substituted peptide that has the ability to activate antibody-producing cells;
Formula (V):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AVF
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).
In the peptide represented by the following formula (VI), or in the formula (VI), one or more amino acids among the amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side are any other amino acids. A substituted peptide that has the ability to activate antibody-producing cells;
Formula (VI):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AEI
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).

(I) Formula (VII) below was created based on the consideration that small amino acids are effective to eliminate hydrophobic amino acids in order to increase the solubility of peptides and stimulate more T cells. .
In the peptide represented by the following formula (VII), or in the formula (VII), one or more amino acids among amino acids consisting of “NN” on the N-terminal side and “NN” on the C-terminal side are any other amino acids. A substituted peptide that has the ability to activate antibody-producing cells;
Formula (VII):
NNYX ₁₂ X ₁₃ VX ₁₄ AAX ₁₅ X ₁₆ NN
(In the formula, X ₁₂ is N or G, X ₁₃ is N or G, X ₁₄ is N or G, X ₁₅ is N or G, and X ₁₆ is A or K.) .

Further, among the T cell epitope peptides consisting of the amino acid sequences represented by the above formulas (I) to (VII), 9 consecutive amino acids are essential sequences as T cell epitopes. Any amino acid can be substituted. That is, a substituted peptide having the following amino acid sequence can also be used as the T cell epitope peptide of the present invention.
In the formula (I), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “QYI” on the N-terminal side and “TEL” on the C-terminal side,
In the formula (II), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “V” on the N-terminal side and “AHKVE” on the C-terminal side,
In the formula (III), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side;
In the formula (IV), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “FQDA” on the N-terminal side and “VF” on the C-terminal side,
In the formula (V), among the amino acids consisting of “RGI” on the N-terminal side and “VF” on the C-terminal side, one or more amino acids are substituted with any other amino acid,
In the formula (VI), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side;
In the formula (VII), an amino acid sequence in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “NN” on the N-terminal side and “NN” on the C-terminal side.
In the present invention, when a T cell epitope peptide is used for activating target antigen-specific antibody-producing cells, one type of peptide may be used, or two or more types of peptides may be used in appropriate combination.

In the present invention, when T cell epitope peptides are used for the activation of target antigen-specific antibody-producing cells, recombinant DNA containing DNA encoding these T cell epitope peptides can be used instead of adding the peptides themselves. It can also be used as a component.
In the present invention, the T cell epitope peptide may be further included in the culture medium in at least one of the antigen sensitization step or the culture step in either the first immunization step or the second immunization step. The antigen-specific antibody-producing cells are activated more specifically and proliferate. Usually, in the culture step of the antigen sensitization step word in the first immunization step or the second immunization step, it is added to the culture solution together with the cytokine. The amount of T cell epitope peptide added is 1 to 10 μg / ml, preferably 2 to 3 μg / ml. In that case, you may introduce | transduce the said DNA into an immune cell using the recombinant DNA containing DNA which codes T cell epitope peptide.
In the present invention, activated antibody-producing cells may be detected by detecting the presence of an antigen-specific antibody in the culture supernatant in the well.

Next, the obtained antigen-specific antibody-producing cells are fused with myeloma cells or other cancer cells by adding a cell fusion agent such as polyethylene glycol.
A hybridoma that produces a high amount of an antigen-specific IgG monoclonal antibody can be obtained by a conventional method. In the embodiment of the present invention, the following method was used.
That is, the hybridomas were selected with a HAT medium, and the culture supernatant of each hybridoma clone obtained by limiting dilution was measured by enzyme immunoassay (ELISA) using the above antigen to produce KLH-specific IgG antibodies. The hybridoma clones in the wells that showed high values were measured. Further, with respect to the obtained clones, a hybridoma clone showing a positive reaction specifically with KLH was obtained, and a clone with good antibody production ability and proliferation was further screened to obtain an anti-KLH monoclonal antibody-producing hybridoma.
The monoclonal antibody of the present invention obtained from the hybridoma is of the IgG class and can specifically recognize KLH.

(Example 1) Preparation of immune cells (1-1) Preparation of spleen cells The spleen was removed from BALB / c mice (♀), and the spleen cells were dispersed in a phosphate buffer (PBS), and a cell strainer (BD Contaminants were removed and washed with RPMI liquid medium (Sigma).

