WO2020129838A1 - Screening method for cells producing specific antigen-specific antibody - Google Patents

Abstract

The present invention addresses the problem of providing a method for efficiently screening culture supernatant including cells that produce a specific antigen-specific antibody from a culture supernatant of cells that produce human antibodies. The present invention provides a method for screening culture supernatant including cells that produce a specific antigen-specific antibody from a culture supernatant of cells that produce human antibodies, the method involving using antibody-dependent cytotoxic activity as an indicator, wherein the method comprises selecting culture supernatant that satisfies prescribed conditions defined in the specification as the culture supernatant including cells that produce a specific antigen-specific antibody.

Description

Method for screening cells producing specific antigen-specific antibody

The present invention relates to a method for screening a culture supernatant containing cells producing a specific antigen-specific antibody from the culture supernatant of cells producing a human antibody using the antibody-dependent cytotoxic activity as an index. The present invention further relates to a method for producing a culture supernatant containing cells producing a specific antigen-specific antibody, which comprises screening as described above. The present invention further relates to a method for producing cells producing a specific antigen-specific antibody, which comprises screening as described above. The present invention further relates to a method for producing a specific antigen-specific antibody, which comprises screening as described above.

Monoclonal antibodies that show high selectivity for specific antigens are expected to be developed as antibody drugs, and in particular, antibody drugs targeting cancer cells are under development. In order to develop an antibody drug, it is necessary to obtain a desired antibody by screening a specific antigen-specific antibody.

Patent Document 1 describes a method for identifying an immunobinder (eg, scFv antibody) capable of specifically binding to a cell surface antigen. The method described in Patent Document 1 describes labeled antigen expression. The step of contacting the cells with the labeled immunobinder-expressing cells and the step of isolating the immunobinder-expressing cells that bind to the antigen-expressing cells using a cell sorter are included.

Further, Patent Document 2 includes a step of contacting a B cell population with a capturing agent, a step of separating captured B cells from uncaptured B cells, and a step of culturing a plurality of captured B cells. The B cells are not sorted into uniform B cells immediately before culturing, and a plurality of cultured cells are screened to identify cells capable of producing an antibody having a desired function. , A step of obtaining a desired antibody from them, and a method of obtaining an antibody having a desired function are described.

On the other hand, in Patent Document 3, a step of contacting a target cell with an antibody selected from the group consisting of a human antibody, a humanized antibody, and a human chimeric antibody that recognizes the target cell; NK expressing a human Fc receptor on the cell surface A step of contacting a cell-derived cell line with the antibody; a step of detecting whether or not cytotoxicity has occurred in the target cell, wherein the antibody is fluorescently labeled, and the step (c) is performed in the target cell Alternatively, an assay method for antibody-dependent cellular cytotoxicity is described, in which at least one of effector cells is fluorescently labeled, and both cells are identified and observed/counted in observation using a fluorescence microscope.

Japanese Patent No. 5764071 Japanese Patent No. 4624357 Japanese Patent No. 5282040

As a cell culture supernatant for antibody screening, a hybridoma culture supernatant is generally used. In producing a hybridoma, an animal is immunized with a large amount of the produced antigen. As described above, since a large amount of antigen is produced in the production of hybridoma, the screening of the culture supernatant of the hybridoma is performed by the ELISA method ((Enzyme-Linked ImmunoSorbent Assay: Enzyme-Linked Immunosorbent Assay). In the screening by ELISA, it is necessary to purify the antigen, and a method for screening an antibody against the antigen which is difficult to purify is desired.

In addition, for immunization of animals, an immunization method using cells expressing an antigen or a plasmid DNA for expressing an antigen of interest is known, and in such an evaluation method (screening method) In some cases, a method using cells expressing an antigen called cell-based ELISA (CELISA) is used (see Patent Document 1). When using this method, it is not necessary to purify the antigen. However, in this method, cells expressing the antigen are seeded on a cell culture plate and then fixed with 1% paraformaldehyde to denature the antigen protein. By denaturing the antigen, the three-dimensional structure of the antigen may change. Moreover, this method is not suitable for high-throughput screening because it involves complicated steps such as cell fixation.

An object of the present invention is to provide a method for efficiently screening a culture supernatant of cells producing a specific antigen-specific antibody from a culture supernatant of cells producing a human antibody. The present invention further provides a method for screening a culture supernatant containing cells that produce a specific antigen-specific antibody without requiring concentration of the culture supernatant and labeling with a fluorescent dye. To do. Another object of the present invention is to provide a method for screening a culture supernatant containing cells producing a specific antigen-specific antibody without requiring purification of the antigen. Another object of the present invention is to provide a method for producing a culture supernatant containing cells that produce a specific antigen-specific antibody, which comprises screening as described above. The present invention further provides a method for producing a cell producing a specific antigen-specific antibody, which comprises screening as described above, and a method for producing a specific antigen-specific antibody, which comprises screening as described above. It is a problem to be solved.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have identified a target cell expressing a specific antigen, an attacking cell, and an antibody-dependent cytotoxic activity in the case of culturing by mixing the culture supernatant, Target cells that do not express antigen, attacking cells, and target cells that express a specific antigen and have higher antibody-dependent cellular cytotoxicity in the case of culturing by mixing the culture supernatants, attacking cells, and the culture supernatants The antibody-dependent cytotoxic activity in the case of mixing and culturing is an indicator that the target cell expressing a specific antigen and the antibody-dependent cytotoxic activity in the case of culturing by mixing the culture supernatant are higher than the index. By doing so, it was found that a culture supernatant containing cells producing a specific antigen-specific antibody can be screened from a culture supernatant of cells producing a human antibody, and the present invention has been completed.

That is, according to the present invention, the following inventions are provided.
(1) A method of screening a culture supernatant of cells producing a specific antigen-specific antibody from the culture supernatant of cells producing a human antibody using the antibody-dependent cytotoxic activity as an index, under the following conditions: A method of selecting a culture supernatant satisfying the following as a culture supernatant containing cells producing a specific antigen-specific antibody:
Target cells that express a specific antigen, attacking cells, and antibody-dependent cytotoxicity in the case of culturing by mixing the culture supernatants are mixed with target cells that do not express a specific antigen, attacking cells, and the culture supernatants. Antibody-dependent cytotoxic activity higher than the antibody-dependent cytotoxic activity in the case of culturing in a specific manner, and the antibody-dependent cytotoxic activity in the case of culturing by mixing the target cells expressing the specific antigen, the attacking cells, and the culture supernatant, It is higher than the antibody-dependent cytotoxic activity when the target cells expressing the antigen and the culture supernatant are mixed and cultured.
(2) The method according to (1), wherein the culture supernatant of human antibody-producing cells is a culture supernatant of human B cells.
(3) The method according to (2), wherein the culture supernatant of human B cells is the culture supernatant of human B cells cultured on feeder cells expressing an antigen.
(4) The method according to (2) or (3), wherein the culture supernatant of human B cells is the culture supernatant of human B cells cultured in the presence of Fas-mediated stimulation.
(5) The culture supernatant of human B cells is the culture supernatant obtained by culturing human B cells in the presence of IL-2 and IL-21. (2) to (4) The method described.
(6) The method according to any one of (1) to (5), wherein the specific antigen is a membrane antigen expressed on the cell membrane surface of target cells.
(7) The method according to any one of (1) to (6), wherein the specific antigen is a transmembrane receptor.
(8) The method according to any one of (1) to (7), wherein the specific antigen is a G protein-coupled receptor.
(9) The method according to any one of (1) to (8), wherein the human antibody is human IgG.
(10) The method according to any one of (1) to (9), wherein the antibody-dependent cytotoxic activity is measured by detecting an enzyme released by dead cells.
(11) A cell producing a specific antigen-specific antibody, which comprises screening a culture supernatant containing a cell producing a specific antigen-specific antibody by the method according to any one of (1) to (10). A method for producing a culture supernatant containing the same.
(12) A method for producing a specific antigen-specific antibody, comprising screening a culture supernatant containing a cell producing a specific antigen-specific antibody by the method according to any one of (1) to (10). Production method.
(13) A method for producing a specific antigen-specific antibody, which comprises screening a culture supernatant containing cells producing the specific antigen-specific antibody by the method according to any one of (1) to (10).

