JP2016146768A - Production method of antibodies in which ability is modified - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide production method of modified antibodies.SOLUTION: Monoclonal antibodies are produced by the following i)-iii). i) A mutation is introduced into a H chain gene of an original monoclonal antibody (0), it is hybridized with a L chain gene of the original monoclonal antibody (0) to be expressed, and a mutant (H1) having objective ability is selected by screening. ii) A mutation is introduced into a L chain gene of the original monoclonal antibody (0), it is hybridized with a H chain gene of the original monoclonal antibody (0) to be expressed, and a mutant (L1) having objective ability is selected by screening. iii) The H chain gene of the mutant (H1) selected in above-mentioned i) and the L chain gene of the mutant (L1) selected in above-mentioned ii) is hybridized and a monoclonal antibody (HL) is expressed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing an antibody with altered performance.

Vertebrates have an immune system that protects the living body from external enemies. The immune system is a biological protection system that recognizes, attacks, and eliminates foreign substances such as pathogens that have entered the body. It is antibodies that play an important role here. Antibodies are mainly in the blood and circulate in the body along the bloodstream. When a foreign substance enters the body, it matures and the affinity and specificity for the antigen are improved. Therefore, even if a very small amount of foreign matter enters the body, it is immediately captured by the antibody.
An immunodiagnostic reagent diagnoses a disease by detecting a specific substance in a biological sample using such characteristics of an antibody. A monoclonal antibody used as an immunodiagnostic reagent requires high affinity and specificity for an antigen. However, high performance antibodies are not always easily isolated. In particular, a low molecular compound has a small site of interaction with an antibody. For this reason, such molecules have low immunogenicity, and the antibody titer does not easily increase even when immunization is performed on experimental animals.

  Among low molecular compounds, steroid hormones are metabolized in the body in complex ways. As a result, there are many compounds having a similar structure. For example, testosterone represented by the following formula (1) is metabolized in the body and changes into compounds represented by formulas (2) to (5).

そのため、血中のテストステロンをを精度よく検出するためには、僅かな構造の違いを見分けられる抗体が必要となる。しかし低分子化合物を抗原とした場合、抗体の単離自体がそもそも難しい。このような状況において、水素原子数個の有無を見分けられる程の特異性をもつ抗体の単離は非常に困難であり、まして高い親和性を有する抗体の単離はさらに困難であった。
抗体の性能を改変する方法としてはこれまで、Ｈ鎖とＬ鎖の遺伝子を繋いでからその配列に変異を導入する方法などがある（特許文献１−４、非特許文献１）。しかしこの方法では、望ましい性能を有する変異体がスクリーニングで得られた場合でも、繋がれていたＨ鎖とＬ鎖の遺伝子を元に戻してそれぞれ個別に発現させた時にその性能が発揮されないこともあった。

Therefore, in order to accurately detect testosterone in blood, an antibody that can distinguish a slight difference in structure is required. However, when a low molecular weight compound is used as an antigen, isolation of the antibody itself is difficult in the first place. Under such circumstances, it has been very difficult to isolate an antibody having such a specificity that it can distinguish the presence or absence of several hydrogen atoms, and even more difficult to isolate an antibody having a high affinity.
As a method for modifying the performance of an antibody, there has heretofore been a method of connecting a gene of H chain and L chain and then introducing a mutation into the sequence (Patent Documents 1-4 and Non-Patent Document 1). However, in this method, even when a mutant having the desired performance is obtained by screening, the performance may not be exhibited when the connected H chain and L chain genes are restored and individually expressed. there were.

US Pat. No. 5,223,409 US Pat. No. 5,821,047 US Pat. No. 5,885,793 US Pat. No. 5,969,108

Toyota Central R & D Review 34, 31-38, (1999)

  An object of the present invention is to provide a method for producing a modified antibody.

