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  • C12N2800/00—Nucleic acids vectors
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  • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
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  • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)

Definitions

• the present disclosure relates to methods for producing cells, methods for producing heteromultimeric proteins, methods for producing bispecific antibodies, vector sets, mammalian cells, CHO cells, and methods for producing cell pools.
• Patent Document 1 discloses a method for selecting bispecific antibody-expressing cells, which involves transducing eukaryotic cells using a lentivirus.
• Patent Document 2 discloses producing a bispecific anti-HER2 antibody by culturing host cells containing an expression vector for the bispecific anti-HER2 antibody.
• Patent Document 3 describes (1) a domain containing an antibody variable region having glypican 3-binding activity, (2) a domain containing an antibody variable region having T cell receptor complex binding activity, and (3) a domain for Fc ⁇ receptor Disclosed is a multispecific antigen-binding molecule comprising a domain including an Fc region with reduced binding activity, and in which the variable region (1) and the L chain variable region contained in the variable region (2) have a common amino acid sequence. has been done.
• Patent Document 4 discloses a first antigen-binding site that recognizes blood coagulation factor IX and a second antigen-binding site that recognizes blood coagulation factor X, and has a function to replace the function of blood coagulation factor VIII.
• a multispecific antigen binding molecule is disclosed that has the following properties.
• Patent Document 5 discloses a bispecific antibody that includes an antigen-binding domain that binds to CD40 and an antigen-binding domain that binds to EpCAM.
• Patent Document 6 discloses a bispecific anti-HER2 antibody having a common light chain and two different heavy chains.
• Patent Document 7 discloses a bispecific antibody that binds to the extracellular portion of PD-1 and the extracellular portion of TIM-3.
• Patent Document 8 discloses a bispecific antibody that binds to hPD-L1 and TIGIT or LAG-3.
• Patent Document 9 discloses a bispecific antibody that binds to CD38 and PD-L1.
• Patent Document 10 discloses a fibronectin secretion leader that promotes secretion of a target polypeptide produced in host cells.
• expression vectors for subunits constituting heteromultimeric proteins have been introduced into host cells for the purpose of producing cells that produce heteromultimeric proteins.
• a heteromultimeric protein consists of one subunit A, one subunit B, and one subunit C
• the expression cassette of subunit A, the expression cassette of subunit B, and the expression of subunit C Expression vectors containing each cassette are introduced into host cells.
• a simple prediction would be that the three types of subunits described above would be expressed in an expression ratio of 1:1:1, and a heteromultimeric protein would be produced.
• the expression level or purity of the heteromultimeric protein may be low, and the production amount of the heteromultimeric protein may not reach the expected value. Possible reasons for this include the fact that each subunit has a different transcription rate or translation rate; some subunits are easily degraded; and all subunits aggregate when a certain type of subunit is in excess. It will be done.
• the optimal ratio of the expression levels of subunits As a means of increasing the expression level or purity of a heteromultimeric protein, it is possible to know in advance the optimal ratio of the expression levels of subunits and to load expression cassettes of subunits into one expression vector in a number ratio according to this ratio. Conceivable. However, it is not always easy to know the optimal ratio of subunit expression levels. Furthermore, when the optimal ratio of the expression levels of subunits is a relatively large integer (for example, 1:2:3), the total number of expression cassettes to be carried on one expression vector increases. As a result, the size of the expression vector increases and the rate of introduction into host cells decreases.
• Another possible means of increasing the expression level or purity of a heteromultimeric protein is to place each subunit expression cassette on a separate expression vector and introduce multiple types of expression vectors into host cells.
• expression vector A for subunit A, expression vector B for subunit B, and expression vector C for subunit C are Introduce into cells. Since the numbers of expression vectors A, B, and C introduced into host cells vary from host cell to host cell, diversity occurs in the number of expression cassettes possessed by host cells. Cells with a high expression level or purity of the heteromultimeric protein are selected from a variety of host cells. However, it is necessary to construct the same number of expression vectors as the number of subunits constituting the heteromultimeric protein, and it is necessary to prepare the same number of selection markers as the number of subunits.
• the present disclosure provides a means different from the above two means as a means for increasing the expression level or purity of a heteromultimeric protein.
• One embodiment of the present disclosure aims to provide a method for producing cells with excellent productivity of heteromultimeric proteins or bispecific antibodies.
• An object of an embodiment of the present disclosure is to provide a method for producing a heteromultimeric protein or a bispecific antibody with excellent productivity.
• One embodiment of the present disclosure aims to provide a vector set for use in producing cells that produce heteromultimeric proteins or bispecific antibodies.
• One embodiment of the present disclosure aims to provide mammalian cells and CHO cells that produce heteromultimeric proteins or bispecific antibodies.
• One embodiment of the present disclosure aims to provide a method for creating a cell pool for use in selecting cells that produce heteromultimeric proteins or bispecific antibodies.
• a first expression vector and a second expression vector that is, two types of expression vectors, are introduced into a host cell to express a heteromultimeric protein.
• the expression cassette of the subunit of interest (usually the subunit whose expression is desired to be enhanced) is placed on both the first expression vector and the second expression vector.
• the number of subunits of interest may be one type or two or more types.
• Expression cassettes for the remaining subunits other than the subunit of interest are placed on either the first expression vector or the second expression vector for each type. Expression of all subunits is ensured by introducing both the first expression vector and the second expression vector into the host cell.
• the expression cassette of the subunit of interest is present in both the first expression vector and the second expression vector, the amount introduced is relatively large, and the expression of the subunit of interest can be enhanced. Since the number of the first expression vector and the second expression vector introduced into the host cell varies from host cell to host cell, diversity occurs in the number of each expression cassette possessed by the host cell. Cells that meet evaluation criteria (eg, expression level or purity of heteromultimeric protein) are selected from a variety of host cells.
• a method for producing cells that produce a heteromultimeric protein consisting of n types of subunits (n is an integer of 2 or more), introducing the vector set into a host cell; selecting cells that produce a heteromultimeric protein from host cells into which the vector set has been introduced; How to create cells: Vector set: a set of a first expression vector and a second expression vector, an expression cassette for at least one type of subunit X that is part of the n types of subunits is included in both the first expression vector and the second expression vector, An expression cassette of subunit Y remaining after subunit X is removed from n types of subunits is contained in one of the first expression vector and the second expression vector for each type.
• Vector set a set of a first expression vector and a second expression vector, an expression cassette for at least one type of subunit X that is part of the n types of subunits is included in both the first expression vector and the second expression vector, An expression cassette of subunit Y remaining after subunit X is removed from n types of subunits
• the vector set is the following vector set, Method for producing cells described in ⁇ 1>:
• Vector set a set of a first expression vector and a second expression vector, an expression cassette for at least one type of subunit X that is part of the n types of subunits is included in both the first expression vector and the second expression vector, All expression cassettes for subunit Y remaining after subunit X is removed from the n types of subunits are contained in one of the first expression vector and the second expression vector.
• Vector set a set of a first expression vector and a second expression vector, an expression cassette for at least one type of subunit X that is part of the n types of subunits is included in both the first expression vector and the second expression vector, All expression cassettes for subunit Y remaining after subunit X is removed from the n types of subunits are contained in one of the first expression vector and the second expression vector.
• the heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of 2 to 6 types of subunits, at least one type of subunit X is one type, two types, or three types of subunits X; The method for producing cells according to ⁇ 1> or ⁇ 2>.
• the heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of 3 types or 4 types of subunits, at least one type of subunit X is one type, two types, or three types of subunits X; The method for producing cells according to ⁇ 1> or ⁇ 2>.
• a heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of three types of subunits, at least one type of subunit X is one or two types of subunits
• the heteromultimeric protein is an antibody, The method for producing cells according to any one of ⁇ 1> to ⁇ 5>.
• the heteromultimeric protein is a bispecific antibody, The method for producing cells according to any one of ⁇ 1> to ⁇ 5>.
• All of the expression cassettes of the subunits constituting the heteromultimeric protein contain the same type of promoter. The method for producing cells according to any one of ⁇ 1> to ⁇ 7>.
• the promoter is hEF-1 ⁇ promoter, The method for producing cells according to ⁇ 8>.
• At least one of the expression cassettes of the subunits constituting the heteromultimeric protein contains a coding sequence for a fibronectin secretion leader, In the expression cassette containing the coding sequence of the fibronectin secretory leader, the coding sequences of the subunits constituting the heteromultimeric protein are located downstream of the coding sequence of the fibronectin secretory leader in the same reading frame.
