JP2012029685A - In vitro immunization and method of producing antibody using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an in vitro immunization that makes it possible to mass-produce an antigen-specific human antibody in a short time, and a method of producing an antibody using the same.SOLUTION: The in vitro immunization includes: a step for immunization in a culture solution containing a group of human blood cells, an antigen, a stimulant, and a human serum; a step for separating genes from a group of cells obtained in the step for immunization, and performing a PCR treatment using a predetermined antibody primer for the genes separated; a step for performing quantitative separation of the genes after the PCR treatment to select an antibody gene; and a step for culturing a single-chain antibody in a host cell.

Description

  The present invention relates to an in vitro immunization method and an antibody production method using the same method.

Elucidation of the immune function, which is the center of the body defense functions, is becoming an important issue in the diagnosis and treatment of diseases.
For example, among methods used for diagnosis of various diseases other than lifestyle-related diseases such as cancer and diabetes, it is simpler like the immunochromatography method that visually detects the concentration and amount of marker substances deeply related to the disease. The research field, diagnostic reagent, reagent for monitoring various substances, immunological diagnosis, and therapeutic fields will be further expanded. The stable securing of will become more important in the future.
In addition, antibody library production technology using a phage display system has been developed, and human antibodies can be isolated.

Preparation of a monoclonal antibody necessary for immunological measurement requires about 3 months in the step of first injecting (immunizing) an antigen into a mouse. Thereafter, the B cell group producing the antibody is taken out and fused with myeloma cells. By this step, a group of antibody-producing cells (hybridoma) that can proliferate indefinitely is constructed. Finally, cells producing the desired antibody are selected (cloning step) from this hybridoma group, and mass production of antibodies is performed using these cells. In this cloning step, the hybridoma group is diluted and cultured from 1 cell with only 1 cell per well. This is grown to a cell concentration where the properties of the antibody can be examined, and the properties of the resulting monoclonal antibody are examined. The wells that become positive in this test are diluted again, and the test is performed as described above. This operation is performed a plurality of times to separate and obtain hybridomas that can withstand use. This process takes about 2 weeks, and may take more than 3 months in total.
Since labor and time-consuming work is necessary in this way, the production cost becomes high when a specialized contractor is requested.
International Publication No. WO2009 / 072660 describes a technique of taking out a cell piece from mouse spleen cell tissue and immunizing it. Although spleen cells are rich in immunocompetent cells and are often used tissues, they need to be adjusted to some extent for excision work from mice and subsequent immunization.

For application to human medicine, it is complicated to eliminate all antigenicity derived from mice.
On the other hand, a screening method using a phage display method or the like from an enormous size human antibody gene naive library requires several weeks and needs to be repeated many times, which is complicated and requires time and labor.
Japanese Patent Application Laid-Open No. 2004-121237 describes that human peripheral blood lymphocytes are exempted from the body. However, a step of separating monocytes is necessary, and an operation of separating and selecting lymphocytes from white blood cells or the like is required. Therefore, a laborious work is required.
JP-A-4-281799 discloses a method for in vitro immunization of peripheral blood lymphocytes that does not contain a polyclonal activator such as LPS. In in vitro immunity, a polyclonal activator that is essential for peripheral blood lymphocytes is disclosed. In vitro immunization is described to reduce immune efficiency.

JP 2004-121237 A Japanese Patent Laid-Open No. 10-282097 JP-T-2006-516408 JP 2006-180708 A International Publication WO2009 / 072660 JP-A-4-281799 JP 2011-92142 A

It is intended to propose a means for simplifying in vitro immunization work, and for producing a stable human antibody in a shorter time and more easily to produce an antibody that requires labor and time.
As described in the above-mentioned prior art, the use of peripheral blood is more convenient in the production of human antibodies, but either method can be used to reliably produce antibodies with specificity and affinity. There are many unexplained parts that have not been reached.

