# Patent

## Title
Acetyllysine-recognizing monoclonal antibody and process for producing the same

## Abstract

Anti-acetyllysine monoclonal antibody capable of recognizing Nε-acetyllysine regardless of the types of the adjacent amino acids. Namely, a monoclonal antibody having a light chain comprising a constant region having the amino acid sequence represented by SEQ ID NO:1 and a variable region having the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one to several amino acids, and a heavy chain comprising a constant region having the amino acid sequence represented by SEQ ID NO:3 and a variable region having the amino acid sequence represented by SEQ ID NO:4 or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one to several amino acids, and being capable of recognizing Nε-acetyllysine in a protein regardless of the types of the adjacent amino acids, i.e., being capable of accepting adjacent amino acids over a broad range; and a process for producing this monoclonal antibody characterized by using a chemically acetylated protein as an antigen.

## Background
TECHNICAL FIELD
The present invention relates to a novel monoclonal antibody and a method for producing the same. Specifically, the present invention relates to a monoclonal antibody capable of recognizing an Nε-acetyllysine residue in a protein without depending on the type of an adjacent amino acid, and a method for producing the same.
BACKGROUND ART
In recent years, it has become apparent that an Nε-acetylation of the lysine residue in N-terminal region of a core histone plays an important role in the control of gene expression of eukaryotes. The enzyme of a histone acetyltransferase responsible for the acetylation and the enzyme of a histone deacetylase responsible for deacetylation were cloned in 1996 for the first time, and after that, a plurality of molecules having similar activities have been found. In recent years, furthermore, beside histone, it has been found that various kinds of non-histone proteins such as p53, TCF, and HMG-1 could be acetylated. It has been pointed out that the acetylation may be post-translation modification that plays various roles as equally as phosphorylation.
To search for an unknown novel acetylation protein as described above, there is no need to discuss the usefulness of a probe molecule that recognizes an Nε-acetyllysine residue specifically and irrespectively of the adjacent amino acid. An antibody has been considered as the most suitable molecule for the object. However, the antibody capable of recognizing acetyllysine under various conditions irrespective of the types of the adjacent amino acids has been hardly reported.
DISCLOSURE OF THE INVENTION
The present invention has been completed in view of the present circumstances and aims to provide an Nε-acetyllysine-recognizing anti-acetyllysine monoclonal antibody which does not particularly depend on the types of the adjacent amino acids and is capable of allowing wide varieties of adjacent amino acids.
The present inventors have been dedicated to the study of solving the above problems and finally attained success to produce an anti-acetyllysine monoclonal antibody capable of allowing wide varieties of adjacent amino acids. Furthermore, the present invention has been completed by determining cDNA sequences of variable regions of the respective monoclonal antibodies being produced to make it clear that the produced antibodies have their characterized structures which are similar to each other.
In other words, the present invention relates to a monoclonal antibody that recognizes Nε-acetyllysine. In particular, the present invention relates to a monoclonal antibody which does not particularly depend on the types of the adjacent amino acids and is capable of allowing wide varieties of adjacent amino acids.
More specifically, the present invention relates to a monoclonal antibody comprising: (1) a light chain comprising a constant region having the amino acid sequence represented by SEQ ID NO.: 1 and a variable region having the amino acid sequence represented by SEQ ID NO.: 2, or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one or several amino acids; (2) a heavy chain comprising a constant region having the amino acid sequence represented by SEQ ID NO.: 3 and a variable region having the amino acid sequence represented by SEQ ID NO.: 4, or an amino acid sequence derived from this amino acid sequence by deletion, substitution or addition of one or several amino acids.
Furthermore, the present invention relates to a method for producing the monoclonal antibody characterized in that the chemically acetylated protein being is used as an antigen.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the comparison (ELISA method) between the reactivities of four kinds of anti-acetyllysine monoclonal antibodies of the present invention with respect to various acetyllysine-containing peptides.
FIG. 2 shows the results of detecting acetylated proteins in the total cell lysates of various kinds of cells using four kinds of anti-acetyllysine monoclonal antibodies.
FIG. 3 shows the results of investigating whether the ELISA reactivities of four kinds of anti-acetyllysine monoclonal antibodies of the present invention are competed with Nε-acetyllysine and Nα-acetyllysine.
FIG. 4 shows the results of comparing between amino acid sequences of the variable regions of heavy chain (SEQ ID NOS 23–26, respectively in order of appearance) and light chain (SEQ ID NOS 19–22, respectively in order of appearance) of four kinds of anti-acetyllysine monoclonal antibodies of the present invention.