• the expression cassettes of the subunits that make up the heteromultimeric protein contain the coding sequence for the fibronectin secretory leader;
• the coding sequences of the subunits constituting the heteromultimeric protein are located downstream and in the same reading frame of the coding sequence of the fibronectin secretory leader.
• the first expression vector further comprises an expression cassette for a first selection marker;
• the second expression vector further comprises an expression cassette for a second selection marker;
• the first selection marker and the second selection marker are different types of selection markers, the first selection marker is at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene;
• the second selection marker is at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene.
• ⁇ 15> The method for producing a cell according to any one of ⁇ 1> to ⁇ 13>, wherein the host cell is a CHO cell (Chinese hamster ovary cell).
• the host cell is a CHO-DG44 cell, a CHO-K1 cell, a CHO-DXB11 cell, a CHOpro3 ⁇ cell, or an established cell line derived from these cells.
• a method for producing cells is any one of ⁇ 1> to ⁇ 13>, wherein the host cell is a CHO-DG44 cell, a CHO-K1 cell, a CHO-DXB11 cell, a CHOpro3 ⁇ cell, or an established cell line derived from these cells.
• the heteromultimeric protein is a bispecific antibody consisting of a first H chain, a second H chain, and an L chain common to the first H chain and the second H chain, Subunit X is a L chain, subunit Y is a first H chain and a second H chain,
• the vector set is the following vector set, Method for producing cells described in ⁇ 1>: Vector set: a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include a light chain expression cassette; one of the first expression vector and the second expression vector comprises a first heavy chain expression cassette; one of the first expression vector and the second expression vector comprises a second heavy chain expression cassette; Vector set.
• a method for producing a cell that produces a bispecific antibody consisting of a first H chain, a second H chain, and an L chain common to the first H chain and the second H chain introducing the vector set into a host cell; selecting cells that produce bispecific antibodies from host cells into which the vector set has been introduced; How to create cells: Vector set: a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include a light chain expression cassette; one of the first expression vector and the second expression vector comprises a first heavy chain expression cassette; one of the first expression vector and the second expression vector comprises a second heavy chain expression cassette; Vector set.
• one of the first expression vector and the second expression vector comprises a first H chain expression cassette and a second H chain expression cassette;
• the first expression vector includes an L chain expression cassette, a first H chain expression cassette, and a second H chain expression cassette, the second expression vector comprises a light chain expression cassette;
• the bispecific antibody is a tetramer consisting of one first H chain, one second H chain, and two L chains, The method for producing cells according to any one of ⁇ 17-1> to ⁇ 17-4>.
• the first expression vector and the second expression vector each contain one L chain expression cassette, one of the first expression vector and the second expression vector contains one first H chain expression cassette; one of the first expression vector and the second expression vector contains one second H chain expression cassette; The method for producing cells according to any one of ⁇ 17-1> to ⁇ 17-5>.
• the heteromultimeric protein is a bispecific antibody consisting of an H chain, a L chain, and a scFv-Fc, Subunit X is a L chain and scFv-Fc, subunit Y is a H chain,
• the vector set is the following vector set A, Method for producing cells described in ⁇ 1>: Vector set A: a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include an expression cassette for the light chain and an expression cassette for the scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; Vector set.
• the heteromultimeric protein is a bispecific antibody consisting of an H chain, a L chain, and a scFv-Fc, Subunit X is scFv-Fc, subunit Y is H chain and L chain,
• the vector set is the following vector set B, Method for producing cells described in ⁇ 1>: Vector set B: a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector contain an expression cassette for scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; one of the first expression vector and the second expression vector contains an expression cassette for the L chain; Vector set.
• ⁇ 18-4> A method for producing cells that produce bispecific antibodies consisting of H chain, L chain, and scFv-Fc, introducing vector set A or B into a host cell; selecting cells that produce bispecific antibodies from host cells into which vector set A or B has been introduced; How to create cells: Vector set A: a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include an expression cassette for the light chain and an expression cassette for the scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; Vector set.
• Vector set B a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector contain an expression cassette for scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; one of the first expression vector and the second expression vector contains an expression cassette for the L chain; Vector set.
• the promoter is hEF-1 ⁇ promoter, The method for producing cells according to ⁇ 19>.
• At least one of the expression cassettes of the subunits constituting the bispecific antibody contains a coding sequence for a fibronectin secretion leader, In the expression cassette containing the coding sequence of the fibronectin secretory leader, the coding sequences of the subunits constituting the bispecific antibody are arranged downstream of the coding sequence of the fibronectin secretory leader in the same reading frame.
• All of the subunit expression cassettes that make up the bispecific antibody contain the coding sequence for the fibronectin secretory leader, The coding sequences of the subunits constituting the bispecific antibody are located downstream of and in the same reading frame as the coding sequence of the fibronectin secretory leader.
• the first expression vector further comprises an expression cassette for a first selection marker; the second expression vector further comprises an expression cassette for a second selection marker; The method for producing a cell according to any one of ⁇ 17-1> to ⁇ 17-5>, ⁇ 18-1> to ⁇ 18-5>, and ⁇ 19> to ⁇ 22>.
• the first expression vector includes an L chain expression cassette, a first H chain expression cassette, a second H chain expression cassette, and a first selection marker expression cassette
• the second expression vector comprises an expression cassette for the light chain and an expression cassette for the second selection marker; The method for producing cells according to ⁇ 23>.
• the first selection marker and the second selection marker are different types of selection markers,
• the first selection marker is a dihydrofolate reductase gene or a glutamine synthetase gene, The method for producing cells according to ⁇ 24>.
• the first selection marker is a dihydrofolate reductase gene or a glutamine synthetase gene, the second selection marker is an antibiotic resistance gene; The method for producing cells according to ⁇ 24>.
• ⁇ 27> The cell according to any one of ⁇ 17-1> to ⁇ 17-5>, ⁇ 18-1> to ⁇ 18-5> and ⁇ 19> to ⁇ 26>, wherein the host cell is a mammalian cell. Creation method.
• ⁇ 28> Any one of ⁇ 17-1> to ⁇ 17-5>, ⁇ 18-1> to ⁇ 18-5>, and ⁇ 19> to ⁇ 26>, wherein the host cell is a CHO cell (Chinese hamster ovary cell).
• the method for producing cells described in . ⁇ 29> The host cell is a CHO-DG44 cell, a CHO-K1 cell, a CHO-DXB11 cell, a CHOpro3 ⁇ cell, or an established cell line derived from these cells, ⁇ 17-1> to ⁇ 17-5>, ⁇ 18
• a method for producing a heteromultimeric protein comprising culturing cells produced by the method for producing cells according to any one of ⁇ 1> to ⁇ 16>.
• ⁇ 31> Cultivating cells produced by the method for producing cells according to any one of ⁇ 17-1> to ⁇ 17-5>, ⁇ 18-1> to ⁇ 18-5>, and ⁇ 19> to ⁇ 29>.
• a method for producing a bispecific antibody comprising:
• Vector set that expresses a heteromultimeric protein consisting of n types of subunits (n is an integer of 2 or more)
• a set of a first expression vector and a second expression vector, an expression cassette for at least one type of subunit X that is part of the n types of subunits is included in both the first expression vector and the second expression vector, All expression cassettes for subunit Y remaining after subunit X is removed from the n types of subunits are contained in one of the first expression vector and the second expression vector.
• the heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of 2 to 6 types of subunits, at least one type of subunit X is one type, two types, or three types of subunits X;
• the heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of 3 types or 4 types of subunits, at least one type of subunit X is one type, two types, or three types of subunits X;
• a heteromultimeric protein consisting of n types of subunits is a heteromultimeric protein consisting of three types of subunits, at least one type of subunit X is one or two types of subunits
• the heteromultimeric protein is an antibody, The vector set according to any one of ⁇ 32> to ⁇ 36>.
• the heteromultimeric protein is a bispecific antibody, The vector set according to any one of ⁇ 32> to ⁇ 36>. ⁇ 39> All of the expression cassettes of the subunits constituting the heteromultimeric protein contain the same type of promoter. The vector set according to any one of ⁇ 32> to ⁇ 38>. ⁇ 40> The promoter is hEF-1 ⁇ promoter, The vector set described in ⁇ 39>.
• At least one of the expression cassettes of the subunits constituting the heteromultimeric protein contains a coding sequence for a fibronectin secretion leader, In the expression cassette containing the coding sequence of the fibronectin secretory leader, the coding sequences of the subunits constituting the heteromultimeric protein are located downstream of the coding sequence of the fibronectin secretory leader in the same reading frame.
