WO2020171020A1 - 細胞の選抜方法、核酸の製造方法、組換え細胞の製造方法、目的物質の製造方法、医薬組成物の製造方法、及び試薬 - Google Patents
細胞の選抜方法、核酸の製造方法、組換え細胞の製造方法、目的物質の製造方法、医薬組成物の製造方法、及び試薬 Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
- C12N2529/10—Stimulation by light
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- the present invention relates to a cell selection method, a nucleic acid production method, a recombinant cell production method, a target substance production method, a pharmaceutical composition production method, and a reagent.
- Cell membrane proteins such as G protein-coupled receptors (GPCRs), transporters, ion channels, and cytokine receptors are known to be involved in various diseases, and have attracted attention as target molecules for diagnostic agents and medical drugs. There is.
- the cell membrane protein expresses a cell function or a pharmacological function by binding or binding to an extracellular substance, for example, a ligand composed of a low molecular weight compound, a peptide, a protein, or the like to activate or inhibit the function.
- -Multiple transmembrane proteins are more difficult to isolate and purify because they contain more hydrophobic structures than soluble proteins (eg cytokines, hormones, enzymes, nuclear receptors).
- soluble proteins eg cytokines, hormones, enzymes, nuclear receptors.
- purification examples that have been proved to be capable of maintaining the structure existing in nature and maintaining the function of the multiple transmembrane protein.
- a multi-pass transmembrane protein exerts its functionality in the state of being present in the lipid bilayer of cells.
- a technique for searching for a substance that specifically binds to a cell membrane protein for example, there is a method of using a large amount of a soluble domain or a partial peptide, which is a part of the cell membrane protein exposed outside the cell, and is purified.
- the purified soluble domain or partial peptide may be immobilized on a 96-well plate and the binding to a candidate specific binding substance may be evaluated by an ELISA method or the like.
- the substance selected by such a method binds specifically to the target cell membrane protein present in the living body with high affinity.
- the target cell membrane protein is produced in order to mimic the three-dimensional structure of the cell membrane protein in vivo. It is more effective to use mammalian cells that are expressed on the cell membrane of the cell.
- substances such as peptide ligands, protein ligands and specific antibodies can be secreted into the culture supernatant by culturing cells derived from human or non-human animals or recombinant cells to which gene recombination technology is applied.
- culturing cells derived from human or non-human animals or recombinant cells to which gene recombination technology is applied.
- production cell populations for example, antibody-producing hybridoma library
- Non-Patent Document 1 a method for identifying a specific antibody-producing cell applying a single cell analysis technique is being developed.
- one antibody-producing cell and an antigen protein are enclosed in a hydrophobic microdroplet.
- the presence or absence of binding between the antibody secreted from the antibody-producing cells and the antigen protein can be visualized, and the microdroplets containing the positive cells producing the specific antibody can be separated in an analyzer having a microchannel.
- Non-Patent Document 2 describes the principle of a method for identifying specific antibody-producing cells by applying a microdroplet and a microchannel.
- Non-Patent Document 1 Non-Patent Document 1
- Patent Literature 2 describes a technique of contacting a cell population expressing a target cell membrane protein with a candidate cell population on a slide glass, and identifying a cell producing an antibody against the target cell membrane protein from the candidate cell population. Has been done. However, since this method also cannot perform the above-mentioned washing step, when targeting a cell membrane protein that is extremely expressed on the cell membrane surface, it is necessary to confirm the presence or absence of binding between the antibody and the cell membrane protein. Is difficult.
- Patent Document 3 a candidate antibody-producing cell is introduced into a microwell coated with purified soluble cytokine receptor protein, and a desired antibody-producing cell having a binding property between the antibody secreted by the cell and the soluble cytokine receptor protein is disclosed.
- a technique for selecting is disclosed.
- the isolated and purified receptor protein does not always maintain the structure that exerts the function in the living body. Furthermore, it is extremely difficult to apply this method to a multiple transmembrane protein that is difficult to purify.
- the present invention provides a technique for more rapidly and efficiently selecting cells that produce a target substance that specifically binds to a cell membrane protein, and the use of cells selected by the technique to achieve the purpose of antibodies and the like. It is intended to provide a technique for manufacturing a substance.
- the target cells can be selected in a very short time.
- One aspect of the present invention is a method for selecting cells from a population of second cells, which is a method for selecting target cells that produce a target substance that specifically binds to a desired cell membrane protein. a) providing a substrate having a plurality of microwells, b) attaching first cells expressing the cell membrane protein on the cell surface to each of the microwells, c) Subsequent to step b), one or two second cells isolated from the population are introduced into each of the microwells, and the first cells and the second cells are introduced into the microwells.
- the step d) includes a visualization step of visualizing that the target substance has bound to the first cells.
- the visualization step includes adding a labeling substance that specifically binds to the target substance to the microwell.
- the labeling substance is a labeling antibody against the target substance.
- the label in the labeling substance is a fluorescent label.
- the labeling substance is an antibody labeled with a first fluorescent substance
- the first cell label for labeling the first cells adhering to the microwell with a second fluorescent substance is different from the fluorescence wavelength of the fluorescence emitted by the second fluorescent substance.
- the visualizing step includes visualizing a change in an intracellular signal transduction substance that accompanies activation of the cell membrane protein, which occurs when the target substance binds to the first cell.
- the cell membrane protein is a multiple transmembrane protein.
- the first cell is a cell into which a vector expressing the cell membrane protein has been introduced.
- the first cell is a tumor cell that expresses the cell membrane protein.
- the first cell is a non-tumor cell expressing the cell membrane protein.
- the target substance is an antibody.
- the second cell is derived from bone marrow, spleen, lymphoid tissue, or blood cell derived from a non-human animal immunized with the cell membrane protein or a nucleic acid encoding the cell membrane protein.
- the second cell is an immortalized cell.
- the second cell is a hybridoma.
- the second cell is derived from human lymphatic tissue or blood.
- the second cell is a cell immortalized by infection with Epstein-Barr virus.
- the second cell is a recombinant cell having an exogenous antibody gene and expressing the antibody.
- the antibody is a complete antibody, a functional antibody fragment, a single chain antibody, or a multispecific antibody.
- the antibody is a fully human antibody, a humanized antibody, or a chimeric antibody.
- the antibody is a feline antibody or a canine antibody.
- Another aspect of the present invention is a method for producing a nucleic acid, in which a nucleic acid encoding the target substance is obtained from a target cell selected from the second cell population by the above method.
- the target substance is an antibody.
- Another aspect of the present invention is a method for producing a recombinant cell in which a nucleic acid produced by the above method is introduced into a host cell to obtain a recombinant cell expressing the target substance.
- Another aspect of the present invention is a method for producing a target substance, which comprises culturing the recombinant cell produced by the above method and obtaining the target substance from the culture.
- Another aspect of the present invention is a method for producing a target substance, which comprises culturing a target cell selected from a population of second cells by the above method and obtaining the target substance from the culture.
- the target substance is an antibody.
- Another aspect of the present invention is to obtain a pharmaceutical composition containing the nucleic acid as an active ingredient by combining the nucleic acid produced by the above method with a pharmaceutically acceptable carrier or additive. Is a manufacturing method.
- Another aspect of the present invention is to obtain a pharmaceutical composition containing the target substance as an active ingredient by combining the target substance produced by the above method with a pharmaceutically acceptable carrier or additive. It is a method of manufacturing a product.
- Another aspect of the present invention is a reagent for detecting the desired cell membrane protein containing the target substance produced by the above method.
- cells producing a target substance that specifically binds to a cell membrane protein can be selected more quickly and efficiently. Furthermore, a target substance that specifically binds to a cell membrane protein, such as an antibody, can be easily produced.
- FIG. 6 is a photograph showing an example of an image of positive microwells in Example 4, (a) is transmitted light, (b) is fluorescence derived from CytoRed, and (c) is a result obtained by observing fluorescence derived from Alexa Fluor 488.
- 5 is a photograph showing an example of an image of a negative microwell in Example 4, where (a) is transmitted light, (b) is fluorescence from CytoRed, and (c) is fluorescence from Fluor 488.
- FIG. 9 is a diagram showing the results of subjecting a recombinant antibody derived from a hybridoma recovered from positive microwells in Example 5 to flow cytometry.
- FIG. 8 is a diagram showing the results of subjecting a hybridoma-derived recombinant antibody recovered from negative microwells in Example 5 to flow cytometry.
- 9 is a photograph showing an example of an image of a positive microwell in Example 6, where (a) is transmitted light, (b) is fluorescence derived from CytoRed, and (c) is a result obtained by observing fluorescence derived from Alexa Fluor 488.
- FIG. 9 is a photograph showing an example of an image of a negative microwell in Example 6, where (a) is transmitted light, (b) is fluorescence from CytoRed, and (c) is the result of observing fluorescence from Alexa Fluor 488.
- FIG. 7 is a diagram showing the results of subjecting a lymphocyte-derived gene recombinant antibody recovered from positive microwells in Example 7 to flow cytometry.
- FIG. 7 is a diagram showing the results of subjecting a lymphocyte-derived gene recombinant antibody collected from negative microwells in Example 7 to flow cytometry.
- Example 9 is a photograph showing an example of an image of a microwell in Example 9, where (a) is the result of observation of transmitted light, (b) is the fluorescence derived from Alexa Fluor 488, and (c) is the fluorescence derived from DyLight 650.
- the cell selection method of the present invention is to select a target cell that produces a target substance that specifically binds to a desired cell membrane protein from a second cell population.
- the target cell may be referred to as a positive cell.
- the desired cell membrane protein may be referred to as "target cell membrane protein”.
- the target substance may be referred to as a "specific binding substance for cell membrane protein” or simply "specific binding substance”.
- the phrase “selecting cells” can be restated as “identifying cells”.
- FIG. 1 shows an outline of a cell selection method according to one embodiment of the present invention.
- A shows a substrate 1 having a plurality of microwells 2.
- B shows a state in which the first cells 3 are attached to the microwells 2.
- C shows a state in which the first cells 3 and the second cells 5 coexist in the microwell 2.