According to the present invention, a culture supernatant containing cells producing a specific antigen-specific antibody can be efficiently screened from a culture supernatant of cells producing a human antibody. Since the screening method of the present invention does not require concentration of the culture supernatant and labeling with a fluorescent dye, high throughput (96 well, 384 well, etc.) detection is possible. Further, the screening method of the present invention does not require purification of the antigen, and is particularly effective in screening an antibody against a membrane antigen having a complicated structure which is difficult to purify. According to the present invention, a culture supernatant containing cells producing a specific antigen-specific antibody, cells producing a specific antigen-specific antibody, and a specific antigen-specific antibody can be produced.

FIG. 1 shows the outline of the screening method using ADCC activity. FIG. 2 shows the results of ADCC activity measurement using the Trastuzumab type antibody. FIG. 3 shows the results of ADCC activity measurement using the culture supernatant of anti-CXCR2 antibody-expressing cells. FIG. 4 shows the results of examination of the antigen-binding ability of the antibody prepared from HEK293T cells. FIG. 5 shows the results of ADCC activity measurement using the culture supernatant of anti-CXCR2 antibody-expressing cells. FIG. 6 shows the results of ADCC activity measurement using the culture supernatant of anti-CXCR2 antibody-expressing cells. FIG. 7 shows the results of examination of the antigen binding ability of the antibody prepared with HEK293T cells.

Hereinafter, the present invention will be described in more detail.
The present invention is a method for screening a culture supernatant containing cells that produce a specific antigen-specific antibody from the culture supernatant of cells that produce a human antibody, using the antibody-dependent cytotoxic activity as an index, which comprises: The present invention relates to a method for selecting a culture supernatant satisfying the conditions as a culture supernatant containing cells producing a specific antigen-specific antibody.
Target cells that express a specific antigen, attacking cells, and antibody-dependent cytotoxicity in the case of culturing by mixing the culture supernatants are mixed with target cells that do not express a specific antigen, attacking cells, and the culture supernatants. Antibody-dependent cytotoxic activity higher than the antibody-dependent cytotoxic activity in the case of culturing in a specific manner, and the antibody-dependent cytotoxic activity in the case of culturing by mixing the target cells expressing the specific antigen, the attacking cells, and the culture supernatant, It is higher than the antibody-dependent cytotoxic activity when the target cells expressing the antigen and the culture supernatant are mixed and cultured.

The present invention further relates to a method for producing a culture supernatant containing cells producing a specific antigen-specific antibody, which comprises screening by the above-mentioned method of the present invention. The present invention further relates to a method for producing cells producing a specific antigen-specific antibody, which comprises screening by the above-mentioned method of the present invention. The present invention further relates to a method for producing a specific antigen-specific antibody, which comprises screening by the above-mentioned method of the present invention.

In the present invention, screening is performed using antibody-dependent cellular cytotoxicity as an index, so that an antibody having high antibody-dependent cellular cytotoxicity can be obtained. Antibodies having high antibody-dependent cellular cytotoxicity are useful in the prevention and treatment of various diseases including cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, and viral or bacterial infections. Is.

The method for producing a culture supernatant according to the present invention may include producing a culture supernatant by culturing cells producing a human antibody, and collecting the culture supernatant obtained above.

The method for producing cells producing a specific antigen-specific antibody according to the present invention comprises the step of preparing cells producing human antibodies, the antibody-dependent cytotoxic activity from the culture supernatant of the cells producing human antibodies prepared above. It may include a step of screening a culture supernatant containing cells that produce a specific antigen-specific antibody, and a step of selecting cells corresponding to the culture supernatant selected by the screening using the above as an index.

The method for producing a specific antigen-specific antibody according to the present invention is a step of preparing cells that produce human antibodies, from the culture supernatant of cells that produce human antibodies prepared above, using the antibody-dependent cytotoxic activity as an index, It may include a step of screening a culture supernatant containing cells producing a specific antigen-specific antibody, and a step of obtaining a specific antigen-specific antibody from the culture supernatant selected by the screening.

Further, the method for producing a specific antigen-specific antibody according to the present invention comprises a step of preparing cells producing a human antibody, the culture supernatant of the cells producing a human antibody prepared above, which is used as an indicator of the antibody-dependent cytotoxic activity. As a step of screening a culture supernatant containing cells producing a specific antigen-specific antibody, a step of selecting cells corresponding to the culture supernatant selected by the screening, culturing the cells selected above and culturing the specific antigen It may include a step of producing a specific antibody.

In the present invention, the cell culture supernatant can be used as it is, and concentration of the culture supernatant, concentration measurement, and labeling of fluorescent dye are not required. In the present invention, since it is not necessary to prepare the culture supernatant, high throughput (96 wells, 384 wells, etc.) can be detected. Further, in the present invention, since the target antigen is directly expressed in cells, it is not necessary to purify the antigen. Since the target antigen is expressed on living cells and is stably expressed on the membrane surface, the three-dimensional structure as the antigen is maintained and is not denatured. Therefore, the method of the present invention is particularly effective in screening an antibody against a membrane antigen having a complicated structure that is difficult to purify (for example, G protein-coupled receptor (7-transmembrane receptor)). is there.

<Using antibody-dependent cellular cytotoxicity as an index>
In the present invention, a culture supernatant containing cells producing a specific antigen-specific antibody is screened from the culture supernatant of cells producing a human antibody using the antibody-dependent cytotoxic activity as an index. Specifically, a culture supernatant satisfying the following conditions is selected as a culture supernatant containing cells producing a specific antigen-specific antibody.

Target cells that express a specific antigen, attacking cells, and antibody-dependent cytotoxicity in the case of culturing by mixing the culture supernatants are mixed with target cells that do not express a specific antigen, attacking cells, and the culture supernatants. Antibody-dependent cytotoxic activity higher than the antibody-dependent cytotoxic activity in the case of culturing in a specific manner, and the antibody-dependent cytotoxic activity in the case of culturing by mixing the target cells expressing the specific antigen, the attacking cells, and the culture supernatant, It is higher than the antibody-dependent cytotoxic activity when the target cells expressing the antigen and the culture supernatant are mixed and cultured.

(Specific antigen)
In the present invention, the specific antigen is not particularly limited, but is preferably a membrane antigen, more preferably a membrane antigen expressed on the cell membrane surface of the target cell.

The specific antigen may be a cancer-specific membrane antigen, and examples thereof include HER2 (abbreviation of human EGFR-related 2), carcinoembryonic antigen (CEA), mucin 1 (MUC-1), epithelial cell adhesion molecular (EPC), and Examples include growth factor receptor (EGFR) and cancer antigen 125 (CA125).

-The specific antigen may be a transmembrane receptor. As the transmembrane receptor, for example, an ion channel-linked receptor or a receptor that is not linked to an ion channel can be mentioned, and further, for example, a kinase type receptor or a non-kinase type receptor can be mentioned. You can

The ion channel-linked receptor is a small pore-shaped receptor that penetrates the cell membrane, and upon binding of a ligand molecule that is a signal, the small pore is opened and closed to form Na ⁺ , K ⁺ inside and outside the cell, Alternatively, ion transport such as Ca ²⁺ is performed. Examples of the ion channel-linked receptor include each receptor having acetylcholine, glutamic acid, serotonin, GABAa, or glycine as a ligand. For example, an ion channel type receptor having acetylcholine, glutamic acid or serotonin as a ligand transports Na ⁺ and an ion channel type receptor having GABAa or glycine as a ligand transports Cl ⁻ .

A kinase-type receptor is a receptor with a single-transmembrane structure in which a kinase is bound within the cell membrane. Examples of the kinase type receptor include the growth factor type receptor family or the TGF (transforming grow factor)-β receptor family.