The present invention has found that a human antibody having specificity and affinity can be obtained by in vitro immunization with a stimulating substance containing LPS, an antigen and human serum on blood cells collected from human peripheral blood. And the present invention has been reached.
Furthermore, the present invention provides a culture solution obtained by adding a specific stimulating substance and human-related culture components to human blood cells, sensitizing with an antigen, and without selecting the immunized immunocompetent cells from this blood cell group. , A PCR amplification operation using a primer specific to the human antibody gene is performed, and after the PCR treatment, only the amplified gene is taken out by quantitative separation means such as electrophoresis, and a single chain antibody ( scFv) and an expression vector having the scFv antibody gene inserted therein is inserted into a host cell for transformation, and this is cultured to allow mass production of antibody genes in a short time.

Examples of blood cells include mononuclear cells, lymphocytes, macrophages, dendritic cells, erythrocytes, etc., but according to the present invention, blood cells are separated from an individual such as after centrifugation. It may be a starting material and may not require sorting of red blood cells and the like, and the sorting process can be simplified.
The quantitative separation method in the present invention refers to a separation method based on a difference in migration distance by electrophoresis, which performs separation based on a quantitative difference such as the molecular weight of the target gene. In addition to electrophoresis, other methods using chromatography such as HPLC can be used.

In order to obtain desired antibody-producing cells in vitro, a stimulating substance is added to human blood cells to stimulate and activate specific blood cells, and germinal center-like B cells or antibody-producing cells (plasma) Cell) and memory B cells.
As stimulating substances in the present invention, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-21, surface antigens Fas ligand, INF-γ (Interferon-gamma), TGF-β (Transforming Growth Factor-beta), BAFF (B cell Activating Factor), APRIL (A Proliferation-Inducing Ligand), CD40 ligand, CD38 ligand, BCDF (B Cell Differentiation Factor), BCAF (B Cell Activation Factor), LPS (Lipopolysaccharide) CpG and the like are exemplified as stimulating substances for receptors involved in signal transmission.

These stimulating substances are divided into somatic cell mutagenesis factor, B cell proliferation activator, and class switch inducer, and the somatic mutagenesis factor is IL-21, LPS (Lipopolysaccharide), B cell, respectively. Growth activator is IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-11, IL-13, IL-21, IL-22, BAFF (B cell Activating Factor), CD30 Ligand, anti-CD30 antibody, CD40 Ligand, anti-CD40 antibody, INF-γ (Interferon-gamma), Fas Ligand, LPS, the class switch inducer is IL-4, IL-5 , IL-6, IL-13, INF-γ, TGF-β (Transforming Growth Factor-beta), BAFF, APRIL (A Proliferation-Inducing Ligand), and these stimulating substances are effective for the present invention Used for.
The concentration used depends on the concentration of cells to be immunized, but LPS is preferably 10 ng / mL to 500 μg / mL, and a concentration of 20 to 80 μg / mL is particularly recommended. For substances other than LPS, a concentration of 1 ng / mL to 50 μg / mL is generally good, and a concentration of 5 ng / mL to 40 ng / mL is particularly recommended.
Moreover, there is no problem even if the agonist antibody substitutes for the function of these substances.
In the process of inducing differentiation, it may be more preferable to induce somatic mutation and class switching of the antibody, particularly by stimulation with IL-4, IL-13, IL-21 or LPS.

The relationship between various stimulating substances and various differentiations, inductions, etc. in the in vitro immunization technique in the present invention will be described below.
The antigen shown in the present invention is sufficient once just after the preparation of blood cells for immunization, but there is no limitation even if it is performed several times at intervals of 2 to 3 days. Further, the antigen concentration at that time is preferably 1 pmol to 1 mmol, and 1 nmol is exemplified.
For the purpose of assisting antigen stimulation, a substance that stimulates TLR (Toll Like Receptor) represented by LPS may be added. This stimulation is preferably added at each antigen stimulation, and the amount added is preferably 10 ng / mL to 500 μg / mL, particularly 20 to 80 μg / mL.