• ⁇ 42> All of the expression cassettes of the subunits that make up the heteromultimeric protein contain the coding sequence for the fibronectin secretory leader; The coding sequences of the subunits constituting the heteromultimeric protein are located downstream and in the same reading frame of the coding sequence of the fibronectin secretory leader.
• the first expression vector further comprises an expression cassette for a first selection marker; the second expression vector further comprises an expression cassette for a second selection marker; The vector set according to any one of ⁇ 32> to ⁇ 42>.
• the first selection marker and the second selection marker are different types of selection markers, the first selection marker is at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene;
• the second selection marker is at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene.
• the heteromultimeric protein is a bispecific antibody consisting of a first H chain, a second H chain, and an L chain common to the first H chain and the second H chain, Subunit X is a L chain, subunit Y is a first H chain and a second H chain, both the first expression vector and the second expression vector include a light chain expression cassette; one of the first expression vector and the second expression vector comprises a first heavy chain expression cassette; one of the first expression vector and the second expression vector comprises a second heavy chain expression cassette; The vector set described in ⁇ 32>.
• a set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include a light chain expression cassette; one of the first expression vector and the second expression vector comprises a first heavy chain expression cassette; one of the first expression vector and the second expression vector comprises a second heavy chain expression cassette;
• the first expression vector includes an L chain expression cassette, a first H chain expression cassette, and a second H chain expression cassette, the second expression vector comprises a light chain expression cassette;
• the bispecific antibody is a tetramer consisting of one first H chain, one second H chain, and two L chains, The vector set according to any one of ⁇ 45-1> to ⁇ 45-4>.
• the first expression vector and the second expression vector each contain one L chain expression cassette, one of the first expression vector and the second expression vector contains one first H chain expression cassette; one of the first expression vector and the second expression vector contains one second H chain expression cassette;
• the heteromultimeric protein is a bispecific antibody consisting of an H chain, a L chain, and a scFv-Fc
• Subunit X is a L chain and scFv-Fc
• subunit Y is a H chain
• both the first expression vector and the second expression vector include an expression cassette for the light chain and an expression cassette for the scFv-Fc
• one of the first expression vector and the second expression vector contains an H chain expression cassette
• the heteromultimeric protein is a bispecific antibody consisting of an H chain, a L chain, and a scFv-Fc, Subunit X is scFv-Fc, subunit Y is H chain and L chain, both the first expression vector and the second expression vector contain an expression cassette for scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; one of the first expression vector and the second expression vector contains an expression cassette for the L chain; The vector set described in ⁇ 32>.
• a vector set that expresses a bispecific antibody consisting of an H chain, an L chain, and a scFv-Fc A set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector include an expression cassette for the light chain and an expression cassette for the scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; Vector set.
• a vector set that expresses a bispecific antibody consisting of an H chain, an L chain, and a scFv-Fc A set of a first expression vector and a second expression vector, both the first expression vector and the second expression vector contain an expression cassette for scFv-Fc; one of the first expression vector and the second expression vector contains an H chain expression cassette; one of the first expression vector and the second expression vector contains an expression cassette for the L chain; Vector set.
• the bispecific antibody is a trimer consisting of H chain, L chain, and scFv-Fc, The vector set according to any one of ⁇ 46-2> to ⁇ 46-5>.
• All of the subunit expression cassettes that make up a bispecific antibody contain the same type of promoter.
• the promoter is hEF-1 ⁇ promoter, The vector set described in ⁇ 47>.
• At least one of the expression cassettes of the subunits constituting the bispecific antibody contains a coding sequence for a fibronectin secretion leader, In the expression cassette containing the coding sequence of the fibronectin secretory leader, the coding sequences of the subunits constituting the bispecific antibody are arranged downstream of the coding sequence of the fibronectin secretory leader in the same reading frame.
• All of the subunit expression cassettes that make up the bispecific antibody contain the coding sequence for the fibronectin secretory leader, The coding sequences of the subunits constituting the bispecific antibody are located downstream of and in the same reading frame as the coding sequence of the fibronectin secretory leader.
• the first expression vector further comprises an expression cassette for a first selection marker; the second expression vector further comprises an expression cassette for a second selection marker; The vector set according to any one of ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 50>.
• the first expression vector includes an L chain expression cassette, a first H chain expression cassette, a second H chain expression cassette, and a first selection marker expression cassette, the second expression vector comprises an expression cassette for the light chain and an expression cassette for the second selection marker; The vector set described in ⁇ 51>.
• the first selection marker and the second selection marker are different types of selection markers,
• the first selection marker is a dihydrofolate reductase gene or a glutamine synthetase gene, The vector set described in ⁇ 52>.
• the first selection marker is a dihydrofolate reductase gene or a glutamine synthetase gene, the second selection marker is an antibiotic resistance gene; The vector set described in ⁇ 52>.
• ⁇ 55> The vector set according to any one of ⁇ 32> to ⁇ 44>, ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• Transfected mammalian cells ⁇ 56> The vector set according to any one of ⁇ 32> to ⁇ 44>, ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• Transfected CHO cells Choinese hamster ovary cells).
• ⁇ 57> In mammalian cells transfected with the vector set according to any one of ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• ⁇ 58> In mammalian cells transfected with the vector set according to any one of ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• a mammalian cell wherein when the first H chain mRNA amount is 1, the second H chain mRNA amount is 0.625 to 1.6, and the L chain mRNA amount is 3 to 8.
• CHO cells transfected with the vector set described in any one of ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• ⁇ 60> CHO cells transfected with the vector set described in any one of ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• ⁇ 61> The vector set according to any one of ⁇ 32> to ⁇ 44>, ⁇ 45-1> to ⁇ 45-5>, ⁇ 46-1> to ⁇ 46-6>, and ⁇ 47> to ⁇ 54>.
• a method of producing a cell pool comprising introducing it into a host cell.
• ⁇ 62> The method for producing a cell pool according to ⁇ 61>, wherein the host cells are mammalian cells.
• ⁇ 63> The method for producing a cell pool according to ⁇ 61>, wherein the host cells are CHO cells (Chinese hamster ovary cells).
• ⁇ 64> The method for producing a cell pool according to ⁇ 61>, wherein the host cells are CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHOpro3 ⁇ cells, or established cell lines derived from these cells.
• a method for producing cells with excellent productivity of heteromultimeric proteins or bispecific antibodies is provided.
• a method for producing a heteromultimeric protein or a bispecific antibody with excellent productivity is provided.
• a vector set is provided for use in producing cells that produce heteromultimeric proteins or bispecific antibodies.
• mammalian cells and CHO cells that produce heteromultimeric proteins or bispecific antibodies are provided.
• a method for creating a cell pool for use in selecting cells that produce a heteromultimeric protein or a bispecific antibody is provided.
• FIG. 1 is a vector map of expression vector 1 constructed in Examples. This is a vector map of expression vector 2 constructed in Examples. This is a vector map of expression vector 3 constructed in Examples. This is a vector map of expression vector 4 constructed in Examples.
• FIG. 2 is a conceptual diagram showing the structure of bispecific antibodies (BiAb1 and BiAb2) produced in Examples.
• Embodiments of the present disclosure will be described below. These descriptions and examples are illustrative of the embodiments and do not limit the scope of the embodiments.
• the mechanism of action described in this disclosure includes speculation, and its validity does not limit the scope of the embodiments.
• a numerical range indicated using " ⁇ " indicates a range that includes the numerical values written before and after " ⁇ " as the minimum and maximum values, respectively.
• the upper limit or lower limit described in one numerical range may be replaced with the upper limit or lower limit of another numerical range described step by step.
• the upper limit or lower limit of the numerical range may be replaced with the values shown in the Examples.
• each component may contain multiple types of corresponding substances.
• the amount of each component in the composition in this disclosure if there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, multiple types of substances present in the composition means the total amount of
• nucleic acids include all nucleic acids (e.g., DNA, RNA, analogs thereof, natural products, artificial products), and all nucleic acids, including low-molecular compounds, groups (for example, methyl groups), molecules and structures other than nucleic acids. This term includes nucleic acids to which substances are linked. Nucleic acids may be single-stranded or double-stranded.
• a vector is a substance that has the effect of transporting a foreign nucleic acid into cells, and is itself a nucleic acid.
• Expression vector refers to a vector that expresses a polypeptide based on its nucleic acid sequence. There are no restrictions on the size and base sequence of vectors and expression vectors. Vectors and expression vectors are preferably double-stranded DNA.