The growth factor type receptor family possesses a tyrosine kinase inside the cell membrane and is activated by phosphorylation of tyrosine in the cell membrane domain upon binding of a ligand. The growth factor type receptor family includes, for example, insulin receptor, M-CSF (macrophase colony stimulating factor) receptor, EGF (epidemal growth factor) receptor, PDGF (platelet derivatized GFrobothrofactor). Examples thereof include a factor (receptor) receptor, an IGF (insulin-like growh factor) receptor, and an HGF (hepatocytogrowth factor) receptor.

The TGF-β receptor family has serine-threonine kinase inside the cell membrane and is activated by phosphorylation of serine or threonine. Examples of the TGF-β receptor family include TGF receptor, activin receptor, BMP (bone morphogenic protein) receptor and the like.

Examples of non-kinase type receptors include cytokine receptor (type I cytokine receptor) superfamily, IFN (interferon) receptor (type II cytokine receptor) family, TNF (tumor necrosis factor) receptor (type III) The cytokine receptor) family, the immunoglobulin superfamily, or the G protein-coupled receptor family can be mentioned.

The cytokine receptor superfamily is a single transmembrane receptor, which has a characteristic amino acid sequence called a repeat of four cysteine residues or WS box at the N-terminal side, and extracellularly has a fibronectin ( It is a receptor having a repeating sequence of FN)-like structure. Examples of the cytokine receptor superfamily include IL (interleukin)-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL -9 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-15 receptor, GM-CSF (granulocyte-macrophase colony stimulating factor) receptor, CNTF (Cirical neurotrophotic factor) receptor Body, EPO (erythropoietin) receptor, LIF (Leukemia inhibitory factor) receptor, OSM (oncostatin M) receptor, or G-CSF (granulocyte-colony stimulating factor) receptor. Often made up of a few subunits, the subunits involved in signal transduction are shared among multiple receptors.

The IFN receptor family has no WS box, but is a receptor structurally similar to the cytokine receptor superfamily, such as a single-transmembrane structure and repeated cysteine residues. Examples of the IFN receptor family include IFNα receptor, IFNβ receptor, IFNγ receptor, and IL-10 receptor.

The TNF receptor family is a receptor whose extracellular domain is composed of units rich in cysteine residues. This unit has homology between receptors of each TNF receptor family. A sequence called a death domain exists in the intracellular domains of TNF receptors I and Fas. Examples of the TNF receptor family include TNF receptor, NGF (nerve growth factor) receptor, Fas, CD27, CD30, CD40, HVEM (Herpes virus entry mediator), LTB (Lymphotoxin βor), NOX (K40) and OX. Activator of NF-κB), CD137, etc. can be mentioned, and many are associated with cell activation and cell death.

The immunoglobulin superfamily is a receptor with a structure that has three extracellular immunoglobulin-like domains. Examples of the immunoglobulin superfamily include IL-1 receptor and IL-18 receptor. In addition, structurally similar Toll or Toll-like receptors can be mentioned. These include TLR1 to TLR12, and TLR4, which transmits information when endotoxin acts on cells, is also a receptor of this family.

The G protein-coupled receptor (GPCR) family is a receptor having a structure that works in conjugation with a G protein and penetrates the cell membrane seven times. Examples of the GPCR family include rhodopsin receptors (Gt), catecholamine receptors [β ₁ , β ₂ , β ₃ (above, Gs), α ₁ (Gq), α ₂ (Gs, Gi, Gz, Gq)]. , Dopamine receptors [D1, D5 (or more, Gs), D2(Gi)], muscarinic receptors [m1, m3 (or more, Gq), m2, m4, m5 (or more, Gi, Go], adenosine receptors [ A1, A3 (or more, Gi), A2 (Gs)], PGE receptor [EP2, EP4 (or more, Gs), EP3 (Gi), EP1], leukotriene receptor [BLT, Cys-LT1 (or more, Gi, Gq)], sphingosine 1-phosphate receptor [S1P ₁ (Gi), S1P ₂ , S1P ₃ (above, Gi, Gq, G ₁₃ ], lysophosphatidic acid receptor [LPA ₁ (Gi, G _12/13 )] , LPA ₂ , LPA ₃ (above, Gs, Gq)], nociceptin receptor, hGPCR44(Gi), hGPCR4, hGPCR10, hGPCR17, hGPCR19, hGPCR39, hGPCR48, etc. After each receptor, The following description in parentheses indicates the receptor subtype and the type of G protein α subunit that works in a coupled manner.

(Target cell)
In the present invention, target cells that express a specific antigen and target cells that do not express a specific antigen are used. As the target cells in the same screening, it is preferable to use the same cells for the target cells expressing the specific antigen and the target cells not expressing the specific antigen.

The type of target cells is not particularly limited, and animal cells expressing a specific antigen can be used. As the target cells, for example, feeder cells can be used.
Examples of the origin of the feeder cells include mammalian primates, ungulates, small mammal rodents, birds, and the like, preferably those derived from rodents and mammals, and examples thereof include mice and humans. can do. Examples of cells that can be used as feeder cells include fibroblasts, epithelial cells (for example, HeLa cells), fetal kidney cells (for example, HEK293), follicular dendritic cells and the like. Among them, fibroblasts are preferable from the viewpoints of rapid growth rate, large cell surface area, and easy removal of feeder cells.

As the feeder cells, preferably, CD40L, BAFF, and optionally, FasL-displayed feeder cells can be used. The above feeder cells can be prepared by those skilled in the art using gene recombination technology or the like, based on the known sequences of CD40L, BAFF, and FasL.

CD40L is a ligand for CD40, and the amino acid sequence of CD40L is known (eg, Nature, Vol. 357, pp. 80-82 (1992) and EMBO J., Vol. 11, pp. 4313-). 4321 (1992)). The CD40L sequence may be conserved to the extent that the binding ability of the active domain involved in receptor binding ability is not lost. For example, if 80% or more amino acid sequence homology of the active domain is present, the present invention Is available at. Such CD40L may be isolated from cells that naturally express, or may be synthesized based on a known amino acid sequence. Further, CD40L may be in a form capable of giving a signal corresponding to the presence of CD40L to B cells in the culture system, and may be in a free form or a membrane-bound form.

BAFF (B cell activator: B cell activation factor belonging to the tumor necrosis factor family) is a TNF-related molecule and is known to be involved in the proliferation and differentiation of B cells that have reacted with antigens. It is a molecule, and the amino acid sequence of BAFF is already known (for example, J Exp Med, Vol. 189, pp. 1747-1756 (1999) and Science, Vol. 285, pp. 260-263 (1999)). And J Bio Chem, Vol. 274, pp. 15978-15981 (1999)). It is sufficient that the BAFF sequence is conserved to the extent that the binding ability of the active domain involved in receptor binding ability is not lost. For example, if 80% or more amino acid sequence homology of the active domain is present, the present invention Is available at. Such BAFF may be isolated from cells that naturally express, or may be synthesized based on a known amino acid sequence. BAFF may be in any form as long as it can give a signal corresponding to the presence of BAFF to IgG-positive B cells in the culture system. Even if it is in a free form (ie, secretory form), it is a membrane-bound form. May be

Fas ligand (FasL) is a death factor belonging to the TNF family, that is, a cytokine showing an apoptosis-inducing activity, and its amino acid sequence is publicly known (eg, Cell, Vol. 75, pp. 1169-1178 (1993)). )reference. In the present invention, the sequence of FasL may be conserved to the extent that the binding ability of the active domain involved in receptor binding ability is not lost, and for example, 80% or more amino acid sequence homology of the active domain may be present. For example, it can be used in the present invention. Such FasL may be isolated from cells that naturally express, or may be synthesized based on a known amino acid sequence. Further, FasL may be in a form capable of giving a signal corresponding to the presence of Fas to IgG-positive B cells in the culture system, and may be in a free form as long as it can generate an intracellular signal, It may be membrane-bound. FasL should generally be present in the culture system at a concentration that can induce apoptosis in B cells.

Examples of the origins of CD40L, BAFF and FasL include mammalian primates, ungulates, small mammal rodents, birds and the like, and preferably those derived from rodents and mammals, such as humans and mice. Can be illustrated.