- D shows a state in which the target substance 6 secreted by the second cell 5 is bound to the surface of the first cell 3.
- E represents a state in which the labeling substance 7 is bound to the target substance 6 on the surface of the first cell 3.
- a substrate having a plurality of microwells is used.
- a microwell refers to a micro-sized well (recess, recess) in which approximately 1 to 3 mammalian cells or avian cells are contained.
- the microwell is a small hole having a bottom.
- the inner diameter of the opening is about 10 ⁇ m to 50 ⁇ m, and the depth is about the same as the inner diameter of the opening.
- the shape of the microwell is typically cylindrical.
- it may be a cylindrical shape composed of a plurality of flat surfaces such as a polygonal prism such as a quadrangular prism or a hexagonal prism, or a mortar shape such as an inverted cone shape or an inverted pyramid shape.
- a shape in which two or more of these shapes are combined and connected may be used.
- the bottom surface of the cone serves as the opening of the microwell and a part of the apex is cut off (that is, a truncated cone shape or a truncated pyramid shape). ..
- the diameter (inner diameter) of the opening can be appropriately determined in consideration of the type and number of cells stored in the microwell.
- the diameter is preferably about 20 ⁇ m to 40 ⁇ m.
- the depth of the microwell is preferably about the same as the diameter of the opening.
- the number of microwells (density) per unit area on the substrate is not particularly limited, and can be appropriately determined in consideration of, for example, the total number of second cells to be searched per time and the expression frequency of target cells.
- the number may be in the range of 20,000 to 200,000 per cm 2 .
- the distance (pitch) between the microwells on the substrate is not particularly limited, and can be set as appropriate within a range that does not affect adjacent microwells.
- the distance between the centers of the openings of adjacent microwells is preferably about 1.5 to 3 times the diameter of the openings.
- the material of the substrate is not particularly limited, but when performing the visualization step described later, it is preferable that it is a transparent material that does not have autofluorescence.
- Substrates with multiple microwells are available from the market.
- a substrate (microwell chamber) having a plurality of microwells having a diameter of 10 ⁇ m, 20 ⁇ m, or 30 ⁇ m is commercially available from As One.
- the target cell membrane protein in the present invention is not particularly limited, and all cell membrane proteins represented by multiple transmembrane proteins can be targeted.
- GPCRs G protein-coupled receptors
- ion channels transporters
- CD antigens CD antigens
- cell adhesion molecules CD antigens
- cancer antigens cell adhesion molecules
- cancer antigens viral antigens, etc.
- animal species from which the cell membrane protein is derived is not particularly limited.
- a first cell expressing a desired cell membrane protein on the cell surface is used by adhering it to a microwell.
- the first cell is not particularly limited as long as the desired cell membrane protein is expressed on the cell surface.
- One embodiment is a recombinant cell into which a vector expressing a target cell membrane protein has been introduced.
- the gene for the full-length target cell membrane protein is inserted into an appropriate expression vector (eg pcDNA, pEF/FRT/V5-DEST, etc.).
- this vector is introduced into cells such as CHO cells, COS cells, HEK293 cells, and NIH3T3 cells to obtain recombinant cells that transiently or stably express the target cell membrane protein on the cell membrane.
- This recombinant cell can be used as the first cell.
- the expression level of the target cell membrane protein on the cell membrane is preferably increased by a factor of 5 or more as compared with cells without the expression vector.
- a tumor cell in which a target cell membrane protein is overexpressed as compared with a normal cell can be used as the first cell.
- the tumor cell for example, a single cell obtained from a surgically excised organ can be used.
- tumor cells can be obtained from ATCC and cell sales companies.
- various cells derived from a normal tissue expressing a target cell membrane protein, for example, a non-human or human tissue can be used as the first cell.
- a normal tissue expressing a target cell membrane protein for example, a non-human or human tissue
- blood cells nerve cells, vascular endothelial cells, vascular smooth muscle cells, immune cells, adipocytes, skeletal muscle cells, lymphocyte cells, skin cells, etc.
- blood cells may be converted into iPS by a known method, and then differentiated into specific tissue cells may be used as the first cells.
- a target cell that produces a target substance that specifically binds to a desired cell membrane protein is selected from the second cell population.
- the target substance includes a polypeptide having a known or unknown structure, a cyclic peptide, or a protein that selectively binds to a specific cell membrane protein. More specifically, specific binding substances consisting of peptide hormones, cytokines, antibodies, artificial polypeptides, artificial cyclic peptides, etc. are included.
- the first cell and the second cell are allowed to coexist in the microwell.
- the second cell will be specifically described below.
- the second cell is not particularly limited as long as it is a cell expected to produce a desired target substance.
- various cells derived from non-human or human tissues such as blood cells, nerve cells, vascular endothelial cells, vascular smooth muscle cells, immune cells, adipocytes, skeletal muscle cells, lymphocyte cells, skin cells, etc. It can be used as a cell.
- the tissue of a non-human animal can be separated, treated with collagenase, and then filtered through a mesh of 30 to 100 ⁇ m to form a single cell, which can be used as the second cell.
- a single cell obtained from human blood or a surgically excised organ can be used as the second cell.
- tumor cells can be used as the second cells.
- the tumor cell for example, a single cell obtained from a surgically excised organ can be used.
- tumor cells can be obtained from ATCC and cell sales companies.
- Recombinant cells can be used as the second cells.
- a cDNA library containing a gene encoding a target substance is incorporated into an expression vector such as pcDNA, pEF/FRT/V5-DEST, or Mammalian PowerExpress System. Then, this vector is transiently introduced into cells such as CHO cells, COS cells, HEK293 cells and NSO cells to obtain recombinant cells. This recombinant cell can be used as the second cell.
- a recombinant cell that has survived using the drug resistance gene retained in the vector and that constantly expresses a gene can be used as the second cell.
- a cDNA library may be incorporated into a viral vector derived from adenovirus, lentivirus, etc., and this may be used to infect CHO cells, HEK293 cells, NIH3T3 cells, etc. as the second cells.
- a gene encoding one kind of target substance is introduced into the recombinant cell.
- the target substance (specific binding substance) is an antibody
- the second cell is an antibody-producing cell
- the term “antibody” can be replaced with “immunoglobulin”.
- the antibody in the present invention includes a functional fragment thereof.
- the “functional fragment of an antibody” refers to a partial fragment of an antibody (that is, an immunoglobulin) that retains at least one action on an antigen.
- the partial fragment include F(ab′) 2 , Fab, Fv, disulfide bond Fv, single chain antibody (scFv, VH-VL), VH, and polymers thereof, and heavy chain CH3 thereof. Examples include fusion with regions.
- the antibody in the present invention may be a multispecific antibody. An example thereof is a diabody (for example, WO 93/11161), which is a type of bispecific antibody.
- the class (isotype) of the antibody in the present invention is not particularly limited.
- it may be any class such as IgG, IgM, IgA, IgD, and IgE.
- the subclass of the antibody may be any subclass such as IgG1, IgG2, IgG3, and IgG4.
- the antibody may be a fully human antibody, a humanized antibody or a chimeric antibody.
- Antibody-producing cells such as B cells and plasma cells derived from human lymphoid tissue or blood can be used as the second cells.
- B cells and plasma cells collected from healthy people, patients with cancer, patients with known or unknown infectious diseases, patients with autoimmune diseases, vaccinated persons, etc. It can be used as a cell.
- cell concentration may be performed in order to more efficiently identify antibody-producing cells for the target cell membrane protein.
- activated B cells or plasma cells obtained from bone marrow, spleen, lymphoid tissue, or blood cells derived from a non-human animal immunized with a target cell membrane protein can be concentrated and used as the second cell population.
- activated B cells or plasma cells obtained from human lymphoid tissue or blood-derived cells can be concentrated and used as a second cell population.
- Enrichment of activated B cells or plasma cells can be performed using, for example, the cell surface CD antigen as an index.
- antibody magnetic beads against a specific CD antigen can be used.
- CD antigens examples include CD2, CD3, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD34, CD40, CD43, CD45R, CD49b, CD56, CD61, CD79a, CD90.2, CD138, CD235a.
- degree of enrichment examples include enrichment of activated B cells or plasma cells by 50 times or more from a population of about 10,000,000 lymphoid cells derived from lymphoid tissue.
- DNA immunization a method of immunizing with an artificial bilayer membrane or virus-like nanoparticles presenting a target cell membrane protein as an antigen.
- DNA immunization a method of immunizing a vector in which a cDNA sequence encoding a target cell membrane protein is inserted into a protein expression vector.
- DNA immunization is preferable because it is possible to obtain a more specific antibody with high affinity.
- Immortalized cells such as hybridomas can be used as the second cells.
- a hybridoma is obtained by collecting immune cells from a non-human animal immunized with a target cell membrane protein and cell fusion of the immune cells with myeloma. This hybridoma can be used as the second cell.
- Known methods can be used for cell fusion, selection of hybridomas, and cloning.
- cell fusion can be performed by a method using polyethylene glycol or a method of applying a voltage to a mixed solution of immune cells and myeloma.
- selection of hybridomas can be performed by culturing using a HAT selection medium.
- Cells can be immortalized by methods other than the hybridoma method.
- cells immortalized by Epstein-Barr virus infection can also be used as the second cells.
- Recombinant cells into which the antibody gene has been introduced can be used as the second cells.
- a cDNA library is prepared from B cells or plasma cells derived from the lymph tissue or blood cells of an immunized animal.
- An antibody or antibody fragment gene is selectively amplified from this cDNA library.
- the amplified genes are modified to produce an antibody gene library so as to express various forms of antibody molecules such as complete antibody, functional antibody fragment, single chain antibody, or multispecific antibody.
- This gene library is incorporated into a vector such as pcDNA, pEF/FRT/V5-DEST, Mammalian Power Express System.
- this vector is transiently introduced into cells such as CHO cells, COS cells, HEK293 cells and NSO cells to obtain recombinant cells.
- This recombinant cell can be used as the second cell.
- a recombinant cell that has survived using the drug resistance gene retained in the vector and that constantly expresses a gene can be used as the second cell.