(1-2) Removal of Unnecessary Cells The spleen cells prepared in (1-1) above were coated with magnetic beads (CD8a (Ly-2)) that were coated with CD8a, which is a CD8-specific antibody, and CD49b, which was a CD49-specific antibody, respectively. Microbeads and CD49b (DX5) microbeads, Miltenyi) were mixed, and CD8 positive T cells and NK cells were labeled with magnetic beads. Next, magnetic separation was performed, and the negative fraction was collected. (Cell separation by CD8a (Ly-2) microbead (mouse), cell separation by CD49b (DX5) microbead (mouse)-Miltenyi Biotech Co., Ltd.)

Example 2 Primary Immune Stimulation The immune cells obtained in Example 1 were suspended in RPMI medium (Sigma) at a concentration of 5.0 × 10 ⁶ cells / ml, and 1 ml was dispensed into a 24-well plate. In each well, 1 μg of KLH (Keyhole Limpet Hemocyanin), 0.25 μM of immunostimulatory substance ODN1826 (Invivogen) and 10 μg of MDP (Sigma) were further added as antigens. After incubating for 2 days in a 37 ° C. CO ₂ incubator (5% CO ₂ ), the immune cells in the wells in which the presence of activated immune cells was confirmed were collected by centrifugation.
The recovered immune cells were then suspended in RPMI medium, and the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) were added at 37 ° C. Immune cells were grown by incubating in a CO ₂ incubator (5% CO ₂ ) for 2 days.

(Example 3) Measurement of production of antigen-specific IgM antibody by ELISA Antigen KLH was immobilized on a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocking solution (Roche). Was used for blocking. 100 μl of the cell culture medium obtained in Example 2 was added to each well, and after the reaction, the culture medium was discarded and washed. Further, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then production of KLH-specific IgM antibody was detected by a color development system using p-nitrophenyl phosphate as a substrate. The results are shown in FIG. At this time, antigen-specific IgG was not produced.

Example 4 Secondary Immune Stimulation Immune cells obtained in Example 2 were collected by centrifugation, suspended in RPMI medium, and dispensed into a 24-well plate at a concentration of 5.0 × 10 ⁶ cells / ml. Further, 0.3 μg of antigen KLH, 0.08 μM of immunostimulatory substance ODN1826 (Invivogen) and 3.3 μg / ml of MDP (Sigma) were further added to each well. After overnight incubation in a 37 ° C. CO ₂ incubator (5% CO ₂ ), activated immune cells were collected by centrifugation. After recovery, the cells are suspended in RPMI medium, and cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) are added to the solution in a 37 ° C. CO ₂ incubator. And incubated for 2-4 days to allow the cells to grow. During this period, 40-60% of the medium was changed daily.

(Example 5) Measurement of production of antigen-specific IgG antibody by ELISA Antigen KLH was immobilized on a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocking solution (Roche). Was used for blocking. 100 μl of the cell culture solution obtained in Example 3 was added to each well, and after the reaction, the culture solution was discarded and washed. Furthermore, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then KLH-specific IgG antibody production was detected with a color development system using p-nitrophenyl phosphate as a substrate. The results are shown in FIG.

(Example 6) Hybridoma production and quantification of positive clones The immune cells obtained in Example 1 were suspended in RPMI medium at a concentration of 5.0 × 10 ⁶ cells / ml, and 5 ml was dispensed into 6-well plates. To each well, 5 μg of KLH, 1.25 μM of immunostimulatory substance ODN1826, and 50 μg of MDP were added as antigens. After culturing in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 2 days, activated immune cells were collected by centrifugation. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained cells were collected by centrifugation, suspended in RPMI medium, and dispensed in 5 wells at a concentration of 5.0 × 10 ⁶ cells / ml in a 6-well plate. To each well, 1.67 μg of KLH, 0.42 μM of immunostimulating substance ODN1826, and 16.67 μg of MDP were added as antigens. After overnight culture in a 37 ° C. CO ₂ incubator (5% CO ₂ ), the activated immune cells were recovered by centrifugation. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained activated immune B cells were mixed with an equal amount of 8-azaguanine-resistant mouse myeloma cells, P3UI (P3-X63-Ag8-U1), and 50% polyethylene glycol (Roche) was used for sedimentation after centrifugation. Was added and allowed to react gently at 37 ° C. for 2 minutes to perform cell fusion. The resulting hybridoma-containing solution was dispensed into a 96-well microculture plate and cultured for 1 week using a HAT medium. The culture supernatant of the wells is collected and screened by enzyme immunoassay (ELISA). The number of wells in which the cells have formed colonies (number of cell growth wells) and IgG specifically reacting with KLH used as the antigen The number of wells contained (number of antigen-specific wells) was measured. The results are shown in (Table 3). As a control, the case of immunization without adding an antigen is shown simultaneously.

