JP2008099602A - Method for regulating distribution situation of mutated site in variable region of antibody gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transducing mutations in the whole variable region of an antibody gene. <P>SOLUTION: The method for transducing a variety of mutations in the antibody gene region existing on the chromosome of an immunocyte is provided, comprising selectively depressing or losing the function of histone deacetylase(hereafter, referred to as HDAC)-2-gene, or selectively depressing or losing the activity or function of HDAC-2-protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for introducing a mutation into an antibody gene region. More specifically, the present invention relates to a method for obtaining various antibody molecules by introducing various mutations into an antibody gene region.

  Somatic cell homologous recombination plays an important role in vivo as one of the factors generating gene diversity. In particular, antibody genes will encode antibody molecules with a great many different specificities, with the variable regions resulting from VDJ recombination being further rearranged. If it becomes possible to freely induce such a series of phenomena in the cell, particularly the rearrangement of the variable region, it is possible to obtain an antibody having desired characteristics.

  In addition, somatic mutation is one of the phenomena that brings diversity to antibodies, and there is a report that the frequency of somatic mutation increases in the DT40 cell line lacking XRCC2 and XRCC3 (non-patented). Literature 1), attempts have been made to obtain antibodies having various characteristics using DT40 cells knocked out of XRCC2 or XRCC3 (Patent Literature 1, Non-Patent Literature 2). Furthermore, a technique has also been reported in which the expression of an AID (Activated Induced Cytidine Deminase) gene involved in both somatic gene conversion and somatic mutation is suspended by a Cre-loxP system, and an antibody-producing clone is isolated ( Patent Document 2, Non-Patent Document 3). However, the antibodies obtained by these techniques still have insufficient points in terms of antigen recognition specificity and affinity.

  On the other hand, the inventors have developed a technique for obtaining various antibodies in vitro by paying attention to the possibility that the rearrangement of variable regions is induced by a homologous recombination mechanism (particularly, somatic gene conversion) ( Patent Document 3, Non-Patent Document 4). This technique significantly promotes somatic gene conversion by treatment with a histone acetylase inhibitor or the like (for example, trichostatin A) at the antibody gene locus of a cultured cell line DT40 derived from chicken B cells. To provide a way to acquire diversity. This technology makes it possible to obtain a variety of antibody molecules compared to previously reported technologies, and is a highly useful technology. There was a tendency that gene conversion was selectively induced in the region (hypervariable region 1). For this reason, there is a possibility that the diversity of antibodies to be obtained is narrowed.

Sale et al., Nature 412: 921-926, 2001 Cumbers et al., Nature Biotech. 20: 1129-1134, 2002 Kanayama et al., Nucleic Acids Res. 34: e10. , 2006 Seo et al., Nature Biotech. 23: 731-735, 2005 JP 2003-503750 A JP 2004-298072 A International Publication No. WO2004 / 011644

In view of the above circumstances, the present inventors have conducted extensive research on methods for regulating the distribution range of mutations introduced into antibody gene variable regions. As a result, the functions of the histone deacetylase 2 (HDAC2) gene are selectively selected. It has been clarified that it can be achieved by suppressing to the above, and the present invention has been completed.
Therefore, an object of the present invention is to provide a method for introducing a mutation into the entire region of the antibody gene variable region.
The present invention also provides a method for producing antibody molecules having various characteristics by introducing mutations into the entire region of the antibody gene variable region.