(Attacking cell)
The attacking cells (also referred to as effector cells) used in the present invention are not particularly limited as long as they express FcγR (Fcγ receptor) that recognizes an antibody, and include T cells, natural killer (NK) cells, natural killer T( NKT) cells, macrophages, monocytes (peripheral blood mononuclear cells (PBMC) and the like), dendritic cells, neutrophils, mast cells and the like can be used. Among the above, preferably, natural killer (NK) cells can be used. Examples of natural killer (NK) cells include KHYG-1, iNK-92, NK-YS, NK-YT, MOTN-1, NKL, HANK-1 and NKG cell lines, but are not particularly limited. Particularly preferably, KHYG-1 can be used.

(Antibody-dependent cytotoxic activity)
In the present invention,
Antibody-dependent cytotoxic activity in the case of culturing the mixture of the attacking cells and the culture supernatant depends on the antibody in the case of culturing by mixing the target cells not expressing a specific antigen, the attacking cells, and the culture supernatant Cells having a higher specific cytotoxic activity and expressing a specific antigen, an attacking cell, and an antibody-dependent cytotoxic activity in the case of culturing by mixing the culture supernatant, a target cell expressing a specific antigen, and Higher than antibody-dependent cytotoxic activity when the culture supernatant is mixed and cultured;
This is used as an index.

Antibody-dependent cellular cytotoxicity (ADCC activity) means that when a specific antibody is attached to a cell surface antigen of a target cell, an attacking cell (effector cell having an Fcγ receptor) is mediated by an Fcγ receptor at the Fc part thereof. Binding and damaging the target cells.

The antibody-dependent cytotoxic activity can be measured by a known method. Specifically, it can be performed by the following method.
A target cell that does not express a specific antigen, a target cell that expresses a specific antigen, an attacking cell, and a culture supernatant are prepared.
Target cells that do not express the specific antigen and target cells that express the specific antigen are seeded in a 96-well plate. Then, the medium is replaced with a culture supernatant for screening, and the culture is performed. Afterwards, challenge cells are added to each well. An experimental system using a target cell that expresses a specific antigen, an attacking cell, and a culture supernatant, an experimental system that uses a target cell that does not express a specific antigen, an attacking cell, and a culture supernatant, and a specific antigen An experimental system using the target cells to be expressed and the culture supernatant is performed. After culturing for an appropriate time, ADCC activity can be evaluated using the culture supernatant by measuring the enzyme released by dead cells (for example, LDH).

"Antibody-dependent cytotoxic activity in the case of mixing and culturing the attacking cells and the culture supernatant, the target cells that do not express a specific antigen, the attacking cells, and the antibody in the case of mixing and culturing the culture supernatant Target cells that have higher specificity-dependent cytotoxicity and that express a specific antigen, attack cells, and antibody-dependent cellular cytotoxicity in the case of culturing by mixing the culture supernatant, target cells that express a specific antigen, And "higher" and "higher" than "higher than antibody-dependent cellular cytotoxicity when the culture supernatant is mixed and cultured" means an enzyme released by dead cells as described above (for example, LDH and the like). It means that the antibody-dependent cellular cytotoxicity evaluated by measuring The degree is such that the ratio of absorbance in enzyme measurement is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1. It may be 7 times or more, 1.8 times or more, 1.9 times or more, or 2.0 times or more.

<Culture supernatant of human antibody-producing cells>
In the present invention, the culture supernatant of cells producing human antibodies is screened.
The type of human antibody is not particularly limited, and may be any of human IgG, human IgA, human IgM, human IgD, and human IgE, but human IgG is preferable.

The culture supernatant of human antibody-producing cells is not particularly limited. For example, the culture supernatant of human B cells or the culture supernatant of established animal cell lines (CHO-K1 cells, etc.) can be used. You can
As the culture supernatant of human B cells, preferably, the culture supernatant obtained by culturing human B cells on feeder cells expressing an antigen can be used.
As the culture supernatant of human B cells, preferably, the culture supernatant obtained after culturing human B cells in the presence of Fas-mediated stimulation can be used.
As the culture supernatant of human B cells, preferably, the culture supernatant obtained by culturing human B cells in the presence of IL-2 and IL-21 can be used.

As human B cells, B cells in which the expression of Bach2 gene is increased may be used. B cells in which the expression of the Bach2 gene is increased include cells obtained by introducing the Bach2 gene into B cells, or by increasing the expression amount or abundance of the Bach2 gene originally possessed by the B cells. Any of the cells obtained may be used. B cells in which the expression of the Bach2 gene is elevated are described in paragraphs 0020 to 0035 of International Publication WO2016/002760, and the content described in International Publication WO2016/002760 is incorporated herein by reference. It

As a method for increasing the expression level or abundance of the Bach2 gene originally possessed by B cells, a method for introducing the Menin gene (Nature Communications, 5:3555, DOI:10:1038.ncommms4555) into B cells, lept Examples include a method of inhibiting the nuclear export of Bach2 by adding mycin B (LMB) to localize it in the nucleus, or a method of adding an inhibitor of ubiquitination of Bach2 and its degradation by proteasome. Not limited.

When introducing the Bach2 gene into B cells, the Bach2 gene is as described in paragraphs 0022 to 0031 of International Publication WO2016/002760.
In order to introduce the Bach2 gene into B cells, a recombinant vector having the Bach2 gene may be constructed and this recombinant vector may be introduced into B cells.

The level of increase in Bach2 gene expression is not particularly limited, but the expression level of Bach2 gene is preferably 1.5 times or more, and more preferably 2 times or more, as compared with the original B cells.

The human antibody-producing cells (preferably B cells) are preferably cells having human antibodies on the cell surface. Cells having human antibodies on the cell surface can be obtained from cell populations based on the presence or absence of human antibodies.

The cell population containing human antibody-producing cells is not particularly limited as long as it is generally derived from peripheral blood cells, bone marrow cells or lymphoid organs such as spleen cells. The IgG-positive B cell may be a naive B cell that has not reacted with the antigen or a B cell after contact with the antigen. Here, the “naive B cell” in the present specification generally refers to a mature B cell that has not reacted with an antigen, and corresponds to a cell showing a CXCR5-positive and CD40-positive surface antigen.

-It may contain B cells of various stages in the differentiation stage and various types of cells. From the viewpoint of efficiency of selection by culture, B cells other than IgG positive B cells, for example, IgE positive cells, CD138 positive (plasma) cells, and cells other than B cells, for example, T cells, monocytes, NK cells are removed. Preferably.

The B cell population may be a cell population derived from an organism having an established immune system, and may include rodents of small mammals such as mammalian primates such as humans and monkeys and ungulates such as pigs, cows and horses. Such species include, for example, mice, rats, rabbits, birds, such as chickens and quails. The origin of this cell population is preferably rodents and primates, and examples thereof include mice and humans. As a method for preparing a cell population from a biological tissue such as spleen, ordinary conditions for preparing a B cell population may be applied as they are. Further, it is not limited to a cell population derived from a living body, and may be an established B cell line.

Culturing of a cell population containing B cells may be carried out under usual culture conditions with a medium used for culturing B cells. Examples of such a medium include Dulbecco's modified Eagle medium (DMEM) and RPMI1640. Various additives applicable to ordinary cell culture such as serum, various vitamins and various antibiotics may be added to these media. As the culture conditions such as the culture temperature, the culture conditions used for general B cells can be applied as they are, and examples thereof include a condition of 37° C. and 5% CO ₂ . The seeding density of the cell population in the medium varies depending on the origin of the cell population, the state of cells prepared from the tissue, and the number of days of culturing in the same culture system, but is generally 1×10 ² to 1×10 ⁷ cells/cell. cm ² and preferably 1×10 ³ to 1×10 ⁷ cells/cm ² , and in particular, human B cells preferably have a growth rate of 1×10 1 because the proliferation rate is better when the culture is started at a high density. It should be ⁴ to 1×10 ⁷ pieces/cm ² . Within this range, it is possible to prevent a hyperproliferation state after culturing for about 4 days.