(Reference Example 1) Increased antibody titer of T cell epitope in in vivo immunization QYIKANSKFIGITEL (pTT, 830-844th of tetanus toxin) and VTYDNESLLSAHKVE (Ovm100S, 100th to 114th of chicken ovomucoid) which are known T cell epitope peptides Was mixed with an antigen (KLH, Keyhole Limpet Hemocyanin), and subcutaneously administered to mice (Balb / c, sputum) for immunization. Thereafter, the same booster was performed 20 days later. After immunization, 10 microliters of blood was collected after a certain period, diluted 1000 times with PBS, and then antigen-specific IgG in the serum was measured by ELISA (FIGS. 4 and 5).
Antigen KLH was fixed to a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocked using a blocking solution (Roche). 100 μl of serum dilution was added to each well, and after the reaction, the culture was discarded and washed. Furthermore, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then production of KLH-specific IgG antibody was detected by a color development system using p-nitrophenyl phosphate as a substrate.
When no peptide was added, no increase in antibody titer was observed, but with the simultaneous addition of peptides, a significant increase in antibody titer was observed. As a negative control, an experiment was conducted in which only KLH and KLH and non-T cell epitope peptide Ovm52S: TNISKEHDGESKETV (52th to 66th of chicken ovomucoid) were added. When no T cell epitope peptide was added or when a non-T cell epitope peptide was added, no increase in antibody titer was observed, but a significant increase in antibody titer was observed with the addition of T cell epitope peptide.

(Example 7) Preparation of immune cells in in vitro immunization T cell epitope peptide was added (3 μg / ml) in in vitro immunization. Subsequent production of antigen-specific IgG was measured.
(7-1) Preparation of spleen cells The spleen was removed from the BALB / c mouse (♀), the spleen cells were dispersed in a phosphate buffer (PBS), and impurities were removed through a cell strainer (BD). Washing was performed using RPMI liquid medium (Sigma).

(7-2) Removal of Unnecessary Cells The spleen cells prepared in (7-1) above were coated with magnetic beads (CD8a (Ly-2)), each coated with CD8a, a CD8-specific antibody, and CD49b, a CD49-specific antibody. Microbeads and CD49b (DX5) microbeads, Miltenyi) are mixed, and CD8 positive T cells and NK cells are labeled with magnetic beads. Subsequently, magnetic separation was performed using an LD column (Miltenyi), and the negative fraction was collected. (Cell separation using CD8a (Ly-2) microbeads (mouse), cell separation using CD49b (DX5) microbeads (mouse)-Miltenyi Biotech Co., Ltd.)

Example 8 Primary Immune Stimulation in In Vitro Immunization The immune cells obtained in Example 7 were suspended in RPMI medium (Sigma) at a concentration of 5.0 × 10 ⁶ cells / ml, and 1 ml was dispensed into a 24-well plate. . In each well, 1 μg of KLH (Keyhole Limpet Hemocyanin) as an antigen, 0.25 μM of immunostimulatory substance ODN1826 (Invivogen) and 10 μg of MDP (Sigma), and pTT (QYIKANSKFIGITEL), which is a T cell epitope peptide: Along with tetanus toxin 830-844), a known T cell epitope peptide p25 (FQDAYNAAGGHNAVF) derived from a sequence in the M. tuberculosis protein, typical novel peptides APL (FQDAYNAVGGHNAVF) and CH3 (FQDAYNAVGGAANAVF) corresponding to formula (IV) Added (3 μg / ml). After incubating for 2 days in a 37 ° C. CO ₂ incubator (5% CO ₂ ), the immune cells in the wells in which the presence of activated immune cells was confirmed were collected by centrifugation.
The recovered immune cells were then suspended in RPMI medium and the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) were added, Immune cells were grown by incubating in a CO ₂ incubator (5% CO ₂ ) for 2 days.

(Example 9) Secondary immune stimulation in in vitro immunization The immune cells obtained in Example 7 were collected by centrifugation, suspended in RPMI medium, and 1 ml each in a 24-well plate at a concentration of 5.0 × 10 ⁶ cells / ml. In each well, 0.3 μg of antigen KLH, 0.08 μM of immunostimulating substance ODN1826 (Invivogen) and 3.3 μg / ml of MDP (Sigma) were further added. After overnight incubation in a 37 ° C. CO ₂ incubator (5% CO ₂ ), activated immune cells were collected by centrifugation. After recovery, the suspension is suspended in RPMI medium, and the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) are added to the solution in a 37 ° C. CO ₂ incubator. And incubated for 2-4 days to allow the cells to grow. During this period, 40-60% of the medium was changed daily.