As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.
(1) A method for producing a modified monoclonal antibody, comprising the following steps i) to iii):
i) Mutation is introduced into the heavy chain gene of the original monoclonal antibody (0), and this is expressed by crossing with the light chain gene of the original monoclonal antibody (0) to obtain a mutant (H1) having the desired performance. Select by screening.
ii) introducing a mutation into the L chain gene of the original monoclonal antibody (0), expressing it by multiplying it with the H chain gene of the original monoclonal antibody (0), and obtaining a mutant (L1) having the desired performance Select by screening.
iii) Multiplication of the H chain gene of the mutant (H1) selected in i) above with the L chain gene of the mutant (L1) selected in ii) above to express a monoclonal antibody (HL) Let
(2) The production method according to (1), wherein a mutation is introduced by an error-prone PCR method.
(3) The production method according to (1) or (2), wherein the original monoclonal antibody (0) is derived from a rabbit.
(4) The production method according to any one of (1) to (3), wherein the original monoclonal antibody (0) has reactivity with testosterone.
(5) A modified monoclonal antibody obtained by the production method according to any one of (1) to (4) above.

  Hereinafter, the present invention will be described in more detail with reference to FIG.

  In the present invention, “monoclonal antibody” means a monoclonal antibody expressed in a separate state in which the genes of the H chain and the L chain are not connected to each other unless otherwise specified.

  In the present invention, the initial monoclonal antibody (0) is not particularly limited. For example, low molecular weight compounds such as steroid compounds such as testosterone, progesterone, and cortisol, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) And antibodies having reactivity with peptides such as brain natriuretic peptide (BNP) and insulin.

  The type of the initial monoclonal antibody (0) is not particularly limited. For example, antibodies derived from mammals such as rabbits, mice, rats, sheep, and humans, antibodies derived from amphibians such as Xenopus, Examples include antibodies derived from birds such as chickens, and antibodies derived from fish. In addition, it is preferable to use the monoclonal antibody (0) used in the present invention that is closer to the intended performance.

  In the present invention, the order in which steps i) and ii) are performed is not particularly limited, and either may be performed first or may be performed simultaneously. In addition, the method for introducing a mutation in steps i) and ii) is not particularly limited. For example, an error-prone PCR method can be used, and any other technique that can introduce a mutation into a target gene sequence can be used. It can be anything. Specifically, there are a method using a chemical substance such as hydroxylamine hydrochloride, a method of irradiating ultraviolet rays, and a method of using E. coli modified so that mutations are frequently introduced into genes (Anal Biochem, 388, 71). -80 (2009), Comb Chem High Throughput Screen, 9, 271-288 (2009)).

  In the present invention, a monoclonal antibody having the desired performance is selected by screening, but the performance at this time is not particularly limited, and examples thereof include sensitivity and specificity. Further, in steps i) and ii), screening may be performed based on the same performance, or screening may be performed based on different performance. There is no particular limitation on the screening method, and for example, ELISA or the like is used.

  The method of the present invention may be repeated. In particular, if screening is performed from the viewpoint of different performance from the second time onward, a more suitable monoclonal antibody may be obtained.

  According to the present invention, antibody performance can be easily and reliably improved. In particular, the present invention is expressed in a separate state where the antibody H chain and L chain genes are not joined together, so that the performance at the time of screening is easily reflected as the performance of the finally obtained monoclonal antibody. Has an effect.

It is a figure which shows the outline | summary of this invention. It is a figure which shows the outline of an Example. In the screening by Example ELISA, it is a figure which shows the format of three kinds of experiment. It is a figure which shows the result of the screening by ELISA of an Example. It is a figure which shows the specificity evaluation result of the antibody in an Example.

Hereinafter, the present invention will be described in detail using examples of rabbit anti-testosterone antibodies. However, the present invention is not limited to this. An outline of the embodiment is shown in FIG.
(1) Cloning of Rabbit Monoclonal Antibody Anti-testosterone rabbit monoclonal antibodies were obtained by the following method (JP 2009-240300 A, JP 2013-083448 A, JP 2013-124224 A). First, a linker was grown on testosterone and bound to BSA. Then, rabbits were immunized with this BSA-testosterone as an antigen. Thereafter, antibody-producing cells were isolated from the immunized rabbit. The antibody gene was cloned from the cells to obtain a monoclonal antibody reactive with testosterone.
(2) Mutation introduction into antibody gene Mutation was introduced into the gene of an antibody reactive with testosterone. The experimental procedure is as follows.

