WO2008035463A1

WO2008035463A1 - Antibody class switching method

Info

Publication number: WO2008035463A1
Application number: PCT/JP2007/001029
Authority: WO
Inventors: Hidetaka Seo
Original assignee: Chiome Bioscience Inc.
Priority date: 2006-09-22
Filing date: 2007-09-21
Publication date: 2008-03-27
Also published as: JP2009291073A

Abstract

Disclosed is a method for switching an antibody class. Specifically disclosed is a means for transforming a given B cell capable of producing a monoclonal antibody into a B cell capable of producing an antibody having a class switched to a desired different one by preparing a chimeric heavy chain antibody gene wherein a heavy chain constant region gene of a monoclonal antibody produced by the antibody-producing B cell is substituted by a heavy chain constant region gene of a desired different class, and transforming the original antibody-producing cell with the chimeric gene. Also disclosed is an antibody having a class switched to a desired different one, which is produced by the means.

Description

Specification

How to convert antibody classes

Technical field

[0001] The present invention relates to a method for converting a class of an antibody.

Background art

[0002] Antibodies recognize specific antigens, cause various in vivo phenomena, and play an important role as in-vivo defense players. In particular, antibody-dependent cell — mediated cytotoxicity (ADCC) activity and complement-dependent cell I (CDC) activity are effective in removing cancer cells. It is used as an anticancer agent. Focusing on the action of such antibodies, many antibody preparations have been put to practical use, and many preparations have good therapeutic effects. In addition to its use as a pharmaceutical formulation, it is widely used as, for example, various diagnostic tests or effective tools for research and development. Therefore, a technique for easily providing a large amount of various antibodies according to each application is required.

[0003] As a technique for easily and easily producing a large amount of an antibody having high specificity for an arbitrary antigen, a method called AD Lib system is attracting attention (Patent Document 1, Non-Patent Document 2). See). According to this method, an antibody having the desired specificity and affinity can be provided by a simple method. The antibody produced by this method is mainly IgM, but other classes (IgG, IgA, IgD, other than IgM) can be used for various applications of the antibody described above. It will also be necessary to prepare antibodies of IgE).

[0004] As a method for converting the class of an antibody, a method of expressing an IgA class with high efficiency by stimulating B cells with a specific composition (Patent Document 2), using a transgenic non-human animal Various methods have been reported, such as a method for producing a desired isotype (Patent Document 3). Few things have been put to practical use. Since B cells are essentially floating cells that produce antibodies, they have advantages over other animal cell expression systems in terms of high-concentration antibody production, high-density culture, etc., and B cells produce the desired class of antibodies. It will be possible to improve the efficiency of antibody production. However, no effective method has been developed to transduce antibody heavy chain constant region genes into monoclonal antibody-producing B cells that have already been screened to convert the produced monoclonal antibodies to other classes.

[0005] Non-Patent Document 1: Seo et al., Nature Biotec h. 23: 731-735, 2005

Patent Document 1: International Publication Gazette WO 2004/01 1 644

Patent Document 2: International Publication WO 96/27390

Patent Document 3: International Publication WO 99/03991

Disclosure of the invention

Problems to be solved by the invention

[0006] In view of the above circumstances, the present inventors have conducted intensive research on a method for easily converting antibody classes, and as a result, the antibody heavy chain variable region genes produced from antibody-producing B cells and By simply transducing a chimeric heavy chain gene with a heavy chain constant region gene of a desired class mainly into the antibody-producing B cell, an antibody retaining the desired class of the constant region is converted into the antibody-producing B cell. It was revealed that it can be produced from Although it was unclear whether the modified chimeric antibody heavy chain and the already existing antibody light chain would be assembled in the cell by such a method alone, the results of the analysis unexpectedly retain effective functions. The production of chimera IgG was confirmed. According to this technique, it is not necessary to transform both the light chain and the heavy chain as is done in CHO cells and the like, so the procedure for producing a chimeric antibody is at least 1/2. In addition, since floating B cells that originally produce antibodies are used, it has been found that there are advantages such as easy availability of cell lines suitable for the production of large amounts of antibodies. Of antibody-producing B cells converted by different antibody classes The purpose is to provide a manufacturing method.

Means for solving the problem

That is, the present invention relates to the following (1) to (9).

(1) A first aspect of the present invention is a method for improving or preparing an antibody-producing B cell that produces an antibody of a class different from the class of a monoclonal antibody originally produced, comprising the following (a) and ( A method comprising the step of b).

(a) producing a chimeric gene operatively linking a variable region gene of a heavy chain gene of a monoclonal antibody originally produced by an B cell and a constant region gene of another class;

(b) a step of inserting the chimeric gene prepared in step (a) into an expression vector so as to allow expression and transducing the antibody-producing B cell ”.