(Example 10) Measurement of production of antigen-specific IgG antibody in in vitro immunization (ELISA)
Antigen KLH was fixed to a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocked using a blocking solution (Roche). 100 μl of the cell culture solution obtained in Example 9 was added to each well, and after the reaction, the culture solution was discarded and washed. Further, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then production of KLH-specific IgG antibody was detected by a color development system using p-nitrophenyl phosphate as a substrate. The results are shown in FIG. It can be seen that the production of KLH-specific IgG antibody is more when the T cell epitope is added.

(Example 11) Hybridoma production and quantification of positive clones The immune cells obtained in Example 9 were suspended in RPMI medium at a concentration of 5.0 × 10 ⁶ cells / ml, and 5 ml was dispensed into 6-well plates. To each well, 5 μg of KLH, 1.25 μM of immunostimulatory substance ODN1826, and 50 μg of MDP were added as antigens. Simultaneously, 3 μg of T cell epitope peptide: pTT (QYIKANSKFIGITEL) and CH3 (FQDAYNAVGAANAVF) were prepared. After culturing in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 2 days, activated immune cells were collected by centrifugation. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained cells were collected by centrifugation, suspended in RPMI medium, and dispensed in 5-well plates at a concentration of 5.0 × 10 ⁶ cells / ml in 6-well plates. To each well, 1.67 μg of KLH, 0.42 μM of immunostimulating substance ODN1826, and 16.67 μg of MDP were added as antigens. After overnight incubation at 37 ° C. for a CO ₂ incubator (5% CO _2), by the activated immune cells centrifuged and collected. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained activated immune B cells were mixed with an equal amount of 8-azaguanine-resistant mouse myeloma cells, P3UI (P3-X63-Ag8-U1), and 50% polyethylene glycol (Roche) was used for sedimentation after centrifugation. Was added and allowed to react gently at 37 ° C. for 2 minutes to perform cell fusion. The resulting hybridoma-containing solution was dispensed into a 96-well microculture plate and cultured for 1 week using a HAT medium. The culture supernatant of the wells is collected and screened by enzyme immunoassay (ELISA). The number of wells in which the cells have formed colonies (number of cell growth wells) and IgG specifically reacting with KLH used as the antigen The number of wells contained (number of antigen-specific wells) was measured. The results are shown in Table 4. As a control, the case of immunization without adding an antigen is shown simultaneously.
Thus, the addition of T cell epitopes increased the number of activated immune B cells, and the proportion of IgG in the total globulin obtained also increased.

(Reference Example 2)
The optimal T cell epitope-like peptide for immune system activation was searched by in silico calculation. Peptides with high binding ability to MHC-II were screened by calculation using the X-ray crystal structure of MHC-II. For the MHC-II that displays T cell epitopes, IA ^d , which is a Balb / c mouse MHC-II that efficiently produces antibodies, was used. Based on the crystal structure of this complex of IA ^d and Ovalbumin peptide 323-339 (PDB, 1IAO, 3D structure of MHC class II + OVA peptide (1IAO) CA Scott et al Immunity, Vol. 8, 319-329, 1998) In addition, the design of an optimal epitope for IA ^d was advanced.
Using AutoLudi, which can perform automatic calculations on Accelrys's analysis software Cerius 2, docking simulations were performed on a large number of candidates. In AutoLudi, binding was assessed using the Ludi score. The Ludi score provides a score that correlates with the dissociation constant Ki in the ligand-receptor complex. The correlation between the dissociation constant Ki and the Ludi score is Ludi score = 100 logKi. Free energy of bond: ΔG is expressed by the following equation.
By calculating the free energy of binding, the Ludi score is determined. Represents the Ludi score and the strength of the bond.