That is, the present invention relates to the following (1) to (11).
(1) The first aspect of the present invention is as follows: “The coding region selectively reduces or eliminates the function of a gene consisting of the base sequence shown in (a) or (b) below, A method of introducing various mutations into the antibody gene variable region present above.
(A) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5
(B) a polynucleotide that hybridizes with a polynucleotide comprising a complementary base sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 under highly stringent conditions, and the encoded protein has histone deacetylation activity The nucleotide sequence of ".
(2) The second aspect of the present invention is achieved by introducing the mutation or deletion into the gene comprising the base sequence represented by the above (a) or (b). The method according to (1) above, which is characterized in that
(3) The third aspect of the present invention is characterized in that “the decrease in function or loss of function is achieved by destroying the entire gene comprising the base sequence represented by (a) or (b) above”. The method according to (1) above.
(4) The fourth aspect of the present invention is as follows: “Antibody gene that is selectively reduced or lost in the activity or function of the protein shown in (a) or (b) below and exists on the chromosome of an immune cell. A method of introducing various mutations into the variable region.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 (b) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 “A protein comprising an amino acid sequence having amino acid substitutions, deletions or insertions and having histone deacetylation activity”.
(5) The fifth aspect of the present invention is as described in any one of (1) to (4) above, wherein the immune cell is additionally contacted with an HDAC inhibitor for treatment. Method.
(6) A sixth aspect of the present invention is “the method according to (5) above, wherein the HDAC inhibitor is trichostatin A”.
(7) A seventh aspect of the present invention is “the method according to (6) above, wherein the immune cell is a DT40 cell”.
(8) The eighth aspect of the present invention is “the method according to any one of (1) to (7) above, wherein the antibody gene is an antibody heavy chain gene”.
(9) A ninth aspect of the present invention is “the method according to any one of (1) to (7) above, wherein the antibody gene is an antibody light chain gene”.
(10) A tenth aspect of the present invention is an “immune cell having a mutation introduced into an antibody gene variable region by the method according to any one of (1) to (9) above”.
(11) The eleventh aspect of the present invention is “an antibody molecule comprising at least one polypeptide encoded by an antibody gene into which a mutation has been introduced by the method according to any one of (1) to (9) above”. It is.
(12) A twelfth aspect of the present invention is “the antibody molecule according to (11) above, wherein the antibody molecule is IgM”.

  According to the method of the present invention, mutations can be introduced in the entire region of the antibody gene variable region.

  According to the method of the present invention, antibody molecules having various characteristics can be prepared by introducing mutations into the entire antibody gene variable region.

In one embodiment of the present invention, the function of a histone deacetylase (hereinafter referred to as HDAC) 2 gene is selectively reduced or lost, or the activity or function of HDAC2 protein is selectively reduced or lost. In this method, various mutations are introduced into the antibody gene region present on the chromosome of immune cells.
“Histone deacetylase (HDAC)” is a metalloenzyme containing zinc at the active center and catalyzes a reaction for eliminating an acetyl group from acetylated histone. As main functions of HDAC in cells, there are known control of chromatin structure, activation or suppression of transcription and the like. HDACs are roughly classified into several classes, for example, in the case of humans, class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II (HDAC4, HDAC5, HDAC6, HDAC7). These isoforms constitute large complexes by interacting with many other proteins. For example, HDAC1 and HDAC2 are present in the SIN3 complex and the NURD / Mi2 complex.

The “HDAC2 gene” in the present invention is not only a DNA whose coding region consists of a base sequence represented by SEQ ID NO: 1, 3, or 5, but also a coding region represented by SEQ ID NO: 1, 3 or 5. Also included are DNAs that hybridize under high stringency conditions with DNA consisting of a base sequence complementary to DNA consisting of a base sequence and that encode a protein having a histone deacetylation activity. The “gene” includes cDNA and genomic DNA. In the case of genomic DNA, not only exons and introns but also transcription regulatory regions such as promoters and enhancers are included.
The “antibody gene” in the present invention is synonymous with an immunoglobulin gene and includes both a heavy chain (H chain) antibody gene and a light chain (L chain) antibody gene.

As a DNA that can hybridize under stringent conditions with a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, 3 or 5, preferably about 70% or more with the base sequence represented by SEQ ID NO: 1, 3 or 5; More preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, most preferably about 99% of the DNA containing a nucleotide sequence having a polynucleotide sequence homology.
Here, stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical conditions that depend on the probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment near or below its melting point.
Specifically, for example, as a low stringency condition, in a filter washing step after hybridization, washing is performed in a 0.1 × SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. Can be raised. Examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By making the stringent conditions higher, a highly homologous polynucleotide can be obtained.