(2) A second aspect of the present invention is “the method according to (1) above, wherein the class of antibody originally produced from the monoclonal antibody-producing B cell is IgM”.

(3) A third aspect of the present invention is “the method according to (1) or (2) above, wherein the other class is IgG”.

(4) A fourth aspect of the present invention is “the method according to any one of (1) to (3) above, wherein the antibody-producing B cells are DT40 cells”.

(5) The fifth aspect of the present invention is the above (1) to (4), wherein the antibody-producing B cell has lost the function of all antibody heavy chain genes present in the cell. The method according to any one of the above).

(6) A sixth aspect of the present invention is “the method according to any one of (1) to (5) above, wherein the constant region gene is derived from a mouse”.

(7) A seventh aspect of the present invention is “the method according to any one of (1) to (5) above, wherein the constant region gene is derived from human”.

(8) The eighth aspect of the present invention is “the method according to any one of (1) to (5) above, wherein the constant region gene is derived from a rabbit.”

(9) A ninth aspect of the present invention is as described in any one of (1) to (8) above. “Class-changed antibodies obtained from cells prepared by the method”. The invention's effect

[0008] By using the method according to the present invention, a monoclonal antibody against a target antigen is obtained.

IgM antibody producing B cells can be readily converted to B cells producing antibodies with the desired class of properties.

Brief Description of Drawings

[0009] Fig. 1 shows the results of confirming the presence or absence of production of a chimeric antibody of anti-FITC antibody produced by DT40 cells and mouse IgG2a.

[FIG. 2] FIG. 2 shows the results of E L ISA for a purified chimeric antibody using an HRP-labeled anti-mouse IgG secondary antibody.

[FIG. 3] FIG. 3 shows the results of Western blotting for a purified chimeric antibody using an HRP-labeled anti-mouse IgG secondary antibody.

[Fig. 4] Fig. 4 shows the presence or absence of production of a chimeric antibody between anti-FITC antibody and mouse Ig G 2a produced by DT 40 cells into which the chimeric antibody gene has been introduced using a single retrovirus vector. Results are shown.

[FIG. 5] FIG. 5 shows the results of confirming the presence or absence of the production of a chimeric antibody of anti-EG FR antibody and mouse Ig G 2a.

FIG. 6 shows the results of confirming the presence or absence of production of a chimeric antibody of anti-EG FR antibody and human IgG1.

[Figure 7] Figure 7 shows the F ACS analysis by staining A 43 1 cells and HEK 293 T cells with avian anti-EG FR antibody, anti-EG FR mouse chimeric antibody, and anti-EG FR human one-trichimeric antibody. The results are shown.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] 1. Antibody-producing cells

The cells used in the present invention can be any B cells that produce antibodies, and the type of animal from which they are derived may be any animal. In addition, cells that are established or not established can be used, but preferably, established cells are used, particularly preferably derived from chickens. It is DT 40 cell which is a cell culture cell line of B cell.

In addition, antibody-producing cells also include derivative strains and sublines in which some modification to the chromosome (for example, recombination, insertion, deletion, etc. of specific genes) has been made. For example, it may be a cell that has been modified to reduce or lose the function of the antibody heavy chain gene that was originally retained. A method of reducing or losing the function of an antibody heavy chain gene can be easily carried out based on the common general technical knowledge. For example, a method of introducing a mutation into an antibody heavy chain gene originally present in an antibody-producing cell. Methods known to those skilled in the art can be used, such as a method of destroying the entire target gene, a method using RNA interference (RNA i), a method of introducing antisense to the target gene into cells. Preferably, a method for introducing a mutation into a target gene, a method for destroying the entire target gene, or a method using RNA interference (RNA i), more preferably a method for destroying the entire target gene or RNA It is a method using interference (RNA i), and most preferably a method of destroying the entire target gene.

[0011] The culture conditions of the cells used in the present invention are carried out by methods well known in the art. Medium and culture conditions suitable for the antibody-producing B cells to be selected (culture temperature, CO 2 concentration) Needless to say, it is done below. However, when the antibody-producing cells selected are DT 40 cells, for example, the medium is IMDM (Invitrogen), and the culture temperature is 39.5 ° C, 5% CO. 2 Perform under concentration conditions. Culturing is carried out while keeping the cell concentration constant, and the class of antibodies produced from the target cells is checked at appropriate intervals (for example, every day or every week).