The interaction energy with IA ^d was calculated when all amino acids of Ovalbumin peptide 323-339 were exchanged for glycine and each position from residue 1 to residue 14 was replaced with another amino acid. . Since there were also conformers with different rotation angles, the interaction energy was calculated by replacing 94 components at each position. Listed were those in which each of positions 1 to 14 of the T cell epitope was replaced with 94 components and the interaction energy obtained was higher than that of glycine. Table 1 shows a summary of amino acids in which the same calculation was performed for all residues and an increase in binding energy was observed.
Since there are 5 × 6 × 9 × 5 × 4 × 4 × 3 × 8 × 1 × 1 × 8 × 2 × 5 × 2 = 82944000 combinations of all candidates shown in Table 1, it is impossible to calculate all of these energies. Therefore, instead of automatically selecting those with high scores, we examined the interaction status of candidate residues with IA ^d (hydrogen bonding, space filling, aromatic interaction, etc.) The promising one was selected while confirming the change in the interaction. In the AutoLudi program, the link sites that can be set simultaneously in Scafold are up to 5 residues. In this case, there are 9 combination candidates (residues 4 to 12). Therefore, it was necessary to create a separate program for 14mer peptide construction work, and a Perl script was created for peptide construction (a script for comprehensively constructing 14mer peptides by combining respective residue candidates). After that, scoring was calculated by AutoLudi. The candidates were narrowed down and the following candidates were obtained. Here, Orig represents the original amino acid of “peptide 323-339” derived from Ovalbumin, which is the starting point of the calculation. Hit represents an amino acid having a high Ludi score in the above calculation.

1st amino acid 1 way Orig (Arg)
2nd amino acid 1 Orig (Gly)
3rd amino acid 1 Orig (Ile)
4th amino acid 4 ways Orig (Ser) + Hit (Phe-1, Leu-2, Ile-3) 1
5th amino acid 3 ways Orig (Gln) + Hit (Phe-1, Lys-2)
6th amino acid 3 ways Orig (Ala) + Hit (Tyr-2, Gln-3)
7th amino acid 2 ways Orig (Val) + Hit (Thr-1)
8th amino acid 3 ways Orig (His) + Hit (Phe-4, Lys-8)
9th amino acid 2 ways Orig (Ala) + Gly
10th amino acid 2 ways Orig (Ala) + Gly
11th amino acid 2 ways Orig (His) + Hit (Tyr-2)
12th amino acid 2 ways Orig (Ala) + Hit (Lys-1)
13th amino acid sequence Orig (Gln)
14th amino acid Orig (Leu)

Combining all these candidates results in 1x1x1x4x3x3x2x3x2x2x2x2x1x1 = 3456 ways. About 3456 peptides were prepared using a linked library, and the binding energy was calculated and scored. By the above operation, by performing the original computational 96 ¹⁴ narrows down candidates could be reduced to 3456. The amino acid sequence having a high Ludi score among the calculation results is as shown in Table 2 above.

The original peptide used to produce the crystal structure (Ovalbumin's peptide 323-339) had a Ludi score of 968 and had the 3363th binding energy. It was characteristic that a peptide with a lysine having a positive charge at the 12th residue that seems to bind to MHCII gave a high score. In addition, the amino acids at other MHCII binding sites were in good agreement with findings from previous experiments.

(Example 12) Antibody titer-increasing effect of T cell epitope-like peptide in in vivo immunization Antigen (KLH) 10 μg and the peptide obtained in Reference Example 2 above were mixed with Freund's imcomplete adjuvant, and mice (Balb / c, ♀) It was administered subcutaneously and immunized. As peptides, 10 μg each of Ovalbumin peptide 323-339 (RGISQAVHAAHAEI), which is the starting point of calculation, and a peptide (No. 1 RGIFFYVFAAYKEI in Table 2) calculated to have the highest binding energy were used. Twenty days after immunization, 10 microliters of blood was collected, diluted 1000 times with PBS, and then antigen-specific IgG in the serum was measured by ELISA (FIG. 7).
Antigen KLH was fixed to a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocked using a blocking solution (Roche). 100 μl of serum dilution was added to each well, and after the reaction, the culture was discarded and washed. Furthermore, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then production of KLH-specific IgG antibody was detected by a color development system using p-nitrophenyl phosphate as a substrate.
When the peptide was not added (peptide-) and when the antigen was not added (NC: Negative Control), the antibody titer was not increased, but the antibody titer was significantly increased by the simultaneous addition of peptides. . The degree of increase was greater for the peptide (No. 1 RGIFFYVFAYKEI in Table 2) calculated to have the highest binding energy than Ovalbumin's peptide 323-339 (RGISQAVHAAHAEI), which is the starting point of the calculation.