- the animal species from which the second cells are derived is not particularly limited, but mammalian cells or avian cells are preferably used.
- mammals include mouse, rat, guinea pig, rabbit, monkey, cow, horse, dog, cat, goat, sheep, pig, camel, alpaca and the like.
- birds include chickens, ducks and turkeys.
- the above-mentioned first cells are adhered to microwells. This allows the first cell to be stored and immobilized in the microwell without impairing its cell function.
- the number of the first cells adhered to the microwell is not limited as long as it can secure a space for receiving the second cells, but is preferably 1 to 2.
- first cell and second cell ⁇ Coexistence of first cell and second cell>
- one or two second cells are introduced into the microwell to which the first cells adhere, and the first cells and the second cells coexist.
- the target substance produced (secreted) by the second cell comes into contact with the surface of the first cell. It is preferable to introduce one second cell into the microwell.
- the incubation conditions can be determined, for example, in consideration of the properties of the target substance produced by the second cells and the survival time of the second cells.
- a cytokine eg, IL-
- the mixture is incubated at 25 to 37° C. for 15 minutes to 6 hours.
- the microwell containing the first cell to which the target substance is bound (positive microwell) is specified. In other words, the presence or absence of binding between the target cell membrane protein expressed on the surface of the first cell and the substance secreted from the second cell in each microwell is detected.
- a visualization method is preferably used. That is, a preferred embodiment includes a visualization step of visualizing that the specific binding substance has bound to the first cell.
- the visualization method There is no particular limitation on the visualization method.
- One example is the direct technique of visualizing the surface of the first cells.
- Examples of the direct method include a method using a labeling substance that specifically binds to a target substance. That is, the labeling substance is brought into contact with the first cells that coexist with the second cells in the microwell.
- the labeling substance is, for example, a labeled antibody.
- a tag eg, FLAG, V5
- Fc portion of the antibody is added to the specific binding substance.
- a recombinant cell obtained by introducing this cDNA library into a CHO cell or the like is used as the second cell.
- a labeled antibody eg, labeled anti-FLAG antibody, labeled anti-V5 antibody
- a labeled anti-Fc antibody eg, labeled anti-IgG antibody
- the first cell and the second cell are allowed to coexist in the microwell, and then incubated under predetermined conditions as necessary, and then the labeling substance is added.
- a label with a fluorescent substance fluorescent complex
- a fluorescent protein an enzyme, or the like
- the fluorescent substance include Alexa Fluor (registered trademark), Aqua, Texas Red (registered trademark), fluorescein and its derivative, rhodamine and its derivative, Cascade Blue (registered trademark), phycoerythrin, and DyLight (registered trademark).
- Alexa Fluor 488 is preferably used.
- fluorescent proteins include green fluorescent protein (GFP).
- the enzyme include alkaline phosphatase, horseradish peroxidase, luciferase, and the like.
- the primary signal bound to the target substance can be detected using the strong signal emitted by the labeled substance (eg, labeled antibody) as an index. Then, a microwell containing a positive signal (positive microwell) can be specified according to the characteristics of the label using a fluorescence microscope, a light emission microscope, an inverted microscope, or a device including these micromanipulation devices.
- the labeled substance eg, labeled antibody
- the indirect method is, for example, a substance in which a specific binding substance promotes the function of a cell membrane protein (for example, an endogenous ligand, a polypeptide considered to have an agonist activity, a cyclic peptide, an antibody, a protein, etc.).
- a reporter gene capable of visualizing changes in intracellular signal transduction substances accompanying activation of the target cell membrane protein is previously introduced into the first cells expressing the target cell membrane protein. Then, when the target substance binds to the target cell membrane protein, the change in the expression of the reporter gene can be visualized. As a result, the binding between the cell membrane protein and the target substance can be indirectly visualized.
- a positive microwell can be identified using a light emission microscope or an instrument including a light emission microscope.
- Another example that can be applied to the indirect method is to visualize changes in intracellular cAMP.
- the fluctuation of intracellular cAMP amount was caused by the specific phosphorylation of CREB (cAMP response element binding protein) observed when a substance that activates a cell membrane protein was added, which was fluorescently labeled with an antiphosphorylation antibody. It can be detected with the following antibody.
- visualization of changes in intracellular Ca can be mentioned.
- the fluctuation of Ca in the cell can be indirectly visualized by the change in the fluorescence intensity of the intracellular fluorescent Ca indicator.
- first fluorescent substance an antibody labeled with a fluorescent substance (first fluorescent substance) is adopted as the labeling substance, and the first cells are labeled with another fluorescent substance (second fluorescent substance).
- second fluorescent substance a substance that emits a fluorescence different from the fluorescence wavelength of the fluorescence emitted by the first fluorescent substance is adopted. That is, the fluorescence wavelength of the fluorescence emitted by the first fluorescent substance is different from the fluorescence wavelength of the fluorescence emitted by the second fluorescent substance.
- the second fluorescent substance Calcein-AM, Fluorescein diacetate (FDA), Carboxyfluorescein diacetate (CFDA), CytoRed, Propidium iodide (PI), Ethidium bromide (EB), Acridine orange (AO), DAPI, Hoechst 33342. Use Hoechst 33258.
- Alexa Fluor 488 is used as the first fluorescent substance. Thereby, the first cell to which the labeled antibody is bound and the first cell to which the labeled antibody is not bound can be easily distinguished by the difference in the emitted fluorescence. Then, the positive microwell can be identified using a fluorescence microscope or an instrument including the fluorescence microscope.
- -Antibodies are usually secreted extracellularly, but it is known that there are membrane-type antibodies that are not secreted extracellularly. Therefore, it is possible that non-secreted membrane-type antibody is present on the surface of the second cell. In this case, when the labeled anti-IgG antibody is added, not only the antibody bound to the first cell but also the membrane-type antibody on the second cell may be bound. However, according to the above-described embodiment using the first fluorescent substance and the second fluorescent substance, only the antibody bound to the first cell can be specifically detected.
- the washing step after adding the labeling substance, it is preferable to perform a washing step to remove the excess labeling substance.
- the washing is not particularly limited as long as it is performed under the condition that the first cell and the second cell are retained in the microwell (the condition that the first cell and the second cell are not washed out).
- the microwells may be gently washed several times with phosphate buffer, HBSS, or cell culture medium.
- the signal in which the target substance binds to the target cell membrane protein can be detected with high sensitivity even when the expression level of the target cell membrane protein on the cell membrane of the first cell is extremely low.
- the second cells are collected as target cells.
- the recovery of the second cells from the microwell can be performed using, for example, a micromanipulator.
- a capillary having a diameter of several ⁇ m to 50 ⁇ m can be inserted into a positive microwell, and the second cells can be aspirated and collected alive.
- the operation by the micromanipulator may be automatic or manual.
- cell picking system As One
- CellCelector Automated Lab Solution
- the recovered second cells are preferably recovered in an appropriate medium for cell culture or in a cell lysate (Lysis buffer) for rapid extraction without degrading mRNA.
- nucleic acids encoding two kinds of target substances are obtained and isolated by the method described below, and one of the nucleic acids is adopted as the target nucleic acid (target gene). can do.
- the present invention includes a method for producing a nucleic acid that obtains a nucleic acid (gene) encoding a target substance from a target cell selected from a second cell population by the above method.
- the present invention also includes a method for producing a recombinant cell in which the nucleic acid is introduced into a host cell to obtain a recombinant cell expressing the target substance.
- the present invention includes a method for producing a target substance in which the recombinant cell is cultured and the target substance is obtained from the culture.
- the target substance is an antibody.
- a known method can be used. For example, a reverse transcription reaction and a PCR method are combined to synthesize cDNA. Then, the target nucleic acid can be isolated from the cDNA.
- a known method can be used as a method for obtaining a recombinant cell that expresses the target substance.
- the isolated target nucleic acid is incorporated into an appropriate vector.
- This vector can be introduced into host cells such as Escherichia coli, yeast and mammalian cells (for example, CHO cells, HEK293 cells or NSO cells) to obtain the target recombinant cells.
- the recombinant cell is cultured, and the target substance can be obtained from the culture (for example, culture supernatant).
- Isolation of the antibody gene from the second cells can be performed, for example, by a combination of methods described in WO 2009/091048, WO 2009/110606, and WO 2011/027808, or Nobuyuki Kurosawa. , Megumi Yoshioka, Rika Fujimoto, Fuminori Yamagishi and Masaharu Isobe, "Rapid production of antigen-specific monoclonal antibodies from a variety of animals", BMC Biology, 10:80, 2012 method) (MAGrahd) it can.
- a recombinant cell expressing a complete antibody, a functional antibody fragment, a single chain antibody, or a multispecific antibody.
- recombinant cells expressing fully human, humanized, or chimeric antibodies can be constructed.
- recombinant cells expressing feline or canine antibodies can be constructed.
- the present invention includes a method for producing a target substance, which comprises culturing the target cell selected from the second cell population by the above method and obtaining the target substance from the culture.
- a known method can be used. For example, various chromatographies such as affinity, ion exchange and gel filtration can be adopted. Examples of the ligand in affinity chromatography include protein A, protein G, anti-FLAG antibody, anti-V5 antibody and the like.
- the present invention includes a method for producing a pharmaceutical composition, which comprises combining the nucleic acid produced by the above method with a pharmaceutically acceptable carrier or additive to obtain a pharmaceutical composition containing the nucleic acid as an active ingredient. To do.
- the present invention also provides a method for producing a pharmaceutical composition, which comprises combining the target substance produced by the above method with a pharmaceutically acceptable carrier or additive to obtain a pharmaceutical composition containing the target substance as an active ingredient. Including a method.
- the target substance produced by the present invention is useful as an active ingredient of a pharmaceutical composition (therapeutic agent).
- the pharmaceutical composition may include a target substance such as the antibody produced by the present invention and a pharmaceutically acceptable carrier or additive.
- the pharmaceutical composition blocks or activates a target cell membrane protein-specific intracellular signal transduction mechanism.
- the pharmaceutical composition can be administered orally or parenterally, systemically or locally.