[0012] 2. Preparation of chimeric antibody heavy chain gene

In practicing the present invention, the heavy chain gene of an antibody originally produced from a target antibody-producing B cell can be prepared based on common general technical knowledge in the technical field. For example, the target antibody heavy chain gene region can be amplified from the target antibody-producing B cells using RT_PCR and cloned into an appropriate vector or the like. In this case, the primer used for R T_PC R is The sequence information of the antibody heavy chain gene can be obtained from a known database related to the gene possessed by the antibody-producing cell, and designed easily based on this information.

In addition, in order to replace the constant region of the heavy chain gene of the originally produced antibody with the constant region of another class, it is necessary to prepare a gene encoding the constant region of another class. This gene can be obtained from a library such as a cell from which the constant region of the other class is derived, or based on known gene sequence information, the gene of interest can be obtained from an appropriate cDNA library. It can be amplified by PCR.

[0013] The obtained antibody production B cell-derived antibody heavy chain gene variable region gene and other classes of constant region genes must be operably linked. Here, “being able to act” means that when a variable region gene and a constant region gene of another class are linked, the constant region gene can express a desired polypeptide. This means that the gene reading frame (frame) is linked with the variable region gene reading frame. The variable region gene and the other region of the constant region gene may be directly linked, or an amino acid sequence that serves as a spacer may be inserted. Even when a spacer is inserted, it goes without saying that the reading frame (frame) must be adjusted so that the constant region gene can express the desired polypeptide. In addition, other classes of constant region genes encode additional genes, for example, peptides that serve as tags for easy purification (for example, epitope tags such as His tags, peptide tags, GST, etc.) Genes to be operably linked may be linked.

The chimeric gene prepared as described above is a polypeptide that codes for the variable region gene of the antibody heavy chain gene derived from antibody-producing B cells, and a gene that codes for the other region of the constant region gene. A chimeric antibody heavy chain polypeptide linked with peptides is coded.

[0014] 3. Introduction of chimeric antibody gene expression vector into cells

By incorporating the prepared chimeric gene into an appropriate expression vector, Mela-state antibody heavy chain polypeptides can be expressed. The expression vector preferably has a component such as a plug motor or an enhancer that allows the target protein to be expressed in the antibody-producing B cells used.

Introducing the prepared expression vector into antibody-producing B cells uses known methods such as the DEAE dextran method, electrorevolution method, calcium phosphate method, cationic lipid method, infection using retroviral vectors, etc. It can be done easily. In particular, a method using a retroviral vector that can reduce the total number of steps is most desirable.

[0015] 4. Introduction of chimeric antibody gene into cassette vector

The plasmids p L PCX_F I TC_m I g G 2a and p L PCX-EG FR-h I gG 1 prepared in the examples of the present invention represent the sequence of the variable region of the avian antibody in the gene common to the region. By converting by an engineering method, it can be easily used to create chimeric genes of different types of avian antibodies. This makes it possible to shorten the total process.

[0016] Examples are shown below, but the present invention is not limited thereto.

Example

[0017] 1. Cell culture

Cell culture of DT 40 cells was basically performed by the following method. The incubator was a C02 thermostatic chamber and cultured at 39.5 ° C in the presence of 5% C02. The medium is IMMD medium (Invitrogen), 10% FBS, 1% chicken serum, penicillin 100 units / mI, streptomycin 100 g I, 2_mercaptoethanol 55 M Used in addition. Trichostatin A (Wako Pure Chemical Industries) is dissolved in DMSO 2 mg / m I as stock, and diluted appropriately in the medium so that the final concentrations are 1.25 ng / mL and m2.5 ng / mL. Used.

[0018] 2. Obtain anti-F I TC antibody-producing B cells

Antibody producing cells were obtained by the AD Lib system using FITC-conjugated BS A as an antigen. Specifically, the following experimental process was taken. 2-1. Preparation of FITC-conjugated BSA magnetic beads:

Dy na b e a d s M—280 Tos y l a c t i va a t e d (D y n a I company) was used for the mirage and D y n a I M PC (D y n a I company) was used for the magnetic stand. Wash the beads 200 UI three times with 500 I buffer A (0. 1 MN a-Phosphatep H 7.4) and then buffer — A 200 UI with 1 FITC-conjugated BSA (SI GMA) and 3 The reaction was carried out at 7 ° C while stirring by rotation. The beads were then washed twice with buffer C (P B S + 0.1% B S A) 200 U I. Then buffer D (0.2 MT ris-HCI pH 8.5, 0.1% BSA) and 200 μI were added, and the mixture was allowed to react at 37 ° C for 4 hours with rotation and stirring, followed by blocking. It was. After washing twice with 500 I buffer C, the suspension was suspended in 200 I buffer containing 0.02 <½ sodium azide.