(Example 13) Activation effect of antigen-specific antibody-producing cells of the peptide of the present invention in in vitro immunization (13-1) Primary immune stimulation in in vitro immunization The immune cells obtained in Example 1 were 5.0 × 10 ⁶ cells / ml. The suspension was suspended in RPMI medium (Sigma) at a concentration, and 1 ml was dispensed into a 24-well plate. In each well, 1 μg of KLH (Keyhole Limpet Hemocyanin) as an antigen, 0.25 μM of immunostimulatory substance ODN1826 (Invivogen) and 10 μg of MDP (Sigma), and the T cell epitope-like peptide of the present invention Of these, typical of formula (V), APL2 (RGIYNAVAAANAVF) APL3 (RGIYNAVAGAAAHAVF), APL4 (RGIYNAVAAAHAVF) APL5 (RGIYNAVAHAANAVN), and typical of formula (VII), P1 (NNYNNNVN, NNNNNVN) 3 μg of each of P6 (NNYNNVNAANKNN) and P7 (NNYNNVGAAGKNN) was added. After incubating for 2 days in a 37 ° C. CO ₂ incubator (5% CO ₂ ), the immune cells in the wells in which the presence of activated immune cells was confirmed were collected by centrifugation.
The recovered immune cells were then suspended in RPMI medium and the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) were added, Immune cells were grown by incubating in a CO ₂ incubator (5% CO ₂ ) for 2 days.

(13-2) Secondary immune stimulation in in vitro immunization The immune cells obtained in (13-1) above were collected by centrifugation, suspended in RPMI medium, and in a 24-well plate at a concentration of 5.0 × 10 ⁶ cells / ml. In each well, 0.3 μg of antigen KLH, 0.08 μM of immunostimulatory substance ODN1826 (Invivogen) and 3.3 μg / ml of MDP (Sigma) were further added. After overnight incubation in a 37 ° C. CO ₂ incubator (5% CO ₂ ), activated immune cells were collected by centrifugation. After recovery, the suspension is suspended in RPMI medium, and the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml) are added to the solution in a 37 ° C. CO ₂ incubator. And incubated for 2-4 days to allow the cells to grow. During this period, 40-60% of the medium was changed daily.

(13-3) Measurement of production of antigen-specific IgG antibody in in vitro immunization (ELISA)
Antigen KLH was fixed to a 96-well microplate using carbonate buffer (pH 9.6), washed with 0.05% Tween-PBS solution, and then blocked using a blocking solution (Roche). 100 μl of the cell culture solution obtained in Example 3 was added to each well, and after the reaction, the culture solution was discarded and washed. Further, alkaline phosphatase-labeled anti-mouse IgG antibody was added and reacted, and then production of KLH-specific IgG antibody was detected by a color development system using p-nitrophenyl phosphate as a substrate. The results are shown in FIG. It can be seen that the production of KLH-specific IgG antibody is more when the T cell epitope-like peptide is added.

(Example 14) Hybridoma production and quantification of positive clones The immune cells obtained in Example 13 (13-2) were suspended in RPMI medium at a concentration of 5.0 × 10 ⁶ cells / ml, and each 5 ml was dispensed into a 6-well plate. Noted. To each well, 5 μg of KLH, 1.25 μM of immunostimulatory substance ODN1826, and 50 μg of MDP were added as antigens. At the same time, T cell epitope-like peptides were prepared by adding 3 μg of typical APL5 (RGIYNAVHAANAVF) of formula (V) and typical P7 (NNYNNVGAAGKNN) of formula (VII). After culturing in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 2 days, activated immune cells were collected by centrifugation. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained cells were collected by centrifugation, suspended in RPMI medium, and dispensed in 5 wells at a concentration of 5.0 × 10 ⁶ cells / ml in a 6-well plate. To each well, 1.67 μg of KLH, 0.42 μM of immunostimulating substance ODN1826, and 16.67 μg of MDP were added as antigens. After overnight culture in a 37 ° C. CO ₂ incubator (5% CO ₂ ), the activated immune cells were recovered by centrifugation. The cells were cultured for 2 days in the presence of the cytokines mrIL-2 (10 ng / ml), mrIL-4 (2.5 ng / ml) and mrIL-21 (10 ng / ml). The obtained activated immune B cells were mixed with an equal amount of 8-azaguanine-resistant mouse myeloma cells, P3UI (P3-X63-Ag8-U1), and 50% polyethylene glycol (Roche) was used for sedimentation after centrifugation. Was added and allowed to react gently at 37 ° C. for 2 minutes to perform cell fusion. The resulting hybridoma-containing solution was dispensed into a 96-well microculture plate and cultured for 1 week using a HAT medium. The culture supernatant of the wells is collected and screened by enzyme immunoassay (ELISA). The number of wells in which the cells have formed colonies (number of cell growth wells) and IgG specifically reacting with KLH used as the antigen The number of wells contained (number of antigen-specific wells) was measured. The results are shown in Table 4. As a control, the case of immunization without adding an antigen is shown simultaneously.
Thus, by adding the T cell epitope-like peptide, activated immune B cells increased, and the proportion of IgG in the total globulin obtained also increased.