Antigens used in the present invention include, for example, h (human) S100A10, he (chicken) EL, h (human) Ras, h (human) rap74, h (human) TOPO2B, b (bovine) SA, b (bovine ) Casein, proteins such as m (mouse) derived from mouse S100A10, mSA, mMapk1, and peptides are exemplified, and the present invention further includes low molecular weight compounds (fluorescent substances such as rhodamine, FITC) can be used.
The present invention makes it possible to use low molecular weight antigens by the step of inducing differentiation into antibody-producing cells by introducing stimulating substances according to the purpose outside the body, and obtains preferred antibodies by class switching and antibodies by affinity maturation. In some cases can be obtained.

The gene obtained from blood cells can be quantitatively separated by introducing a primer for the desired antibody gene into the desired antibody and amplifying it by PCR (polymerase chain reaction) method. By combining the step of obtaining an antibody by preferably colonizing with a host cell such as a cell, many human antibodies having affinity can be produced in a short time.
Examples of host cells include E. coli, Streptomyces, Bacillus subtilis, yeast, CHO cells, COS cells, HEK293 cells, HeLa cells, and the like.
In the present invention, cell lines obtained by transforming human fetal kidney cells such as HepG-2 cells, RPMI8226, HS-sultan, HeLa cells, HEK293 cells with the E1 gene of adenovirus, HEK293T cells, HEK293S cells, HEK293EBNA cells In addition, other human-related cells may be used as host cells, which may be a preferable form of antibody medicine, such as promoting the expression of antibodies with higher affinity.

Any expression vector can be used as long as it can be expressed in a host cell. Examples thereof include vectors derived from bacterial plasmids, yeast plasmids, chromosomes, episomes, viruses, and retroviruses. Here, the vector is well known to those skilled in the art, and is not particularly limited as long as it has an appropriate promoter for expressing the polypeptide from the DNA incorporated in the animal cell to be administered. And pcDNA3 expression vector and pMX expression vector.
Examples of the antibody selection method in the present invention include DNA such as Phage, E. coli / yeast / animal cell surface display, 2-hybrid method using yeast, E. coli, etc., IVV method, ribosome display method. For example, a method of presenting to mRNA, BIACORE method using surface plasmon resonance, ELISA method, Western blot method, antibody or plateau immobilized on magnetic beads and selection using magnetic force (MACS) method, etc. The technique is not particularly limited as long as it is a technique capable of examining the interaction between the two.

As described above, the present invention enables an in vitro immunization method capable of producing a human antibody with high specificity and affinity and an antibody production method incorporating this in vitro immunization method.
The concentration of serum used for in vitro immunization in the present invention is, for example, in the range of 5% to 60% (preferably 20% to 50%).

The stimulating substance is as described above, but IL-4 (5 ng / mL to 40 ng / mL), IL-6 (5 ng / mL) can be used to obtain antibodies with further specificity and affinity. -40 ng / mL), IL-10 (5 ng / mL-40 ng / mL), IL-13 (5 ng / mL-40 ng / mL), IL-21 (5 ng / mL-40 ng / mL), BAFF (5 ng / mL) -40 ng / mL), anti-CD40 antibody (50 ng / mL to 5 [mu] g / mL), LPS (20 to 80 [mu] g / mL), and a combination of all of these stimulating substances is more preferable.
Unlike other stimulating substances such as interleukins, LPS generally used in in vitro immunity has multiple actions, so the use of LPS can reduce the types of stimulating substances, and the production cost However, the use of LPS for in vitro immunization of blood cells may reduce the immunity efficiency depending on the components of other culture solutions, but in combination with the process of obtaining human antibodies as a whole, If a reasonable scFv type human antibody is obtained, it may be included in the present invention.
In the present invention, the LPS is combined with the culture fluid components in the in vitro immunization in combination with human serum, antigen and stimulating substance at a predetermined concentration, and as a result, it is possible to produce a human antibody with high specificity and affinity.