[0019] 2-2. Selection of antibody-producing clones using FITC-conjugated BSA magnetic beads About 1 X 10 8 wild-type DT 40 cells treated with trichostatin A, 2.5 ng / m I were washed with a buffer (1% 83) Including 83) After washing once with 1 Om I and once with 1 m I, mix with 5 times 106 FITC-shared BSA magnetic beads in 1 m I wash buffer, 4 ° Incubate at C for 30 minutes with gentle rotation. The plate was then washed 5 times with 1 ml wash buffer. Finally, the cells bound to the magnetic beads were suspended in 500 I, added to 30 m I medium, dispensed 300 I each into a 96-well plate, and cultured at 39.5 ° C.

[0020] 2-3. Screening for anti-FITC antibody producing clones

ELISA was performed as follows. Six days after step 2_2 above, dispense 1 00 L each into 9 6-hole immunoplate U— 9 6 M axisorp (Nunc) at 1 g of F 1 〇0 conjugate 曰 38 2.5 g / m L I left it. In order to examine the specificity of the antibody, BSA not labeled with FITC was also immobilized on the plate as a control. Discard the contents the next day and block the buffer (PBS containing 0.5% skim milk) 200 UL was added and incubated for 2 hours at room temperature. ELISA wash buffer

(P B S containing 0.05% T ween 2 0) Washed 3 times with 2 0 0 L. 100 L of each culture supernatant derived from the colonies generated by the above selection in 2-2 was added and incubated at room temperature for 1 hour. After washing 5 times with 200 mL of ELISA wash buffer, Λ 00 UL was diluted with the secondary antibody diluted 20 times with PBS, and incubated at room temperature for 45 minutes. As the secondary antibody, anti-chicken IgM_HRP (BET HYL) was used. After washing 5 times with 200 liters of ELISA wash buffer, TMB + (Dako Ltd.) was added and incubated for 10 minutes. After that, the reaction is stopped with 1 N sulfuric acid 1 00 1 1_ and 6 5 0 nm (or 4 5 0 nm) (* 4 5 50 nm is deleted, which is consistent with the previous application. If there seems to be no problem, you should leave the description of 45 nm as well. It was measured.

3. Preparation of anti-FITC chicken IgM and mouse IgG2a chimeric antibody (hereinafter, chimeric antibody) gene

3-1. I Tar R N A Extraction

For extraction of Ital RNA from anti-FITC antibody-producing cells, RNeasyPluin Minik (QIAGEN) was used, and extraction was performed according to the manual attached to the kit. At that time, QIA shr d der (QIA AG) was used to prepare a cell lysate.

3-2.? ^ -Don’t you? ^ Isolation of anti-ITC chicken IgM heavy chain gene Anti-FITC antibody heavy chain gene was isolated by RT_PCR. RT_PCR was Invitrogen's One Step RT-PCR. I Tal RNA 1 I was in a saddle type, and two types of primers_ (VH_F1: self-sequence number 1, VH—R: SEQ ID NO: 2) were used. The reaction conditions are as follows. 5 After 5 min at 30 ° C, treatment for 2 min at 94 ° C, 9 4 ° C for 15 sec, 60 ° C for 30 sec, 68 min for 1 min 28 cycles It was. The mixture was then incubated for 5 minutes at 68 ° C. The heavy chain fragment obtained by RT_PCR was obtained using the DH5 strain. TA cloning to pCR 2.1—TOPO vector (Invitrogen) gave pANTI_FIT C_HC. The sequence was confirmed using an ABI prism 377 sequencer.

[0022] 3-3. Isolation of mouse I gG2 a

Mouse IgG2a cloning uses Mouse Spleen BD Ma ratho n-Re adyc DNA (Clontech) as a saddle, and two primers (mIgG2aF-1: SEQ ID NO: 3, mlg G 2 a R-1: 酉自歹¹ J number 4) was used for PCR with Pyrobestpolylase (SSia). The reaction conditions are as follows. After 98 ° C for 2 minutes, 98 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes were reacted for 30 minutes at 72 ° C for 5 minutes. The mouse I g G 2 a F c region fragment obtained here was added with “A” by adding E x T aq (Takara Shuzo) and reacting at 72 ° C for 15 minutes, and then using the DH 5 strain. PCR2. 1— TOPO vector (Invitrogen) was TA-cloned to obtain pM IgG2a. The sequence was confirmed using AB I prism 377 sequencer.

