JP2009060850A - Method for converting antibody gene variation form of b cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a B cell-derived cell having an increased frequency of point mutation of a gene encoding an antibody, and a method for selecting and producing a gene having a desired specificity from an antibody library by increasing the frequency of the point mutation of a gene encoding an antibody in a B cell-derived cell being an antibody-forming cell and forming the antibody library having various specificities. <P>SOLUTION: The cell is a B cell-derived cell in which only one of two alleles of XRCC3 gene is inactivated, and has an increased frequency of transfer of point mutation to an antibody gene in comparison with a cell having both XRCC3 genes. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing an antibody having a point mutation by converting the antibody gene mutation mode of B cells.

  In vivo, after immunization with an antigen, the affinity of the produced antibody improves with time, and a high-affinity antibody is efficiently produced. This process is called affinity maturation. In affinity maturation, mutant B cells that have acquired high affinity are strictly selected from the B cell population diversified by high frequency mutations that occur in the antibody gene variable region of antigen-stimulated B cells, and survive. Other unwanted cells progress by dying (FIG. 1). This clever mechanism can be regarded as an ideal molecular evolution system. According to this principle, a highly specific antibody can be obtained by repeated immunization to a living body, but obtaining an antibody by this method requires time and labor.

If the process of antibody production in vivo as shown in FIG. 1 can be reproduced by an in vitro cultured cell system, a rapid and efficient antibody creation system can be constructed. Several B cell lines derived from humans, mice, and chickens are known as B cell lines having a high frequency mutation function that can be used for this purpose. The chicken B cell line DT40 has the following excellent characteristics: Has characteristics.
1) A variety of antibody libraries can be formed by spontaneously introducing mutations into antibody genes during culture.
2) IgM antibody is expressed as a membrane-bound type and secreted on the cell surface.
3) The growth rate is 2-3 times faster than normal animal cells and easy to culture.
4) Exceptionally for animal cells, since the frequency of homologous recombination with a foreign gene is high, site-specific gene manipulation such as gene knockout can be easily performed.

  Mutations in antibody genes in DT40 cells are mainly performed by a mechanism called gene conversion. This proceeds by incorporating a large number of pseudo V gene partial sequences arranged upstream into the antibody variable region (V) gene by gene conversion (FIG. 2). Point mutations due to single base substitutions occur but are infrequent. Thus, an antibody library having various specificities is formed in the cultured cell population.

  Therefore, it is considered that necessary antibodies can be rapidly and efficiently obtained by isolating cells that produce antibodies that bind to the target antigen from the cultured population of DT40 cells (FIG. 3).

  Based on such an idea, the present inventor has developed an in vitro antibody production method using the DT40 strain DT40-SW whose function has been modified previously (see Patent Document 1). Another important feature of this technique is that when a cell producing the target antibody can be selected, mutations are introduced again into the cell clone as shown in FIG. It can be evolved into an antibody with high affinity (affinity maturation). In order to allow this affinity maturation to proceed efficiently, it is necessary to further modify the cell at the gene level as described below.

  On the other hand, in DT40 cells where gene conversion mutations predominate, there has already been reported a method for converting the mutation mode from gene conversion type to point mutation type, and factors XRCC2 and XRCC3 involved in repairing DNA damage are reported. It has been known that this can be realized by deleting (see Non-Patent Documents 1 and 2).

JP 2006-109711 A Sale, J. E. et al. Nature 412, 921-926 (2001) Arakawa, H. et al., PLOS Biol. 2, 967-974 (2004)

  The present invention provides a cell derived from a B cell in which the frequency of point mutation of a gene encoding an antibody is increased, and the frequency of point mutation of a gene encoding an antibody in a cell derived from a B cell that is an antibody producing cell. It is an object of the present invention to provide a method for forming an antibody library having various specificities, selecting and producing an antibody having a desired specificity from among them.

  In order to efficiently perform the affinity maturation described above, it is important that diversification of antibody libraries by mutation proceeds in a form suitable for the purpose. As shown in FIG. 4 (left), in DT40 cells, gene conversion in which sequences of several tens of bases are replaced at a time is dominant as a mutation mode, and this is a mechanism that generates antibody diversity of B cells in chickens. Since it is the same, it can be said that it is advantageous for expansion of the initial antibody library. However, in chickens, it is known that the frequency of point mutations increases with gene conversion in the process of affinity maturation, which produces high-affinity antibodies after immunization, and this mutation pattern creates high-affinity antibodies. It is considered suitable. This is because a point mutation that is a single base substitution can be expected to change the antigen specificity with a smaller deviation from the original specificity, so it is considered possible to make a more precise modification of the antigen specificity. (Right side of FIG. 4).