The present invention can be used without separation of blood cells of peripheral blood, and enables production and acquisition of human antibodies suitable for medicines quickly by producing antibodies by genetic engineering techniques.
Furthermore, the present invention can be an in-vitro immunization method in which a human antibody having high specificity and affinity can be obtained, and can be a method that focuses on antibody drugs in addition to a method for producing a human antibody.

It is a drawing substitute photograph for explaining one Example of this invention. It is a drawing substitute photograph for explaining one Example of this invention. It is a drawing substitute photograph for explaining one Example of this invention. It is a drawing substitute photograph for explaining one Example of this invention. It is a drawing substitute photograph for explaining one Example of this invention. It is a figure for demonstrating one Example of this invention. It is a figure for demonstrating one Example of this invention.

The present invention performs in vitro immunization on blood cells obtained from human peripheral blood or the like without separating immunocompetent cells, and then performs PCR treatment with a predetermined antibody to make it quantitatively separable. Obtain an antibody gene.
In addition, human antibodies with higher specificity and affinity are obtained by performing in vitro immunization with a stimulating substance containing LPS, an antigen and human serum on blood cells collected from human peripheral blood.
Obtaining H chain and L chain genes from this antibody gene by PCR, modifying them into single chain antibodies scFv so that they can be expressed in host cells, and then expressing them in host cells to obtain antibodies If so, the antigen is not specified.

Hereinafter, examples of the present invention will be described in detail with respect to a method for producing a single chain antibody using chicken egg white lysozyme protein as an antigen example.

Preparation of peripheral blood mononuclear cells (PBMC)
PBMC (VERITAS) was purchased and obtained as a frozen sample. This was dissolved and recovered according to the manufacturer's recommended protocol. Prior to immunization, the cells were washed with serum-free medium and resuspended in serum-free medium to 1 × 10 ⁷ cells / mL.

In vitro immunity
Hen egg Lysozyme (SIGMA) was prepared to 1 nmol / μL with PBS (−). The solution was sterilized by filtration using a 0.22 μm filter and used for in vitro immunization as an antigen. 5 μL of 1 nmol of HEL and 10 mg / mL of N-acetylmuramyl-L-alanyl-D-isoglutamine as an adjuvant were added to a 15 mL centrifuge tube. To this, 1.5 × 10 ⁷ cells of PBMC were added and sensitized for 15 minutes at room temperature. Add stimulant (IL-4 (final concentration 10ng / mL), IL-13 (final concentration 10ng / mL), anti-CD40 antibody (final concentration 1μg / mL), LPS (final concentration 40μg / mL)) Then, 3.5 mL of RPMI1640 medium containing 40% FBS was added and cultured at 5% CO ₂ · 37 ° C. for 2 days. After the culture, IL-21 (final concentration 10 ng / mL) was added, and further cultured for 3 days. The cultured cells were observed with a microscope, and the generation of cell clusters thought to be generated by immunization and the increase in cell size accompanying cell blastogenesis were observed. (Figure 1)
In FIG. 1, microscopic images of cells before and after immunization. The generation of cell clusters thought to be generated by immunization and the increase in cell size accompanying cell blastogenesis were observed.

DNA extraction and preparation of single chain antibody scFv plasmid Total RNA was obtained from 2.5 × 10 ⁶ cells after immunization using Isogen (manufactured by Nippon Gene). Single-stranded cDNA was synthesized using the obtained total RNA as a template and a Quantitect Reverse Transcription Kit (manufactured by QIAGEN) reagent. 1st PCR was performed by PrimeSTAR HS Polymerase (TaKaRa) using the prepared cDNA as a template and using primers VH forward primer mix / VH reverse primer mix or VL forward primer mix / VL reverse primer mix. The primer sequences used were those introduced in Antibody Engineering Methods and protocols (pp. 120-121). Then, it confirmed by 2% Agarose Gel electrophoresis. (Figure 2)
2% Agarose Gel electrophoresis image after 1st PCR. Amplification of VH gene (about 400 bp) and VL gene (about 350 bp) was confirmed.