First, 10 μl of error-prone PCR buffer (100 mM Tris-Cl, 500 mM KCl, 70 mM MgCl ₂ , 0.1% (W / V) gelatin) and dNTP mix solution (2 mM dATP, 2 mM dGTP, 10 mM dCTP, 10 mM dTTP), 10 μl, 3 μl of 10 mM MnCl ₂ solution, 1 μl of 50 μM Fw / Rv Primer mix solution, 1 μl of vector solution (10 ng / μl) containing the cloned antibody gene (H chain or L chain), 1 μl of Taq DNA polymerase solution (5 units / μl), A reaction solution was prepared by adding 74 μl of sterilized water. Then, 20 μl of this reaction solution was dispensed and PCR was carried out to randomly introduce mutations over the entire length of the antibody gene. PCR reaction conditions are 94 ° C., 10 seconds → 94 ° C., 30 seconds → 60 ° C., 1 minute → 72 ° C., 2 minutes → 4 ° C., 94 ° C., 30 seconds → 60 ° C., 1 minute → 72 ° C., 2 After performing the minute portion 35 times, the temperature was changed to 4 ° C. After completion of the PCR reaction, agarose gel electrophoresis was performed to confirm that the gene was amplified. Thereafter, the dispensed reaction solutions were combined into one, and the PCR product was purified with an illustra GFX PCR purification kit (manufactured by GE Life Sciences).

Next, this PCR product was treated with a restriction enzyme. Thereafter, this gene was inserted into an antibody expression vector (including an ampicillin resistance gene) treated with the same restriction enzyme. Then, Escherichia coli JM109 strain was transformed with the expression vector into which the antibody gene was inserted. This E. coli was seeded on LB agar medium (containing ampicillin) and cultured at 37 ° C. overnight. E. coli colonies formed after the culture were picked one by one and inoculated into LB medium (containing ampicillin) and cultured overnight at 37 ° C. Then, the expression vector of the antibody gene into which the mutation was introduced was purified by the alkali miniprep method.
(3) Expression of antibody introduced with mutation The prepared vector was introduced into COS-1 cells to express the antibody introduced with the mutation. The experimental procedure is as follows.

First, COS-1 cells were suspended in D-MEM medium (Life Technologies) containing 10% bovine serum and seeded at 100 μl / well in a 96-well plate (Falcon). This plate was placed under conditions of 37 ° C. and 5% CO ₂ , and the culture was continued until cells grew on the entire surface of the plate. Once the cells grew across the plate, the medium used was removed. Then, Opti-MEM medium (manufactured by Life Technologies) was newly added to each 100 μl / well. The plate was then allowed to stand for 3 hours at 37 ° C. and 5% CO ₂ .

  The following work was performed 30 minutes before the completion of the three-hour standing. First, 120 μl of lipofectamine 2000 (manufactured by Life Technologies) was added to 12 ml of Opti-MEM medium and mixed well. Then, 1920 ng of a wild type H chain gene expression vector or 3840 ng of a wild type L chain gene expression vector was added to this solution and mixed well. Thereafter, each 50 μl / well of this solution was dispensed into a 96-well plate. A mutation-introduced L-chain gene expression vector (16 ng / well) or a mutation-introduced H-chain gene expression vector (8 ng / well) was added, respectively, and wild type H-chain gene × mutant L-chain gene or mutation The solution was adjusted to be a combination of type H chain gene × wild type L chain gene. Thereafter, this transfection solution was allowed to stand at 37 ° C. for 30 minutes.

Three hours after the medium was changed to the Opti-MEM medium, 50 μl / well of the transfection solution prepared previously was added to the plate in which COS-1 cells were cultured. Thereafter, this plate was placed under conditions of 37 ° C. and 5% CO ₂ and cultured for 1 week to express the antibody.
(4) Screening by ELISA The culture supernatant was collected from the cells into which the antibody gene had been introduced, and the performance (sensitivity and specificity) of the antibody contained therein was examined and screened. In this experiment, the antigen is a low molecular weight compound. Therefore, antibody performance was evaluated by a competitive method. This competition method is an experimental system in which an antigen labeled with alkaline phosphatase is reacted with an unlabeled antigen or a crossover substance. In order to evaluate the sensitivity and specificity performance of an antibody by this method, three experimental formats are required (FIG. 3, plates A to C). Therefore, in this experiment, three ELISA plates (96 wells) were prepared for one transfected COS-1 culture plate (96 wells). Then, an equal amount of each culture supernatant was added to the corresponding well of the ELISA plate, and the performance of each mutant was evaluated. Detailed conditions are as follows.