The “HDAC2 protein” in the present invention is a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. Here, the “polypeptide comprising substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, more preferably about 80%, with the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, and most preferably a protein having an amino acid sequence having about 99% amino acid identity and having histone deacetylation activity.
Alternatively, the polypeptide containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 is preferably one or several (preferably, the amino acid sequence represented by SEQ ID NO: 2, 4 or 6). A protein having an amino acid sequence in which 1 to 30 amino acids, more preferably about 1 to 10 amino acids, and further preferably 1 to 5 amino acids are deleted, substituted or added, and having histone deacetylation activity. is there.
In the present specification, unless otherwise specified, “protein” and “polypeptide” are used interchangeably.

The “immune cell” in the present invention refers to a B cell having antibody-producing ability, and for example, cells derived from human, mouse, rat, sheep, chicken, etc. can be used. Preferably, established cells are used, and DT40 cells are particularly preferably used as immune cells.
“DT40 cell” is a cultured cell of a chicken-derived B cell, and a derivative strain in which some modification (for example, recombination, insertion, deletion, etc. of a specific gene) is added to the chromosome possessed by the cell. , Including sublines.
The culture conditions of the cells used in the present invention are carried out by methods well known in the art, but it goes without saying that they are carried out under a medium and culture conditions (culture temperature, CO ₂ concentration) suitable for the selected immune cells. Thus, when the immune cells to be selected are DT40 cells, for example, IMDM (Invitrogen) is used as a medium, and the culture temperature is, for example, 39.5 ° C. and 5% CO ₂ concentration. Culturing is carried out while keeping the cell concentration constant, and the presence or absence of somatic homologous recombination at the antibody gene locus of the target cell is confirmed every appropriate period (for example, every day or every week).

    HDAC2 gene function can be altered in order to reduce or lose function of the HDAC2 gene. The method for modifying the function of the gene is not limited, but for example, a method for introducing a mutation into the HDAC2 gene present in the cell, a method for destroying the entire HDAC2 gene, and an expression by modifying the transcription promoter of the HDAC2 gene Methods well known to those skilled in the art such as a method of controlling the amount, a method of utilizing RNA interference (RNAi), a method of introducing antisense to the HDAC2 gene into cells can be used. Preferred are a method for introducing a mutation into the HDAC2 gene, a method for disrupting the entire HDAC2 gene, or a method using RNA interference (RNAi), and a method for disrupting the entire HDAC2 gene.

As a method of destroying the entire gene, there is a method of transforming a cell with DNA having a marker gene inserted into the essential region DNA sequence (region to be disrupted) of the cloned target gene (HDAC2 gene). Since the DNA introduced into the cell induces homologous recombination via both flanking sequences of the HDAC2 gene, the HDAC2 gene on the chromosome can be replaced with a marker gene, and as a result, the HDAC2 gene is destroyed. be able to.
Alternatively, RNA interference (RNAi) can be used to lose gene function. In this case, based on the base sequence relating to the functional domain of the protein encoded by the HDAC2 gene, a function or a loss of function of the HDAC2 gene is reduced by introducing a short RNA duplex or a vector producing the RNA into the cell. Can bring.
In addition, an inducible transcription promoter is placed in the upstream region of the HDAC2 gene to conditionally control the expression of the HDAC2 gene, or a site-specific recombinase system such as Cre-loxP is used to control the HDAC2 gene and the transcription promoter. There is a method of controlling the expression by changing the relative position.
Furthermore, as a method for introducing a mutation in vitro, a method known in the art such as site-directed mutagenesis can be used. When functional changes are made by introducing mutations into the HDAC2 gene, mutations are introduced so that the activity of the HDAC2 protein is lost, and mutations are made at sites necessary for the interaction between the protein and other proteins. It is desirable to introduce a mutation that eliminates the interaction by introducing.