BRIEF MODE FOR CARRYING OUT THE INVENTION
In the monoclonal antibody of the present invention, the variable region of the light chain having the amino acid sequence, represented by SEQ ID NO.: 2, where one or several amino acids are substituted includes, for example, an amino acid sequence of SEQ ID NO.: 5, SEQ ID NO.: 6, or SEQ ID NO.: 7.
Furthermore, in the monoclonal antibody of the present invention, the variable region of a heavy chain, having the amino acid sequence represented by SEQ ID NO.: 4, where one or several amino acids are substituted, includes, for example, an amino acid sequence represented by SEQ ID NO.: 8; where one or several amino acids are deleted, includes, for example, an amino acids sequence, represented by SEQ ID NO.: 9; where one or several amino acids are added, includes, for example, an amino acid sequence represented by SEQ ID NO.: 10, respectively.
The monoclonal antibodies of the present invention are antibodies that recognize Nε-acetyllysine and have the properties of allowing wide varieties of adjacent amino acids without depending on the types of their adjacent amino acids when recognizing Nε-acetyllysine residue existing in protein. These antibodies can be prepared by using various kinds of acetyllysine-containing molecules as antigens. The antibody having particularly excellent properties is obtained by a method of preparation in which a chemically acetylated protein having a plurality of lysine residues is used as an antigen.
The antibodies of the present invention include those obtained by binding synthetic peptides containing acetyllysine with carrier proteins such as purple-limpet hemocyanin, or those produced by antibody-producing immortalized cells, obtained by immobilizing the antibody-producing cells such as spleen cells are fused with myeloma cells or the like, where the antibody-producing cells are obtained by immunizing an animal such as a mouse using a protein in which a plurality of lysine residues are chemically acetylated, for example purple-limpet hemocyanin acetylated by acetic anhydride, as an antibody.
Among the above antigen molecules, A method using a protein containing a plurality of lysine residues in the molecule thereof is chemically acetylated, is more preferable than a method using a peptide containing a single lysine residue as an antigen. Therefore, it is easily thinkable that using a mixture of various kinds of acetyllysine-containing peptides is preferable for obtaining excellent antibodies.
Alternatively, antibody-producing cells can be obtained by various methods in which antibody libraries displayed on phages are screened by means of their affinities with antigens.
For obtaining such antibody-producing immortalized cells, all the monoclonal antibody producing technologies, which have been conventionally used, and which will be newly developed in future, can be used.
The screening of antibody-producing cells can be performed by selecting a clone that produces an antibody allowing adjacent amino acids as much as possible by using a protein, different from an antigen in which a plurality of lysine residues are acetylated, such as acetylated bovine serum albumin or a acetyllysine-containing peptide having various kinds of adjacent amino acids. In addition, the produced antibody molecules can be purified by an affinity column in which acetyllysine is immobilized or an affinity column using protein A.
It can be detected whether the produced antibody corresponds to the antibody of the present invention by analyzing a DNA sequence of the variable region of the antibody gene in the antibody-producing immobilized cell, and translating it into a protein, and determined by whether it has a high similarity of the sequential characteristics of the protein with the above sequence or not.
Furthermore, the present invention provides a gene, preferably DNA, which encodes a monoclonal antibody that recognizes Nε-acetyllysine of the present invention, which has been mentioned above. Examples of the DNA of the present invention are shown in SEQ ID Nos. 11 to 18 in the sequence table. SEQ ID Nos. 11 to 14 are related to light chains, and SEQ ID Nos. 15 to 18 are related to heavy chains, respectively. The DNA of the present invention includes complement chains thereof, or the base sequences capable of hybridizing with these base sequences under stringent conditions.
In the monoclonal antibodies of the present invention, as sensitized animals, various species of mammals such as mice, rats, rabbits, and dogs, and birds such as chickens can be used. In addition, it is also possible to make chimeric antibodies or human-type antibodies using variable regions and/or a hypervariable region of the monoclonal antibody of the present invention.
EXAMPLES
Hereinafter, the present invention will be explained in details with reference to the examples. However, the present invention is not limited to these examples at all.
Example 1
Preparation of Anti-Nε-acetyllysine Monoclonal Antibody
The anti-Nε-acetyllysine monoclonal antibody was prepared with a combination of three kinds of immunological antigens and screening antigens as described in Table 1. Furthermore, the acetylation of bovine serum albumin and purple-limpet hemocyanin was performed using acetic anhydride by the following method. 10 mg of protein was dissolved in 1 ml of a borate buffer (20 mM Na2B4O7, pH 9.3), and then 250 μmol of acetic anhydride (about 22.6 μl) and 500 μl of 1M NaOH were added therein while cooling with ice, followed by incubating for 30 minutes with stirring occasionally. After the reaction, a solvent was changed using a G-25 gel filtration (PD-10, Pharmacia Co., Ltd.) and a phosphate buffer solution (PBS) of acetylated protein was obtained.