- the administration form include injection type, nasal administration type, pulmonary administration type, transdermal administration type and the like.
- it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the target substance is an antibody
- the dose of the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per 1 kg of body weight. Alternatively, for example, the dose can be selected within the range of 0.001 to 100000 mg antibody/body per patient. However, the dose of the antibody is not limited to these ranges.
- the aforementioned pharmaceutical composition can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A).
- the carrier or additive include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavors, preservatives, stabilizers, buffers (phosphoric acid). , Citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrating agents, lubricants, fluidity promoters, and corrigents.
- Examples include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like. It may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin and immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine and lysine.
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. It may be used in combination with a suitable solubilizing agent such as alcohol (ethanol or the like), polyalcohol (propylene glycol, PEG or the like), nonionic surfactant (polysorbate 80, HCO-50) or the like.
- ethanol ethanol or the like
- polyalcohol propylene glycol, PEG or the like
- nonionic surfactant polysorbate 80, HCO-50
- the active ingredient, antibody may be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly[methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions). , Nanoparticles, nanocapsules, etc.) (see “Remingto's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.). Further, a technique is known in which an antibody is directly fused to another drug to enhance the therapeutic effect, and it can be applied to the pharmaceutical composition.
- nucleic acid (gene) obtained in the present invention for example, an antibody gene
- a gene therapy vector to prepare a gene therapy drug.
- retrovirus vector in addition to direct administration by Naked plasmid, administration by packaging in liposome or the like, retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, Method of incorporating into various viral vectors such as adeno-associated virus vector and HVJ vector (see Adolph “Viral Genome Method”, CRC Press, Florid (1996)), coating on a bead carrier such as colloidal gold particles (International Publication No. 93 /17706) and the like, and the like.
- the gene therapeutic agent may be administered by any method as long as an antibody as an active ingredient is expressed in vivo and the action can be exerted.
- a sufficient amount is administered by a suitable parenteral route.
- Parenteral routes include intravenous, intraperitoneal, subcutaneous, intracutaneous, adipose tissue, mammary gland tissue, inhalation, or intramuscular route via injection, infusion, or gas-induced particle bombardment (electron gun). Etc.), a method via a mucosal route such as a nasal drug, and the like.
- the gene therapeutic agent is administered to cells using ex vivo liposome transfection, particle bombardment (US Pat. No. 4,945,050), or viral infection, and the cells are reintroduced into the animal. May be administered by administration.
- the present invention includes a reagent for detecting a desired cell membrane protein containing the target substance produced by the above method.
- the antibody is brought into contact with blood cells of human origin or non-human mammal origin using a reagent containing the antibody (target substance) produced by the method of the present invention.
- a labeling substance composed of a fluorescent substance or a dye is brought into direct or indirect contact.
- the expression of the desired cell membrane protein can be detected by flow cytometry or a plate reader.
- the expression of a desired cell membrane protein can be detected by bringing the above antibody into contact with a pathological tissue piece derived from human or non-human mammal.
- kits for detecting a cell membrane protein containing the above reagents.
- a kit for detecting a cell membrane protein can be constructed by combining the above reagent with a labeling substance or the like.
- the present invention includes a method for detecting the desired cell membrane protein, which uses the target substance produced by the above method.
- the present invention includes the use of the target substance produced by the above method for detecting the desired cell membrane protein.
- Examples 1 to 8 below cells that produce a specific binding antibody to an apelin receptor (hereinafter sometimes abbreviated as APLNR), which is one of human GPCRs, were mainly selected. Furthermore, an antibody gene was isolated from the selected cells to construct a recombinant cell expressing the antibody. Furthermore, the functionality of the antibody expressed by the recombinant cells was evaluated.
- APLNR apelin receptor
- APLNR expression vector An artificial synthetic gene (SEQ ID NO: 1) was prepared by optimizing the human APLNR gene sequence (NM_005161.4) registered in Gene Bank to the mouse amino acid codon. Using this artificially synthesized gene, a vector pCI-APLNR-GroEL containing a fusion gene of human APLNR gene and GroEL gene was constructed in accordance with the method described in International Publication No. 2012/043533 (Japanese Patent No. 5315495). did.
- Flp-In-CHO cells were cultured in Ham's F-12 medium (Invitrogen) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 ⁇ g/mL streptomycin. Lipofectamin 2000 was used to simultaneously introduce pEF-FRT-APLNR and pOG44 plasmid (Invitrogen) into these cells. From the day after the introduction, the medium was changed to Ham's F-12 medium containing 500 ⁇ g/mL hygromycin (Invitrogen), and the cells were cultured for 2 weeks while changing the medium every 3rd day. Hygromycin resistant cells were cloned from the formed colonies by the limiting dilution method.
- Phycoerythrin (PE)-labeled anti-mouse IgG antibody (Beckman Coulter, Inc.) was used as a secondary antibody, and the obtained hygromycin-resistant cells and anti-apelin antibody (R&D) or anti-V5 tag antibody (Invitrogen) were used. Binding was analyzed by flow cytometer. As a result, it was confirmed that the obtained hygromycin-resistant cells were PE-positive and stably expressed human APLNR. Hereinafter, this cell is referred to as a human APLNR stably expressing CHO cell line (first cell).
- (2-2) Preparation of cell population (second cell) containing antibody-producing cells from spleen
- the spleen is excised from the mouse immunized with DNA in (2-1) and collected in a 6-well plate containing refrigerated HBSS. did. After removing the attached connective tissue and adipose tissue, the spleen was loosened in fresh HBSS to release lymphocytes. The cells were harvested and resuspended in 10 mL HBSS. After the unbroken tissue was separated with a cell strainer, the cells were collected by centrifugation at 2000 rpm for 5 minutes. The collected cells were suspended in 1 mL of hemolysis solution and incubated at 37° C. for 5 minutes to remove red blood cells.
- Lymphocyte cells were collected by centrifugation at 1000 rpm for 5 minutes. Using the EasySep Mouse Biotin Positive Selection Kit (STEMCELL TECHNOLOGIES), approximately 1.3 ⁇ 10 5 cells containing the candidate of the desired antibody-producing cells (target cells) from 2.5 ⁇ 10 7 of the above lymphocyte cells. The cell population (second cell) was isolated.
- Cell fusion of splenocytes and myeloma cells was carried out using a cell fusion device ECFG21 (Neppagene). After cell fusion, RPMI1640 medium (FBS-containing, antibiotic-free) in an amount twice that of the cell solution was added, and the mixture was allowed to stand in a CO 2 incubator for 1 hour. The cells were collected by centrifugation and suspended in HAT medium (RPMI1640 with 10% FBS, 2-mercaptomethanol (x500), HFCS (x100), HAT (x50)). Antibody-producing hybridoma cells were cloned and cultured by a conventional method using a 96-well plate, a 24-well plate and a 10 cm dish.
- HAT medium RPMI1640 with 10% FBS, 2-mercaptomethanol (x500), HFCS (x100), HAT (x50)
- a microwell chamber ASMC30-20P (Azuwan) was prepared.
- This microwell chamber is a substrate in which 84,640 microwells having a diameter of 30 ⁇ m are arranged at equal intervals in an area of about 1.5 cm ⁇ about 2.4 cm.
- the depth of each microwell is equal to the diameter of the microwell.
- the pitch between the microwells is twice the diameter of the microwells.
- it is general to store and use one cell in a microwell. However, in this example, the experiment was performed by storing the first cell and the second cell, that is, two or more cells in the microchamber. This will be described below.
- Human APLNR stable expression CHO cells (first cells) were suspended in F-12 medium (containing 10% FBS, Penicillin/Streptomycin) to prepare a cell suspension of 3 ⁇ 10 5 cells/500 ⁇ L. This cell suspension was filled in each microwell. The microchamber was centrifuged twice at 300 rpm for 2 minutes to prepare 1 or 2 first cells in each microwell. After washing the microchamber with F-12 medium, 500 ⁇ L of F-12 medium was added. After incubating at 37° C. for 1 hour in a CO 2 incubator, the first cells were allowed to adhere to the bottom surface of the microwell while maintaining the functionality as cells.
- F-12 medium containing 10% FBS, Penicillin/Streptomycin
- CytoRed solution adjusted to a concentration of 10 nM with F-12 medium was added, and the mixture was further incubated at 37°C for 1 hour to stain the first cells. After washing with F-12 medium three times to remove excess CytoRed, 1 mL of F-12 medium was filled in the microchamber.
- the hybridoma (second cell) population prepared in Example 3 was cultured to prepare a cell suspension of 3 ⁇ 10 5 cells/500 ⁇ L. This cell suspension was filled in each microwell. The microchamber was centrifuged twice at 300 rpm for 2 minutes to prepare 1 or 2 second cells in each microwell. After washing the microchamber with a medium, an appropriate amount of the medium was added and incubated at 37° C. for 30 minutes to allow the antibody to be secreted from the hybridoma.
- Alexa Fluor 488-labeled anti-mouse IgG antibody (secondary antibody; labeling substance) diluted 500 times with RPMI1640 (containing 10% FBS) was applied, and the mixture was incubated at 37°C for 30 minutes. Incubated. After washing three times with RPMI1640 (phenol red-free, 1% FBS-containing), 1 mL of RPMI1640 was added. A microchamber was set in a cell picking system (As One Co., Ltd.), and information of transmitted light images of all microwells and two types of fluorescence images were acquired.
- CytoRed fluorescence was detected under the conditions of an excitation wavelength of 543 nm and a fluorescence wavelength of 593 nm.
- the fluorescence detection of Alexa Fluor 488 was performed under the conditions of excitation wavelength 482 nm and fluorescence wavelength 536 nm.
- FIGS. 2(a) to 2(c) are examples of images of hybridomas that produce an antibody that specifically binds to APLNR on the surface of the first cell, that is, positive microwells determined to contain the target cells.
- FIGS. 3(a) to 3(c) are images of a hybridoma producing an antibody that does not specifically bind to APLNR on the surface of the first cell, that is, an image of a negative microwell in which it was determined that cells of the non-target were stored. This is an example. 2 and 3, (a) shows transmitted light, (b) shows fluorescence derived from CytoRed, and (c) shows fluorescence derived from Alexa Fluor 488.