Hereinafter, for reference, SEQ ID NOS: 1 to 21 used in the present invention are shown in contrast with the origin.

SEQ ID NO: 1 QYIKANSKFIGITEL Formula (I) Tetanus toxin pTT (830-844)
Sequence number 2 VTYDNESLLSAHKVE formula (II)
SEQ ID NO: 3 RGIFFX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ KEI Formula (III) X ₁ = YorQ, X ₂ = VorT, X ₃ = F, HorK, X ₄ = AorG, X ₅ = AorG, X ₆ = YorH
SEQ ID NO: 4 FQDAYNAX ₇ X ₈ X ₉ X ₁₀ X ₁₁ AVF Formula (IV) X ₇ = AorV, X ₈ = G, AorH, X ₉ = GorA, X ₁₀ = HorA, X ₁₁ = NorH
SEQ ID NO: _{5 RGIYNAVX 8 X 9 X 10 X} 11 AVF formula _{(V) X 8 = G,} AorH, X 9 = GorA, X 10 = HorA, X 11 = NorH
SEQ ID NO: 6 RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AEI Formula (VI) X ₈ = G, AorH, X ₉ = GorA, X ₁₀ = HorA, X ₁₁ = NorH
Sequence number 7 NNYX ₁₂ X ₁₃ VX ₁₄ AAX ₁₅ X ₁₆ NN Formula (VII) X ₁₂ = NorG, X ₁₃ = NorG, X ₁₄ = NorG, X ₁₅ = NorG, X ₁₆ = AorK
Sequence number 8 TNISKEHDGESKETV Ovm52S (52-66)
Sequence number 9 FQDAYNAAGGHNAVF Formula (IV) original Mycobacterium tuberculosis origin / p25
SEQ ID NO: 10 FQDAYNAVGAANAVF Formula (IV) CH3
Sequence number 11 RGISQAVHAAHAEI Ovalbmin (323-339)
Sequence number 12 RGIFFYVFAAYKEI Formula (III)-(1)
SEQ ID NO: 13 RGIYNAVAAANAVF Formula (V) APL2
Sequence number 14 RGIYNAVGAAHAVF Formula (V) APL3
Sequence number 15 RGIYNAVAAAHAVF Formula (V) APL4
Sequence number 16 RGIYNAVHAANAVF Formula (V) APL5
SEQ ID NO: 17 NNYNNVNAANANN formula (VII), P1
SEQ ID NO: 18 NNYNNVGAAGANN formula (VII), P2
SEQ ID NO: 19 NNYNNVNAANKNN Formula (VII), P6
SEQ ID NO: 20 NNYNNVGAAGKNN Formula (VII), P7
Sequence number 21 FQDAYNAVGGHNAVF Formula (IV) APL

2 is a flowchart showing an exemplary protocol of the present invention. It is the result of having tested the induction | guidance | derivation of the antigen specific IgM production cell after the primary stimulation of this invention by ELISA method. In the figure, KLH + is the case where the antigen KLH is added, ODN + is the case where the immunostimulatory substance ODN is added, MDP + is the case where the immunostimulatory substance MDP is added, and ODNc + is the substance ODNc which does not stimulate the immune system. This is the case when it is added. Moreover,-shows the case where the said substance was not added. An increase in OD405 indicates that antigen-specific IgM is being produced. It is the result of having tested the induction | guidance | derivation of the antigen specific IgG production cell of this invention by ELISA method. It is the result of having measured the activation of the antibody production ability when administering a peptide with an antigen (KLH) to a mouse | mouth as a time change from antigen administration. In the figure, pTT is a case where a tetanus toxin-derived T cell epitope is added. Ovm100S is when the chicken ovomucoid-derived T cell epitope is added, and Ovm52S is when the non-T cell epitope is added. KLH shows the case of antigen alone as a control. An increase in OD405 indicates that antigen-specific IgG is being produced. It is the result of having tested the activation of the antibody production ability when administering a peptide with an antigen (KLH) to a mouse | mouth by ELISA method. In the figure, the meanings of the terms pTT, Ovm100S, Ovm52S, and KLH are the same as in FIG. The result of having tested the activation of the antibody production ability by four types of T cell epitope peptides (pTT, p25, APL, and CH3) by in vitro immunization method by ELISA method is shown. It is the result of having tested by ELISA method activation of the antibody production ability when a peptide is administered to a mouse | mouth with an antigen (KLH). In the figure, Original represents Ovalbumin's peptide 323-339 (RGISQAVHAAHAEI), which is the starting point of the calculation, and No. 1 represents the peptide having the highest binding energy (No. 1 RGIFFYVFAAYKEI in Table 2). Is the case where the same non-T cell epitope was added. KLH shows the case where only the antigen was administered, and NC shows the case where the peptide and the antigen were not administered as a negative control. An increase in OD405 indicates that antigen-specific IgG is being produced. In in vitro immunization methods, T cell epitope peptides (APL2: RGIYNAVAAANAVVF, APL3: RGIYNAVGAAHAVF, APL4: RGIYNAVAAAANAVVF, APL5: RGIYNAVANANNF, P1: NNYNNNVN, P2: NNYNNNV, N The result of having been tested by the ELISA method is shown. Moreover, KLH shows the case where only an antigen is administered, NC shows the case where a peptide and the antigen KLH are not administered as a negative control. An increase in OD405 indicates that antigen-specific IgG is being produced.