[0023] 3-4. Preparation of anti-FTIC chicken I gM_mouse I g G 2 a fusion gene Anti-FI TC chicken I gM and mouse I gG2 a chimeric antibody (hereinafter referred to as chimera rod) Regressor is P yrobestpo It was prepared by PCR using I ymerase. First, (i) pAN TI _F I TC—HC is of a saddle type, and the primer is VH—F 1 and Gd I gM-CH 1 + m I g G 2 a -R: a combination of SEQ ID NO: 5 and (ii ) pM I g G 2 a is a saddle type Primer was performed with Gd I gM-CH 1 + m I g G 2 a-F: SEQ ID NO: 6 and m I g G 2 a R-1: SEQ ID NO: 4 . The conditions were 98 ° C for 2 minutes, 98 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute after 15 cycles, followed by reaction at 72 ° C for 5 minutes. The band amplified in (i) and (ii) was purified by Qiaquick Gel Extraction Minikit (QI AGEN), and the mixture of fragments 1 L each was used as a saddle type, and (i), (ii PCR was carried out under the same reaction conditions as in). Note that the primer is V H_ F 1 and , Ml gG2 a R_1 was used under the following conditions. The obtained chimera gene fragment is added with “A” by adding E x T aq (Takara Shuzo) and reacting at 72 ° C for 15 min, and using DH 5 strain, p CR 2.1 vector Cloned and obtained pANTI-FIT C-mG 2a. The sequence was confirmed by ABI AB I pri sm377 sequencer.

[0024] 4. Construction of chimeric antibody gene expression construct and its introduction

4- 1. Construction of chimeric antibody expression construct

P EG FP—C 1 (Clontech) was used as the expression vector. p EG F PC 1 is digested with the restriction enzymes N he I and B am H I, and the end of the vector is smoothed with Klenow Fragment (Takara Shuzo) and then removed with BAP (Takara Shuzo). Phosphorylated. The chimeric gene to be inserted was obtained by blunting the EcoR I fragment derived from pAN T I — F I TC — mG2a. Both the vector 1 and the insert were purified by Q i aq u i c k G e l e x t r a c t i o n k i t (Q i a g e n company), and then subjected to ligation using L i gat i o n k i t v e r 2 (Takara Shuzo). Transformation was performed on DH 5 strains.

[0025] 4-2. Gene transfer

The expression construct of the chimeric antibody obtained in 4_1 above was introduced into an anti-FITC antibody production strain. Gene transfer was performed using electroporation. About 20 g equivalent is digested with the restriction enzyme M I u I to make a single strand, and after purification, PBS, 500! Dissolved in _. About 107 anti-FITC antibody-producing strains were electroporated under conditions of about 550 V and 25 F, then left on ice for 10 minutes, transferred to 20 mL of medium, and cultured at 37 ° C for 1 hour. . The next day, the culture solution is centrifuged (190 xg, 10 min), and the cells are collected in a pellet and then suspended in 80 mL of medium containing 2 mg / mL G41 8 (Invitrogen). After dispensing at 200 L / well, the cells were cultured at 39.5 ° C.

[0026] 5. Confirmation of chimeric antibody expression and antigen recognition ELISA was performed as follows. FI TC conjugated BS A 10 U L / 96 L was dispensed into a 96-well immunoplate U—96 Maxisorp (Nunc) in 100 L portions and allowed to stand for a while. In addition, in order to examine the specificity of the antibody, BSA not labeled with FITC as a control was similarly fixed to the plate. The next day, the contents of the plate were discarded, and blocking buffer (PBS containing 0.5% skim milk) 200 1_ was added and incubated at room temperature for 2 hours. Washed 3 times with 200 L of ELISA wash buffer (BS containing 0.05% Ding 6 ^ 20). Thereafter, 100 L of each culture supernatant derived from the chimeric antibody production candidate clone 18 clone was added and incubated at room temperature for 1 hour. After washing 5 times with 200 L of ELISA wash buffer, 100 L of secondary antibody diluted 2000 times with PBS was added and incubated at room temperature for 45 minutes. As the secondary antibody, anti-mouse IgG_HRP (BETH YL) was used. After washing 5 times with 200 L of ELISA wash buffer, 100 ml of TMB + (Dako) was added and incubated for 10 minutes. Thereafter, the reaction was stopped with 100 L of 1 N sulfuric acid, and the absorbance at 650 nm was measured. The results are shown in Figure 1. It was confirmed that the number 3 clone expressed a chimeric antibody with mouse Ig 2a.

6. Biochemical verification of purified chimeric antibody

The chimeric antibody was purified from the No. 3 clone prepared in 5 above, and the characteristics were further examined in detail.