First, goat-anti-rabbit antibody was dissolved in a solid phase buffer (12 mM Na ₂ CO ₃ , 38 mM NaHCO ₃ , pH 9.6) so as to be 0.5 μg / ml. Then, 100 μl / well of this was dispensed into an ELISA plate (96 well, manufactured by Greiner). After allowing the plate to stand at room temperature for 1 hour, each well was washed three times with a washing buffer (1 mM Tris-Cl, 7.5 mM NaCl, 0.05% tween 20, pH 7.4). Then, blocking buffer (8.1 mM Na ₂ HPO ₄ · 12H ₂ O, 1.5 mM K ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl, 1% skim milk, pH 7.5) was added at 200 μl / well and 1 at room temperature. The well was allowed to stand for 1 hour, and each well was washed once with a washing buffer. Then, 70 μl / incubation buffer (8.1 mM Na ₂ HPO ₄ · 12H ₂ O, 1.5 mM K ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl, 0.1% skim milk, pH 7.5) was added to the plate. Wells were added one by one. Thereafter, 30 μl / well of culture supernatant was added to each well position corresponding to the culture plate. Here, in the same manner, two other ELISA plates to which the culture supernatant was added were duplicated. The plates were then allowed to stand at room temperature for 1 hour and each well was washed 3 times with wash buffer.
Then, 100 μl / well of an incubation buffer containing testosterone labeled with alkaline phosphatase (hereinafter referred to as CJ) was added to the first of the three ELISA plates prepared. This plate is referred to as plate A. Next, 100 μl / well of an incubation buffer containing CJ and unlabeled testosterone was added to the second plate. This plate is referred to as plate B. Finally, the third plate contains 100 μl / ml of incubation buffer containing CJ and nothing labeled crossover material (mixture of dehydroepiandrosterone sulfate, dehydroepiandrosterone-glucuronite, androsterone sulfate). Wells were added one by one. This plate is referred to as plate C.
These plates were then allowed to stand at room temperature for 1 hour, and each well was washed 3 times with wash buffer. Finally, 100 μl / well of alkaline phosphatase buffer (1M diethanolamine, 0.5 mM MgCl ₂ , 1 mM 4-methylumbelliferyl phosphate, pH 9.8) was added to these plates and allowed to stand for 30 minutes, and the fluorescence intensity was measured. (Excitation light 360 nm / fluorescence 465 nm). The above experiment was performed on all the culture plates subjected to transfection, and the performance of each mutant was evaluated.

Based on the measurement results obtained, the performance of each mutant was evaluated as follows. First, the fluorescence intensity (B0) emitted from each well of plate A was defined as 100%. And the fluorescence intensity (test B) emitted from each well of the plate B corresponding to this was converted into the relative value with respect to the plate A [(test B / B0) × 100]. At this time, the smaller this value, the higher the antigen detection sensitivity of the antibody. The same calculation was performed for the plate C, and the relative fluorescence intensity [(Cross B / B0) × 100] with respect to the plate A was obtained. At this time, the larger the value, the higher the specificity of the antibody. After performing the above calculation, the fluorescence intensity emitted from each well of plate A is the size of the circle, the antigen detection sensitivity [(test B / B0) × 100] is the horizontal axis, and the specificity [(Cross B / B0) The measurement results for each well were plotted in FIG. Based on this figure, the performance of each mutant was evaluated and screened. As a result of the experiment, it was confirmed that the performance of the antibody was remarkably changed by introducing the mutation (FIG. 4). In FIG. 4, black circles indicate the original monoclonal antibody (wild type H chain × wild type L chain) into which no mutation has been introduced.
(5) Crossing of the H chain gene into which the mutation has been introduced and the L chain gene into which the mutation has been introduced As shown in FIG. 4, as a result of the introduction of the mutation, the antigen detection sensitivity and specificity of the antibody have changed significantly. However, none of the mutants whose two performances improved simultaneously were found. Therefore, it was decided to express a mutant having improved sensitivity performance and a mutant having improved specificity performance. Then, it was investigated whether this cross-linking could improve the antigen detection sensitivity and specificity of the antibody at the same time. Therefore, this time, it was decided to express the H chain mutant whose sensitivity was improved by screening by ELISA and the L chain mutant whose specificity was improved.