  Therefore, when a library for antibody search is formed, mutations proceed in a gene-converted form, and after the target antibody-producing clone has been selected, DT40 can be modified to increase the frequency of point mutations. It is valid. The present inventor conducted the following study to overcome this problem and found an effective solution.

  In DT40 cells where gene conversion mutations are dominant, there are already reports on how to change the mutation mode from gene conversion type to point mutation type, and factors involved in repairing DNA damage XRCC2 and XRCC3 (Sale, JE et al. Nature 412, 921-926 (2001); Arakawa, H. et al., PLOS Biol. 2, 967-974) (2004)).

  However, if these genes are completely deficient, 1) the cell growth rate is reduced, 2) non-functional genes are efficiently generated by point mutations, and many cells that do not produce antibodies are generated. 3) DT40 A problem arises that homologous recombination, which is the greatest advantage, cannot be performed and genetic manipulation becomes difficult. As a result of various studies to overcome this problem, the present inventor has surprisingly found that a hetero knockout cell in which only one of the two alleles of the XRCC3 gene is deleted in the intermediate stage of producing the XRCC3 deficient cell. The XRCC3 (+/-) strain was found to be useful in overcoming the above three problems. In addition, since only one of the alleles needs to be destroyed, it is extremely easy to perform an experiment. Therefore, this method is extremely useful for improving the performance of an antibody production system using DT40.

That is, the aspects of the present invention are as follows.
[1] Cells derived from B cells in which only one of the two alleles of the XRCC3 gene is inactivated, and the frequency of point mutations to the antibody gene is increased compared to cells having both XRCC3 genes .
[2] The cell according to [1], wherein the sixth exon of one of the two alleles of the XRCC3 gene is inactivated.
[3] The cell according to [1] or [2], wherein inactivation of only one of the two alleles of the XRCC3 gene is performed by homologous recombination.
[4] The cell according to any one of [1] to [3], wherein the B cell-derived cell is a chicken B cell line DT40 or a mutant thereof.
[5] A variant of chicken B cell line DT40 is
Expression of exogenous Cre recombinase gene is induced by extracellular stimulation, and by inducing and stopping AID expression by reversing the direction of exogenous AID (activation induced cytidine deaminase) gene by expressed Cre recombinase Possible variants with the following characteristics:
1) The endogenous AID gene is functionally disrupted, and no AID protein is produced by endogenous AID gene expression.
2) having an exogenous AID gene sandwiched between two loxP sequences in opposite directions, and a promoter capable of functioning in the animal cell existing upstream of the region sandwiched between the two loxP sequences, When the AID gene is placed in the forward direction with respect to the promoter, the AID gene can be expressed by the promoter, and when the AID gene is placed in the reverse direction with respect to the promoter 3) The Cre recombinase gene has been introduced in a form capable of activating Cre recombinase by extracellular stimulation, and the above-mentioned exogenous AID gene is contained by activating Cre recombinase 2 The direction of the region between the two loxP arrays is reversed,
[4] The cell according to [4], wherein the AID gene expression is stopped by extracellular stimulation and the mutation function is stopped.
[6] The cell according to any one of [1] to [5], into which a gene encoding a foreign antibody has been introduced.
[7] A method for selecting an antibody-producing cell that produces an antibody having desired characteristics,
(i) a step of preparing a cell according to any one of [1] to [6],
(ii) culturing the cell for a certain period of time;
(iii) isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation; and
(iv) selecting a cell producing an antibody having a desired mutation.
[8] A method for producing an antibody having desired characteristics,
(i) a step of preparing a cell according to any one of [1] to [6],
(ii) culturing the cell for a certain period of time;
(iii) isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation;
(iv) selecting a cell producing an antibody having the desired mutation, and
(v) culturing cells having the selected desired mutation to produce antibodies.