• polypeptide refers to a molecule in which amino acids are linked by peptide bonds. There is no limit to the number of amino acid residues in a polypeptide, and the term polypeptide includes proteins. It is desirable that the polypeptide according to the present disclosure has six or more amino acid residues.
• Polypeptides include polypeptides in which amino acids have been post-translationally modified. Post-translational modifications of amino acids include phosphorylation, methylation, acetylation, and the like.
• a heteromultimeric protein refers to a protein formed by an assembly of at least two types of subunits.
• One subunit refers to a single polypeptide that constitutes a multimeric protein.
• a heteromultimeric protein is a protein made up of at least two coding sequences that are expressed independently of each other.
• a heteromultimeric protein composed of n types of subunits is expressed by n types of coding sequences that are expressed independently of each other (n is an integer of 2 or more).
• heteromultimeric proteins include antibodies, Fc fusion proteins, enzymes, interleukins, cytokines, chemokines, peptide hormones, growth factors, transcription factors, receptors, receptor fragments, therapeutic proteins, viruses, virus-like particles, and vaccines. can be mentioned.
• antibodies are not limited to immunoglobulins, and may be any molecules that bind to antigens.
• antibody is a term that includes antibody fragments and antigen-binding molecules.
• Antibodies include both monovalent antibodies and multivalent antibodies.
• Antibodies include intact immunoglobulin molecules, Fab antibodies, Fab' antibodies, F(ab') 2 antibodies, single chain antibodies (scFv), diabodies, and variants thereof.
• Antibodies have antigen recognition sites called paratopes, and paratopes bind to antigens.
• Typical IgG contains two heavy chains (HC) with the same amino acid sequence and two light chains (LC) with the same amino acid sequence. It has a structure connected by bonds.
• HC contains a variable region (VH) and constant region (CH1, CH2, and CH3)
• LC contains a variable region (VL) and a constant region (CL).
• antibodies examples include human antibodies that have human variable regions and human constant regions; mouse antibodies that have mouse variable regions and mouse constant regions; chimeric antibodies that combine regions derived from multiple types of animals; mouse variable regions and human constant regions.
• bispecific antibodies refers to an antibody that can bind to two types of epitopes simultaneously.
• Multispecific antibody refers to an antibody that can bind to three or more types of epitopes simultaneously.
• the structure of a bispecific antibody and the number of epitope binding sites are not limited.
• the structure of a multispecific antibody and the number of epitope binding sites are not limited.
• bispecific antibodies include bispecific antibodies consisting of a first H chain, a second H chain, and an L chain common to the first H chain and the second H chain.
• an L chain common to two types of H chains means one type of L chain that pairs with two types of H chains.
• An example of this bispecific antibody is a tetramer in which one first H chain, one second H chain, and two L chains are connected by disulfide bonds.
• the first H chain, the second H chain, and the L chain each need only contain at least a region for recognizing an antigen and a region for forming an antibody.
• bispecific antibodies include bispecific antibodies consisting of an H chain, an L chain, and an scFv-Fc.
• scFv is a fusion protein of immunoglobulin heavy chain variable region and light chain variable region
• scFv-Fc is a fusion protein of scFv and Fc region.
• An example of this bispecific antibody is a trimer in which one H chain, one L chain, and one scFv-Fc are connected by a disulfide bond.
• the antigen and structure of the bispecific antibody are not limited as long as it has an arm that recognizes the first antigen and an arm that recognizes the second antigen.
• Examples of bispecific antibodies include Emicizumab, Blinatumomab, Vanucizumab, Istiratumab, Pasotuxizumab, Duligotuzumab, Duvortuxizumab, Faricimab. can be mentioned.
• One embodiment of the present disclosure is a method for producing cells that produce heteromultimeric proteins.
• the method for producing cells includes introducing a vector set of a first expression vector and a second expression vector into a host cell, and selecting cells that produce a heteromultimeric protein from the host cells into which the vector set has been introduced. including.
• a "vector set” means a set of a first expression vector and a second expression vector.
• expression vector when describing matters common to the first expression vector and the second expression vector, the first expression vector and the second expression vector are collectively referred to as "expression vector.”
• the host cell may be a prokaryotic cell or a eukaryotic cell.
• prokaryotic cells include bacterial cells.
• eukaryotic cells include yeast, insect cells and mammalian cells. As the host cell, eukaryotic cells are preferred, and mammalian cells are more preferred.
• bacterial cells examples include Gram-negative bacterial cells such as Escherichia coli, Salmonella typhimurium, Serratia marcescens, Pseudomonas putida, Pseudomonas aeruginosa; gram-positive bacterial cells such as P. subtilis).
• Preferred bacterial cells are Enterobacteriaceae, more preferably E. coli, especially strain B or K12.
• yeast examples include Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.
• insect cells examples include BmN cells from Bombyx mori, Sf9 and Sf21 cells from Spodoptera frugiperda, S2 cells from Drosophila melanogaster, and Pv11 cells from Polypedilum vanderplanki. Can be mentioned.
• mammalian cells examples include Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), human embryonic kidney cell lines (e.g. HEK293 cells), and human retinoblast-derived cell lines (e.g. PER.C6 cells). and mouse myeloma cell lines (eg, NS0 cells and SP2/0 cells).
• CHO cells Chinese hamster ovary cells
• BHK cells baby hamster kidney cells
• human embryonic kidney cell lines e.g. HEK293 cells
• human retinoblast-derived cell lines e.g. PER.C6 cells
• mouse myeloma cell lines eg, NS0 cells and SP2/0 cells.
• the host cells are preferably Chinese hamster ovary cells (CHO cells).
• CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHOpro3 ⁇ cells, and established cell lines derived from these cells.
• Examples of means for introducing expression vectors into host cells include electroporation, lipofection, microinjection, and cell infection with viral vectors. Electroporation is preferred from the viewpoints of high safety, high introduction efficiency, and low cytotoxicity. Further, when introducing multiple types of expression vectors into cells, electroporation is preferred from the viewpoint of highly uniform introduction into cells and chromosomes without bias depending on the type of expression vector.
• first expression vector and the second expression vector are introduced into the host cell.
• the first expression vector and the second expression vector may be introduced into a host cell together or separately. From the viewpoint of shortening the steps and period required for producing the target cells, it is preferable to introduce the first expression vector and the second expression vector into the host cell together.
• a vector solution containing both expression vectors is prepared.
• the molar concentration ratio of the first expression vector and the second expression vector is, for example, 1:5 to 5:1, 1:2 to 2:1, or 1:1.
• a vector solution containing the first expression vector and a vector solution containing the second expression vector are prepared.
• the molar concentration ratio of the expression vectors in both vector solutions is, for example, 1:5 to 5:1, 1:2 to 2:1, or 1:1.
• the molar ratio of the first expression vector and the second expression vector to be brought into contact with the host cell is, for example, 1:5 to 5:1, 1. :2 to 2:1 or 1:1.
• the sizes of the first expression vector and the second expression vector are not limited.
• the size of the first expression vector is, for example, 400 bp to 50,000 bp (base pair), 600 bp to 20,000 bp, or 800 bp to 10,000 bp.
• the size of the second expression vector is, for example, 400 bp to 50,000 bp (base pair), 600 bp to 20,000 bp, or 800 bp to 10,000 bp.
• the expression vector introduced into a host cell may be integrated into the genome of the host cell, may be integrated into an extrachromosomal element such as a plasmid, or may be contained in the cell as an independent extrachromosomal element. From the viewpoint of enabling long-term expression of the heteromultimeric protein of interest, the expression vector is preferably integrated into the genome of the host cell.
• Selecting cells that produce a heteromultimeric protein from host cells into which a vector set has been introduced involves, for example, setting standards for the expression level and/or purity of the heteromultimeric protein, and selecting cells that meet the standards; This is carried out by selecting cells with relatively high expression levels and/or purity of multimeric proteins. Specifically, for example, the following (a) to (d) are performed.
• the expression level of a heteromultimeric protein is the amount of the heteromultimeric protein itself.
• the purity of a heteromultimeric protein refers to the ratio of the heteromultimeric protein of interest to the total amount of multiple types of proteins.
• the expression level and/or purity of a protein can be measured by a known method. For example, the amount of protein separated by electrophoresis or the like is detected by a known method such as a fluorescence method or absorbance measurement. Measuring equipment such as the icIEF analyzer Maurice (Protein Simple) may be used.