- the fluorescence of Alexa Fluor 488 was observed at the same position as the CHO cells labeled with CytoRed. That is, the CHO cells in the microwell coexisting with the target cells were co-stained with Alexa Fluor 488 and Cyto Red. From this, it was confirmed that the Alexa Fluor 488-labeled anti-IgG antibody was bound to the surface of the CHO cells.
- hybridoma was aspirated using a capillary having a diameter of several ⁇ m to several tens of ⁇ m and collected in the cell lysate. Finally, at least 3 independent hybridomas were selected.
- the selection of hybridoma which normally takes about 60 days, could be completed within 7 days.
- the oligo dT magnet was washed with a washing solution, and then cDNA synthesis was performed by a reverse transcription reaction. After cleaning the magnet, a 5'terminal translational reaction was performed.
- the gene of the antibody heavy chain variable region (VH region) and the gene of the antibody light chain variable region (VL region) were isolated and amplified by the 5'race PCR method. The PCR was performed twice in order to increase the specificity of the amplification product.
- the first forward primer (SEQ ID NO: 3) that commonly amplifies the VH region and the VL region, the first reverse primer (SEQ ID NO: 4) that specifically amplifies the VH region, and the VL region were A second reverse primer (SEQ ID NO: 5) that specifically amplifies was mixed and used.
- a second forward primer SEQ ID NO: 6
- a third reverse primer SEQ ID NO: 7
- a second forward primer SEQ ID NO: 6
- a fourth reverse primer SEQ ID NO: 8
- the gene for the VL region amplified in (5-1), the gene for the antibody light chain constant region, and the promoter region necessary for gene expression are fused using PCR to express a full-length antibody light chain.
- An antibody expression unit was constructed. By introducing these antibody expression units into mammalian cells, recombinant cells that transiently express the desired antibody (IgG) can be obtained.
- HEK293FT cells were co-administered with the above two antibody expression units. Introduced. That is, HEK293FT cells were seeded on a collagen-coated 96-well plate at 1.5 ⁇ 10 4 cells/100 ⁇ L/well. Lipofectamine 2000 was used to co-introduce the two antibody expression units constructed in (5-2) into HEK293FT cells. Cell supernatants were collected 3 days after the introduction and used for the binding evaluation of the expressed antibody.
- This cell suspension was mixed with the cell supernatant recovered in (5-3) in a 96-well plate and incubated at 4° C. for 1 hour. After incubation, the cells were washed twice with 100 ⁇ L FACS buffer. A dilution of a fluorescently labeled anti-IgG antibody (secondary antibody) was added to each well in an amount of 50 ⁇ L and incubated at 4° C. for 1 hour to bind the secondary antibody to the antibody bound to the CHO cell surface. After washing the cells twice with 100 ⁇ L of FACS buffer, the cells were suspended in 80 ⁇ L of FACS buffer, and the fluorescence intensity on the cell surface was measured by the flow cytometry method.
- a fluorescently labeled anti-IgG antibody secondary antibody
- FIG. 4 shows the results of flow cytometry. That is, the antibody expressed by the recombinant cells obtained in (5-3) had binding properties to human APLNR-expressing CHO cells. The antibody did not show binding to wild type CHO cells that did not express APLNR. On the other hand, as shown in FIG. 5, in the comparative example using the unintended hybridoma recovered from the negative microwell, the antibody expressed by the recombinant cell did not have the binding property to the human APLNR stably expressing CHO cell. .. From the above, it was shown that the antibody obtained in this example has specific binding properties to human APLNR-expressing CHO cells.
- CHO cells stably expressing human APLNR (first cells) were adhered to the bottom surface of the microwell in the same manner as in Example 4. Further, the first cells were stained with CytoRed, and 1 mL of F-12 medium was filled in the microchamber.
- the lymphocyte (second cell) population prepared in Example 2 was suspended in RPMI1640 to prepare a cell suspension of 3 ⁇ 10 5 cells/500 ⁇ L. This cell suspension was filled in each microwell. The microchamber was centrifuged twice at 300 rpm for 2 minutes, and the second cell was prepared so that one or two secondary cells were stored in each microwell. After washing the microchamber with RPMI1640, 1 mL of RPMI1640 was added and incubated at 37° C. for 30 minutes to promote antibody production from lymphocytes.
- Alexa Fluor 488-labeled anti-mouse IgG antibody (secondary antibody; labeling substance) diluted 500 times with RPMI1640 (containing 10% FBS) was applied, and the mixture was incubated at 37°C for 30 minutes. Incubated. After washing three times with RPMI1640 (phenol red-free, 1% FBS-containing), 1 mL of RPMI1640 was added. A microchamber was set in a cell picking system (As One Co., Ltd.), and information of transmitted light images of all microwells and two types of fluorescence images were acquired.
- FIGS. 6(a) to 6(c) are examples of images of positive microwells in which it is determined that lymphocytes that produce an antibody that specifically binds to APLNR on the surface of the first cell, that is, target cells are stored. ..
- FIGS. 7(a) to 7(c) are images of lymphocytes that produce antibodies that do not specifically bind to APLNR on the surface of the first cell, that is, images of negative microwells in which it was determined that cells of the non-target were stored. Is an example. 6 and 7, (a) shows transmitted light, (b) shows fluorescence derived from CytoRed, and (c) shows fluorescence derived from Alexa Fluor 488.
- Alexa Fluor 488 fluorescence was observed at the same position as the CHO cells labeled with CytoRed. That is, the CHO cells in the microwell coexisting with the target cells were co-stained with Alexa Fluor 488 and Cyto Red. In particular, the fluorescence of Alexa Fluor 488 was strongly observed near the target cells (lymphocytes). From this, it was confirmed that the Alexa Fluor 488-labeled anti-IgG antibody was bound to the surface of the CHO cells.
- the fluorescence of AlexaFluor488 is observed at the same position as the CHO cells labeled with CytoRed. None, it was only observed from the cell membrane surface of lymphocytes. That is, the Alexa Fluor 488-labeled anti-IgG antibody did not bind to the surface of the CHO cells, and the CHO cells were not co-stained with Alexa Fluor 488 and CytoRed. From this, it was confirmed that the Alexa Fluor 488-labeled anti-IgG antibody was not bound to the surface of the CHO cells.
- lymphocytes were aspirated from the selected microwell using a capillary having a diameter of several ⁇ m to several tens ⁇ m and collected in a cell lysate. Finally, at least 14 independent antibody-producing lymphocytes confirmed to bind to the target substance by flow cytometry were selected.
- selection of desired lymphocytes from direct lymph tissue could be completed in only one day without using the conventional hybridoma method that takes about 60 days.
- FIG. 8 shows the result of flow cytometry. That is, the antibody expressed by the recombinant cells obtained in (7-3) had binding properties to human APLNR-expressing CHO cells. The antibody did not show binding to wild type CHO cells that did not express APLNR. On the other hand, as shown in FIG. 9, in a comparative example using unintended lymphocytes collected from negative microwells, the antibody expressed by the recombinant cells did not have binding ability to CHO cells stably expressing human APLNR. It was
- Example 6 The same examination was performed for the other lymphocytes (13 types) selected in Example 6. As a result, all the antibodies had binding properties to human APLNR-expressing CHO cells. Furthermore, 12 of these did not show binding to wild-type CHO cells that did not express APLNR.
- the antibody obtained in this example has a specific binding property to human APLNR-expressing CHO cells.
- cells expressing a target substance that specifically binds to a cell membrane protein that is difficult to purify can be selected more rapidly and efficiently regardless of immortalization.
- a specific binding substance eg, antibody
- CHO cells stably expressing human GLP-1 receptor prepared by a method similar to that in Example 1 were suspended in F-12 medium (containing 10% FBS, Penicillin/Streptomycin) to give 3 ⁇ 10 5 cells. /500 ⁇ L of cell suspension was prepared. This cell suspension was filled in each microwell. The microchamber was centrifuged twice at 300 rpm for 2 minutes to prepare 1 or 2 first cells in each microwell.
- Rats were immunized in the same manner as in Example 2.
- An antibody-producing hybridoma fused with mouse myeloma was prepared in the same manner as in Example 3, and the antibody was purified from the medium.
- a solution was prepared by diluting the purified antibody in F-12 medium (containing penicillin/streptomycin) to 500 nM. 400 ⁇ L of this solution was added to the microwells.
- a solution containing no purified antibody was similarly added to another microwell. After the addition of the solution, the mixture was allowed to stand at room temperature for 30 minutes to allow the reaction between the first cells and antibody binding.
- a ligand (GLP-1) adjusted to 500 pM with F-12 medium (containing penicillin/streptomycin) was added (GLP-1 final concentration 250 pM), and the mixture was incubated at 37° C. for 1 hour to human GLP-1 receptor Was activated.
- 600 ⁇ L of 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) was added, and the mixture was allowed to stand at room temperature for 15 minutes to immobilize the first cells.
- 600 ⁇ L of 90% methanol that had been ice-cooled was added and left standing on ice for 15 minutes to permeabilize the first cells.
- rabbit anti-phosphorylation-CREB (Clone 87G3) that recognizes phosphorylation of Ser133 diluted 100 times with antibody diluent (1X PBS, 1% BSA, 0.3% Triton X-100). ) 500 ⁇ L of antibody (Cell Signaling TECHNOLOGY) was added. The mixture was allowed to stand at room temperature for 1 hour to carry out a primary antibody reaction.
- 200 fluorescence-labeled secondary antibodies for detecting rabbit anti-phospho-CREB antibody are Alexa Fluor 488-labeled anti-rabbit IgG antibody and DyLight 650-labeled anti-rat IgG antibody for detecting rat-derived antibody, respectively.
- 500 ⁇ L of a secondary antibody solution was prepared by diluting the antibody with a diluting solution of 500 times. After the completion of the primary antibody reaction, the microwell was washed with PBS, and 500 ⁇ L of the prepared secondary antibody solution was added. After standing at room temperature for 1 hour, each antibody was visualized. After washing the microwell with PBS, 1 mL of PBS was added.