  The present invention is an antibody production system using DT40 cells derived from chicken B cells, and makes it possible to obtain a high affinity antibody more efficiently. In addition, the antibody of the present invention makes it possible to improve an existing antibody to a higher performance antibody in the XRCC3 (+/−) mutant. In particular, the use of the cells of the present invention makes it easier to obtain antibodies useful for the treatment of intractable diseases such as cancer.

  Hereinafter, the present invention will be described in detail.

  As long as the cell derived from B cell used in this invention can produce an antibody, origin animal species and cell strain species are not limited. B cells such as humans, mice, sheep, rats, rabbits and chickens, or cell lines thereof, or mutants thereof can be used, and a DT40 cell line derived from chicken B cells is preferably used. The DT40 cell line is a cell derived from B lymphoma, and the second chromosome is characterized by trisomy (Baba, T.W., Giroir, B.P. and Humphries, E.H .: Virology 144: 139-151,1985). The DT40 strain is a wild-type DT40 strain or the DT40-SW strain originally established by the present inventors (by reversibly switching the expression of the gene AID that controls the mutation function, the antibody mutation function is ON / OFF controlled. Mutants (for details, see Kanayama, N., Todo, K., Reth, M., Ohmori, H. Biochem. Biophys. Res. Commn. 327: 70-75 (2005) and JP-A-2006-109711). Described)).

  XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3) is a gene involved in maintaining chromosome stability and repairing DNA damage, and is involved in homologous recombination of cells. The chicken XRCC3 gene has 8 exons. The base sequence of the chicken XRCC3 gene is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the positions of 8 exons are as follows. Exon 1: 1-215, Exon 2: 216-284, Exon 3: 285-422, Exon 4: 423-635, Exon 5: 636-790, Exon 6: 791-1003, Exon 7: 1004-1050, Exon 8: 1051-1935.

  In the cell of the present invention, only one of the two XRCC3 alleles present in each of the cell's homologous chromosomes is inactivated. The cells of the present invention are represented by the genotype XRCC3 (+/−). Here, gene inactivation means that partial deletion, substitution, insertion, addition, or the like has occurred in the base sequence of the gene, resulting in suppression of gene expression. “Suppressing the expression of a gene” includes a case where a gene encoding a protein having a normal function is not expressed although the gene itself is expressed. “A gene is inactivated” may be “a gene is destroyed”, “a gene is deficient”, or “a gene is knocked out”. In addition, gene inactivation includes not only the case where gene expression is completely suppressed, but also the case where it is partially suppressed. In the present invention, in order to inactivate only one of the alleles, the XRCC3 gene is sometimes knocked out heterogeneously, and the resulting cells are sometimes referred to as heteroknockout cells.

  Gene inactivation can be performed, for example, by homologous recombination. Homologous recombination refers to a method of modifying a target gene by carrying out homologous genetic recombination between a gene on a chromosome and foreign DNA. In homologous recombination, another DNA sequence is inserted into an exon of the gene for the purpose of disrupting the sequence encoding the protein. Homologous recombination is performed using a gene targeting vector. After homologous recombination, a selectable marker gene is used as a sequence that disrupts the target gene in order to identify cells having a gene targeting vector. As a selectable marker gene, an antibiotic resistance gene such as a Blasticidin resistance gene, a fluorescent protein gene such as GFP, a chromoprotein gene, or the like can be used.

  In the present invention, any of the 8 exons of the chicken XRCC3 gene may be disrupted. A plurality of exons may be divided. Preferably, the sixth exon is split.

  The targeting vector is designed based on the sequence information of the chicken XRCC3 gene. Kenichi Yamamura et al. Transgenic Animals Kyoritsu Shuppan Co., Ltd. Shinichi Aizawa, Generating Mutant Mouse Using Gene Targeting ES Cell Bio Manual Series March 1, 1997 8 Yodosha 1995, Hogan et al., Manipulating the Mouse Embryo, Cold Spring Hoarbor Laboratory Press (1944), Joyner, AL, Gene Targeting, A Practical Approach Series, IRL Press (1993), Masamura Matsumura et al. It can be prepared according to the method described in New Genetic Engineering Handbook (Revised 3rd Edition) Yodosha 1999 etc. The targeting vector may be either an insertion type or a substitution type. Furthermore, recombination can be caused by targeting using the Cre-lox system. Targeting using the Cre-lox system can be performed, for example, according to the method described in JP-T-11-503015. As a method for selecting a homologous recombinant having undergone homologous recombination, a known selection method such as positive selection, promoter selection, negative selection, poly A selection may be used. Cells that have undergone homologous recombination can be identified by a known method such as PCR or Southern blotting.