A target band of about 400 bp was cut out from the agarose gel after electrophoresis, and the VH gene and VL gene were extracted and purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). Using 200 ng each of the VH gene and VL gene, PrimeSTAR HS Polymerase (manufactured by TaKaRa) was used to perform Link PCR without adding a primer to construct an scFv gene. Then, it confirmed by 1.8% Agarose Gel electrophoresis. (Figure 3)
1.6% Agarose Gel electrophoresis image after Link PCR. The construction of the scFv gene (about 800 bp) was confirmed.

A target band of about 800 bp was cut out from the agarose gel after electrophoresis, and the scFv gene was extracted and purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). 2nd PCR was performed by PrimeSTAR HS Polymerase (TaKaRa) using 2 nd PCR Primer Premix with 10 ng of the scFv gene as a template. The primer sequences used were those introduced in Antibody Engineering Methods and protocols (pp. 121-122). Then, it confirmed by 1.6% Agarose Gel electrophoresis. (Figure 4)
1.6% Agarose Gel electrophoresis image after 2nd PCR. The construction of the scFv gene (about 800 bp) was confirmed.

The scFv PCR product is digested with the restriction enzymes EcoRI and HindIII at 37 ° C for 2 hours to digest the target band from the agarose gel after electrophoresis, and the scFv gene is extracted using the QIAquick Gel Extraction kit (QIAGEN). Extracted and purified. Similarly, the plasmid vector pMAL-c2E is digested with restriction enzymes EcoRI, HindIII and CIAP at 37 ° C for 1 hour, and the target band is cut out from the agarose gel after electrophoresis, and the QIAquick Gel Extraction kit (QIAGEN) is prepared. The plasmid vector gene was extracted and purified. These were mixed at a ratio of 10 mol of scFv gene to 1 mol of plasmid vector gene, and ligated using DNA Ligation kit <Mighty Mix> (TaKaRa). 10 μL of the ligation solution was mixed with 100 μL of competent cells of E. coli JM109 strain to transform E. coli JM109 strain. This transformant was applied to an LB agar medium containing 100 μg / mL ampicillin and cultured at 37 ° C. for 18 hours.
In order to select the formed colonies of E. coli clones having a plasmid to which the scFv gene was linked, colony PCR was performed and confirmed by 1.6% Agarose Gel electrophoresis. (Figure 5)
Example of 1.6% Agarose Gel electrophoresis image after colony PCR. An E. coli clone having a plasmid to which the scFv gene (about 800 bp) was linked was confirmed.

Expression of single chain antibody scFv by E. coli After colony formation, the colonies formed were streaked and cultured at 30 ° C with LB liquid medium containing 500 μL of 100 μg / mL ampicillin and Overnight Express Auto induction system (Novagen). Cultured for 18 hours. The medium was changed by centrifugation, suspended in LB liquid medium containing 500 μL of 100 μg / mL ampicillin, Overnight Express Auto induction system (Novagen) and 1 mM IPTG, and cultured at 30 ° C. for 3 hours. After completion of the culture, E. coli cells were collected by centrifugation (2000 rpm, 15 min, 4 ° C.). Here, glass beads were added, and E. coli was disrupted by shaking at 1300 rpm, 20 min, 15 ° C. Uncrushed cells and glass beads were precipitated by centrifugation (2500 rpm, 15 min, 4 ° C.) to obtain a culture supernatant containing scFv.

Preparation of ELISA Plate
HEL antigen was diluted to 20 μg / mL with PBS (−), and 50 μL each was added to a 96 well plate (Nunc-Immuno module F8 maxisoap, manufactured by Nunc). After standing at 4 ° C. for 1 day, the supernatant was discarded, and 400 μL of Protein Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added. After standing at room temperature for 2 hours, the plate was washed 3 times with PBS (−) to prepare a HEL ELISA Plate. Similarly, a plate on which BSA and anti-FLAG antibody were immobilized was also prepared.