6- 1. Preparation of purified chimeric antibody

6— 1— 1. Preparation of chicken serum components

Centrifugation was performed at 500 X g, and 10 ml of chicken serum (In Vitrogen) from which the precipitate was removed was added to 10 ml of saturated ammonium sulfate solution, and ammonium sulfate precipitation was performed at 4 ° C. The next day, the mixture was centrifuged at 9100 xg for 15 minutes, and the supernatant was collected. The mixture was centrifuged again at 91 00 X g for 15 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45 m filter (Zartorius), transferred to a dialysis tube (Spectrapore, MWCO: 3 500, 9.3 mL / cm), and 3 times against 1 L of PBS. (Of which 2 times Dialysis was performed at 4 ° C for 4 hours once. The sample was taken out from the dialysis tube the next day and concentrated to 1 OmL at 2330 xg with Centrip Ius YM3 (Mii II ipore). After concentration, the solution was sterilized with a 0.22 m filter (Sartorius), dispensed in appropriate amounts, and stored frozen at 30 ° C.

[0028] 6- 1 -2. Production on culture

A medium prepared by adding the anti-FITC chimeric antibody production number 3 clone to serum-free medium AIM _V (In Vitrogen) with 5% chicken serum component (hereinafter referred to as “quasi-serum free medium”) In culture. Cells diluted with semi-serum-free medium to 2 X 106 L were prepared in 50 mL (total 200 mL) for four 14 cm dishes (Nunc Corporation). This was cultured in a CO 2 incubator at 39.5 ° C and 5% CO 2 for 5 days. The obtained culture solution was centrifuged at 2330 X g for 15 minutes, and the supernatant was collected. The supernatant was again centrifuged at 2330 Xg for 15 minutes and used as the culture supernatant.

[0029] 6- 1 -3. Purification of chimeric antibody

The chimeric antibody was purified using MabTra ap kit (GE Healthcare) according to the manual. Using a peristaltic pump, almost the entire culture supernatant was applied to M ab T r a p (only 10 mL was collected for control). 10 ml_ of b i n d i n g b u f f er scrambled and eluted with 5 ml_ of e l u t i o n b u f f e r. At that time, 1 mL each was collected in 5 fractions. The elution fraction (N o. 2) with the highest protein concentration was dialyzed against PBS using S I id e -A-L i z e r (10 K) (P I E RC E).

[0030] 6-2. Examination by ELISA using purified chimeric antibody

Purified chimeric antibody diluted with x5, x25, x125, x625, x3125, x1 5625 times with HRP labeled anti-mouse Ig g secondary antibody and ELISA against FITC went. ELI SA was performed in the same manner as “5. Confirmation of expression and antigen recognition of chimeric antibody”. The result is shown in figure 2. It shows that the purified chimeric antibody reacts with FITC at the ELISA level. [0031] 6-3. Purification of chimeric antibody by Western plot method

The purified chimeric antibody was analyzed by Western plot. Electrophoresed on 4% polyacrylamide gel (Bio R ad), transferred to nylon membrane (M i II ipore), blocked with 5% skim milk, reacted with HRP-labeled anti-mouse IgG secondary antibody I let you. It was washed 3 times for 15 minutes each with 曰 3 containing 0.1% T ween 20 and a signal was obtained by chemiluminescence with EC L p lus (GE Healthcare). The signal was detected with LAS-1 000 (Fuji Photo Film). The results are shown in Figure 3. The purified chimeric antibody reacts with the anti-mouse IgG secondary antibody to produce a specific band. In the lane of the culture supernatant, the chimera antibody band has a higher mobility than that of the purified chimera antibody. This is because the amount of protein in the AI M_V medium that has a molecular weight close to that of the heavy chain is very large. it is conceivable that.

[0032] 7. Expression of anti-FITC chimeric antibody by retrovirus expression vector

7-1. Insertion of anti-FITC chimeric antibody gene into expression vector pL PCX (Takara Bio Inc.) was used as the expression vector. pL PCX was treated with restriction enzymes Not I and C I a I and treated with C I A P. On the other hand, the chimeric antibody gene was amplified by pCR from pATNTI_FITC-mG2a. Two primers, VH_F 1_N o t 1 (SEQ ID NO: 7) and mT g G 2 a R-1 _C I a I (SEQ ID NO: 8), were used as primers. The amplified DNA fragment was treated with NotI and CIaI, separated by agarose electrophoresis, and purified by Qiaquick ^ IExtrtactiot \ agen). p L PCX and the chimeric antibody gene fragment were used for lysation with a ligation kit (Niibon Scene). The transformation was performed on the DH5 strain. The plasmid thus prepared was pLP CX— FIT Cm I g G 2 a

[0033] 7-2. Production of retrovirus and infection with D T 40

p LPC X- FIT C- m I g G 2 a, Fu GEN E6 (Roche Diag P C L_amp h by the lipofusion method using Nostax. It was introduced into 293 T cells together with Plasmid (Takara Bio). Two days later, the culture supernatant was collected, and the anti-FITC antibody-producing DT 40 cell line was treated in this culture supernatant for 2 days. By treating the treated cells in a medium containing puromycin (0.5 g / ml), only those cells into which Vector I had been introduced were selected.