In the presence of IL-21, B cells produce an intracellular signal by stimulation through CD40, BAFF receptor, and thus IL-21 receptor (IL-21R), CD40, and BAFF receptor ( BAFF-R) is preferred. There is no limitation on the stimulation to these molecules as long as they recognize these molecules from the outside and generate intracellular signals inside IgG positive B cells having CD40 and BAFF receptors.

In the present invention, preferably, when B cells having an increased expression of the Bach2 gene are cultured in the presence of a means for acting on CD40 and/or BAFF receptors, in the presence of a means for acting on IL-21 receptors. Incubate.

As a means for acting on the IL-21 receptor, IL-21 or anti-IL-21 receptor antibody can be used.
IL-21 may be naturally-occurring or recombinantly obtained by biotechnology. Examples of the origin of IL-21 include mammalian primates, ungulates, small mammalian rodents, birds and the like, as in the above-mentioned cell population. Each of these molecules is preferably derived from rodents and mammals, and examples thereof include mice and humans. Further, it may be a molecule derived from the same species as the above-mentioned cell population to be presented, or cells derived from a different species.

The concentration of IL-21 and/or anti-IL-21 receptor antibody contained in the medium for culturing B cells may be any amount capable of proliferating IgG-positive B cells having an affinity for a specific antigen, Generally, it is preferably 1 ng/ml to 1 μg/ml, more preferably 5 ng/ml to 100 ng/ml.

In the present invention, preferably, when B cells having an elevated expression of the Bach2 gene are cultured in the presence of a means for acting on CD40 and/or BAFF receptor, in the presence of IL-4 and/or IL-2. You may culture in.

The IgG-positive B cells having the IL-21 receptor (IL-21R), CD40, and BAFF receptor (BAFF-R) in the present invention show the presence of these molecules, for example, the reactivity when an antibody or the like is used. However, from the viewpoint of preparation time and the desired cell density of IgG-positive B cells, the cell population containing B cells can be stimulated via CD40 and BAFF receptors while IL- It may be obtained by a method comprising culturing in the presence of 4 in the primary culturing step and culturing in the presence of IL-21 in the secondary culturing step (culturing step).

In the above culturing process, both the primary culture and the secondary culture can be performed while applying stimulation via the CD40 and BAFF receptors. Stimulation via the CD40 and BAFF receptors may be performed using antibodies against these molecules, or CD40L and BAFF may be used, as in the selection step. From the viewpoint of surely stimulating the cell population to be cultured with these antibodies and molecules, a carrier having these antibodies or CD40L and BAFF, such as feeder cells, may be used. With respect to CD40L and BAFF, carriers and the like, the matters described in the selection step can be applied as they are. The carrier or feeder cell used in the culturing step may be referred to as a culturing carrier or culturing feeder cell, respectively.

IL-4 used in the primary culture may be of natural origin or bioengineered recombinant. From the viewpoint of effective proliferation of B cells, the concentration of IL-4 is preferably 1 ng/ml to 100 ng/ml, more preferably 5 ng/ml to 100 ng/ml, and 10 ng/ml to 50 ng/ml. More preferably, it is ml.

In the primary culture, other cytokines may be used together with IL-4 depending on the type and origin of the B cell population to be cultured. For example, when IL-2 is used in combination with IL-4, the concentration of IL-2 is preferably 1 unit/ml to 1000 unit/ml, more preferably 2.5 unit/ml to 1000 unit/ml, It is more preferably from 2.5 unit/ml to 500 unit/ml, particularly preferably from 10 unit/ml to 100 unit/ml.

The seeding density of cells at the time of starting the primary culture is not particularly limited, but it varies depending on the origin of the cell population, the state of cells prepared from the tissue, and the number of days of culture in the same culture system. ^It may be ² to 1×10 ⁶ pieces/cm ² , preferably 1×10 ³ to 1×10 ⁶ pieces/cm ² . Although the primary culture may vary depending on the seeding density, it may be generally 2 to 8 days after the start of the culture from the viewpoint of the growth rate of the B cell population, and 3 from the viewpoint of the density of IgG-positive B cells in the cell population. It is preferably from day to 6 days, more preferably from 3 to 5 days.

When starting the secondary culture, a predetermined amount of IL-21 may be added to the culture system after the primary culture, and the cells are recovered from the culture system after the primary culture to obtain IL-4-free IL. It may be started by transferring to a -21 containing medium. From the viewpoint of suppressing the proliferation rate of IgG-positive B cells in the secondary culture and the contamination of IgE-positive B cells in the obtained cell population, the secondary culture is performed with a medium containing IL-21 and not IL-4. Most preferably it is done.

The origins of IL-4 and IL-21 used in the present invention include mammalian primates, ungulates, small mammal rodents, birds, etc., as in the above-mentioned cell population. Each of these molecules is preferably derived from rodents and mammals, and examples thereof include mouse and human. Further, it may be a molecule derived from the same species as the above-mentioned cell population to be presented, or cells derived from a different species.

After completion of secondary culture, it is preferable to remove cells other than IgG-positive B cells in order to surely increase the target B cell concentration. Examples of cells to be removed include IgE-positive B cells, CD138-positive plasma cells, feeder cells (if present), and the like. These cells can be removed by a known technique using an antibody against a specific surface antigen present on the surface.

In the secondary culture, other cytokines may be used together with IL-21 depending on the type and origin of the B cell population to be cultured. For example, when IL-2 is used in combination with IL-21, the concentration of IL-2 is preferably 1 unit/ml to 1000 unit/ml, more preferably 2.5 unit/ml to 1000 unit/ml. , 2.5 unit/ml to 500 unit/ml is more preferable, and 10 unit/ml to 100 unit/ml is particularly preferable.

In the method for producing a B cell population of the present invention, it may be carried out for a period of time necessary for selecting a desired antigen-specific IgG-positive cell, and the number of IgG-positive B cells at the time of starting the selection step, It can be appropriately changed depending on the type or the state of feeder cells. For example, in order to efficiently obtain a target cell population, the selection step may be carried out for 1 day to 2 days. When the culture step is included, the primary culture is carried out for 3 days to 5 days, and the secondary culture is carried out for 2 days. It may be 5 days, and the selection step may be 1 to 2 days.

The production method of the present invention may include a proliferation step for further proliferating the selected antigen-specific IgG-positive B cells after the selection step. This growth step may be performed under culture conditions that allow the antigen-specific IgG-positive B cells to grow against the specific antigen selected in the selection step, but from the viewpoint of efficient growth of the selected IgG-positive B cells. Therefore, it is preferable to culture with CD40L and BAFF in the presence of IL-21. Regarding IL-21 that is preferably used in the proliferation step, the conditions applied in the secondary culture or selection step described above can be applied as they are. Further, the above-mentioned matters can be directly applied to CD40L and BAFF which are preferably used in the proliferation step.

The proliferating step may be continued for a period corresponding to the number of target IgG-positive B cells in the obtained cell population, and can be generally 1 day or longer, preferably 3 days or longer, but it is included in the culture system. It may be appropriately adjusted depending on the growth rate and density of the cell population.

Examples of the present invention will be described below, but the invention is not limited thereto.
A detailed description of conventional methods such as those used herein can be found in the cited references. Unless otherwise indicated below, specificities of interest and their recombinant expression and molecular cloning of anti-CXCR2 antibodies are described in International Application No. PCT/EP2008/000053, and WO2010/000053, published as WO 2008/081008. Has been, or can be, carried out as described in the examples and capture methods of International Application No. PCT/EP2009/009186, published as 069603, the disclosure content of which is hereby incorporated by reference in its entirety. Incorporated into the book.
Regarding the antigen-specific B cell selection method (FAIS method), the methods described in Japanese Patent No. 5550132 and International Publication No. WO2018/131698 can be referred to, and the disclosure content thereof is entirely incorporated by reference. Incorporated herein.

(1) Cell culture Unless otherwise specified, RPMI-1640 (10% (v/v) FCS (fetal bovine serum), penicillin/streptomycin, 2 mM L-glutamine, 55 nM 2-mercaptoethanol, 10 mM HEPES and 1 mM sodium pyruvate was added) as a B cell culture medium, and 5% (v/v) CO ₂ was performed at 37°C.