  In the XRCC3 (+/−) mutant of B cell-derived cells obtained by the above method, point mutations of antibody genes occur frequently. In addition, in the cell XRCC3 (+/−) of the present invention, gene conversion that occurs frequently in the wild type cell XRCC3 (+ / +) also occurs. On the other hand, in the cell XRCC3 (− / −) in which both of the two XRCC3 alleles are inactivated, the frequency of gene conversion is significantly reduced. Accordingly, the cells of the present invention are susceptible to both gene conversion and point mutation, and various mutant antibodies can be obtained. In addition, the growth rate of the cell XRCC3 (+/−) of the present invention is slightly lower than that of the wild type, but is not so low as to cause a problem, and the cell density is the same as that of the wild type. On the other hand, in the cell XRCC3 (− / −) in which both of the two XRCC3 alleles are inactivated, the proliferation rate and the cell density are remarkably reduced as compared to the wild type cell XRCC (+ / +). Furthermore, in XRCC3 (− / −), strains that cannot produce antibodies frequently appear, whereas in XRCC3 (+/−), the incidence of strains that cannot produce antibodies is low.

  By continuing subculture of the cell XRCC (+/−) of the present invention, cell populations having various point mutations in the antibody gene can be obtained. That is, an antibody library having various characteristics is formed in the cell population. An antibody having desired characteristics may be selected from this cell population. Since B cells express an antibody on the surface or secrete an antibody outside the cell, a desired antibody can be selected by examining the characteristics of the cell surface antibody or the secreted antibody. In order to select and isolate the antibody on the cell surface, for example, magnetic beads on which an antigen is immobilized may be used. In other words, the antigen is immobilized on the magnetic beads by adsorption, the antigen-immobilized magnetic beads and the cell population are mixed, the cells producing the desired antibody are bound to the magnetic beads, and the magnetic beads are collected using magnetism. By doing so, cells that produce the desired antibody can be recovered and isolated. On the other hand, the antibody secreted into the culture solution can be detected by ELISA (enzyme-linked immunosorbent assay) or the like.

  By isolating cells that produce antibodies that can bind to the antigen of interest, further culturing them to cause mutation, and selecting cells that produce antibodies with superior characteristics from among them, it is further possible to further An antibody having excellent characteristics can be obtained. Here, the characteristics of the antibody include specificity to the target antigen and binding force, as well as cytotoxic activity such as optimal temperature, optimal pH, ADCC (antibody-dependent cellular cytotoxicity), CDC (complement-dependent cytotoxicity), etc. Etc. Specificity and binding power are improved by the occurrence of mutations in variable regions such as antibody complementarity determining regions (CDRs). In addition to obtaining antibodies with improved specificity for the target antigen, it is also difficult to produce proteins with conserved amino acid sequences between species that were difficult to produce using conventional methods of immunizing animals. However, highly specific antibodies can be obtained.

  The present invention includes a method for producing an antibody having a desired mutation and an antibody library having various mutations using the cells of the present invention.

A method for producing an antibody having a mutation includes:
Preparing a B cell inactivated in only one of the two XRCC3 alleles of the B cell;
Culturing the cells for a period of time;
Isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation;
Selecting a cell that produces an antibody having the desired mutation, and culturing the selected cell having the desired mutation to produce an antibody.

  Furthermore, the antibody of the present invention can be mutated by using the cell of the present invention to obtain an antibody having more excellent characteristics. That is, a gene encoding an existing foreign antibody is introduced into a cell in which only one of the two alleles of the XRCC3 gene of the present invention is inactivated, integrated into the genome of the cell, What is necessary is just to make a mutation in an antibody gene. In this case, the antibody need not be a complete antibody molecule, it may be a cell capable of producing an antibody fragment containing the variable region necessary for binding to the antigen, and may be a non-natural type such as a chimeric antibody or a humanized antibody. It may be a cell capable of producing molecules capable of binding to antigen and fragments thereof.