• a multimeric protein that is not in its original form e.g., a multimeric protein lacking some subunits, or a multimeric protein in which one subunit is replaced by another subunit
• the proportion of multimeric proteins that are not in their original form is low.
• the cells that produce the heteromultimeric protein may be transiently expressing cells or stably expressing cells, and are preferably stably expressing cells.
• One embodiment of the present disclosure is a vector set used to create cells that produce heteromultimeric proteins.
• the vector set is a set of a first expression vector and a second expression vector, and these two types of expression vectors contain all the expression cassettes of the subunits constituting the heteromultimeric protein.
• a heteromultimeric protein consisting of n types of subunits is expressed from n types of expression cassettes in which the coding sequences of the subunits are different from each other.
• the vector set includes n types of expression cassettes whose subunit coding sequences differ from each other.
• the expression cassette for a subunit contains all the nucleic acids necessary for expression of that subunit.
• the size and base sequence of the subunit expression cassette are not limited.
• the size of one subunit expression cassette is, for example, 400bp to 4000bp (base pair ), 600bp to 3000bp, or 800bp to 2000bp.
• subunits constituting the heteromultimeric protein are divided into two groups, subunit X and subunit Y.
• Subunit X is a part of the subunits that constitute the heteromultimeric protein, and is, for example, a subunit whose expression is desired to be enhanced.
• Subunit X is a part, but not all, of the subunits that constitute the heteromultimeric protein. There may be one type of subunit X, or there may be two or more types of subunits.
• the number of types of subunits X is an integer of 1 or more (n-1) or less.
• Subunit Y is the remaining subunit after removing subunit X from the subunits constituting the heteromultimeric protein. There may be one type of subunit Y or two or more types.
• the subunit X expression cassette is included in both the first expression vector and the second expression vector.
• both the first expression vector and the second expression vector contain an expression cassette for subunit X.
• Subunit X is expressed from both the first expression vector and the second expression vector.
• the first expression vector contains the expression cassette for X1, the expression cassette for X2, and the expression cassette for The expression cassette includes an X2 expression cassette and an X3 expression cassette.
• the first expression vector may contain one or more expression cassettes for at least one type of subunit X for each type of subunit.
• the number of expression cassettes for subunits X contained in the first expression vector may be the same or different between the types of subunits. For example, when there are three types of subunits X1, X2, and X3, the number of expression cassettes for X1, the number of expression cassettes for X2, and the number of expression cassettes for X3 contained in the first expression vector are independent of each other, They may all have the same number, or may have different numbers.
• the second expression vector may contain one or more expression cassettes for at least one type of subunit X for each type of subunit.
• the number of expression cassettes for subunits X contained in the second expression vector may be the same or different between the types of subunits. For example, when there are three types of subunits X1, X2, and X3, the number of expression cassettes for X1, the number of expression cassettes for X2, and the number of expression cassettes for X3 contained in the second expression vector are independent of each other, They may all have the same number, or may have different numbers.
• the first expression vector includes at least one expression cassette for each type of subunit X
• the second expression vector includes at least one expression cassette for each type of subunit X.
• An example is a form containing one each.
• At least one type of expression cassette for subunit Y is included in one of the first expression vector and the second expression vector for each type. At least one type of subunit Y is expressed from one of the first expression vector and the second expression vector for each type.
• one of the first expression vector and the second expression vector may contain the entire expression cassette of subunit Y, or the first expression vector and the second expression vector may contain the entire expression cassette of subunit Y.
• the vector may contain expression cassettes for subunit Y divided for each type of subunit.
• the first expression vector when there are three types of subunits Y, Y1, Y2, and Y3, A configuration in which only the first expression vector contains an expression cassette for Y1, an expression cassette for Y2, and an expression cassette for Y3; A configuration in which the first expression vector contains an expression cassette of Y1, and the second expression vector contains an expression cassette of Y2 and an expression cassette of Y3; A configuration in which the first expression vector contains an expression cassette of Y2, and the second expression vector contains an expression cassette of Y1 and an expression cassette of Y3; The first expression vector may contain the Y3 expression cassette, and the second expression vector may contain the Y1 expression cassette and the Y2 expression cassette.
• the first expression vector may contain one or more expression cassettes for each type of subunit.
• the number of expression cassettes for subunit Y contained in the first expression vector may be the same or different between the types of subunits.
• the first expression vector contains three types of subunits Y, Y1, Y2, and Y3, the number of expression cassettes for Y1, the number of expression cassettes for Y2, and the number of expression cassettes for Y3 are included in the first expression vector.
• the numbers are independent from each other, and may be the same number or different numbers.
• the second expression vector may contain one or more expression cassettes for each type of subunit.
• the second expression vector may contain two or more types of expression cassettes for subunit Y
• the number of expression cassettes for subunit Y contained in the second expression vector may be the same or different between the types of subunits.
• the second expression vector contains three types of subunits Y, Y1, Y2, and Y3, the number of expression cassettes for Y1, the number of expression cassettes for Y2, and the number of expression cassettes for Y3 are included in the second expression vector.
• the numbers are independent from each other, and may be the same number or different numbers.
• An example of an embodiment is a configuration in which one of the first expression vector and the second expression vector contains one expression cassette for each type of subunit Y.
• Another example of an embodiment is a configuration in which the first expression vector and the second expression vector contain expression cassettes for subunit Y, one for each type.
• At least one expression cassette for subunit X is included in both the first expression vector and the second expression vector, and all expression cassettes for at least one subunit Y are included in , a vector set included in one of the first expression vector and the second expression vector.
• both the first expression vector and the second expression vector contain the expression cassette for subunit X
• one of the first expression vector and the second expression vector contains the expression cassette for subunit Y.
• the first expression vector includes one expression cassette for each type of subunit X
• the second expression vector includes one expression cassette for each type of subunit
• Examples include a vector set in which one of the first expression vector and the second expression vector includes one expression cassette for each type of subunit Y.
• a preferred form of using a vector set is one in which the heteromultimeric protein is an antibody.
• a more preferred form of using a vector set is one in which the heteromultimeric protein is a bispecific antibody.
• a preferred form of using a vector set includes a form in which the heteromultimeric protein consists of two or more types and six or less types of subunits, and the number of subunits X is one, two, or three types.
• This form includes, for example: A form in which the heteromultimeric protein consists of two types of subunits, with one type of subunit X; A form in which the heteromultimeric protein consists of three types of subunits and one type of subunit X: A form in which the heteromultimeric protein consists of three types of subunits, and there are two types of subunits X; A form in which the heteromultimeric protein consists of four types of subunits, with one type of subunit X; A form in which the heteromultimeric protein consists of four types of subunits, and there are two types of subunits X; A form in which the heteromultimeric protein consists of five types of subunits, with one type of subunit X; A form in which the heteromultimeric protein consists of six types of subunit
• a particularly preferable form of using a vector set includes a form in which the heteromultimeric protein consists of three types of subunits, and the number of subunits X is one or two types.
• Both the first expression vector and the second expression vector contain a light chain expression cassette, one of the first expression vector and the second expression vector contains a first heavy chain expression cassette, and one of the first expression vector and the second expression vector contains a first heavy chain expression cassette;
• the vector set related to the above bispecific antibody is such that the first expression vector and the second expression vector each contain one L chain expression cassette, and the first expression vector and the second expression vector each contain one L chain expression cassette.
• one of the second expression vectors contains one expression cassette for the first H chain
• one of the first expression vector and the second expression vector contains one expression cassette for the second H chain.
• a further example of a form that utilizes a vector set is a form in which the heteromultimeric protein is a bispecific antibody consisting of an H chain, a L chain, and an scFv-Fc.
• the expression level of scFv-Fc is preferably 0.3 times or more, more preferably 0.5 times or more, the expression level of H chain.
• the amount of scFv-Fc mRNA is preferably 0.3 times or more, more preferably 0.5 times or more, the amount of H chain mRNA.
• the amount of mRNA is determined by quantitative PCR as described below. Examples of embodiments of vector sets related to this bispecific antibody include vector sets A and B below.
• Vector set A Both the first expression vector and the second expression vector contain an expression cassette for the L chain and an expression cassette for the scFv-Fc, and one of the first expression vector and the second expression vector contains the expression cassette for the H chain.
• Vector set B both the first expression vector and the second expression vector contain the expression cassette of scFv-Fc, one of the first expression vector and the second expression vector contains the expression cassette of H chain, and the first expression vector and the second expression vector contain the expression cassette of H chain.