TABLE 1






Immunological antigen
Screening antigen







Case 1
Conjugate of acetylated N-terminal
Acetylated N-terminal



peptide of histone H4 with
peptide of histone H3



purple-limpet hemocyanin


Case 2
Conjugate of acetylated N-terminal
Bovine serum albumin



peptide of histone H4 with
being acetylated with



purple-limpet hemocyanin
acetic anhydride


Case 3
Purple-limpet hemocyanin being
Bovine serum albumin



acetylated with acetic anhydride
being acetylated with




acetic anhydride, and




acetylated peptide









The immunization was performed on female Balb/c mice every week for three weeks intraperitoneally, using Freund's complete adjuvant at the first time, and Freund's incomplete adjuvant at the second and third times. The amount of immunization is 0.1 mg/mouse.
From the mouse on which the immunization was completed, hybridoma that produces an anti-acetyllysine monoclonal antibody was cloned using the conventional method. As a result, Clone-1 from Case 1, Clone-2 from Case 2, and Clone-3 and Clone-4 from Case 3 were established. Using various kinds of acetyllysine-containing peptides covalently bonded to an ELISA plate (Iwaki Glass Co., Ltd., AquaBind Plate) through their C-terminal cysteine, the results of comparing reactivities thereon are shown in FIG. 1. In FIG. 1, “A” presents the results of Clone-1, “B” represents those of Clone-2, “C” represents those of Clone-3, and “D” represents those of Clone-4, respectively. The right side of each graph denotes the concentration of each antibody at the time of providing an absorbance of 0.5. In addition, a list of peptides used in the figure is as shown in Table 2. As shown in FIG. 1, it was found that each of the antibodies of Clone-1 to Clone-4 showed the binding reactivity to acetyllysine under the conditions in which various kinds of adjacent amino acids were present. From this experiment, three clones, Clone-2 to Clone 4, showed their reactivities at almost same level as the respective peptides investigated at this time and it was found that they accept adjacent amino acids widely.
In addition, as a result of determining isotypes of the respective antibodies, it was confirmed that all of them were Ig G1κ.








TABLE 2





Name of



Peptides
Amino Acid Sequence

















H2A-5-GKQ
SGRGK(Ac)QGGKC
(SEQ ID NO: 27)


H2B-5-AKS
PEPAK(Ac)SAPAC
(SEQ ID NO: 28)


H2B-12-KKG
PAPKK(Ac)GSKKC
(SEQ ID NO: 29)


H2B-15-SKK
KKGSK(Ac)KAVTC
(SEQ ID NO: 30)


H3-9-RKS
TARK(Ac)STGGKAC
(SEQ ID NO: 31)


H3-14-GKA
STGGK(Ac)APRKC
(SEQ ID NO: 32)


H3-18-RKQ
KAPRK(Ac)QLATC
(SEQ ID NO: 33)


H3-23-TKA
LATK(Ac)AARKSAC
(SEQ ID NO: 34)


H4-5-GKG
SGRGK(Ac)GGKGLC
(SEQ ID NO: 35)


H4-16-AKR
KGGAK(Ac)RHRKVC
(SEQ ID NO: 36)


H4RC
SGRGKGGKGLGKGGAKRHRKVC
(SEQ ID NO: 37)


p53-320
SPQPKK(Ac)KPLC
(SEQ ID NO: 38)


p53-373
HLKSKK(Ac)GQSC
(SEQ ID NO: 39)


p53-382
TSRHKK(A)LMFC
(SEQ ID NO: 40)





Numbers added on the right side of each name denotes the position of the corresponding acetyllysine residue in each protein. H4RC represents a non-acetylated peptide. In addition, amino acid sequences in the table are represented by means of a one-character notation.