In addition to the methods that can be used to reduce or lose the function of the HDAC2 gene described above, methods well known to those skilled in the art can be used to reduce or lose the activity or function of the HDAC2 protein. Without limitation, for example, a method of introducing an antibody that decreases or loses the activity or function of HDAC2 protein into the target cell, an inactive form (dominant negative) of HDAC2 protein, or And a method of introducing a small molecule inhibitor that inhibits the activity of the HDAC2 protein into the target cell.
In order to introduce an inactive form of an antibody or HDAC2 protein into a cell, for example, a cell membrane-permeable peptide may be linked to an inactive form of an antibody or HDAC2 protein and introduced into the cell, or a commercially available kit Can also be used.
In order to express an inactive form of HDAC2 protein in a cell, it can be easily carried out according to the common technical knowledge of those skilled in the art. Select an expression vector (having a component necessary for expression induction such as a promoter corresponding to the target cell) according to the target cell, and inactivate HDAC2 protein under appropriate culture conditions Can be expressed.

In another embodiment of the present invention, the function of the HDAC2 gene is reduced or lost, or the activity or function of the HDAC2 protein is reduced or lost, and an immune cell in which the HDAC2 gene, HDAC2 protein is present is treated with an HDAC inhibitor. In which various mutations are introduced into the antibody gene region present on the chromosome of the immune cell.
As the “HDAC inhibitor”, any protein factor such as an antibody having an activity of suppressing the activity of HDAC, small molecule compounds such as trichostatin A, butyric acid and valproic acid, and the like known to those skilled in the art can be used. Most likely, trichostatin A is used. When contacting immune cells with an HDAC inhibitor, the treatment concentration and treatment time with the HDAC inhibitor can be used as long as the cells to be contacted are within the range not causing death. Specifically, in the case of trichostatin A, the treatment concentration is preferably 1 to 20 nM, and the treatment time is preferably 2 weeks to 4 months.

  Examples are shown below, but the present invention is not limited thereto.

Example 1: Production of chHDAC2 − / − strain and diversification of variable region of antibody gene (heavy chain / light chain) by continuous culture <Method>
1. Production of chHDAC2 − / − strain (homozygous gene disruption strain) Amplification of BamHI (6949) -SacI (13140), 6.2 kbp fragment of chHDAC2 gene by PCR using DT40 genomic DNA as a template using the following primer set Then, after cleaving with a restriction enzyme, cloning was performed by inserting the pBluescript SK (-) vector into the BamHI-SacI site. From this plasmid, a StuI (7543) -StuI (10129), 2.6 kbp fragment (including a part of the third exon and the fourth and fifth exons) is excised and loxP blanked expressed in the β-actin promoter there. A blastidin R (or puromycin C) selection marker sequence was inserted (FIG. 1). Thereafter, 30 μg of the recombinant DNA was cleaved with SacI, and then transformed into the CL18 strain of DT40 cells by electroporation (Bio-Rad Gene Pulser, 550 V, 25 uFD). First, a heterozygous gene disruption strain (chHDAC2 +/−) was selected from the blasticidin-resistant colonies, and further transformed with a gene disruption marker containing a puromycin resistance marker. Finally, two homozygous transformants were transformed. (ChHDAC2 − / −) was isolated.

These two strains of chHDAC2 gene disruption were named WL3.1, and the original strain was stored frozen. These strains were confirmed by RT-PCR using chHDAC2 primer (FIG. 2). RT-PCR reaction was performed using “Superscript III One-Step RT-PCR with Platinum Taq Polymerase” (Invitrogen) and 200 ng template total RNA, and 10 pmol of chHDAC2 and β-actin (control) PCR primer set Was carried out in a 50 μl reaction solution. The reaction conditions were 55 ° C for 30 minutes and 94 ° C for 2 minutes, followed by 25 cycles of 94 ° C for 15 seconds / 60 ° C for 30 seconds / 68 ° C for 1 minute. Finally, the reaction was carried out at 68 ° C for 5 minutes. As a precaution, we confirmed gene disruption by genomic Southern hybridization.
chHDAC2 PCR primer set :
Upstream: 5′-CCGCTCCGAGGGGACAAGGGCATCCAATGAAACCCTATAGG-3 ′ (SEQ ID NO: 7)
Downstream: 5′-CCATCGATGTATATACTACAGCACTGGGCTGGTATACATCTCC-3 ′ (SEQ ID NO: 8)
RT-PCR primer set :
(For chHDAC2)
Upstream: 5′-GCTGTCAACTGGAGGCTCTG-3 ′ (SEQ ID NO: 9)
Downstream: 5′-CTGATTTTGCTCCTTTGGGTCCCG-3 ′ (SEQ ID NO: 10)
(For β-actin)
Upstream: 5'-CCCCAAGCTTACTCCCACAGCCAGCCATGG-3 '
(SEQ ID NO: 11)
Downstream: 5'-GGCTCTAGATATAGTCCGTCAGGTCACGGCCA-3 '
(SEQ ID NO: 12)