First, COS-1 cells were suspended in D-MEM medium (Life Technologies) containing 10% bovine serum and seeded 1.6 ml / well on a 6-well plate (Falcon). This plate was placed under conditions of 37 ° C. and 5% CO ₂ , and the culture was continued until cells grew on the entire surface of the plate. Once the cells grew across the plate, the medium used was removed. Then, Opti-MEM medium (manufactured by Life Technologies) was newly added to each 1.6 ml / well. The plate was then allowed to stand for 3 hours at 37 ° C. and 5% CO ₂ .

  The following work was performed 30 minutes before the completion of the three-hour standing. First, 16 μl of lipofectamine 2000 (manufactured by Life Technologies) was added to 1.6 ml of Opti-MEM medium and mixed well. Then, 267 ng of the mutant H chain gene expression vector (clone with improved sensitivity in FIG. 4) and 532 ng of mutant L chain gene expression vector (clone with improved specificity in FIG. 4) were added and mixed well. In addition, since it uses as a control here, in addition to this, the above-mentioned mutant H chain gene x wild type L chain gene, wild type H chain gene x mutant type L chain gene, wild type H chain gene x wild type L chain gene Each solution was also prepared by mixing expression vectors with the above combinations. These solutions were allowed to stand at 37 ° C. for 30 minutes.

Three hours after the medium was changed to the Opti-MEM medium, the solution prepared previously was added to each plate at 800 μl / well in 2 wells on which COS-1 cells were cultured. Thereafter, this plate was placed under conditions of 37 ° C. and 5% CO ₂ and cultured for 1 week to express the antibody.
(6) Performance Evaluation of Antibody Crossed with Mutated Antibody Genes The antibody performance was evaluated by the following method. First, a method for evaluating the antigen detection sensitivity of an antibody will be described. First, CJ was mixed with anti-rabbit IgG antibody-immobilized particles. Next, a calibrator solution (Cal 2-6) having a known testosterone concentration was prepared, and a predetermined amount was added thereto. Further, the culture supernatant in which the antibody was expressed was added here and incubated for 10 minutes. After B / F separation, ALP substrate was added, and a signal emitted from CJ bound to the solid phase was detected. The signal (B0) when the calibrator solution (Cal1) containing no testosterone is added is defined as 100%, and the signal (B) of each calibrator (Cal2-6) is a relative value [(B / B0). × 100]. A calibration curve was prepared by plotting the measurement results when using each calibrator solution, taking the testosterone concentration on the horizontal axis (logarithm) and B / B0 (%) on the vertical axis. And, using the calibrator concentration (C ₅₀ ) when (B / B0) × 100 = 50% as an index, the antigen detection sensitivity of each antibody was quantitatively evaluated (the smaller this value, the higher the antigen detection sensitivity). Meaning). This series of operations was performed fully automatically at 37 ° C. using an AIA-600II manufactured by Tosoh Corporation. When the antigen detection sensitivity of each antibody was evaluated by the above method, the antibody obtained by multiplying the mutagenized H chain and the mutagenized L chain was almost equivalent to the antibody obtained by multiplying the mutagenized H chain and the wild type L chain. It was found to have a level of antigen detection sensitivity.