Unless otherwise specified, the culture of 40LB cells (using the cells described in Japanese Patent No. 5550132), which are feeder cells that present CD40L and BAFF on the cell surface when culturing B cells, is D-MEM (10%). (V/v) FCS, penicillin/streptomycin added) was used as a 40 LB cell culture medium under the conditions of 5% (v/v) CO ₂ and 37°C.

Cultures of KHYG-1 cells were RPMI-1640 (10% (v/v) FCS (fetal calf serum), penicillin/streptomycin, 2 mM L-glutamine, 55 nM 2-mercaptoethanol, 10 mM HEPES and 1 mM unless otherwise specified). Sodium pyruvate was added), and 100 U/mL IL-2 (manufactured by Peprotech) was used as a KHYG-1 cell culture medium under conditions of 5% (v/v) CO ₂ and 37°C.

At the time of B cell culture experiment, 40 LB cells were seeded at 2×10 ⁴ cells/well in a 96-well plate (Thermo Fisher), cultured for 24 hours to form a monolayer, and then irradiated with 120 Gy of X-ray. Used from.

At the time of seeding B cells, the medium was removed from the 384-well plate in which 40 LB cells had been seeded in advance, and then the cells were seeded using a multipipette at 20 cells/well.

(2) Preparation of feeder cells To perform a screening method using ADCC activity, 40LB-Her2 cells and 40LB-CXCR2 cells were used as antigen-expressing cells, and 40LB cells were used as antigen-non-expressing cells.

40LB-Her2 cells and 40LB-CXCR2 cells were cloned by introducing human Her2 or human CXCR2 into 40LB cells by a lentiviral vector according to a conventional method and expressing them constantly.

(3) Examination of Screening Method Utilizing ADCC Activity Using Model Antigen 40LB cells, 40LB-Her2 cells, and KHYG-1 cells were used for examination of screening method utilizing ADCC activity. Further, a trastuzumab type antibody was prepared as an antibody having antigen specificity, and a commercially available human IgG antibody was prepared as an antigen non-specific antibody at 1 μg/mL and used. The trastuzumab type antibody used at this time was a recombinant lentivirus vector CSIV-CMV-MCS-IRES2-Venus vector (Cancer Science, Vol.105, pp.402-408 (2014)), and Her2 antigen-specific B cells. CSIV-CMV-mGK (trastuzumab antibody kappa light chain gene expression vector)-IRES2-mGH (trastuzumab antibody heavy chain gene expression vector) in which a heavy chain and a light chain of a receptor (trastuzumab antibody gene) are incorporated is prepared, and a trastuzumab type is prepared. After constructing an antibody gene expression vector and transfecting the gene into CHO-K1 cells to prepare antibody-producing cells, the culture supernatant was purified with Protein A carrier (Kaneka) and used. 12 hours before measuring ADCC activity, 40LB cells and 40LB-Her2 cells were seeded in a 96-well plate at 1×10 ⁴ cells/well. 12 hours after cell seeding, the medium was exchanged with 50 μL of an antibody solution prepared to 1 μg/mL with KHYG-1 cell culture medium, and cultured for 30 minutes under the conditions of 5% (v/v) CO ₂ and 37° C. .. Then, 1×10 ⁴ cells/50 μL of KHYG-1 cells in KHYG-1 cell culture medium was added to each well. At this time, as a control group, an equal amount of the medium alone was added to the wells to which KHYG-1 cells were not added. 12 hours after the addition, ADCC activity was evaluated by measuring LDH released by dead cells using the culture supernatant. The LDH Cytotoxicity Assay Kit ((manufactured by Dojindo)) was used for the measurement of LDH. The culture plate was centrifuged at 250×g for 2 minutes, 40 μL of the culture supernatant was collected and dispensed into a 96-well Maxisorp plate (Nunc). Next, 40 μL of Working Solution was added, and shaking culture was performed for 30 minutes in a dark place at room temperature. After 30 minutes, 20 μL of Stop Solution was added to stop the reaction, and the absorbance at 490 nm was measured. As shown in FIG. 1, cell death is induced and LDH is released only in the presence of antigen-specific antibodies in addition to antigen-expressing cells and attacking cells, confirming that high absorbance can be measured as shown in FIG. did it.

(4) Preparation of B cell subpopulation containing B cells expressing CXCR2 antigen-specific B cell receptor A B cell subpopulation containing B cells expressing CXCR2 antigen-specific receptor was obtained as follows.
Human B cells were prepared as follows.
That is, mononuclear cells were separated from human peripheral blood of a healthy person using a Lymphoprep tube (manufactured by AXIS SHIELD), and FcR Blocking Reagent (manufactured by Miltenyi Biotec) was used, and then CD2 negative cells and CD235a negative cells were further isolated. , Biotin-anti-human CD2 antibody (manufactured by Biolegend), biotin-anti-human CD235a antibody (manufactured by eBioscience), Streptavidin-Particle Plus-DM (manufactured by BD Pharmingen), and BD iMag CellBene Separation (manufactured by BD Phage). And manufactured by MACS Separation Columns (manufactured by Miltenyi Biotec). Among the collected cells, CD19-positive cells were collected using a PE-anti-human CD19 antibody (manufactured by Biolegend) and a cell sorter (BD FACS Aria III). Furthermore, by introducing a long-term survival gene into the obtained B cells, B cells that can be cultured for a long time were produced. Long-term cultured B cells were obtained as follows.

That is, a CSIV-CMV-MCS-IRES2-Venus vector (Cancer Science, Vol.105, pp.402-408 (2014)) was recombined, and βactin was used as a promoter and CSIV-βactin-Bach2 incorporating Bach2 as a transgene. It was prepared and a Bach2 expression vector was constructed. Using this expression lentivirus vector, a B cell population in which Bach2 was expressed in the recovered CD19-positive B cells was obtained by a conventional method.

(5) Preparation of human B cells (culture step)
The population containing the antigen-specific B cells obtained above was primary cultured for 7 days on 40 LB cells that had formed a monolayer. At the time of culturing, 1×10 ⁵ cells/cm ² of cells were prepared using a B cell culture medium containing human IL-4 (50 ng/mL, manufactured by PEPROTECH) and human IL-2 (50 unit/mL, manufactured by PEPROTECH). The cells were cultured at a density in a CO ₂ incubator.

On day 7 of culture, all cells were collected together with feeder cells using D-PBS containing 2 mM EDTA and 0.5% by mass BSA. The recovered cultured B cells were prepared by using biotin-anti-mouse H-2K ^d antibody (manufactured by Biolegend), biotin anti-human CD138 antibody (manufactured by Diaclone), Streptavidin-Particle Plus-DM (manufactured by BD Pharmingen), and BD. Feeder cells and antibody-producing cells were removed using iMag Cell Separation Magnet (manufactured by BD Bioscience) and MACS Separation Columns (manufactured by Miltenyi Biotec). The B cells from which the feeder cells were removed were human IL-21 (10 ng/mL, manufactured by PEPROTECH) and human IL-2 (50 unit/mL, manufactured by PEPROTECH) in a 90 mm dish on which 40 LB cells were newly prepared. ) Was used to inoculate at a cell density of 5×10 ⁴ cells/cm ² or less, and the cells were cultured.

(6) Selective culture of antigen-specific B cells (selection step)
On the 10th day of culture, in order to carry out selective culture of CXCR2 antigen-specific B cells, feeder cells and antibody-producing cells were removed from the recovered cultured B cells in the same manner as in (5). The B cells from which the feeder cells were removed were human IL-24 (100 ng/mL, manufactured by PEPROTECH) and human IL-2 (50 unit/mL, manufactured by PEPROTECH) in a 90 mm dish in which newly prepared 40 LB cells were seeded. )-Containing B cell medium was seeded at a cell density of 2×10 ⁵ cells/cm ² and pre-stimulation culture was performed.