7- 3. Confirmation of chimeric antibody expression

FIG. 4 shows the result of confirming the expression of the chimeric antibody according to the technique of “5. Confirmation of expression and antigen recognition of the chimeric antibody”.

8. Production of chimeric antibody gene expression vector for EG F receptor (EG FR)

8- 1. Insertion of anti-EGFR antibody gene into expression vector

The anti-FI TC chimeric antibody gene expression vector prepared by the technique described in “7_ 1. Insertion of anti-FI TC chimeric antibody gene into expression vector” above contains a restriction enzyme recognition sequence that is common to all tri-antibody sequences. It can be used for other specific antibody sequences with few operations.

p LPC XF IT Cm Ig G2a was treated with restriction enzymes NotI and PmaCI. An antibody gene was obtained from the anti-EG FR antibody-producing strain by PCR in the same manner as in “3. Preparation of anti-FITC chicken I gM and mouse Ig G 2a chimeric antibody gene”. As the primers, VH—F 1—Not I — Xho I — B g III (SEQ ID NO: 9) and c Ig MCH 1 + h Ig G 1 hinge R (SEQ ID NO: 10) were used. The obtained DNA fragment was treated with Not I and Pma CI. Each DNA fragment was isolated by agarose electrolysis and purified by Qiaquick Gel Extraction kit (Qiagen). Subsequently, Ligation was performed using a Ligation kit (Nibonbon Gene), and the transformation was performed on the DH5 strain. As a result, pLPCXEGFRmIgG2a was obtained. [0035] 8-2. Retrovirus production and DT 40 infection

Production of retrovirus and infection with D は 40 are carried out in the same manner as described in “7—2. Production of retrovirus and infection with D Τ 40” p L PCX—FIT Cm I g G 2 a instead of p L This was done using PCX_EG FR_I g G 2 a.

8- 3. Confirmation of expression of chimeric antibody

FIG. 5 shows the result of confirming the expression of the chimeric antibody according to the technique of “5. Confirmation of expression and antigen recognition of the chimeric antibody”.

[0036] 9. Expression of anti-EG FR human one-trichimeric antibody

9- 1. Preparation of human Ig G 1 constant region gene

The gene of the constant region of human Ig G 1 can be cloned from human RNA by a method common in the art. P FUS E_h I g G 1 vector (Invivogen) has obtained the human Ig G 1 constant region gene from the p FUS E-h I g G 1 vector by PCR. Amplified. As primers, hIgG1 + hinge15bp (SEQ ID NO: 1 1) and hIgG1-3—C Ia1 (SEQ ID NO: 12) were used. This DNA fragment was separated by agarose electrophoresis and purified by Q i aq u i ck G e l E xt r a c t i o n k i t. Let this DNA fragment be h i n g i G 1.

[0037] 9-2. Preparation of anti-EG FR avian I gM and human I gG 1 chimeric antibody genes

The gene for the anti-EG FR avian I gM and human I gG 1 chimeric antibody (hereinafter referred to as human trichimeric antibody) is the same as in “3-4. Preparation of anti-FT IC chicken I gM—mouse I gG2 a fusion gene”. It produced using the method of. 1. Introduction (i) p G EM— EGFR is a saddle type and the primer is VH— F 1— Not I _ X ho I _B g III (SEQ ID NO: 9) and c I g MCH 1 + h I g G 1 The combination of hinge R (SEQ ID NO: 10), and (ii) hinge G 1 DNA fragment prepared in 9_1 as a saddle type primer is G d I g M_C H 1 + h I g G 1 -F (SEQ ID NO: 1 3) and h I g G 1 -3_C I a 1 (SEQ ID NO: 1 2) were used. The conditions were 98 ° C. for 2 minutes, 98 ° C. for 30 seconds, 58 ° C. for 30 seconds, 72 ° C. for 1 minute, 15 cycles, and then reacted at 72 ° C. for 8 minutes. The band amplified in (i) and (ii) was purified by Qiaquick Gel Extraction Minikit (QI AG EN), and the mixture of fragments 1 L each was used as a saddle, and (i), (ii PCR was carried out under the same reaction conditions as in). As the primers, VH_F1_NotI_XhoI_BgIII (SEQ ID NO: 9) and hIgG1_3—CIa1 (SEQ ID NO: 12) were used. The resulting chimeric gene fragment was added with E x T aq (Takara Shuzo) 7