2. chHDAC2 - / - antibody genes (heavy and light chain) variable region diversification the WL3.1 by subculturing strains at 39.5 ° C. with 5% CO ₂ presence in CO ₂ thermostat bath Culture was performed for the indicated period (3 weeks to 2 months). The medium was IMDM medium (Invitrogen), and 10% FBS, 1% chicken serum, penicillin 100 units / ml, streptomycin 100 μg / ml, 2-mercaptoethanol 55 μM were used. Trichostatin A (Wako Pure Chemical Industries) was dissolved in DMSO 2 mg / ml as a stock and diluted with a suitable medium so that the final concentration would be 2.5, 5, 10 nM. Culturing was continued while maintaining the cell concentration at 105-2 × 10 ⁶ cells / ml.

3. After the fluorescent cell sorter (FACS) Cell culture of the estimated cell density ^{to 106} of gene conversion frequencies by the analysis of surface IgM positive clones were recovered 1000 × g, 5 min centrifugation cells in 1.5ml tubes, staining buffer (phosphate Buffer PBS: 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 with 0.3% BSA added) suspended in 200 μl. The cells were collected again by centrifugation, and then FITC-labeled anti-chicken IgM antibody (BETHYL) diluted 1/250 times with a staining buffer was suspended in 200 μl and reacted on ice for 1 hour. The cells were recovered by centrifugation, suspended and centrifuged again in 200 μl of staining buffer, and the cells were washed. After this washing step was repeated once more, the cells were suspended in 100 μl of staining buffer containing 5 μg / ml propidium iodide (Nacalai). The percentage of cells expressing IgM was calculated by measuring about 10,000 cells by EPICS ELITE ESP (Beckman Coulter). At that time, cells stained with propidium iodide were gated out as dead cells.

4). Extraction of genomic DNA / Total RNA Approximately 100,000 cells (for genomic DNA) or 2 million cells (for Total RNA) were collected by centrifugation (1000 g, 5 minutes) in a 1.5 ml tube, and a sample of the cell pellet was collected at −80 ° C. saved. After thawing, genomic DNA and total RNA were purified using Mag Extractor MFX-2000 (Toyobo) and Genome Purification Kit or RNA Purification Kit, and dissolved in 100 μl of sterile water.

5. Analysis of antibody light chain gene variable region sequence PCR (Perkin Elmer 9600) was used for amplification of antibody light chain and heavy chain gene variable regions. Using 1 μl of genomic DNA solution (corresponding to 5000 cells) as a template, 10 pmol of each of the following primers was used.
Light chain variable region :
Upstream: 5′-CACACCTCAGGTACTCGTTTGCG-3 ′ (SEQ ID NO: 13)
Downstream: 5'-TCAGCGACTCACCTAGGACGGG-3 '(SEQ ID NO: 14)
Heavy chain variable region :
Upstream: 5'-CGGGAGCTCCGTCAGCCGCTCTCTGTCC-3 '
(SEQ ID NO: 15)
Downstream: 5′-GGGGTACCCGGAGGAGACGATGAACTTCCG-3 ′
(SEQ ID NO: 16)
The PCR reaction was performed in 50 μl reaction solution using Pyrobest DNA Polymerase (Takara Shuzo). The reaction conditions were 98 ° C. for 2 minutes, followed by 27 cycles of 98 ° C. for 30 seconds, 57 ° C. for 30 seconds, and 72 ° C. for 1 minute, and finally 72 ° C. for 5 minutes. Thereafter, 1 μl of ExTaq DNA Polymerase (Takara Shuzo) was added and reacted at 72 ° C. for 15 minutes, and then 20 μl of the total reaction solution was separated by agarose gel electrophoresis. A band corresponding to the variable region of the light chain gene was excised, DNA was collected by Gel Extraction kit (Qiagen), incorporated into pCR2.1-TOPO vector with TOPO-TA Cloning kit (Invitrogen), and transformed into E. coli. . Thereafter, the plasmid was extracted, and the sequence was analyzed by ABI PRISM 377 DNA Sequencer (Perkin Elmer) or ABI 3730xl (Applied Biosystems).