ELISA
To the Anti-FLAG antibody ELISA plate, 50 μL of the culture supernatant obtained in the section of expression of the single-chain antibody scFv by E. coli was added and allowed to stand in a 37 ° C. incubator for 1 hour. Discard the supernatant,
After washing 4 times with PBS (−), 50 μL of a secondary antibody (anti-MBP-HRP, manufactured by NEB) diluted 2000 times with PBS (−) was added and allowed to stand in a 37 ° C. incubator for 1 hour. The supernatant was discarded, washed 4 times with PBS (−), 100 μL of TMB solution was added, and allowed to stand at room temperature for 10 minutes. Immediately thereafter, 100 μL of 1N HCl (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction. Thereafter, the absorbance at 450 nm of each well was measured with a microplate reader (Model 550, manufactured by Bio-rad).

  According to this example, an antigen-specific human antibody could be obtained in 10 days by in vitro immunization. Conventionally, since a phage display method or the like is used, it takes several weeks, but it was able to be obtained in a short period of time. By using this method, it becomes easy to obtain human antibodies obtained by immunization.

  Next, with respect to another embodiment of the present invention, human S100A10 protein is used as an antigen example and human peripheral blood mononuclear cells are subjected to in vitro immunization using a culture solution containing a specific stimulating substance, antigen, and human serum component Thus, a method for producing a single-chain human antibody having high specificity and affinity, in which somatic mutation is introduced with high efficiency without lowering immune efficiency, will be described in detail.

Preparation of human peripheral blood mononuclear cells (PBMC) Human PBMC (VERITAS) was purchased and obtained as a frozen sample. This was dissolved and recovered according to the manufacturer's recommended protocol. Prior to immunization, the cells were washed with serum-free medium and resuspended in serum-free medium to 1 × 10 ⁷ cells / mL.

In vitro immunized human S100A10 (hS100A10) protein was prepared to 1 nmol / μL with PBS (−). The solution was sterilized by filtration using a 0.22 μm filter and used for in vitro immunization as an antigen. To a 15 mL centrifuge tube, 5 μL of 5 nmol of hS100A10 and N-acetylmuramyl-L-alanyl-D-isoglutamine 10 mg / mL as an adjuvant were added. To this, 1.5 × 10 ⁷ cells of PBMC were added and sensitized for 15 minutes at room temperature. Here, stimulants (IL-4 (final concentration 10 ng / mL), IL-6 (final concentration 10 ng / mL), IL-10 (final concentration 10 ng / mL), BAFF (final concentration 10 ng / mL), IL-13 (Final concentration 10 ng / mL), anti-CD40 antibody (final concentration 1 μg / mL), LPS (40 μg / mL)), and then 4 mL of RPMI1640 medium containing 40% human serum and 5% CO _2. The cells were cultured at 37 ° C for 3 days. After the culture, IL-21 (final concentration 10 ng / mL) was added, and further cultured for 4 days.

DNA extraction, preparation of single chain antibody scFv plasmid Using Isogen (manufactured by Nippon Gene) from 6.8 × 10 ⁶ cells after immunization and 1.2 × 10 ⁶ cells cultured for the same day without immunization. Total RNA was obtained. Single-stranded cDNA was synthesized using the obtained total RNA as a template and a Quantitect Reverse Transcription Kit (manufactured by QIAGEN) reagent. 1st PCR was performed by PrimeSTAR HS Polymerase (TaKaRa) using the prepared cDNA as a template and using primers VH forward primer mix / VH reverse primer mix or VL forward primer mix / VL reverse primer mix. The primer sequences used were those introduced in Antibody Engineering Methods and protocols (pp. 120-121).

A target band of about 400 bp was cut out from the agarose gel after electrophoresis, and the VH gene and VL gene were extracted and purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). Using 500 ng of each of the VH and VL genes, Link PCR was performed with PrimeSTAR HS Polymerase (TaKaRa) without adding a primer to construct an scFv gene.
A target band of about 800 bp was cut out from the agarose gel after electrophoresis, and the scFv gene was extracted and purified using QIAquick Gel Extraction kit (manufactured by QIAGEN).