- a microchamber was set in the cell picking system, and information of a transmitted light image and two types of fluorescence images were acquired.
- the fluorescence detection of Alexa Fluor 488 was performed under the conditions of an excitation wavelength of 482 nm and a fluorescence wavelength of 536 nm.
- the fluorescence detection of DyLight650 was performed under the conditions of an excitation wavelength of 628 nm and a fluorescence wavelength of 692 nm.
- the fluorescence derived from Alexa Fluor 488 is derived from phosphorylated CREB via the increase of intracellular cAMP by the addition of the ligand (GLP-1). That is, the fluorescence derived from Alexa Fluor 488 reflects the presence or absence of activation of cell membrane protein (GLP-1 receptor). When the GLP-1 receptor is activated, the fluorescence intensity derived from Alexa Fluor 488 increases.
- the fluorescence derived from DyLight650 is derived from the antibody on the surface of the first cell. That is, the fluorescence derived from DyLight650 reflects the presence or absence of binding of the added antibody to the surface of the first cell. When the antibody binds to the surface of the first cell, the fluorescence intensity derived from DyLight650 increases.
- Table 1 shows the fluorescence intensity derived from Alexa Fluor 488 and the fluorescence intensity derived from DyLight 650 for 8 microwells.
- FIG. 10 is a photograph showing images of two microwells (No. 1 and No. 5), (a) transmitted light, (b) Alexa Fluor 488-derived fluorescence, and (c) DyLight 650. It is the result of observing the origin fluorescence.
- the fluorescence derived from Alexa Fluor 488 is No. 1 microwell was weaker. Further, as shown in Table 1, No. The fluorescence intensity derived from Alexa Fluor 488 of No. 1 is It was about 1/3 of 5 (84.1 vs. 255). This is No. In the microwell of No. 1, GLP-1 function (activation of GLP-1 receptor) was inhibited, while in No. 1 microwell. It shows that GLP-1 function is not inhibited in 5 microwells.
- the fluorescence derived from DyLight650 is No.
- the microwell of 1 was stronger.
- the fluorescence intensity derived from DyLight 650 of No. 1 is It was about 18 times that of 5 (166.78 vs. 9/11). This is No. In 1 microwell, it is shown that the added antibody is strongly bound to the surface of the first cell. In addition, No. The 5 microwells show that the antibody is not bound to the first cell surface.
- the fluorescence intensity of DyLight 650 was low in the microwells with high fluorescence intensity derived from Alexa Fluor 488 (see No. 5 to No. 8). From the above results, the relative decrease in fluorescence intensity corresponding to the amount of phosphorylated CREB indicates that the increase in intracellular cAMP due to activation of human GLP-1 receptor is inhibited.
- the microwell containing the antibody that specifically binds to the human GLP-1 receptor on the surface of the first cell and inhibits the function of the receptor it is possible to identify the microwell containing the antibody that promotes the function of the receptor by the same principle.
- a second cell such as a hybridoma (eg, Example 4) or an antibody-producing lymphocyte cell (eg, Example 6) was placed in a microwell to give a functional antibody (target substance).
- the microwell containing the target cells to be produced can be identified. Then, the target cell can be separated as a single cell from the identified microwell.
- the functional antibody includes both an antibody that inhibits the function of the receptor and an antibody that promotes the function of the receptor.
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Abstract
Description
非特許文献2には、マイクロドロップレットとマイクロ流路を応用した、特異的抗体産生細胞を同定する方法の原理が記載されている。
しかしながら、この方法では、マイクロドロップレット中で抗体と抗原タンパク質の結合の有無を可視化する過程において、洗浄工程を加えることができない。そのため、細胞膜表面上に発現する量が極めて少ない細胞膜タンパク質を標的とする場合には、バックグラウンドの蛍光シグナルに比べ、抗体と抗原タンパク質の結合による蛍光シグナルが弱いため、前記結合の有無を確認することが困難であるといった欠点が報告されている(非特許文献1)。
加えて、一般的には、陽性細胞を含むマイクロドロップレット中には複数の陰性抗体産生細胞も含まれる。そのため、モノクローナル抗体を樹立するためには、複数回、スクリーニング操作を実施する必要がある。
さらに、抗体と標的細胞膜タンパク質の結合の有無を可視化するための方法は、標的細胞膜タンパク質ごとに最適化する必要があり、汎用的に実用化されるためには改良の余地が残されている。
a)複数のマイクロウェルを有する基板を提供する工程、
b)前記細胞膜タンパク質を細胞表面に発現する第一細胞を、各々の前記マイクロウェルに接着させる工程、
c)工程b)に続いて、各々の前記マイクロウェルに、前記集団から単離された1又は2個の第二細胞を導入し、前記マイクロウェル内で前記第一細胞と前記第二細胞を共存させる工程、
d)工程c)に続いて、前記目的物質が結合した第一細胞を含むマイクロウェルを特定する工程、
e)工程d)で特定されたマイクロウェルから、前記目的細胞として前記第二細胞を回収する工程、
を包含する、細胞の選抜方法である。
本発明では、複数のマイクロウェルを有する基板を用いる。マイクロウェルとは、哺乳動物細胞や鳥類細胞が1~3個程度入る微小サイズのウェル(窪み、凹部)を指す。マイクロウェルは有底の微小な穴であり、例えばその開口部の内径は10μm~50μm程度、深さは開口部の内径と同程度である。
マイクロウェルが円筒形の場合、その開口部の直径(内径)は、マイクロウェルに格納する細胞の種類や数を考慮して適宜決定することができる。第一細胞がCHO細胞、第二細胞が非ヒト動物由来のBリンパ球又は形質細胞である場合は、直径が20μm~40μm程度であることが好ましい。また、マイクロウェルの深さは、開口部の直径と同程度であることが好ましい。
本発明における標的細胞膜タンパク質としては特に限定はなく、複数回膜貫通型タンパク質に代表される全ての細胞膜タンパク質が対象となりうる。例えば、Gタンパク質共役型受容体(GPCR)、イオンチャンネル、トランスポーター、CD抗原、細胞接着分子、癌抗原、ウイルス抗原などが対象となりうる。また細胞膜タンパク質の由来動物種に特に限定はない。また本発明では、精製法が確立していない、大量精製が困難、天然に存在する構造を維持した形での単離精製が困難、等の事情を有する細胞膜タンパク質であって、その一部が脂質2重膜層から細胞外に露出しているタンパク質、が対象となりうる。
本発明では、所望の細胞膜タンパク質を細胞表面に発現する第一細胞を、マイクロウェルに接着させて使用する。