After 48 hours (ie, 12th day of culture), whole cells were treated with biotin-anti-mouse H-2K ^d antibody (Biolegend), biotin anti-human CD138 antibody (Diaclone) and Streptavidin-Particle Plus-DM (BD Pharmingen). The feeder cells and antibody-producing cells were removed using BD iMag Cell Separation Magnet (manufactured by BD Bioscience) and MACS Separation Columns (manufactured by Miltenyi Biotec). The B cell population that has undergone pre-stimulation culture in which feeder cells and antibody-producing cells have been removed is subsequently suspended in a B cell medium containing cytokine under the same conditions, and cultured with 40LB-CXCR2 cells that have formed a monolayer. , Antigen-specific B cells were stimulated.

After 48 hours (that is, on the 14th day of culture), feeder cells and antibody-producing cells were removed from all cells by the same method as above. 40 LB-CXCR2 cells and B cells under each condition subjected to antigen-stimulated culture in which antibody-producing cells were removed were human IL-21 (10 ng/mL, manufactured by PEPROTECH) and human IL-2 (50 unit/mL, manufactured by PEPROTECH). ) Was cultured in the B cell culture medium containing 40 LB-FasL cells in which a monolayer was formed to give a death stimulus to all the cultured B cell populations.

After 24 hours (that is, on the 15th day of culture), whole cells were treated with biotinylated anti-mouse H-2K ^d antibody (manufactured by Biolegend), biotinylated anti-human CD178 (FasL) antibody (manufactured by Biolegend), Streptavidin-Particle Plus-. 40LB-FasL cells were removed using DM (manufactured by BD Pharmingen) and BD iMag Cell Separation Magnet (manufactured by BD Bioscience) and MACS Separation Columns (manufactured by Miltenyi Biotec). Further, in the B cell population from which 40LB-FasL cells were removed, dead cells were removed using ClioCell Pro Kit (manufactured by ClioCell). The B cell population from which feeder cells and dead cells have been removed is suspended in a B cell medium containing human IL-21 (10 ng/mL, PEPROTECH) and human IL-2 (50 unit/mL, PEPROTECH), Recovery cultures were performed on fresh 40LB cells.

After cell death stimulation, recovery culture was performed, cells cultured for 6 days were collected, cells were seeded at 20 cells/well/384-well plate, and diluted culture was performed for 14 days.

After 14 days, antibody production was confirmed by the ELISA method using the culture supernatant of the cells that were diluted and cultured. In the ELISA method, an anti-human IgG+IgM+IgA antibody (manufactured by Kirkegaard & Perry Laboratories (KPL)) was prepared at 1 μg/mL using a carbonate-bicarbonate buffer (pH 9.0), and coated on a 384-well Maxisorp plate. After the addition of the antibody solution, the coating was performed by leaving it at room temperature for 2 hours. After 2 hours, the plate was washed with TBS (TBS-T) containing 0.5% Tween 20 (manufactured by SIGMA). A plate washer was used to wash the plate. After the plate was washed, a 10% skim milk solution (manufactured by Wako) suspended in a TBS solution was added to each well and allowed to stand at room temperature for 1 hour for blocking. After 1 hour, the plate was washed, the above-mentioned B cell culture supernatant was added to each well, and the mixture was allowed to stand at room temperature for 1 hour to carry out an antibody reaction. After 1 hour, the plate was washed, and then an HRP-labeled anti-human IgG-Fc antibody (Jakson ImmunoResearch) was diluted 10,000 times with TBS solution as a secondary antibody and allowed to stand at room temperature for 1 hour. Then, an antibody reaction was performed. After 1 hour, the plate was washed, and then TMB substrate (manufactured by KPL) was added to develop color. Two minutes after the addition, 1N H ₂ SO ₄ was added to stop the enzymatic reaction, and the absorbance at 450 nm was measured.

The cells in the wells in which antibody production was confirmed by the ELISA method were re-seeded to a 96-well plate in which 40 LB cells were newly seeded, and expanded culture was performed. At this time, the B cells were suspended in a B cell medium containing human IL-21 (10 ng/mL, manufactured by PEPROTECH) and human IL-2 (50 unit/mL, manufactured by PEPROTECH), and cultured for 7 days.

ADCC activity was measured using the B cell subpopulation culture supernatant that had been cultured for 7 days. First, 12 hours before the measurement, ⁴⁰ LB-CXCR2 cells were seeded as antigen-expressing cells in a 96-well plate at 1×10 ⁴ cells/well. After 12 hours, 40LB-CXCR2 cell culture supernatant was collected, and 40 μL of B cell subpopulation culture supernatant was added to each well. At this time, the B cell small population culture supernatant collected from the same well was added to 2 wells for ADCC induction and control culture.

KHYG-1 cells for inducing ADCC were prepared by removing dead cells using ClioCell Pro Kit (manufactured by ClioCell), and then RPMI-1640 (10% (v/v) FCS (fetal bovine serum), penicillin/ Streptomycin, 2 mM L-glutamine, 55 nM 2-mercaptoethanol, 10 mM HEPES and 1 mM sodium pyruvate added), and 200 U/mL IL-2 (manufactured by Peprotech), the final concentration of IL-2 at the time of culturing. Was adjusted to 100 U/mL and KHYG-1 cells were adjusted to 1×10 ⁴ cells/40 μL. For control culture, 200 U/mL IL-2-containing RPMI-1640 without KHYG-1 cells (10% (v/v) FCS (fetal calf serum), penicillin/streptomycin, 2 mM L-glutamine, 55 nM 2- Mercaptoethanol, 10 mM HEPES and 1 mM sodium pyruvate were also added).

To 40LB-CXCR2 cells to which the B cell subpopulation culture supernatant was added, 1×10 ⁴ cells/40 μL of KHYG-1 cells were added to the ADCC induction well, and 40 μL of the medium alone was added for the control culture. After the addition, the cells were cultured in a CO ₂ incubator for 12 hours.

After 12 hours, ADCC activity was evaluated by measuring LDH released by dead cells using the culture supernatant. An LDH Cytotoxicity Assay Kit (manufactured by Dojindo) was used for measuring LDH. The culture plate was centrifuged at 250×g for 2 minutes, 40 μL of the culture supernatant was collected and dispensed into a 96-well Maxisorp plate (Nunc). Next, 40 μL of Working Solution was added, and shake culture was carried out at room temperature in a dark place for 30 minutes. After 30 minutes, 20 μL of Stop Solution was added to stop the reaction, and the absorbance at 490 nm was measured. The measurement results are shown in FIG.

The absorbance values obtained for ADCC induction and control culture were compared, and it was determined that the wells having a positive difference contained the antigen-specific antibody, and the difference was the most positive. Antibody genes were obtained from B cells in the wells. The cells cultured in 96 wells were collected, and mRNA was extracted and purified using RNeasy Mini Kit (manufactured by QIAGEN). Next, cDNA was synthesized from the purified mRNA using an antibody gene-specific reverse transcription primer and SuperScript III (Invitrogen).

Regarding the reaction composition, after preparing a mixed solution according to the instructions attached to SuperScript III, reverse transcription reaction PCR (65°C/5 minutes, 55°C/60 minutes, 70°C/15 minutes) was performed. Then, RNase H (manufactured by Invitrogen) was added according to the attached instruction, and the reaction was carried out (37°C/20 minutes). The synthesized cDNA was purified using an Agencourt AMPure XP nucleic acid purification kit (manufactured by BECKMAN COULTER). The purified cDNA was subjected to 3'-tailing reaction (37°C/30 minutes) using Terminal deoxy Transferase (Invitrogen) and dGTP (Invitrogen) according to the attached instructions.

5'RACE method was used for amplification of antibody gene variable region. First, 1st-PCR (94° C./2 min; 1) using KOD-FX-DNA polymerase (manufactured by TOYOBO) for each of Igγ heavy chain, Igκ light chain and Igλ light chain using 3′-tailing cDNA. Cycle, 98°C/10 seconds, 60°C/10 seconds, 68°C/40 seconds; 25 cycles, 68°C/2 minutes; 1 cycle).

Then, using each DNA solution, Nested-PCR (94° C./2 min; 1 cycle, 94° C./15 sec, 60° C./10 sec, 68° C./) using KOD-plus-DNA polymerase (manufactured by TOYOBO) 40 seconds; 35 cycles, 68° C./2 minutes; 1 cycle).