• An example of a bispecific antibody consisting of a H chain, a L chain, and a scFv-Fc is a trimer in which one H chain, one L chain, and one scFv-Fc are assembled. .
• Expression vectors and expression cassettes contain the necessary elements for expression of the polypeptide, depending on the type of host cell.
• the expression vector contains plasmid stability loci such as, for example, origins of replication, restriction enzyme sites, transcription terminators, and cer stability sequences. Good too.
• the expression vector may contain, for example, a promoter, a transcription terminator, a selection marker, and an origin of replication.
• the expression vector may include, for example, a promoter, a transcription terminator, and a selection marker.
• the expression vector includes, for example, a promoter, a polyadenylation sequence (e.g., human beta globin polyA sequence, bovine growth hormone polyA sequence, SV40 early polyA sequence, SV40 late PolyA sequence (SV40 late polyA )) and a selection marker.
• a polyadenylation sequence e.g., human beta globin polyA sequence, bovine growth hormone polyA sequence, SV40 early polyA sequence, SV40 late PolyA sequence (SV40 late polyA )
• a selection marker e.g., human beta globin polyA sequence, bovine growth hormone polyA sequence, SV40 early polyA sequence, SV40 late PolyA sequence (SV40 late polyA )
• Promoters that can be used in prokaryotic cells include the promoters disclosed in J. Mol. Biol. 1986; 189(1): 113-30, phage polymerase promoters, and E. coli polymerase promoters. Specific examples include T7A1, T7A2, T7A3, ⁇ pL, ⁇ pR, lac, lacUV5, trp, tac, trc, phoA and rrnB.
• promoters examples include gal promoter, AOX1 promoter, AOX2 promoter, GAP promoter, GAL1 promoter, and GAL10 promoter.
• promoters examples include polyhedrin promoter, P10 promoter, viral infection early expression protein (IE-1) promoter, MT promoter, COPIA promoter, CMV promoter, RSV promoter, SV40 promoter, heat shock protein promoter, Examples include the OPIE2 promoter and the actin5C promoter.
• promoters that can be used in mammalian cells include virus-derived promoters and housekeeping gene-derived promoters.
• virus-derived promoters include human CMV promoter, rat CMV promoter, SV40 promoter, RSR-LTR promoter and HSK-TK promoter.
• promoters derived from housekeeping genes include the hEF-1 ⁇ promoter, the Chinese hamster EF-1 ⁇ promoter, the ⁇ -actin promoter, and the mouse phosphoglycerate kinase (mPGK) promoter.
• mPGK mouse phosphoglycerate kinase
• a preferred example of a promoter that can be used in mammalian cells is the EF-1 ⁇ promoter, more preferably the hEF-1 ⁇ promoter.
• the subunit expression cassette preferably contains a secretion leader coding sequence for the purpose of promoting transport or secretion of the expressed polypeptide to the outside of the cell.
• a secretory leader is a type of signal peptide that induces the transport or secretion of a polypeptide to the outside of the cell.
• Functional equivalents of the fibronectin secretory leader have an amino acid sequence with 70% or more identity, preferably 75% or more identity, more preferably 80% or more identity, even more preferably 90% or more identity, most preferably It preferably has an identity of 95% or more and has the function of secreting the recombinant polypeptide to the outside of the cell. Sequence identity of amino acid sequences is calculated using, for example, BLAST (Basic Local Alignment Search Tool) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
• the origin organism of the fibronectin secretion leader depends on the type of host cell.
• the host cell is a human cell, it is preferred to utilize a human fibronectin secretory leader in the expression cassette.
• the host cell is a rat cell, it is preferred to utilize a rat fibronectin secretory leader in the expression cassette.
• the host cell is a CHO cell, it is preferable to utilize the Chinese hamster fibronectin secretory leader as the expression cassette.
• fibronectin secretion leader examples include a human fibronectin secretion leader having the amino acid sequence of SEQ ID NO: 1 and a Chinese hamster fibronectin secretion leader having the amino acid sequence of SEQ ID NO: 2.
• An example of the code sequence of SEQ ID NO: 1 is SEQ ID NO: 3.
• An example of the code sequence of SEQ ID NO: 2 is SEQ ID NO: 4.
• Sequence number 3 ATGCTGAGAGGCCCTGGACCTGGACTGCTGCTGCTGGCTGCCGTCTGGGAACCGCCGTGCCTTCTACCGGCGCC
• Sequence number 4 ATGCTCAGGGGTCCGGGACCCGGGCTGCTGCTGGCCGTCCTGTGCCTGGGACAGCGGTGCGCTGTACCGAAGCC
• the expression cassette for a subunit of a heteromultimeric protein comprises an hEF-1 ⁇ promoter, a fibronectin secretion leader coding sequence, a subunit coding sequence, and a polyA sequence, operably linked to each other. .
• the expression vector preferably contains an expression cassette for a selection marker for the purpose of confirming the introduction of the expression vector into host cells or for the purpose of creating stable expression cells.
• the selection marker is a marker that functions to select cells into which an expression vector has been introduced, and refers to a gene integrated into the expression vector and a protein encoded by the gene.
• the selected drug is an enzyme inhibitor
• the selection drug is methotrexate (MTX)
• the selection marker is dihydrofolate reductase (DHFR) and its gene.
• MTX methotrexate
• DHFR dihydrofolate reductase
• the DHFR-MTX system is an effective system in host cells lacking the DHFR gene (eg, CHO-DG44 cells).
• the selected drug is an enzyme inhibitor
• the selection agent is methionine sulfoximine (MSX)
• the selection marker is glutamine synthetase (GS) and its gene.
• MSX methionine sulfoximine
• GS glutamine synthetase
• the GS-MSX system is an effective system in host cells lacking the GS gene (eg, GS knockout CHO cells).
• the selection marker is an antibiotic degrading enzyme and its gene, the antibiotic resistance gene.
• the antibiotic resistance gene For example, hygromycin resistance gene, neomycin resistance gene, puromycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, G418 resistance gene, blasticidin resistance gene , zeocin resistance gene, phleomycin resistance gene, and ampicillin resistance gene.
• An example of an embodiment of a vector set is such that the first expression vector includes an expression cassette for the first selection marker, the second expression vector includes the expression cassette for the second selection marker, and the first expression vector includes the expression cassette for the first selection marker.
• the two selection markers are different types of selection markers.
• the first selection marker is, for example, at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene.
• the second selection marker is, for example, at least one selected from the group consisting of a dihydrofolate reductase gene, a glutamine synthetase gene, and an antibiotic resistance gene.
• An example of an embodiment of the vector set is that one of the first expression vector and the second expression vector contains an expression cassette for a dihydrofolate reductase gene or a glutamine synthetase gene, and the other contains an expression cassette for an antibiotic resistance gene.
• a preferred example of an expression cassette for the dihydrofolate reductase gene includes the mPGK promoter.
• a preferred example of an expression cassette for the glutamine synthetase gene includes the mPGK promoter.
• a preferred example of an antibiotic resistance gene expression cassette includes a viral promoter.
• the following vector set is an example of an embodiment of a vector set related to a bispecific antibody consisting of a first H chain, a second H chain, and an L chain common to the first H chain and the second H chain.
• the first expression vector includes an L chain expression cassette, a first H chain expression cassette, a second H chain expression cassette, and a first selection marker expression cassette;
• a vector set comprising an expression cassette and an expression cassette of a second selection marker.
• the vector set of the above form has a first selection marker and a second selection marker, from the viewpoint of making the amount of subunits expressed from the first expression vector larger than the amount of subunits expressed from the second expression vector.
• the first selection marker is preferably a dihydrofolate reductase gene or a glutamine synthetase gene.
• the first selection marker is a dihydrofolate reductase gene or a glutamine synthetase gene
• the second selection marker is an antibiotic resistance gene.
• Expression vectors containing a dihydrofolate reductase gene or a glutamine synthetase gene tend to increase in copy number within host cells more easily than expression vectors containing an antibiotic resistance gene.
• An example of an embodiment of a vector set related to a bispecific antibody consisting of an H chain, a L chain, and an scFv-Fc includes the following vector set.
• the first expression vector includes an expression cassette for an H chain, an expression cassette for an L chain, an expression cassette for an scFv-Fc, and an expression cassette for a first selection marker
• the second expression vector includes an expression cassette for an L chain, an expression cassette for an scFv-Fc, and an expression cassette for a first selection marker.
• a vector set comprising an expression cassette for Fc and an expression cassette for a second selection marker.