次に、抗体の特異性評価方法について述べる。まずは先程と同様に各抗体の検量線を作成した。その後、Ｃａｌ１に交叉物質（デヒドロエピアンドロステロン硫酸塩、デヒドロエピアンドロステロン−グルクロナイト、または、アンドロステロン硫酸塩）を加えた溶液を作製した。そしてこの溶液をＣＪと一緒に抗ウサギＩｇＧ抗体固定化粒子へ加えた。そして更に抗体を発現させた培養上清をここに加え、１０分間インキュベーションした。その後Ｂ／Ｆ分離を行なった後にＡＬＰの基質を加え、固相に結合しているＣＪから発せられるシグナルを検出した。そしてテストステロンが全く含まれないキャリブレーター溶液（Ｃａｌ１）を加えた時のシグナル（Ｂ０）を１００％として、交叉物質を含むキャリブレーターを加えた時のシグナル（Ｃｒｏｓｓ Ｂ）をそれぞれ相対的な値［（Ｃｒｏｓｓ Ｂ／Ｂ０）×１００］として算出した。この値を先程作成した検量線上にプロットし、テストステロンとして測定される濃度をそれぞれ求めた。そして、交叉物質に対する各抗体の交叉反応率をそれぞれ計算した。この時、野生型抗体が示す各交叉物質との交叉反応率を１００％と設定した。そして各変異体の交叉反応率を野生型抗体に対する相対的な値に換算して特異性の評価を行った（この値が低いほど特異性が高いことを意味している）。尚この一連の操作は、東ソー社製ＡＩＡ―６００ＩＩを用いて３７℃にて全自動で行なわれた。以上の方法で各抗体の特異性を評価したところ、変異型Ｈ鎖と変異型Ｌ鎖とを掛け合わせた抗体は、野生型Ｈ鎖と変異型Ｌ鎖とを掛け合わせた抗体とほぼ同等レベルの特異性を有することが分かった（図５）。

Next, an antibody specificity evaluation method will be described. First, a calibration curve for each antibody was prepared as before. Thereafter, a solution in which a crossover substance (dehydroepiandrosterone sulfate, dehydroepiandrosterone-glucuronite, or androsterone sulfate) was added to Cal1 was prepared. This solution was added to the anti-rabbit IgG antibody-immobilized particles together with CJ. Further, the culture supernatant in which the antibody was further expressed was added here and incubated for 10 minutes. After B / F separation, ALP substrate was added, and a signal emitted from CJ bound to the solid phase was detected. The signal (B0) when the calibrator solution (Cal1) containing no testosterone is added is defined as 100%, and the signal (Cross B) when the calibrator containing the crossover substance is added is a relative value [(Cross B). B / B0) × 100]. This value was plotted on the calibration curve prepared earlier, and the concentration measured as testosterone was determined. And the cross-reaction rate of each antibody with respect to the crossing substance was calculated, respectively. At this time, the cross-reaction rate with each cross-linking substance indicated by the wild-type antibody was set to 100%. Then, the cross-reaction rate of each mutant was converted into a relative value with respect to the wild type antibody, and the specificity was evaluated (the lower the value, the higher the specificity). This series of operations was performed fully automatically at 37 ° C. using an AIA-600II manufactured by Tosoh Corporation. When the specificity of each antibody was evaluated by the above method, the antibody obtained by multiplying the mutant H chain and the mutant L chain was almost the same level as the antibody obtained by multiplying the wild H chain and the mutant L chain. (Fig. 5).

  From the above results, it was revealed that the antibody performance can be improved (antigen detection sensitivity and specificity can be improved at the same time) by combining the H chain gene into which the mutation has been introduced and the L chain gene into which the mutation has been introduced. Therefore, the performance of the antibody can be easily improved by using the present invention.

Claims (5)

A method for producing a modified monoclonal antibody, comprising the following steps i) to iii):
i) Mutation is introduced into the heavy chain gene of the original monoclonal antibody (0), and this is expressed by crossing with the light chain gene of the original monoclonal antibody (0) to obtain a mutant (H1) having the desired performance. Select by screening.
ii) introducing a mutation into the L chain gene of the original monoclonal antibody (0), expressing it by multiplying it with the H chain gene of the original monoclonal antibody (0), and obtaining a mutant (L1) having the desired performance Select by screening.
iii) Multiplication of the H chain gene of the mutant (H1) selected in i) above with the L chain gene of the mutant (L1) selected in ii) above to express a monoclonal antibody (HL) Let The production method according to claim 1, wherein mutation is introduced by error-prone PCR. The production method according to claim 1 or 2, wherein the original monoclonal antibody (0) is derived from a rabbit. The production method according to any one of claims 1 to 3, wherein the original monoclonal antibody (0) has reactivity with testosterone. A modified monoclonal antibody obtained by the production method according to claim 1.

Images