6). Production of rabbit IgG magnetic beads:
As the magnetic beads, Dynabeads M-280 Tosylactivated (Dynal) was used, and as the magnetic stand, Dynal MPC (Dynal) was used. 200 μl of beads were washed three times with 500 μl of buffer A (0.1 M Na-phosphate pH 7.4), and then stirred with 120 μg rabbit IgG (SIGMA) in buffer A, 200 μl at 37 ° C. overnight by rotation. It was made to react. The beads were then washed twice with 200 μl of buffer C (PBS + 0.1% BSA). Thereafter, 200 μl of buffer D (0.2 M Tris-HCl pH 8.5, 0.1% BSA) was added, and the mixture was allowed to react at 37 ° C. for 4 hours with stirring by rotation to perform blocking. Thereafter, the cells were washed twice with 500 μl of buffer C and then suspended in 200 μl of buffer C containing 0.02% sodium azide.

7). Selection of Antibody-Producing Clones with Rabbit IgG Magnetic Beads About 5 × 10 ⁷ wild-type DT40 cells treated with trichostatin A, 2.5 ng / ml, once in 10 ml of washing buffer (PBS containing 1% BSA), After further washing with 1 ml once, mix with 5 × 10 ⁶ streptavidin magnetic beads (rabbit IgG magnetic beads in the case of selection with rabbit IgG magnetic beads) in 1 ml washing buffer and gently at 4 ° C. for 30 minutes. Incubated while rotating. Thereafter, it was washed 5 times with 1 ml of washing buffer. Finally, the cells bound to the magnetic beads were suspended in 500 μl, added to 30 ml of medium, dispensed 300 μl in 96-well plates, and cultured at 39.5 ° C.

<Result>
1. Diversification of antibody gene light chain variable region in chHDAC2 − / − strain ChHDAC2 − / − strain (CL18 strain, surface IgM negative clone) in the presence of 5 nM trichostatin A (after day 14) / 0 in absence The cells were cultured for 14, 21, 28, 35, and 42 days, and the number of IgM positive cells was measured by FACS.
As a result, in the chHDAC2 − / − strain, in the absence of trichostatin A, 28% of the cells showed surface IgM positive after 28 days, but thereafter the ratio of surface IgM positive cells slightly decreased from the steady state. It became clear.
In contrast, in the presence of 5 nM trichostatin A, the ratio of surface IgM positive cells was remarkably increased as in the case of wild type cells, and nearly 70% of the cells were positive after 35 days. However, in the wild type, the positive rate continued to increase, whereas in the chHDAC2 − / − strain, it became clear that the state shifted to a steady state at about 70% (FIG. 3).

Next, the genomic DNA sequence of a region of about 400 bp including CDR 1 to 3 of the light chain variable region was analyzed. As a result, it was shown that the sites where gene conversion occurs were dispersed throughout the variable region, and a considerable number of mutations were introduced into the CDR2 and CDR3 portions as compared with the case where the wild type was treated with trichostatin A (FIG. 4). ).
The diversification pattern is also more complicated than wild type + trichostatin A (16 types for 31 clones, Fig. 5), 38 types for 43 clones in total, or 22 types for 30 clones. Had.

As shown in FIG. 6, in the chHDAC2 − / − strain, 30-45% of all clones have mutations in the CDR1 site, and 30-45 have mutations in regions other than CDR1 including CDR2 and 3 regions. %.
When trichostatin A is added to the chHDAC2 − / − strain and cultured, gene conversion in the CDR1 region is more prominent, and a considerable number of mutations are also found in regions other than CDR1 including CDR2 and CDR3. It was found that the effects of the chHDAC2 − / − strain and the effect of trichostatin A treatment were additive (FIG. 7).