  In the method of the present invention, Kanayama, N., Todo, K., Reth, M., Ohmori, H. Biochem. Biophys. Res. Commn. 327: 70-75 (2005) and Japanese Patent Laid-Open No. 2006-2006. Innovative antibody production technology using a culture system using DT40 cells can be established by combining the method for reversibly ON / OFF controlling the mutation function of an antibody gene described in JP-A-109711. In the DT40-SW cells used in the above method developed by the present inventors, the expression of activation-induced cytidine deaminase (AID) gene, which controls the mutation of the antibody gene, is reversibly turned on / off by stimulation with an estrogen derivative or the like given from the outside. Can be turned off. Even if the target clone can be isolated from the heterologous DT40 cell in which only one of the two alleles of the XRCC3 gene is inactivated, further mutations are introduced in the process of growing the clone In this case, it is difficult to stably maintain a useful mutant. In order to avoid this problem, it is necessary to stop the mutation function of the target clone obtained. Therefore, as described below, one of the XRCC3 genes may be inactivated in the DT40-SW strain. By stopping the AID expression of the clone selected from this XRCC3 gene hetero knockout DT40-SW cell, a genetically stable clone producing the target antibody can be obtained.

That is, the present invention induces AID expression by activating exogenous Cre recombinase by extracellular stimulation, and reversing the direction of exogenous AID (activation induced cytidine deaminase) gene by activated Cre recombinase. And B cell-derived cells that can be arrested, having the following characteristics:
1) The endogenous AID gene is functionally disrupted, and no AID protein is produced by endogenous AID gene expression.
2) having an exogenous AID gene sandwiched between two loxP sequences opposite to each other, and a promoter capable of functioning in the cell located upstream of the region sandwiched between the two loxP sequences, When the gene is arranged in the forward direction with respect to the promoter, the AID gene can be expressed by the promoter, and when the AID gene is arranged in the reverse direction with respect to the promoter, 3) The Cre recombinase gene has been introduced in a form that enables Cre recombinase activation by extracellular stimulation (specifically, a fusion protein of Cre and an estrogen receptor (Cre-ER Cre-ER moves from the cytoplasm to the nucleus when bound by an estrogen derivative and acts on the AID gene sandwiched between the substrate loxP (Zhang, Y. et al. Nucleic Acids Res. 24, 534-5 48 (1996))), Cre recombinase activation reverses the direction of the region between the two loxP sequences containing the exogenous AID gene,
In which only one of the two XRCC3 alleles is inactivated. Examples of the promoter include β-actin promoter, immunoglobulin promoter, cytomegalovirus promoter, CAG promoter and the like. In addition, as an extracellular stimulus that activates Cre recombinase, for example, Cre recombinase can be expressed as an estrogen receptor or a fusion protein with a protein containing an estrogen-binding domain thereof, and estrogen receptor or estrogen binding thereof. When a DNA construct in which a gene encoding a protein containing a domain and Cre recombinase cDNA are linked in-frame is introduced into the cell, activation of Cre recombinase is induced when estrogen derivative stimulation is applied to the cell.

Furthermore, the present invention induces AID expression by reversing the direction of the exogenous AID (activation induced cytidine deaminase) gene by expressing the exogenous Cre recombinase gene by extracellular stimulation. And B cell-derived cells that can be arrested, having the following characteristics:
1) The endogenous AID gene is functionally disrupted, and no AID protein is produced by endogenous AID gene expression.
2) having an exogenous AID gene sandwiched between two loxP sequences in opposite directions, and a promoter capable of functioning in the animal cell existing upstream of the region sandwiched between the two loxP sequences, When the AID gene is placed in the forward direction with respect to the promoter, the AID gene can be expressed by the promoter, and when the AID gene is placed in the reverse direction with respect to the promoter 3) The Cre recombinase gene has been introduced in a form capable of activating Cre recombinase by extracellular stimulation, and the above-mentioned exogenous AID gene is contained by activating Cre recombinase 2 The direction of the region between the two loxP arrays is reversed,
In which only one of the two XRCC3 alleles is inactivated.