Claims (8)

  Obtained after subjecting immune cells to an antigen sensitization step for sensitizing a target antigen in vitro and a first immunization step for culturing antibody-producing cells in a culture solution containing cytokines A two-step in vitro characterized by subjecting a target antigen-specific antibody-producing cell to a second immunization step in which an antigen sensitization step with the same antigen in vitro and a culture step in a culture medium containing a cytokine are performed. Immunization method.   The two-step in vitro immunization method according to claim 1, wherein the immune cells are provided with a T cell removal step in advance of the first immunization step.   The two-step in vitro immunization method according to claim 2, wherein the T cell removal step uses a CD8-specific antibody and a CD49-specific antibody.   The T-cell epitope peptide is further contained in the culture medium used in at least one of the antigen sensitization step or the culture step in any one of the first immunization step and the second immunization step. Item 4. The two-step in vitro immunization method according to any one of Items 1 to 3. The two-step in vitro immunization method according to claim 4, wherein the T cell epitope peptide is a peptide represented by any of the following formulas (I) to (VII):
Formula (I):
QYIKANSKFIGITEL
Formula (II):
VTYDNESLLSAHKVE
Formula (III):
RGIFFX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ KEI (where X ₁ is Y or Q, X ₂ is V or T, X ₃ is F, H or K, X ₄ is A or G, X ₅ is A or G, X ₆ is Y or H).
Formula (IV):
FQDN NAX ₇ X ₈ X ₉ X ₁₀ X ₁₁ AVF
(Wherein X ₇ is A or V, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H).
Formula (V):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AVF
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).
Formula (VI):
RGIYNAVX ₈ X ₉ X ₁₀ X ₁₁ AEI
(Wherein X ₈ to X ₁₁ are the same as in formula (IV), that is, X ₈ is G, A or H, X ₉ is G or A, X ₁₀ is H or A, and X ₁₁ is N or H. ).
Formula (VII):
NNYX ₁₂ X ₁₃ VX ₁₄ AAX ₁₅ X ₁₆ NN
(In the formula, X ₁₂ is N or G, X ₁₃ is N or G, X ₁₄ is N or G, X ₁₅ is N or G, and X ₁₆ is A or K.) . The T cell epitope peptide is
In the formula (I), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “QYI” on the N-terminal side and “TEL” on the C-terminal side,
In the formula (II), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “V” on the N-terminal side and “AHKVE” on the C-terminal side,
In the formula (III), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side,
In the formula (IV), a peptide in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “FQDA” on the N-terminal side and “VF” on the C-terminal side,
In the formula (V), a peptide in which one or more amino acids among amino acids consisting of “RGI” on the N-terminal side and “VF” on the C-terminal side are substituted with any other amino acid,
A peptide in which one or more amino acids are substituted with any other amino acid among the amino acids consisting of “RGI” on the N-terminal side and “EI” on the C-terminal side in the formula (VI),
In the formula (VII), a peptide in which one or more amino acids are substituted with any other amino acid among amino acids consisting of “NN” on the N-terminal side and “NN” on the C-terminal side,
The in vitro immunization method according to claim 5, wherein   A method for producing a hybridoma that produces an antigen-specific monoclonal antibody, comprising a step of fusing an antigen-specific antibody-producing cell obtained by the two-step in vitro immunization method according to any one of claims 1 to 6 with a myeloma cell.   A method for producing a target antigen-specific monoclonal antibody, wherein the hybridoma according to claim 7 is used.