After the PCR, the reaction solution was electrophoresed for a total amount of 100 V for 35 minutes, and the amplified fractions of each of the Igγ heavy chain, Igκ light chain and Igλ light chain were extracted using Gel Extraction Kit (manufactured by QIAGEN).

As the plasmid for transient expression of the antibody gene, a plasmid incorporating a human antibody constant region was used. The Igγ heavy chain and Igκ light chain plasmids were excised using the restriction enzyme PvuII, and the Igλ light chains were excised using the restriction enzyme StuI. The excised plasmid DNA was dephosphorylated by reacting with the dephosphorylating enzyme BAPC75 (manufactured by Takara Bio Inc.) at 65° C. for 30 minutes.

The dephosphorylated Igγ heavy chain, Igκ light chain, and Igλ light chain, and the antibody gene variable region amplified using the Nested-PCR method are the enzyme ligation high ver. 2 (manufactured by TOYOBO Co., Ltd.) was used for bonding at 16° C. for 60 minutes. The ligated DNA was transformed into Escherichia coli JM109 (Takara Bio Inc.). Sequence analysis of the Ig γ heavy chain, Ig κ light chain, and Ig λ light chain plasmids obtained after transformation was carried out using a CMV primer and a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems). Three types of Igγ heavy chain, two types of Igκ light chain, and one type of Igλ light chain were obtained. The combination of heavy and light chains was expressed in HEK293T cells to produce 9 kinds of antibodies. The heavy chain/light chain antibody genes were expressed using Lipofectamine 3000 (manufactured by Invitrogen).

Using the prepared antibody, the antigen binding ability was detected using a flow cytometer (BD FACS Aria III). 40LB-CXCR2 cells were used as antigen-expressing cells, and 40LB cells were used as antigen-unexpressing cells. The prepared antibody was added to 3×10 ⁵ cells of each and reacted at room temperature for 30 minutes. Then, the cells were washed with FACS buffer (D-PBS(-) containing 0.5% BSA and 200 mM EDTA), and APC-labeled anti-human IgG-Fc antibody (manufactured by Biolegend) was used as a secondary antibody at room temperature for 20 minutes. It was made to react. After that, the cells were washed twice with FACS buffer and subjected to flow cytometry analysis. The results are shown in Fig. 4. The antibody obtained from the results was found to be an antibody that recognizes some antigen on 40LB cells that is not the CXCR2 antigen.

From the above results, it was found that the obtained antibody is an antibody that recognizes some antigen on 40LB cells that is not the CXCR2 antigen. The cause is considered to be that the evaluation method had a problem from the result of FIG. At the time of the above-mentioned evaluation, a cell population having high ADCC activity (difference in LDH absorbance) with or without KHYG-1 as an attacking cell was selected to obtain an antibody gene. However, when the ADCC activity at this time was re-evaluated from the viewpoint of the presence or absence of the antigen, it was found that the cell population in which the ADCC activity was high in the presence or absence of KHYG-1 was the cell population in which the ADCC activity was low in the presence or absence of the antigen. From this result, it was considered that ADCC activity needs to be measured not only for antigen-expressing cells but also for cells not expressing antigen.

12 hours before the ADCC activity was measured, ⁴⁰ LB-CXCR2 cells as antigen-expressing cells and ⁴⁰ LB cells as antigen-unexpressing cells were seeded in a 96-well plate at 1×10 ⁴ cells/well. After 12 hours, each cell culture supernatant was collected, and 40 μL of a B cell small population culture supernatant was newly added to each well. At this time, the B cell subpopulation culture supernatant collected from the same well was added to 4 wells of ADCLB induction and control culture for each of 40LB-CXCR2 cells and 40LB cells. Thereafter, in the same manner as in the above-mentioned method, for each of 40LB-CXCR2 cells and 40LB cells to which the B cell subpopulation culture supernatant was added, 1×10 ⁴ cells/40 μL of KHYG-1 cells were added to the well for ADCC induction. For control culture, 40 μL of medium alone was added. After the addition, the cells were cultured in a CO ₂ incubator for 12 hours. After 12 hours, LDH in which dead cells release ADCC activity using the culture supernatant was measured by the same method as described above, and the absorbance at 490 nm was measured. Results are shown in FIG.

Regarding the absorbance obtained by the measurement, first, the difference between the cells expressing the antigen and the cells not expressing the antigen was calculated. Next, the differences obtained with and without the antigen were compared, and those with a large positive difference were judged to have high antigen specificity. Regarding the cells having cell specificity as described above, a difference occurs in both the antigen-expressing cells and the antigen-non-expressing cells, and it is considered that the ratio of both approaches 1. As a result, it was considered that the results obtained by this method were likely to eliminate noise and facilitate selection of wells containing antigen-specific antibodies.

From the wells that showed a large difference from the above results, mRNA was extracted by the same method as the previous time, the antibody gene variable region was amplified, and an antibody gene expression plasmid was prepared. When the sequence was analyzed using CMV primers, 4 types of Igγ heavy chain and 3 types of Igκ light chain were obtained. The combination of heavy and light chains was expressed in HEK293T cells to prepare 12 kinds of antibodies.

Using the prepared antibody, the antigen binding ability was detected using a flow cytometer (BD FACS Aria III). The results are shown in Fig. 7. As a result, the above-mentioned cell-specific antibody did not exist, and it was confirmed that an antigen-specific antibody was obtained.

Claims (13)

1. A method for screening a culture supernatant containing cells that produce a specific antigen-specific antibody using the antibody-dependent cytotoxic activity as an index, from the culture supernatant of cells producing a human antibody, the culture satisfying the following conditions: Method of selecting supernatant as culture supernatant containing cells producing specific antigen-specific antibody:
   Target cells that express a specific antigen, attacking cells, and antibody-dependent cytotoxicity in the case of culturing by mixing the culture supernatants are mixed with target cells that do not express a specific antigen, attacking cells, and the culture supernatants. Antibody-dependent cytotoxic activity higher than the antibody-dependent cytotoxic activity in the case of culturing in a specific manner, and the antibody-dependent cytotoxic activity in the case of culturing by mixing the target cells expressing the specific antigen, the attacking cells, and the culture supernatant, It is higher than the antibody-dependent cytotoxic activity when the target cells expressing the antigen and the culture supernatant are mixed and cultured.
2. The method according to claim 1, wherein the culture supernatant of cells producing a human antibody is a culture supernatant of human B cells.
3. The method according to claim 2, wherein the culture supernatant of human B cells is the culture supernatant of human B cells cultured on feeder cells expressing an antigen.
4. The method according to claim 2 or 3, wherein the culture supernatant of human B cells is the culture supernatant of human B cells cultured in the presence of Fas-mediated stimulation.
5. The method according to any one of claims 2 to 4, wherein the culture supernatant of human B cells is a culture supernatant obtained by culturing human B cells in the presence of IL-2 and IL-21.
6. The method according to any one of claims 1 to 5, wherein the specific antigen is a membrane antigen expressed on the cell membrane surface of target cells.
7. The method according to any one of claims 1 to 6, wherein the specific antigen is a transmembrane receptor.
8. The method according to any one of claims 1 to 7, wherein the specific antigen is a G protein-coupled receptor.
9. The method according to any one of claims 1 to 8, wherein the human antibody is human IgG.
10. The method according to any one of claims 1 to 9, wherein the antibody-dependent cytotoxic activity is measured by detecting an enzyme released by dead cells.
11. A culture supernatant containing cells producing a specific antigen-specific antibody, comprising screening a culture supernatant containing cells producing a specific antigen-specific antibody by the method according to claim 1. Manufacturing method.
12. A method for producing a cell producing a specific antigen-specific antibody, which comprises screening a culture supernatant containing cells producing the specific antigen-specific antibody by the method according to any one of claims 1 to 10.
13. A method for producing a specific antigen-specific antibody, which comprises screening a culture supernatant containing cells that produce a specific antigen-specific antibody by the method according to claim 1.

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