第一細胞としては、所望の細胞膜タンパク質が細胞表面に発現するものであれば、特に限定はない。一つの実施形態として、標的細胞膜タンパク質を発現するベクターを導入した組換え細胞が挙げられる。例えば、完全長の標的細胞膜タンパク質の遺伝子を、適当な発現ベクター(例えば、pcDNA、pEF/FRT/V5-DEST等)に挿入する。そして、このベクターをCHO細胞、COS細胞、HEK293細胞、NIH3T3細胞などの細胞に導入し、標的細胞膜タンパク質を細胞膜上に一過的又は安定的に発現する組換え細胞を取得する。この組換え細胞を第一細胞として用いることができる。この場合、標的細胞膜タンパク質の細胞膜上での発現量が、発現ベクターを有さない細胞と比較して、5倍以上に増強されていることが好ましい。
本発明では、所望の細胞膜タンパク質に対して特異的に結合する目的物質を生産する目的細胞を、第二細胞の集団から選抜する。前記目的物質(特異的結合物質)には、特定の細胞膜タンパク質に選択的に結合する、構造が既知又は未知のポリペプチド、環状ペプチド、タンパク質が含まれる。より具体的には、ペプチドホルモン、サイトカイン、抗体、人工ポリペプチド、人工環状ペプチド、等からなる特異的結合物質が含まれる。
本発明では、マイクロウェル内で第一細胞と第二細胞を共存させる。以下、第二細胞について具体的に説明する。
第二細胞としては、所望の目的物質を生産することが予想される細胞であれば、特に限定はない。例えば、非ヒト又はヒト組織由来の各種の細胞、例えば血液細胞、神経細胞、血管内皮細胞、血管平滑筋細胞、免疫細胞、脂肪細胞、骨格筋細胞、リンパ球細胞、皮膚細胞などを、第二細胞として用いることができる。例えば、非ヒト動物の組織を分離し、コラーゲナーゼ処理等の後、30~100μmのメッシュでろ過し、単一細胞化したものを、第二細胞として用いることができる。例えば、ヒトの血液や手術摘出臓器から単一細胞化したものを、第二細胞として用いることができる。さらに、腫瘍細胞を第二細胞として用いることができる。腫瘍細胞は、例えば、手術摘出臓器から単一細胞化したものを用いることができる。その他、腫瘍細胞は、ATCCや細胞販売会社から入手することができる。
組換え細胞を第二細胞として用いる場合は、組換え細胞には1種類の目的物質をコードする遺伝子が導入されていることが好ましい。
本発明では、上記した第一細胞をマイクロウェルに接着させる。これにより、第一細胞が、その細胞機能を損なうことなくマイクロウェル内に格納及び固定化される。マイクロウェルに接着させる第一細胞の数は、第二細胞を受け入れるスペースを確保できるものであれば限定されないが、好ましくは1~2個である。
本発明では、第一細胞が接着したマイクロウェルに1又は2個の第二細胞を導入し、第一細胞と第二細胞を共存させる。これにより、第二細胞が生産した(分泌した)目的物質が、第一細胞の表面に接触する。マイクロウェルには1個の第二細胞を導入することが好ましい。
マイクロウェル内で第一細胞と第二細胞を共存させ、必要に応じてインキュベートした後、目的物質が結合した第一細胞を含むマイクロウェル(陽性マイクロウェル)を特定する。換言すれば、各マイクロウェルにおける、第一細胞の表面に発現した標的細胞膜タンパク質と、第二細胞から分泌された物質との結合の有無を検出する。
具体的手順を例示すると、まず、上記したcDNAライブラリーを作製する際に、特異的結合物質にタグ(例えば、FLAG、V5)や抗体のFc部分が付与されるように設計する。そして、このcDNAライブラリーをCHO細胞等に導入した組換え細胞を、第二細胞として用いる。タグが付与された目的物質に対しては、当該タグに対する標識抗体(例えば、標識抗FLAG抗体、標識抗V5抗体)を用いることができる。Fc部分が付与された目的物質に対しては、標識抗Fc抗体(例えば、標識抗IgG抗体)を用いることができる。具体的操作としては、マイクロウェル内で第一細胞と第二細胞を共存させた後、必要に応じて所定の条件でインキュベートし、その後、標識物質を添加する。
前記蛍光物質としては、Alexa Fluor(登録商標)、Aqua、Texas Red(登録商標)、フルオレセイン及びその誘導体、ローダミン及びその誘導体、Cascade Blue(登録商標)、フィコエリトリン、DyLight(登録商標)等が挙げられる。好ましくは、Alexa Fluor 488が用いられる。
蛍光タンパク質としては、緑色蛍光タンパク質(GFP)が挙げられる。
前記酵素としては、アルカリフォスファターゼ、西洋ワサビペルオキシダーゼ、ルシフェラーゼ、等が挙げられる。
陽性マイクロウェルを特定した後、目的細胞として第二細胞を回収する。マイクロウェルからの第二細胞の回収は、例えば、マイクロマニュピレーターを用いて行うことができる。例えば、直径が数μmから50μmのキャピラリーを陽性マイクロウェルに挿入し、第二細胞を生きたまま吸引し、回収することができる。マイクロマニュピレーターによる操作は、自動と手動とを問わない。例えば、セルピッキングシステム(アズワン社)やCellCelector (Automated Lab Solution社)を用いて回収することが出来る。
回収した第二細胞は、適切な細胞培養用の培地内、あるいはmRNAを分解させず素早く抽出するための細胞溶解液(Lysis緩衝液)内に回収することが好ましい。
本発明は、上記の方法によって第二細胞の集団より選抜された目的細胞から、目的物質をコードする核酸(遺伝子)を取得する核酸の製造方法を包含する。また本発明は、当該核酸を宿主細胞に導入し、目的物質を発現する組換え細胞を取得する組換え細胞の製造方法を包含する。さらに本発明は、当該組換え細胞を培養し、その培養物から目的物質を取得する目的物質の製造方法を包含する。好ましくは、前記目的物質は抗体である。
そして、当該組換え細胞を培養し、その培養物(例えば、培養上清)から目的物質を取得することができる。
本発明は、上記の方法によって製造された核酸に、薬学的に許容される担体又は添加物を組み合わせて、前記核酸を有効成分として含有する医薬組成物を取得する医薬組成物の製造方法を包含する。また本発明は、上記の方法によって製造された目的物質に、薬学的に許容される担体又は添加物を組み合わせて、前記目的物質を有効成分として含有する医薬組成物を取得する医薬組成物の製造方法を包含する。
さらに、抗体に他の薬剤を直接融合させて治療効果を高める技術が知られており、前記医薬組成物に適用し得る。
すなわち、前記遺伝子治療薬は、生体内において有効成分たる抗体が発現され、その作用を発揮できる限り、いかなる方法により投与してもよい。好ましくは、適当な非経口経路により十分な量が投与される。非経口経路としては、静脈内、腹腔内、皮下、皮内、脂肪組織内、乳腺組織内、吸入、又は筋肉内の経路を介した、注射、注入、またはガス誘導性粒子衝撃法(電子銃等による)、添鼻薬等粘膜経路を介する方法、等が挙げられる。さらに、前記遺伝子治療薬は、ex vivoにおいてリポソームトランスフェクション、粒子衝撃法(米国特許第4,945,050号)、またはウイルス感染を利用して細胞に投与し、該細胞を動物に再導入することにより投与してもよい。
本発明は、上記の方法によって製造された目的物質を含む、所望の細胞膜タンパク質を検出するための試薬を包含する。例えば、本発明の方法によって製造された抗体(目的物質)を含む試薬を用いて、ヒト由来又は非ヒト哺乳動物由来の血液細胞に前記抗体を接触させる。さらに、蛍光物質又は色素からなる標識物質を、直接的又は間接的に接触させる。そして、フローサイトメトリー又はプレートリーダーによって、所望の細胞膜タンパク質の発現を検出することができる。また前記試薬を用いて、ヒト由来又は非ヒト哺乳動物由来の病理組織片に前記抗体を接触させて、所望の細胞膜タンパク質の発現を検出することができる。
ジーンバンクに登録されているヒトAPLNR遺伝子配列(NM_005161.4)をマウスのアミノ酸コドンに最適化した人工合成遺伝子(配列番号1)を作製した。この人工合成遺伝子を用い、国際公開第2012/043533号(日本国特許第5315495号)に記載の方法に準じて、ヒトAPLNR遺伝子とGroEL遺伝子との融合遺伝子を含むベクターpCI-APLNR-GroELを構築した。
pEF5/FRT/V5-DESTベクター(インビトロジェン社)に上記人工合成遺伝子(配列番号1)を導入し、pEF-FRT-APLNRを構築した。pEF-FRT-APLNRから発現されるヒトAPLNRは、C末端にV5と6×HISタグが付加される。
国際公開第2012/043533号(日本国特許第5315495号)に記載の方法に準じて、8週齢のマウスICR(雌)に、ベクターpCI-APLNR-GroELを複数回に分けて注射した(DNA免疫)。
(2-1)でDNA免疫を行ったマウスから脾臓を摘出し、冷蔵HBSSを入れた6ウェルプレートに回収した。付着している結合組織や脂肪組織を除去した後、新しいHBSS内で脾臓をほぐしてリンパ球を遊離させた。細胞を回収し、10mLのHBSSで再懸濁した。セルストレーナーにて未破壊組織を分離後、2000rpmで5分間遠心分離し、細胞を回収した。回収した細胞を1mLの溶血溶液に懸濁して37℃で5分間インキュベートし、赤血球を除去した。1000rpmで5分間遠心分離し、リンパ球細胞を回収した。
EasySep Mouse Biotin Positive Selection Kit(STEMCELL TECHNOLOGIES社)を用いて、2.5×107個の上記リンパ球細胞から、所望の抗体産生細胞(目的細胞)の候補を含む約1.3×105個の細胞集団(第二細胞)を分離した。
細胞融合に使用するミエローマ細胞(SP2/O)は、細胞融合の5日前に起眠し、2日前に一度継代を行ってから使用した。
実施例2で取得した免疫済みマウスの凍結脾細胞を融解し、37℃のRPMI1640培地(10%FBS含有)で懸濁し、細胞数をカウントした。脾細胞とミエローマ細胞(SP2/O)を細胞数比1:1となるように混合した。なおミエローマ細胞は、細胞融合の5日前に起眠し、2日前に一度継代を行ってから使用した。細胞混合物を遠心分離した後、ECFバッファーで細胞を洗浄した。同様の洗浄をさらに2回行った。
マイクロウェルチャンバーASMC30-20P(アズワン社)を準備した。このマイクロウェルチャンバーは、約1.5cm×約2.4cmのエリアの中に直径30μmのマイクロウェル84,640個が等間隔で配置されている基板である。各マイクロウェルの深さは、マイクロウェルの直径と等しい。マイクロウェル間のピッチは、マイクロウェルの直径の2倍である。従来技術では、マイクロウェルに1個の細胞を格納して用いるのが一般的である。しかし本実施例では、マイクロチャンバーに第一細胞と第二細胞、すなわち2個以上の細胞を格納して実験を行った。以下、説明する。
実施例4で選抜したハイブリドーマの1つから、MAGrahd法(Nobuyuki Kurosawa, Megumi Yoshioka, Rika Fujimoto, Fuminori Yamagishi and Masaharu Isobe, "Rapid production of antigen-specific monoclonal antibodies from a variety of animals", BMC Biology, 10:80, 2012)にて抗体遺伝子を取得した。すなわち、実施例4で得た細胞溶解液5μLとオリゴdTマグネット5μgとを混合し、オリゴdTマグネット上に細胞由来のmRNAを捕捉した。MAGrahdリアクタートレーとネオジム磁石を使い、オリゴdTマグネットを洗浄溶液で洗浄後、逆転写反応によるcDNA合成を行った。さらにマグネットを洗浄後、5’ターミナルトランスレーショナル反応を実施した。合成した上記cDNAを用い、5’race PCR法にて、抗体重鎖可変領域(VH領域)の遺伝子と、抗体軽鎖可変領域(VL領域)の遺伝子を単離増幅した。