A method for obtaining an antibody having a mutation using the above-mentioned cells is as follows.
Preparing a B cell in which only one of the two XRCC3 alleles of the B cell is inactivated,
Making the cell capable of expressing AID;
Culturing the cells for a period of time;
Applying an extracellular stimulus capable of activating Cre recombinase to the cell, reversing the direction of the AID gene, and stopping the expression of AID;
Isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation;
Selecting a cell that produces an antibody having the desired mutation, and culturing the selected cell having the desired mutation to produce an antibody.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

1) Establishment of XRCC3 (+/-) and XRCC3 (-/-) strains by disruption of XRCC3 gene
To destroy the XRCC3 gene in DT40 cells, two types of targeting vectors (Bsr and HisD, respectively) with a blasticidin resistance gene or a histidinol dehydrogenase gene inserted into the sixth exon of the XRCC3 gene as shown in FIG. Was prepared by the following procedure. A 5 ′ gene fragment was obtained by PCR using primer1 containing SpeI site and primer2 containing BamHI site. In addition, a 3 ′ gene fragment was obtained by PCR using primer3 containing a BamHI site and primer4 containing an XhoI site. The sequences of Primers 1 to 4 are shown in SEQ ID NOs: 2 to 5 in the sequence listing, respectively. There, HisD and BSR genes were inserted as drug resistance markers, and a targeting vector was prepared.

  First, Bsr was introduced, and one XRCC3 was destroyed by targeted homologous recombination to obtain an XRCC3 (+/−) strain. Subsequently, HisD was introduced into the XRCC3 (+/-) strain, the remaining XRCC3 was destroyed, and the XRCC3 (-/-) strain was established. DNA was extracted from the DT40 cell clone into which the targeting vector was introduced, and it was confirmed by PCR that target homologous recombination was performed correctly. Primers A, B, and C shown in FIG. 6 were used for PCR (the respective sequences are shown in SEQ ID NOs: 6 to 8 in the sequence listing). Homologous recombination was confirmed using primers A and C for Bsr and primers B and C for HisD. As shown in FIG. 7, it was confirmed that the XRCC3 (+/−) and XRCC3 (− / −) strains were correctly produced as a result of gene disruption by targeted homologous recombination of XRCC3. Successful second-stage gene transfer and acquisition of XRCC3 (-/-) indicates that the homologous recombination function is retained in the XRCC3 (+/-) strain, and XRCC3 ( This further supports the usefulness of the +/-) strain.

2) Comparison of mutation patterns in XRCC3 (+ / +), (+/-), (-/-) XRCC3 (+/-) that partially or completely destroyed the original strains XRCC3 (+ / +) and XRCC3 ) And XRCC3 (− / −) cells were continuously cultured for 4 weeks, and the efficiency of mutation introduction and mutation mode were evaluated by sequence analysis of the antibody L chain gene. As shown in FIG. 8 and Table 1, gene conversion occurred predominantly in the original strain XRCC3 (+ / +), but in the XRCC3 (− / −) strain, it was largely converted to the point mutation type. On the other hand, surprisingly, in the XRCC3 (+/-) strain, the point mutation was increased to the same extent as that of XRCC3 (-/-) with only a slight decrease in gene conversion.

  Furthermore, XRCC3 (+/-) was the highest in the overall mutation frequency of gene conversion and point mutation. This indicates that the XRCC3 (+/-) strain is an excellent mutant that combines the advantages of gene conversion and point mutation.

3) Comparison of surface IgM disappearance rate and growth rate
In the XRCC3 (-/-) strain, it is reported that the number of mutant cells (surface IgM-negative cells) that cannot produce antibody protein increases due to the occurrence of stop codons and frameshifts, etc., accompanied by an increase in point mutations. (Sale, JE et al. Nature 412, 921-926 (2001)). It is also known that the cell growth rate decreases with XRCC3 deficiency (the same document). This is a serious drawback for an antibody search system.

  Therefore, three types of cell lines XRCC3 (+ / +), (+/-), (-/-) were cultured for a long period of time as shown in FIG. 9, and the expression of surface IgM antibody was measured by flow cytometry. did. As reported, surface IgM negative cells increased with time in the XRCC3 (− / −) strain, but not with XRCC3 (+ / +). Surprisingly, the appearance rate of surface IgM negative cells was low even in XRCC3 (+/−) where the point mutation efficiency was increased. This suggests that loss of function of the antibody gene due to point mutations has been repaired by gene conversion, further supporting the usefulness of the XRCC3 (+/-) strain.

