Method for generation of multivalent, bispecific antibody expressing cell by targeted integration of multiple expression cassettes in prescribed organization

Abstract

To provide a method for producing a multivalent, bispecific antibody.SOLUTION: The method comprises the steps of: cultivating a mammalian cell comprising a deoxyribonucleic acid encoding the multivalent, bispecific antibody; and recovering the multivalent, bispecific antibody from the cell or the cultivation medium. The deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of the mammalian cell, and comprises, in the 5'- to 3'-direction, a first expression cassette encoding the first heavy chain, a second expression cassette encoding the first light chain, a third expression cassette encoding the first light chain, a fourth expression cassette encoding the second heavy chain, a fifth expression cassette encoding the first light chain, and a sixth expression cassette encoding the first light chain.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of cell line engineering and polypeptide production. More precisely, recombinant mammalian cells obtained by a double recombinase-mediated cassette exchange reaction leading to the integration of specific expression cassette sequences into the genome of mammalian cells are reported herein. Said cells can be used in a method for producing multivalent, bispecific antibodies. [Background technology]

[0002] Secreted glycosylated polypeptides, such as antibodies, are usually produced by recombinant expression in eukaryotic cells, either as stable or transient expression.

[0003] One strategy for generating recombinant cells expressing an exogenous polypeptide of interest involves random integration of a nucleotide sequence encoding the polypeptide of interest, followed by secretion and isolation steps. However, this approach has several disadvantages. First, functional integration of a nucleotide sequence into a cell's genome is not only a rare event, but these rare events, given the randomness of where the nucleotide sequence is integrated, can result in various phenotypes in gene expression and cell growth. Such mutations, known as "position effect mutations," are due, at least in part, to the complex gene regulatory networks present in eukaryotic cell genomes and the availability of specific genomic loci for integration and gene expression. Second, random integration strategies generally do not control the number of nucleotide sequence copies integrated into the cell's genome. In fact, gene amplification methods are often used to achieve high-producing cells. However, such gene amplification can also lead to undesirable cellular phenotypes, such as unstable cell growth and / or product expression. Third, due to the heterogeneity of integration loci inherent in the random integration process, screening thousands of cells after transfection to isolate recombinant cells exhibiting the desired expression level for the polypeptide of interest is time-consuming and labor-intensive. Even after isolating such cells, stable expression of the polypeptide of interest is not guaranteed, and further screening may be required to obtain stable, commercially viable production cells. Fourth, polypeptides produced from cells obtained by random integration exhibit a high degree of sequence variance, which may be due in part to the mutagenicity of the selection agent used to select for high levels of polypeptide expression. Finally, the greater the complexity of the polypeptide to be produced, i.e., the greater the number of different polypeptides or polypeptide chains required to form the polypeptide of interest in cells, the more important it becomes to control the expression ratios of different polypeptides or polypeptide chains relative to each other.Control of the expression ratio is required to allow efficient expression, correct assembly, and successful secretion of the polypeptide of interest with high expression yields.

[0004] Targeted integration by recombinase-mediated cassette exchange (RMCE) is a method for specifically and efficiently directing foreign DNA into predetermined sites in the eukaryotic host genome (Turan et al., J. Mol. Biol. 407 (2011) 193-221 (Non-Patent Document 1)).

[0005] WO 2006 / 007850 discloses a production method using an anti-RhD recombinant polyclonal antibody and site-specific integration into the genome of an individual host cell.

[0006] Crawford, Y. et al. (Biotechnol. Prog. 29 (2013) 1307-1315 (Non-Patent Document 2)) reported that a combination of phiC31 integrase and CRE-Lox technologies was used to rapidly identify reliable hosts for target-directed cell line development through limited genomic screening.

[0007] WO 2013 / 006142 (Patent Document 2) discloses a near-homogeneous population of genetically modified eukaryotic cells that have stably integrated into their genomes a donor cassette comprising a strong polyadenylation site operably linked to an isolated nucleic acid fragment comprising a targeting nucleic acid site and a selectable marker protein-coding sequence, the isolated nucleic acid fragment being flanked by a first recombination site and a second, distinct recombination site.

[0008] WO 2018 / 162517 (Patent Document 3) discloses that large differences in expression yield and product quality were observed depending on i) the expression cassette sequence and ii) the distribution of the expression cassette among different expression vectors.

[0009] Tadauchi, T. et al. disclose the use of a regulated targeting integration cell line development approach to systematically investigate the factors that make antibody expression difficult (Biotechnol. Prog. 35 (2019) No. 2, 1-11 (Non-Patent Document 3)).