Example 2
Detection of Acetylated Protein by Western Blotting Method
Five types of cells, B16/BL6, MOLT-4F, HeLa-S3, COS-1, and COS-7 were treated with, 1 μM of histone-deacetylase inhibitor, CHAP31, for 24 hours, and then cell lysates were prepared and developed on an electrophoresis, followed by detecting acetylated proteins using the above four antibodies as primary antibodies. The results were shown in FIG. 2. In FIGS. 2, A, B, C, and D indicate the results of the detections using Clone-1, Clone-2, Clone-3, and Clone-4, respectively. The concentration of the primary antibodies used for the Western blotting were 107 ng/ml, 65.7 ng/ml, 258 ng/ml, and 158 ng/ml, respectively, and each of these concentration provide the same reactivity (A492=1) in ELISA where acetylated bovine serum albumin was immobilized. The total cell lysates were prepared from the cells (+) after subjecting each cell in the 1 μM CHAP31 treatment for 24 hours or the cells (−) without the treatment. In each lane, 20 μg of protein was loaded. Five types of cells being used were 1: B16/BL6, 2: MOLT-4F, 3: HeLa-S3, 4: COS-1, and 5: COS-7, respectively.
As shown in FIG. 2, the Clone-1 antibody prepared in Case 1 of Example 1 detected only the acetylation of histone increased by CHAP31. In addition, the Clone-2 antibody prepared in Case 2 detected several proteins other than histone, but had an insufficient reactivity to the others. On the other hand, the Clone-3 and Clone-4 antibodies prepared in Case 3 strongly detected acetylated proteins on the position around 50 kDa other than histone in MOT-4F, COS-1, and COS-7 cells. Furthermore, it was found that they can detect a plurality of proteins around 20 kDa and high-molecular portions in the MOLT-4F cell, and much more acetylated proteins in the COS-7 cells. From these results, it was confirmed that in the detection of the acetylated non-histone protein by the Western blotting method, Clone-3 and Clone-4 prepared by using acetylated purple-limpet hemocyanin as an antigen are particularly excellent. In other words, it was found that for preparing the antibody capable of accepting wide varieties of adjacent amino acids of acetyllysine, the use of a molecule, of which a plurality of lysine residues in the protein was acetylated, such as the acetylated purple-limpet hemocyanin, as an antibody is preferable.
Example 3
Confirmation of Specificity to Nε-Acetyllysine
To confirm that the prepared antibody was specifically reacting with Nε-acetylated lysine, acetylated bovine serum albumin was immobilized on an ELISA plate and investigated whether the reactivities of the respective antibodies to the ELISA were competed with Nε-acetyllysine and Nα-acetyllysine. That is, the ELISA plate immobilized with a phosphate buffer solution (50 μl) of 1 μg/ml acetylated bovine serum albumin at 4° C. overnight was used, and then the inhibitions by Nε-acetyllysine and Nα-acetyllysine were investigated under the conditions on which 1 μg/ml of each antibody was reacted. The results are shown in FIG. 3. In the figure, ●, ▴, ▪, and ♦ show the results of the inhibitions by Nε-acetyllysine and ◯, Δ, □, and ⋄ show the results of the inhibitions by Nα-acetyllysine, respectively. Furthermore, ● and ◯ are results of the case using Clone-1 (AL3D5); ▴ and Δ are results of the case using Clone-2 (AL11); ▪ and □ are results of the case using Clone-3 (AKL3H6); and ♦ and ⋄ are results of the case using Clone-4 (AKL5C1), respectively.
As is obvious from FIG. 3, the reactivity of each antibody of Clone-1 to Clone-4 decreased as being competed with Nε-acetyllysine, but not competed with Nα-acetyllysine. Accordingly, it has become obvious that these antibodies specifically react with Nε-acetyllysine.
Example 4
Determination of cDNA and Amino Acid Sequence in Variable Region of Each Antibody
To make clear what kind of amino acid sequence in the variable region has excellent properties as described in the above examples of AKL3H6 and AKL5C1 antibodies, the DNA in variable region of the L and H chains of each antibody using hybridoma was cloned and then the sequence thereof was determined. The cloning was performed by isolating RNA from the hybridoma using RNeasy Mini Kit available from QIAGEN Co., Ltd., performing a reverse transcription reaction using the TrueScript II RT available from Sawady Technology using random-9mer as a template, and amplifying the variable-region cDNA by the PCR method, using SuperTaq 2× kit available from Sawady Technology Co., Ltd., using the mix primer available from Novagen Co., Ltd. as 5′-primer and 5′-ACTGTTCAGGACGCCATTTTGTCGTTCACT-3′ (SEQ ID NO: 41) for the light chain and 5′-GGATCCAGAGTTCCAGGTCACTGT-3′ (SEQ ID NO: 42) for