2nd PCR was performed by PrimeSTAR HS Polymerase (TaKaRa) using 2 nd PCR Primer Premix with 10 ng of the scFv gene as a template. The primer sequences used were those introduced in Antibody Engineering Methods and protocols (pp. 121-122).
The scFv PCR product is digested with the restriction enzymes EcoRI and HindIII at 37 ° C for 2 hours to digest the target band from the agarose gel after electrophoresis, and the scFv gene is extracted using the QIAquick Gel Extraction kit (QIAGEN). Extracted and purified. Similarly, the plasmid vector pMAL-c2E is digested with restriction enzymes EcoRI, HindIII and CIAP at 37 ° C for 1 hour, and the target band is cut out from the agarose gel after electrophoresis, and the QIAquick Gel Extraction kit (QIAGEN) is prepared. The plasmid vector gene was extracted and purified.

These were mixed at a ratio of 10 mol of scFv gene to 1 mol of plasmid vector gene, and ligated using DNA Ligation kit <Mighty Mix> (TaKaRa). 10 μL of the ligation solution was mixed with 100 μL of competent cells of E. coli JM109 strain to transform E. coli JM109 strain. This transformant was applied to an LB agar medium containing 100 μg / mL ampicillin and cultured at 37 ° C. for 18 hours.
Colony PCR was performed to select E. coli clones having a plasmid to which the scFv gene was linked from the formed colonies.

After colonizing colonies with single-chain antibody scFv expression by Escherichia coli, 64 clones from the non-immunized PBMC-derived scFv library and 40 clones from the PBMC-derived library after immunization were streaked, 500 μL of 100 μg / mL ampicillin and The cells were cultured at 30 ° C. for 18 hours in an LB liquid medium containing Overnight Express Auto induction system (Novagen). The medium was changed by centrifugation, suspended in LB liquid medium containing 500 μL of 100 μg / mL ampicillin, Overnight Express Auto induction system (Novagen) and 1 mM IPTG, and cultured at 30 ° C. for 3 hours. After completion of the culture, E. coli cells were collected by centrifugation (2000 rpm, 15 min, 4 ° C.). Here, glass beads were added, and E. coli was disrupted by shaking at 1300 rpm, 20 min, 15 ° C. Uncrushed cells and glass beads were precipitated by centrifugation (2500 rpm, 15 min, 4 ° C.) to obtain a culture supernatant containing scFv.

Preparation of ELISA Plate
The hS100A10 antigen was diluted to 20 μg / mL with PBS (−), and 50 μL was added to a 96-well plate (Nunc-Immuno module F8 maxisoap, manufactured by Nunc). After allowing this to stand at 4 ° C. overnight, the supernatant was discarded, and 400 μL of Protein Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added. After standing at room temperature for 2 hours, the plate was washed 3 times with PBS (−) to prepare an hS100A10 ELISA plate. Similarly, a plate on which Hen Egg Lysozyme (HEL) was immobilized as a negative control and anti-FLAG antibody was immobilized for concentration determination was also prepared.

Screening by ELISA
To the Anti-FLAG antibody ELISA plate, 50 μL of the culture supernatant obtained in the section of expression of the single-chain antibody scFv by E. coli was added and allowed to stand in a 37 ° C. incubator for 1 hour. Discard the supernatant, wash 4 times with PBS (−), add 50 μL of secondary antibody (anti-MBP-HRP, manufactured by NEB) diluted 2000 times with PBS (−), and add 1 in a 37 ° C. incubator. Let stand for hours. The supernatant was discarded, washed 4 times with PBS (−), 100 μL of TMB solution was added, and allowed to stand at room temperature for 10 minutes. Immediately thereafter, 100 μL of 1N HCl (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction. Thereafter, the absorbance at 450 nm of each well was measured with a microplate reader (Model 550, manufactured by Bio-rad).
Similarly, a calibration curve was prepared using purified and quantified MBP-FLAG-hS100A10 protein, and the scFv expression level of each clone was calculated based on this.