• the first expression vector includes an expression cassette for H chain, an expression cassette for L chain, an expression cassette for scFv-Fc, and an expression cassette for the first selection marker
• the second expression vector includes an expression cassette for scFv-Fc and an expression cassette for scFv-Fc.
• a vector set containing expression cassettes for two selection markers The first expression vector includes an expression cassette for the H chain, an expression cassette for scFv-Fc, and an expression cassette for the first expression marker
• a second expression vector includes an expression cassette for the L chain, an expression cassette for scFv-Fc, and an expression cassette for the first expression marker.
• a preferred embodiment of the first selection marker and the second selection marker in the vector set of the above form is common to the first H chain, the second H chain, and the first H chain and the second H chain. This is similar to the vector set related to bispecific antibodies consisting of L chain.
• Embodiments of the present disclosure do not preclude the use of a third expression vector other than the vector set (i.e., the first expression vector and the second expression vector).
• a third expression vector may be utilized, for example, to express polypeptides necessary for host cell growth or metabolism.
• One embodiment of the present disclosure is a mammalian cell transfected with a set of vectors.
• the mammalian cell transfected with the vector set may be a cell that transiently expresses the heteromultimeric protein or a cell that stably expresses the heteromultimeric protein, and is preferably a cell that stably expresses the heteromultimeric protein.
• One embodiment of the present disclosure is CHO cells transfected with a set of vectors.
• Specific examples include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHOpro3 ⁇ cells transfected with the vector set, and cell lines derived from these cells.
• the CHO cells transfected with the vector set may be transiently or stably expressing the heteromultimeric protein, and are preferably stably expressing cells.
• the heteromultimeric protein is a bispecific antibody consisting of a first H chain, a second H chain, and a L chain common to the first H chain and the second H chain
• transfection with a vector set is required.
• the number of copies of the L chain expression cassette in the infected mammalian cells and CHO cells is greater than the sum of the number of copies of the first H chain expression cassette and the number of copies of the second H chain expression cassette.
• it is a cell. This increases the expression level and/or purity of the bispecific antibody.
• the amount of mRNA measured by quantitative polymerase chain reaction (PCR) is useful for predicting the number of copies of an expression cassette present in a cell.
• the amount of mRNA measured by quantitative PCR is the amount of mRNA of the second H chain, when the amount of mRNA of the first H chain is set to 1. It is preferable that the mRNA amount of the chain is 0.5 to 2, and the amount of L chain mRNA is 2 to 10.
• the amount of mRNA measured by quantitative PCR is the amount of mRNA of the second H chain, when the amount of mRNA of the first H chain is set to 1. It is more preferable that the mRNA amount of the chain is 0.625 to 1.6, and the amount of L chain mRNA is 3 to 8.
• Mammalian cells and CHO cells transfected with the vector set are cells that express heteromultimeric proteins and can be used for the production of heteromultimeric proteins. Mammalian cells and CHO cells transfected with the vector set can also be used for administration, infusion, or transplantation into mammals.
• a method for producing a heteromultimeric protein includes culturing cells that produce the heteromultimeric protein. By culturing cells, heteromultimeric proteins are produced within the cells, and the heteromultimeric proteins accumulate in the culture medium and/or cells.
• the cells used in the method for producing a heteromultimeric protein are cells produced by a cell production method that is one of the embodiments of the present disclosure. These cells are selected according to evaluation criteria and/or relative production performance regarding heteromultimeric proteins, and therefore have excellent productivity of heteromultimeric proteins.
• the cell culture method and medium composition for producing a heteromultimeric protein may be selected depending on the type of host cell.
• Culture conditions eg, culture scale, cell density, temperature, and CO 2 concentration
• An example of an embodiment of the method for producing a heteromultimeric protein includes recovering the heteromultimeric protein from a culture solution.
• Methods for recovering heteromultimeric proteins from culture solutions include, for example, centrifugation, filtration, diafiltration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and high performance liquid chromatography (HPLC). ).
• the recovered heteromultimeric protein is used, for example, in the production of pharmaceutical compositions.
• An example of an embodiment of a method for producing a heteromultimeric protein includes recovering cells in which heteromultimeric proteins have accumulated from a culture medium. Methods for recovering cells from the culture medium include, for example, centrifugation and filtration. Depending on their nature, heteromultimeric proteins accumulate inside or on the surface of cells. The collected cells are, for example, administered, infused, or transplanted into a mammal.
• One embodiment of the present disclosure is a method for creating a cell pool.
• the method for creating a cell pool includes introducing a set of vectors into a host cell.
• the created cell pool is subjected to selection of cells that produce heteromultimeric proteins.
• a cell pool refers to a population of cells that has diversity in the copy number of the expression cassette of each subunit.
• the scale of the cell pool is not limited, but includes, for example, 10 to 10,000 types of clones.
• the host cells used in the cell pool production method are preferably eukaryotic cells, more preferably mammalian cells, and even more preferably CHO cells.
• Examples of CHO cells include CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHOpro3 ⁇ cells, and established cell lines derived from these cells.
• the cells contained in the cell pool may be cells that transiently express the heteromultimeric protein or cells that stably express the heteromultimeric protein, and preferably cells that stably express the heteromultimeric protein.
• the cell pool may be maintained by culturing the cells, or may be stored by freezing the cells.
• Expression vectors 1 and 2 were constructed to express a tetrameric bispecific antibody consisting of two types of H chains having mutually different amino acid sequences and one type of L chain common to the two types of H chains.
• BiAb1 the above bispecific antibody
• the two types of H chains will be referred to as “HC1” and “HC2”
• one type of L chain will be referred to as “common LC”.
• HC1, HC2 and common LC are collectively referred to as "antibody subunits.”
• Figure 1 is a vector map of expression vector 1.
• Expression vector 1 is a vector containing one expression cassette for HC1, one expression cassette for HC2, one expression cassette for common LC, and one expression cassette for DHFR gene.
• Each antibody subunit expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretory leader coding sequence, the antibody subunit coding sequence, and the polyA sequence, consecutively downstream and in the same reading frame of the fibronectin secretory leader coding sequence.
• the coding sequences for antibody subunits are located.
• Figure 2 is a vector map of expression vector 2.
• Expression vector 2 is a vector containing one common LC expression cassette and one hygromycin resistance gene expression cassette.
• the common LC expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretory leader coding sequence, the common LC coding sequence and the polyA sequence, and the common LC expression cassette is continuous and in the same reading frame downstream of the fibronectin secretory leader coding sequence.
• the code arrangement is arranged.
• Expression vector 1 alone or expression vector 1 and expression vector 2 were introduced into CHO-DG44 cells by electroporation.
• CHO-DG44 cells introduced with the expression vector were seeded in 10 mL of OptiCHO medium (Lifetechnologies, 12681011) and cultured statically at a temperature of 37° C. in an atmosphere of 5% CO 2 (v/v).
• methotrexate was added as a selective drug to CHO-DG44 cells into which only expression vector 1 had been introduced, and methotrexate and hygro as selective drugs were added to CHO-DG44 cells into which expression vector 1 and expression vector 2 had been introduced.
• Mycin B was added at the same time.
• the culture volume was expanded to 20 mL, transferred to a 125 mL flask for shaking culture, and cultured with shaking.
• ⁇ Selection of BiAb1 producing cells The shake-cultured cells were dispensed one cell per well into a 96-well plate using a droplet cell sorter, and cultured stationary at a temperature of 37° C. in an atmosphere of 10% CO 2 (v/v). After culturing for 14 days, the culture supernatant was collected, and the antibody concentration was measured using an intermolecular interaction analyzer Octet Qke (Sartorius). Clones with high antibody concentrations were selected and cultured in 24-well plates, and then in bioreactor tubes for scale-up. In this way, cells producing BiAb1 were created.
• BiAb1-producing cells created by introducing only expression vector 1 are referred to as BiAb1-TGV cells.
• BiAb1-producing cells created by introducing expression vector 1 and expression vector 2 are referred to as BiAb1-TGV/SGV cells.
• BiAb1-TGV cells and BiAb1-TGV/SGV cells were each suspended in 40 mL of basal medium, transferred to a 125 mL flask for shaking culture, and shaken at a speed of 140 rpm at a temperature of 37°C and an atmosphere of 5% CO 2 (v/v). Cultivation and feeding were performed. A constant amount of feed medium was added every day from the 3rd day to the 13th day of culture. Samples were taken every 1 to 3 days, and cell density, culture medium components, and antibody concentration were measured. On the 14th day after the start of culture, the culture solution was collected, and cells and cell debris were removed using a depth filter (pore size: 0.22 ⁇ m) to obtain a culture supernatant.