なお、増幅産物の特異性を高めるために、PCRは2回行った。1回目のPCRでは、VH領域とVL領域を共通して増幅させる第一フォワードプライマー(配列番号3)と、VH領域を特異的に増幅させる第一リバースプライマー(配列番号4)と、VL領域を特異的に増幅させる第二リバースプライマー(配列番号5)を混合して使用した。2回目のPCRでは、1回目の増幅産物を鋳型とし、VH領域の増幅については、第二フォワードプライマー(配列番号6)と、VH領域を特異的に増幅させる第三リバースプライマー(配列番号7)、VL領域の増幅については第二フォワードプライマー(配列番号6)とVL領域特異的に増幅させる第四リバースプライマー(配列番号8)をそれぞれプライマーとして使用した。
2回目のPCR後のサンプルについてアガロースゲル電気泳動を行ったところ、750bpの位置にVH領域、550bpの位置にVL領域に、それぞれ対応する増幅産物が確認できた。
TS-jPCR法(Megumi Yoshioka, Nobuyuki Kurosawa and Masaharu Isobe, "Target-selective joint polymerase chain reaction: A robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells", BMC Biotechnol. 2011 Jul 21;11:75)にて抗体発現ユニットを構築した。すなわち、(5-1)で増幅したVH領域の遺伝子と、抗体重鎖定常領域の遺伝子と、遺伝子発現に必要なプロモーター領域とをPCRを用いて融合し、完全長抗体重鎖を発現する抗体発現ユニットを構築した。同様に、(5-1)で増幅したVL領域の遺伝子と、抗体軽鎖定常領域の遺伝子と、遺伝子発現に必要なプロモーター領域とをPCRを用いて融合し、完全長抗体軽鎖を発現する抗体発現ユニットを構築した。これらの抗体発現ユニットを哺乳動物細胞に供導入することにより、所望の抗体(IgG)を一過的に発現する組換え細胞を得ることができる。
上記文献(Nobuyuki Kurosawa et al., BMC Biology, 10:80, 2012)に記載の方法で、HEK293FT細胞に上記2種の抗体発現ユニットを共導入した。すなわち、コラーゲンコート96ウェルプレートに、HEK293FT細胞を1.5×104細胞/100μL/ウェルとなるように播種した。リポフェクトアミン2000を用いて、(5-2)で構築した2種の抗体発現ユニットをHEK293FT細胞に共導入した。導入3日目に細胞上清を回収して、発現された抗体の結合性評価に用いた。
ヒトAPLNR安定発現CHO細胞株を、直径10cmのディッシュ内で培養した。細胞をPBSで3回洗浄後、細胞剥離用バッファーを1mL加え、37℃で15分間インキュベートした。剥離された細胞をFACSバッファーで懸濁し、1000rpmで5分間遠心した後、細胞濃度が1×107細胞/mLとなるようにFACSバッファーで再懸濁した。Fc Block(べクトン・ディッキンソン社)を細胞懸濁液の1/500量加え、4℃で30分間ブロッキングを行った。ブロッキング後、2×105細胞/50μLとなるように細胞を懸濁した。96ウェルプレート内で、この細胞懸濁液を(5-3)で回収した細胞上清と混合し、4℃で1時間インキュベートした。インキュベート後、100μLのFACSバッファーで2回、細胞を洗浄した。蛍光標識抗IgG抗体(2次抗体)の希釈液を各ウェルに50μLずつ加え、4℃で1時間インキュベートし、CHO細胞表面に結合した抗体に2次抗体を結合させた。100μLのFACSバッファーで細胞を2回洗浄した後、80μLのFACSバッファーに懸濁し、フローサイトメトリー法にて細胞表面の蛍光強度を測定した。
一方、図5に示すように、陰性マイクロウェルから回収された目的外のハイブリドーマを用いた比較例では、組換え細胞が発現した抗体は、ヒトAPLNR安定発現CHO細胞に対する結合性を有さなかった。
以上より、本実施例によって得られた抗体が、ヒトAPLNR発現CHO細胞に対する特異的結合性を有することが示された。
実施例4の方法に準じて、実施例2で調製した不死化していないリンパ球細胞(第二細胞)の集団から、目的の抗体を産生する細胞を選抜した。以下、説明する。
実施例5と同様の操作を行い、実施例6で選抜したリンパ球の1つから抗体遺伝子を取得した。
実施例5と同様の操作を行い、(7-1)で得られた抗体遺伝子から完全長抗体重鎖と完全長抗体軽鎖を発現する2種の抗体発現ユニットを構築した。
実施例5と同様の操作を行い、(7-2)で得られた抗体発現ユニットをHEK293FT細胞に共導入した。導入48時間後に細胞上清を回収して、発現された抗体の機能性評価に用いた。
実施例5と同様の操作を行い、(7-3)で得られた組換え細胞が発現する抗体の結合性評価を行った。
一方、図9に示すように、陰性マイクロウェルから回収された目的外のリンパ球を用いた比較例では、組換え細胞が発現した抗体は、ヒトAPLNR安定発現CHO細胞に対する結合性を有さなかった。
2 マイクロウェル
3 第一細胞
5 第二細胞
6 目的物質
7 標識物質
Claims (30)
- 所望の細胞膜タンパク質に対して特異的に結合する目的物質を生産する目的細胞を、第二細胞の集団から選抜する細胞の選抜方法であって、下記工程:
a)複数のマイクロウェルを有する基板を提供する工程、
b)前記細胞膜タンパク質を細胞表面に発現する第一細胞を、各々の前記マイクロウェルに接着させる工程、
c)工程b)に続いて、各々の前記マイクロウェルに、前記集団から単離された1又は2個の第二細胞を導入し、前記マイクロウェル内で前記第一細胞と前記第二細胞を共存させる工程、
d)工程c)に続いて、前記目的物質が結合した第一細胞を含むマイクロウェルを特定する工程、
e)工程d)で特定されたマイクロウェルから、前記目的細胞として前記第二細胞を回収する工程、
を包含する、細胞の選抜方法。 - 前記工程d)は、前記目的物質が前記第一細胞に結合したことを可視化する可視化工程を包含する、請求項1に記載の細胞の選抜方法。
- 前記可視化工程は、前記マイクロウェルに、前記目的物質に対して特異的に結合する標識物質を添加することを含む、請求項2に記載の細胞の選抜方法。
- 前記標識物質が前記目的物質に対する標識抗体である、請求項3に記載の細胞の選抜方法。
- 前記標識物質における標識が蛍光標識である、請求項3又は4に記載の細胞の選抜方法。
- 前記標識物質は、第一蛍光物質で標識された抗体であり、
前記工程b)は、前記マイクロウェルに接着している前記第一細胞を第二蛍光物質で標識する第一細胞標識工程を含み、
第一蛍光物質が発する蛍光の蛍光波長は、第二蛍光物質が発する蛍光の蛍光波長と異なる、請求項5に記載の細胞の選抜方法。 - 前記可視化工程は、前記目的物質が前記第一細胞に結合した際に起きる、前記細胞膜タンパク質の活性化に伴う細胞内情報伝達物質の変動を可視化することを含む、請求項2に記載の細胞の選抜方法。
- 前記細胞膜タンパク質が複数回膜貫通型タンパク質である、請求項1~7のいずれかに記載の細胞の選抜方法。
- 前記第一細胞が、前記細胞膜タンパク質を発現するベクターを導入した細胞である、請求項1~8のいずれかに記載の細胞の選抜方法。
- 前記第一細胞が、前記細胞膜タンパク質を発現する腫瘍細胞である、請求項1~8のいずれかに記載の細胞の選抜方法。
- 前記第一細胞が、前記細胞膜タンパク質を発現する非腫瘍細胞である、請求項1~8のいずれかに記載の細胞の選抜方法。
- 前記目的物質が抗体である、請求項1~11のいずれかに記載の細胞の選抜方法。
- 前記第二細胞が、前記細胞膜タンパク質又はそれをコードする核酸で免疫した非ヒト動物由来の骨髄、脾臓、リンパ組織、又は血液細胞由来である、請求項12に記載の細胞の選抜方法。
- 前記第二細胞が不死化された細胞である、請求項13に記載の細胞の選抜方法。
- 前記第二細胞がハイブリドーマである、請求項14に記載の細胞の選抜方法。
- 前記第二細胞が、ヒトのリンパ組織又は血液由来である、請求項12に記載の細胞の選抜方法。
- 前記第二細胞が、エプスタイン・バール・ウイルス感染により不死化された細胞である、請求項16に記載の細胞の選抜方法。
- 前記第二細胞が、外来性の抗体遺伝子を有し、当該抗体を発現する組換え細胞である、請求項12に記載の細胞の選抜方法。
- 前記抗体が、完全抗体、機能的抗体断片、一本鎖抗体、又は多重特異性抗体である、請求項18に記載の細胞の選抜方法。
- 前記抗体が、完全ヒト抗体、ヒト化抗体、又はキメラ抗体である、請求項18又は19に記載の細胞の選抜方法。
- 前記抗体が、ネコ化抗体又はイヌ化抗体である、請求項18又は19に記載の細胞の選抜方法。
- 請求項1~21のいずれかに記載の方法によって第二細胞の集団より選抜された目的細胞から、前記目的物質をコードする核酸を取得する、核酸の製造方法。
- 前記目的物質が抗体である、請求項22に記載の核酸の製造方法。
- 請求項22又は23に記載の方法によって製造された核酸を宿主細胞に導入し、前記目的物質を発現する組換え細胞を取得する、組換え細胞の製造方法。
- 請求項24に記載の方法によって製造された組換え細胞を培養し、その培養物から前記目的物質を取得する、目的物質の製造方法。
- 請求項1~21のいずれかに記載の方法によって第二細胞の集団より選抜された目的細胞を培養し、その培養物から前記目的物質を取得する、目的物質の製造方法。
- 前記目的物質が抗体である、請求項25又は26に記載の目的物質の製造方法。
- 請求項22又は23に記載の方法によって製造された核酸に、薬学的に許容される担体又は添加物を組み合わせて、前記核酸を有効成分として含有する医薬組成物を取得する、医薬組成物の製造方法。
- 請求項25~27のいずれかに記載の方法によって製造された目的物質に、薬学的に許容される担体又は添加物を組み合わせて、前記目的物質を有効成分として含有する医薬組成物を取得する、医薬組成物の製造方法。
- 請求項25~27のいずれかに記載の方法によって製造された目的物質を含む、前記所望の細胞膜タンパク質を検出するための試薬。
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WO2024071374A1 (ja) * | 2022-09-30 | 2024-04-04 | 富士フイルム株式会社 | 目的物質を産生する細胞のスクリーニング方法、核酸の製造方法、及び、目的物質の製造方法 |
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JP2021118722A (ja) | 2021-08-12 |
EP3928794A1 (en) | 2021-12-29 |
KR20210128444A (ko) | 2021-10-26 |
EP3928794A4 (en) | 2022-11-30 |
US11357787B2 (en) | 2022-06-14 |
CN113454457A (zh) | 2021-09-28 |
JPWO2020171020A1 (ja) | 2021-03-11 |
JP6881801B2 (ja) | 2021-06-02 |
JP7456627B2 (ja) | 2024-03-27 |
US20220347202A1 (en) | 2022-11-03 |
US20220040216A1 (en) | 2022-02-10 |
CA3130051A1 (en) | 2020-08-27 |
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