Based on the calculated concentration value, adjust the concentration to 2 pmol / well for each clone, and perform ELISA using the hS100A10 solidified plate and HEL solidified plate to obtain the antigen to hS100A10. The affinity and specificity of each clone was evaluated.
As a result, a large number of clones having affinity and specificity for the antigen were obtained by performing in vitro immunization using this method. In addition, the proportion of antigen-positive clones in the library was significantly increased. (Figure 6)
FIG. 6 shows a comparative example of activity distribution of scFv library in (±) in vitro immunization performed by ELISA, and an hS100A10 solidified plate serving as an index of affinity for antigen was used on the Y axis. The absorbance at 450 nm was shown, and the X-axis shows the absorbance ratio of hS100A10 solidified plate to HEL solidified plate, which is an index of specificity.
According to FIG. 6, by carrying out the in vitro immunization shown in this example, clones having high affinity clones (high Absorbance) and high specificity clones (high hS100A10 / HEL ratio) were obtained. We were able to acquire with a high abundance ratio.

DNA and amino acid sequence analysis About the clones (anti-immune library clone No. n1-n4, post-immunization library clone No. i1-i4) that were reactive to the antigen obtained as a result of screening by the above ELISA, DNA sequencing analysis was performed.
As a result, it was confirmed that somatic mutation was efficiently induced by in vitro immunization using this method. (Fig. 7)
FIG. 7 shows the results of IgBLAST analysis of the DNA sequence analysis results of the clones numbered in FIG. 6 (n1 to n4 and i1 to i4), and each region (FWR1, CDR1, FWR2, CDR2, FWR3) The Germline V gene (database) from which the scFv clone was derived, the number of mismatched bases and the number of amino acid mutations (shown in parentheses in the figure) were shown. According to FIG. 7, somatic cell mutations are not accumulated when cultured for the same number of days without immunization. By performing in vitro immunization using this method, the body can be efficiently produced at both the DNA level and the amino acid level. It was shown that cell mutation was induced.

  The present invention expands the field of antibody medicine because human antibodies with high specificity and affinity that can be used for the treatment of diseases based on antigen-antibody reactions can be produced in large quantities from blood cells of human peripheral blood in a short period of time. Can be encouraged. In addition, since human antibodies with high specificity and affinity can be obtained in a short period of time, it is possible not only to treat diseases but also to expand the fields of diagnosis and immunological marker testing.

Claims (7)

An in vitro immunization method for immunizing human blood cells in a culture solution containing an antigen, a stimulating substance and human serum.   The in vitro immunization method according to claim 1, wherein the serum concentration is 5% to 60%, preferably 20% to 50%. The in vitro immunization method according to claim 1, wherein the stimulating substance comprises a B cell proliferation activator, a somatic mutation inducer, and a class switch inducer. The somatic mutation-inducing factor is IL-21, LPS (Lipopolysaccharide), and the B cell proliferation activating factor is IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL- 10, IL-11, IL-13, IL-21, IL-22, BAFF (B cell Activating Factor), CD30 Ligand, anti-CD30 antibody, CD40 Ligand, anti-CD40 antibody, INF-γ (Interferon-gamma) , Fas Ligand, LPS, the class switch inducer is IL-4, IL-5, IL-6, IL-13, INF-γ, TGF-β (Transforming Growth Factor-beta), BAFF, APRIL (A Proliferation- The in vitro immunization method according to claim 3, wherein one or more of these factors are selected. Separating a gene from cells containing immune cells obtained by the in vitro immunization method, performing a PCR treatment using a predetermined antibody primer on the separated gene, and quantitatively separating the gene after the PCR treatment An antibody production method comprising a quantitative separation step of selecting an antibody gene. The method for producing an antibody according to claim 5, further comprising the steps of forming a single-chain antibody from the antibody gene and culturing the single-chain antibody in a host cell. The antibody production method according to claim 1 or 5, wherein the blood cells are mononuclear cells obtained from peripheral blood.

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