the heavy chain, as 3′-primers. The resulting DNA fragment was ligated using the DNA Ligation kit ver. 2 available from TaKaRa with pT7 Blue T-Vector available from Novagen Co., Ltd, and then it was used for transforming JM 109 competent cells available from TaKaRa Co., Ltd and seeded in X-gal-, ampicillin-, and IPTG-containing plates, followed by picking up white colonies. Plasmids were prepared from each of five species of clones that contains normal-sized inserts, respectively. After that, the DNA sequences were determined using an ABI PRISM 310-type automatic sequencer. The determined sequences showed the same sequences as those of five clones, except that a variation which may be caused by a PCR error was found in a part of them, so that these sequences were regarded as the objective DNA sequences. The results are shown in SEQ ID NO.: 11 to SEQ ID NO.: 18.
Furthermore, on the basis of the DNA sequences determined as described above, the results of the estimations of the amino acid sequences of the L and H chains are shown in SEQ ID NO.: 19 to SEQ ID NO.: 26, respectively.
Furthermore, FIG. 4 shows the results of comparing amino acid sequences in the variable regions of the light and heavy chains in the respective antibodies by aligning the amino acid sequences. The amino acids were represented by means of a one-character notation. In addition, the complementarity determining regions in the variable region were surrounded by squares and represented by CDR 1 to 3, respectively.
As is obvious from this figure, each of four kinds of antibodies obtained by three independent immunologic operations has the common framework structure. Such a commonality seems to have something important with the acetyllysine recognition itself. On the other hand, the difference in the properties among the antibodies shown in Examples 1 and 2 seems to be attributed to a slight difference in their sequences described herein; however, since there is no three-dimensional information at present, it is not obvious which portion is responsible for the difference in the properties of the antibodies.
INDUSTRIAL APPLICABILITY
As the antibody of the present invention can detect acetyllysine without depending on the types of adjacent amino acids of acetyllysine so much, it is useful for detecting the state of acetylation of well-known various acetylated protein. For instance, it can be easily detected the change of acetylating level of histone under the influences of various stimulants by the method such as Western blotting. Furthermore, from the same reason, the antibody of the invention is very useful for detecting an unknown novel acetyllysine-containing protein. Concretely, by using an immunoprecipitation method using the antibody of the invention; or an affinity column, an antibody chip, or the like, on which the antibody of the invention is immobilized, it is expected that an unknown novel acetyllysine-containing protein would be found. Furthermore, as the antibody of the present invention is a monoclonal antibody, it can be altered to a single-chain antibody by an already-established method. Alternatively, as its epitope is acetyllysine and is small, there is a possibility that either a light chain or a heavy chain may have its activity, in this case, by means of a two-hybrid method using DNA encoding this, it can be used for detecting an acetyllysine-containing protein having a weak-affinity to the antibody. Furthermore, it may be used in a functional analysis on an acetyllysine-containing protein by expressing in various kinds of cells. Furthermore, if the presence of an acetylated protein having any connection with a pathologic condition is revealed in future, it will play an important role in establishment of a diagnosis method.

## Claims

1. A monoclonal antibody recognizing an Nε-acetyllysine, comprising:
(1) a light chain comprising a constant region having an amino acid sequence represented by SEQ ID NO. 1 and a variable region having an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7; and
(2) a heavy chain comprising a constant region having an amino acid sequence represented by SEQ ID NO. 3 and a variable region having an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a mouse monoclonal antibody.

3. A monoclonal antibody recognizing an Nε-acetyllysine, comprising:
(1) a light chain comprising a constant region having an amino acid sequence represented by SEQ ID NO. 1 and a variable region having an amino acid sequence represented SEQ ID NO. 7; and
(2) a heavy chain comprising a constant region having an amino acid sequence represented by SEQ ID NO. 3 and a variable region having an amino acid sequence represented by SEQ ID NO. 10.