  When the growth rates of the three cell lines XRCC3 (+ / +), (+/-) and (-/-) were compared, the XRCC3 (+ / +) strain grew the fastest, and XRCC3 (+/-) ) Growth was somewhat slower but not problematic, and the maximum cell density was almost the same as that of the XRCC3 (+ / +) strain. On the other hand, in the XRCC3 (− / −) strain, both the growth rate and the maximum cell density were decreased (FIG. 10). This result also shows the usefulness of XRCC3 (+/-) strain.

It is a figure which shows the principle of production (affinity maturation) of the highly specific antibody in an immune system. It is a figure which shows the diversification mechanism of a chicken antibody gene. It is a figure which shows the antibody preparation system using a chicken B cell strain | stump | stock DT40. It is a figure which shows the conversion of the variation | mutation mode from the gene conversion by the defect | deletion of XRCC3 gene to a point mutation. It is a construction figure of XRCC3 targeting vector. It is a figure which shows the position of the primer for the method of inactivation of XRCC3 gene by target homologous recombination, and homologous recombination confirmation. It is a figure which shows the result of the confirmation of the inactivation of XRCC3 gene by the target homologous recombination of XRCC3. It is a figure which shows the increase in the point mutation by inactivation of a XRCC3 gene. The results of 5 representative clones of each mutant cell are shown, and the thick horizontal line indicates the sequence inserted by gene conversion, and the vertical thick line indicates the location of the point mutation. It is a figure which shows the disappearance rate of surface IgM in XRCC3 (+ / +), XRCC3 (+/-), and XRCC3 (-/-). It is a figure which shows the cell growth rate in XRCC3 (+ / +), XRCC3 (+/-), and XRCC3 (-/-).

SEQ ID NOs: 2-8 primers

Claims (8)

  A cell derived from a B cell in which only one of the two alleles of the XRCC3 gene is inactivated, and the frequency of point mutation introduction into the antibody gene is increased compared to cells having both XRCC3 genes.   The cell according to claim 1, wherein the sixth exon of one of the two alleles of the XRCC3 gene is inactivated.   The cell according to claim 1 or 2, wherein inactivation of only one of the two alleles of the XRCC3 gene is performed by homologous recombination.   The cell according to any one of claims 1 to 3, wherein the B cell-derived cell is a chicken B cell line DT40 or a mutant thereof. A mutant strain of chicken B cell line DT40 is induced by exogenous Cre recombinase gene expression by extracellular stimulation, and by reversing the direction of exogenous AID (activation induced cytidine deaminase) gene by the expressed Cre recombinase, AID A variant capable of inducing and stopping expression, characterized by:
1) The endogenous AID gene is functionally disrupted, and no AID protein is produced by endogenous AID gene expression.
2) having an exogenous AID gene sandwiched between two loxP sequences in opposite directions, and a promoter capable of functioning in the animal cell existing upstream of the region sandwiched between the two loxP sequences, When the AID gene is placed in the forward direction with respect to the promoter, the AID gene can be expressed by the promoter, and when the AID gene is placed in the reverse direction with respect to the promoter 3) The Cre recombinase gene has been introduced in a form capable of activating Cre recombinase by extracellular stimulation, and the above-mentioned exogenous AID gene is contained by activating Cre recombinase 2 The direction of the region between the two loxP arrays is reversed,
The cell according to claim 4, which is a mutant strain in which expression of the AID gene is stopped by extracellular stimulation and the mutation function is stopped.   The cell according to any one of claims 1 to 5, into which a gene encoding an exogenous antibody has been introduced. A method of selecting an antibody producing cell that produces an antibody having a desired property, comprising:
(i) a step of preparing the cell according to any one of claims 1 to 6,
(ii) culturing the cell for a certain period of time;
(iii) isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation; and
(iv) selecting a cell producing an antibody having a desired mutation. A method for producing an antibody having desired properties comprising:
(i) a step of preparing the cell according to any one of claims 1 to 6,
(ii) culturing the cell for a certain period of time;
(iii) isolating an antibody produced by the cell or a gene encoding the antibody and detecting the mutation;
(iv) selecting a cell producing an antibody having the desired mutation, and
(v) culturing cells having the selected desired mutation to produce antibodies.

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