2. Separation of antibody-producing clones from the above-mentioned diversified cell group using magnetic beads to which rabbit IgG is immobilized A cell population obtained by adding trichostatin A to the chHDAC2 − / − strain and culturing for 60 days or longer is used as a rabbit. Magnetic beads conjugated with IgG were added. When cells that bind tightly were collected with a magnet and cultured in a 96-well plate, many clones grew. FIG. 8 shows ELISA data of an antibody production example (clone H11 is a specific antibody) against rabbit IgG.

3. Diversification of antibody gene heavy chain variable region in chHDAC2 − / − strain The results confirmed in the antibody light chain gene region were also confirmed in the antibody heavy chain gene region (FIG. 9).

  The present invention provides a method for introducing a mutation into the variable region of an antibody gene. Furthermore, when this method is used, desired antibodies having various properties (specificity to antigen, affinity to antigen, etc.) can be produced. Therefore, a desired antibody that has been difficult to produce can be obtained by the method of the present invention, and can be used as an effective means for medical treatment, drug discovery, and the like.

A schematic diagram of the HDAC2 gene knockout construct is shown. The result of RT-PCR which confirmed that cHDAC2 gene was destroyed is shown. WT is a wild type strain, (+/−) is a heterozygous gene-disrupted strain, and (− / −) is a result of a homozygous gene-disrupted strain. The result of the gene conversion frequency of a wild type and chHDAC2-/-cell is shown. The horizontal axis represents the culture time (days), and the vertical axis represents the percentage (%) of IgM + cells. The average of three culture results is shown. The results of the sequence diversity of the antibody light chain variable region of the chHDAC2-/-2 month-old cell population are shown. The results of sequence diversity of antibody light chain variable regions of wild type + trichostatin A (5 nM) 2 month old cell population are shown. The ratio in each hypervariable region of the sequence diversification site | part in a chHDAC2 (-/-), chHDAC2 (-/-) + TSA (trichostatin A), TSA (trichostatin A) cell population is shown. The results of the sequence diversity of the antibody light chain variable region of chHDAC2 (− / −) + trichostatin A (5 nM) 1 month old cell population are shown. The result of the riser of the antibody clone produced with respect to rabbit IgG is shown. The result of the sequence diversity of the antibody heavy chain variable region of the chHDAC2 (− / −) 2-month-old cell population is shown.

Claims (12)

The coding region selectively reduces or loses the function of the gene consisting of the base sequence shown in the following (a) or (b), and various mutations are made in the antibody gene variable region present on the chromosome of immune cells. How to introduce.
(A) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5
(B) a polynucleotide that hybridizes with a polynucleotide comprising a complementary base sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 under highly stringent conditions, and the encoded protein has histone deacetylation activity Nucleotide sequence   2. The method according to claim 1, wherein the decrease in function or loss of function is achieved by introducing a mutation or deletion into a gene comprising the base sequence represented by (a) or (b). .   2. The method according to claim 1, wherein the decrease in function or loss of function is achieved by disrupting the entire gene comprising the base sequence represented by (a) or (b). A method for introducing various mutations into the antibody gene variable region existing on the chromosome of immune cells by selectively reducing or losing the activity or function of the protein shown in the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 (b) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 A protein comprising an amino acid sequence having amino acid substitutions, deletions or insertions, and having histone deacetylation activity   The method according to any one of claims 1 to 4, wherein the immune cells are additionally contacted with an HDAC inhibitor for treatment.   6. The method of claim 5, wherein the HDAC inhibitor is trichostatin A.   The method according to any one of claims 1 to 6, wherein the immune cell is a DT40 cell.   The method according to any one of claims 1 to 7, wherein the antibody gene is an antibody heavy chain gene.   The method according to any one of claims 1 to 7, wherein the antibody gene is an antibody light chain gene.   The immune cell which introduce | transduced the variation | mutation into the antibody gene variable region by the method in any one of Claims 1 thru | or 9.   An antibody molecule comprising at least one polypeptide encoded by an antibody gene into which a mutation has been introduced by the method according to any one of claims 1 to 9.   The antibody molecule according to claim 11, wherein the antibody molecule is IgM.