• a depth filter pore size: 0.22 ⁇ m
• the antibody concentration in the culture supernatant was measured by liquid chromatography using a Protein A column.
• the antibody was purified from the culture supernatant using a Protein A column, and the icIEF analyzer Maurice (Protein Simple) was used to quantify BiAb1 and detect mispaired antibodies (antibodies with only HC1 in the H chain and antibodies with only HC2 in the H chain). Quantification was performed to determine the purity of BiAb1. Purity here is BiAb1/(BiAb1+mispaired antibody) ⁇ 100 (%). Table 1 shows the measurement results for the clones with the highest antibody concentrations.
• the amount of mRNA of the antibody subunit in each clone was measured by quantitative PCR.
• Table 2 shows the measurement results for the clones with the highest antibody concentrations.
• the numerical values shown in Table 2 are relative values when the amount of HC1 mRNA in BiAb1-TGV cells is set to 1.
• BiAb1-TGV cells and BiAb1-TGV/SGV cells contain HC1 expression cassettes and HC2 expression cassettes at a ratio of 1:1. It is thought that it is held at 1.
• the expression levels of HC1 and HC2 are comparable, and it is presumed that mispaired antibodies are unlikely to occur.
• the amount of BiAb1 was 2.6 g/L in BiAb1-TGV cells, 4.7 g/L in BiAb1-TGV/SGV cells, and 1.8 times that in BiAb1-TGV/SGV cells. Ta.
• Expression vector 1 contains one HC1 expression cassette, one HC2 expression cassette, and one common LC expression cassette, so BiAb1-TGV cells contain one HC1 expression cassette, one HC2 expression cassette, and one common LC expression cassette. It is thought that they are held in a number ratio of 1:1:1. It is presumed that the difference in mRNA amount between the subunits is due to the difference in transcription efficiency of each expression cassette.
• BiAb1-TGV/SGV cells the common LC mRNA amount was 5.6 times that of HC1 and 8.9 times that of HC2.
• BiAb1-TGV/SGV cells are thought to possess more common LC expression cassettes than HC1 and HC2 expression cassettes. This is considered to be the result of introducing both expression vector 1 and expression vector 2 into the host cell.
• the common LC mRNA amount was 3.4 times the total mRNA amount of HC1 and HC2. If we simply predict, the total expression level of HC1 and HC2 is sufficient for the expression level of common LC, so it can be said that the expression level of common LC is excessive in BiAb1-TGV/SGV cells (three times the theoretically required amount). That's all.) It is presumed that the excessive expression level of common LC contributed to the increase in the amount of BiAb1 in BiAb1-TGV/SGV cells (1.8 times that in BiAb1-TGV cells).
• CHO cells with high expression level and purity of BiAb1 were selected from CHO cells transfected with the vector set of expression vector 1 and expression vector 2, and CHO cells with excellent BiAb1 productivity could be generated.
• Expression vectors 3 and 4 for expressing a trimeric bispecific antibody consisting of one type of H chain, one type of L chain that pairs with the H chain, and one type of single chain antibody scFv-Fc was built.
• BiAb2 the above bispecific antibody
• one type of H chain will be referred to as "HC”
• one type of L chain will be referred to as "LC”
• scFv-Fc the above bispecific antibody
• HC, LC, and scFv-Fc are collectively referred to as "antibody subunits.”
• Figure 3 is a vector map of expression vector 3.
• Expression vector 3 is a vector containing one HC expression cassette, one LC expression cassette, one scFv-Fc expression cassette, and one DHFR gene expression cassette.
• Each antibody subunit expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretory leader coding sequence, the antibody subunit coding sequence, and the polyA sequence, consecutively downstream and in the same reading frame of the fibronectin secretory leader coding sequence.
• the coding sequences for antibody subunits are located.
• Figure 4 is a vector map of expression vector 4.
• Expression vector 4 is a vector containing one expression cassette for LC, one expression cassette for scFv-Fc, and one expression cassette for hygromycin resistance gene.
• the LC expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretory leader coding sequence, the LC coding sequence, and the polyA sequence, and the LC coding sequence is continuously downstream of the fibronectin secretory leader coding sequence and in the same reading frame. It is located.
• the scFv-Fc expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretion leader coding sequence, the scFv-Fc coding sequence and the polyA sequence, and the scFv-Fc expression cassette contains the hEF-1 ⁇ promoter, the fibronectin secretory leader coding sequence, the scFv-Fc coding sequence, and the polyA sequence, and the scFv-Fc is continuously downstream of the fibronectin secretory leader coding sequence and in the same reading frame.
• -Fc code sequence is located.
• Expression vector 3 alone or expression vector 3 and expression vector 4 were introduced into CHO-DG44 cells by electroporation.
• CHO-DG44 cells introduced with the expression vector were seeded in 10 mL of OptiCHO medium (Lifetechnologies, 12681011) and cultured statically at a temperature of 37° C. in an atmosphere of 5% CO 2 (v/v).
• OptiCHO medium Lifetechnologies, 12681011
• methotrexate and hygromycin B were simultaneously added as selective drugs to the CHO-DG44 cells into which expression vectors 3 and 4 had been introduced.
• the culture volume was expanded to 20 mL, transferred to a 125 mL flask for shaking culture, and cultured with shaking.
• BiAb2-producing cells created by introducing only expression vector 3 are referred to as BiAb2-TGV cells.
• BiAb2-producing cells created by introducing expression vector 3 and expression vector 4 are referred to as BiAb2-TGV/DGV cells.
• BiAb2-TGV cells and BiAb2-TGV/DGV cells were each suspended in 40 mL of basal medium, transferred to a 125 mL flask for shaking culture, and shaken at a speed of 140 rpm at a temperature of 37°C and an atmosphere of 5% CO 2 (v/v). Cultivation and feeding were performed. A fixed amount of feed medium was added every day from the 3rd day to the 13th day of culture. Samples were taken every 1 to 3 days, and cell density, culture medium components, and antibody concentration were measured. On the 14th day after the start of culture, the culture solution was collected, and cells and cell debris were removed using a depth filter (pore size: 0.22 ⁇ m) to obtain a culture supernatant.
• a depth filter pore size: 0.22 ⁇ m
• the antibody concentration in the culture supernatant was measured by liquid chromatography using a Protein A column.
• Antibodies were purified from the culture supernatant using a Protein A column, and quantification of BiAb2 and mispaired antibodies (antibodies in which HC and scFv-Fc are not paired) were performed using a capillary electrophoresis analyzer PA800plus (AB SCIEX). The purity of BiAb2 was determined. Purity here is BiAb2/(BiAb2+mispaired antibody) ⁇ 100 (%). Table 3 shows the productivity of each cell.
• the amount of mRNA of the antibody subunit in each BiAb2-producing cell was measured by quantitative PCR.
• Table 4 shows the measurement results for each cell.
• the numerical values shown in Table 4 are relative values when the amount of HC gene mRNA in BiAb2-TGV cells is set to 1.
• BiAb2-TGV cells As shown in Table 3, the purity of BiAb2 in BiAb2-TGV cells was 51%, whereas in BiAb2-TGV/DGV cells, the purity was 95%, significantly improving the purity. Since expression vector 3 contains one HC expression cassette and one scFv-Fc expression cassette, BiAb2-TGV cells and BiAb2-TGV/DGV cells carry the HC expression cassette and the scFv-Fc expression cassette. It is thought that they are held at a number ratio of 1:1.
• Expression vector 1 contains one each of the HC expression cassette, LC expression cassette, and scFv-Fc expression cassette. It is thought that the expression cassettes are contained in a number ratio of 1:1:1. It is presumed that the difference in mRNA amount between subunits is due to the difference in transcription efficiency of each expression cassette, and in particular, it was found that scFv-Fc has significantly lower transcription efficiency than HC and LC.
• BiAb2-TGV/DGV cells the amount of LC mRNA was 4 times that of HC, and the amount of scFv-Fc mRNA was about 0.5 times that of HC.
• BiAb2-TGV/DGV cells are thought to possess more LC and scFv-Fc expression cassettes than HC expression cassettes. This is considered to be the result of introducing both expression vector 3 and expression vector 4 into the host cell.
• CHO cells with high BiAb2 expression level and purity were selected from CHO cells transfected with the vector set of expression vector 3 and expression vector 4, and CHO cells with excellent BiAb2 productivity could be generated.

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