“A” was added by reacting at 2 ° C. for 15 minutes, and TA cloning was performed on the pGEM_T vector using DH5 strain to obtain pANTI_EGFR_hIgG1. The sequence was confirmed by ABI Company A B I pri sm377 sequencer.

pAN TI—EG FR—hl gG I and p 1_ ○ 乂 were treated with 1 \ 10 ₀ 1 and ○ 1 3 I, each DNA fragment was separated by agarose electrophoresis, and Q iaquick ^ e IE xtractionkit Made / Second. After that, Ligation was performed with pG EM_T (Promega) using Ligation kit (Nibonbon Gene). In this way, pLPCX—EGFR-hIgG1 was obtained.

9-3. Retrovirus production and D T 40 infection

Retrovirus production and infection with DT 40 are carried out in the same manner as described in “7—2. Retrovirus production and infection with DT 40” above. P L PCX—FIT I g G 1 was used.

9-4. Confirmation of human atrichimeric antibody expression

ELISA was performed as follows. Anti-avian IgM antibody (Bethy I) was dispensed at 50 g each into 96-well immunoplate U-96 Maxisorp (Nunc) at 10 g / ml and left to stand. Discard the contents of the plate the next day Put 200 L of blocking buffer (PBS containing 1% BSA) and incubate at room temperature for 1 hour. Washed 3 times with ELISA wash buffer (PBS containing 0.05% Tween 20) 200 1_. Thereafter, 100 L of each culture supernatant derived from the chimeric antibody-producing cells was added, and incubated at room temperature for 1 hour. After washing 5 times with 200 μl of ELISA wash buffer, 100 L of secondary antibody diluted 25,000 times with 曰 3 was added and incubated at room temperature for 45 minutes. As the secondary antibody, anti-human Ig G_HRP (BETHYL) was used. After washing 5 times with 200 L of ELISA wash buffer, add 1 00 1_ of TMB + (Dako) and incubate for 5 minutes. Thereafter, the reaction was stopped with 100 L of 1 N sulfuric acid, and the absorbance at 450 nm was measured. The result is shown in FIG. The signal output when using anti-human IgG antibody as a secondary antibody indicates that the chimeric antibody is expressed.

[0039] 1 0. Confirmation of antigen-binding properties of mouse chimeric antibody and human one-trichimeric antibody by FA C S

The cell line A 43 1 derived from squamous cell carcinoma is known to express EGFR. After recovering A 43 1 cells cultured in 10 cm plastic dishes and HEK 29 3 T cells known not to express EGFR by treatment with 0.25% trypsin for 15 minutes This was treated for 30 minutes in a solution containing mouse chimeric antibody and human one-trichimeric antibody. After washing the cells three times, the mouse chimeric antibody treated with FITC-conjugated anti-mouse IgG antibody (eBioscience), the human one-trichimeric antibody treated with FITC-conjugated anti-human Ig Staining was performed with G antibody (Southern Biotech). The stained cells were washed three times and then analyzed by FACSort (Beeton Dickin Son). The results are shown in Fig. 7.

Industrial applicability

[0040] Since the present invention can change the class of antibodies produced by antibody-producing cells to a desired class according to the application, the field of antibody preparations, antibody diagnostics, etc. Is widely available as a research tool.

Claims

The scope of the claims

[1] A method for improving or preparing an antibody-producing B cell that produces an antibody of a class different from that of the originally produced monoclonal antibody, the method comprising the following steps (a) and (b):

(a) producing a chimeric gene operatively linking a variable region gene of a heavy chain gene of a monoclonal antibody originally produced by an antibody-producing B cell and a constant region gene of another class;

(b) a step of inserting the chimeric gene prepared in step (a) into an expression vector so as to allow expression, and transducing the expression vector into the antibody-producing B cell.

[2] The method according to claim 1, wherein the class of the antibody originally produced from the monoclonal antibody-producing B cell is IgM.

[3] The method according to claim 1 or 2, wherein the other class is IgG.

[4] The method according to any one of claims 1 to 3, wherein the antibody-producing B cell is a DT40 cell.

[5] The method according to any one of [1] to [4], wherein the antibody-producing B cell has lost the function of an antibody heavy chain gene present in the cell.

6. The method according to any one of claims 1 to 5, wherein the constant region gene is derived from a mouse.

7. The method according to any one of claims 1 to 5, wherein the constant region gene is derived from a human.

[8] The method according to any one of [1] to [5], wherein the constant region gene is derived from a rabbit.

[9] An antibody with an altered class, obtained from a cell prepared by the method according to any one of claims 1 to 8.