[0010] WO 2017 / 184831 (Patent Document 4) discloses site-specific integration and expression of recombinant proteins in eukaryotic cells, and in particular, a method for improving the expression of antibodies, including bispecific antibodies, in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by using an expression-enhancing gene locus. The data in this document is presented in an anonymized manner, so it is not possible to draw any conclusions about what is actually shown.

[0011] Rajendra, Y. et al. disclose that a single quad vector is a simple yet effective alternative approach for generating stable CHO cell lines, which may accelerate cell line generation for clinical hetero-mAb therapeutics (Biotechnol. Prog. 33 (2017) 469-477 (Non-Patent Document 4)).

[0012] WO 2017 / 060144 (Patent Document 5) discloses a tetravalent bispecific antibody against costimulatory TNF receptors, and WO 2019 / 086497 (Patent Document 6) discloses combination therapy with a targeting Ox40 agonist. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2006 / 007850 [Patent Document 2] International Publication No. 2013 / 006142 [Patent Document 3] International Publication No. 2018 / 162517 [Patent Document 4] International Publication No. 2017 / 184831 [Patent Document 5] International Publication No. 2017 / 060144 [Patent Document 6] International Publication No. 2019 / 086497 [Non-patent literature]

[0014] [Non-Patent Document 1] Turan et al., J.Mol.Biol.407(2011)193~221 [Non-patent document 2] Crawford, Y. et al., Biotechnol.Prog.29(2013)1307~1315 [Non-patent document 3] Tadauchi, T. et al., Biotechnol.Prog.35(2019) No.2,1~11 [Non-patent document 4] Rajendra, Y. et al., Biotechnol.Prog.33(2017)469~477 Summary of the Invention

[0015] Recombinant mammalian cells expressing multivalent bispecific antibodies are reported herein. Multivalent bispecific antibodies are heteromultimeric polypeptides that are not naturally expressed by the mammalian cells. More specifically, multivalent bispecific antibodies are heteromultimeric proteins consisting of three polypeptides or polypeptide chains: one light chain, which is a full-length light chain; one heavy chain, which is an extended heavy chain, comprising an additional heavy chain Fab fragment at its N-terminus and an additional light chain variable domain at its C-terminus; and a further heavy chain, which is an extended heavy chain, comprising an additional heavy chain Fab fragment at its N-terminus and an additional heavy chain variable domain at its C-terminus. To achieve expression of multivalent bispecific antibodies, recombinant nucleic acids containing multiple different expression cassettes in specific and predetermined sequences were integrated into the genome of mammalian cells.

[0016] Also reported herein are methods for making recombinant mammalian cells that express multivalent, bispecific antibodies and methods for producing multivalent, bispecific antibodies using the recombinant mammalian cells.

[0017] In one preferred embodiment, the multivalent bispecific antibody comprises: a first heavy chain comprising, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a second copy of the first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a first light chain variable domain; - a second heavy chain comprising, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a second copy of the first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a second heavy chain variable domain; and a first light chain comprising, from the N-terminus to the C-terminus, a second light chain variable domain and a CL domain; Including, wherein the first heavy chain variable domain and the second light chain variable domain form a first binding site, and the second heavy chain variable domain and the first light chain variable domain form a second binding site.

[0018] In one preferred embodiment, neither the first nor the second light chain of the multivalent bispecific antibody is a common or universal light chain.

[0019] The present invention is based, at least in part, on the discovery that the sequences of the different expression cassettes required for expression of heteromultimeric multivalent bispecific antibodies, i.e., the expression cassette configuration, influence the expression yield of the multivalent bispecific antibody when integrated into the genome of a mammalian cell.

[0020] The present invention is based, at least in part, on the discovery that efficient recombinant expression and production of multivalent bispecific antibodies can be achieved by integrating nucleic acids encoding heteromultimeric multivalent bispecific antibodies with specific expression cassette configurations into the genome of mammalian cells.

[0021] It has been found that a given expression cassette sequence can be advantageously integrated into the genome of a mammalian cell by a dual recombinase-mediated cassette exchange reaction.

[0022] One embodiment according to the present invention provides a method for producing a multivalent bispecific antibody, comprising the steps of: a) culturing mammalian cells comprising deoxyribonucleic acid encoding the multivalent bispecific antibody, optionally under conditions suitable for expression of the multivalent bispecific antibody; b) recovering the multivalent, bispecific antibody from the cells or culture medium wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of a mammalian cell and comprises, in a 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain A method comprising:

[0023] In one embodiment, exactly one copy of the deoxyribonucleic acid is stably integrated into a single site or locus in the genome of the mammalian cell.

[0024] One aspect of the invention is a deoxyribonucleic acid encoding a multivalent, bispecific antibody, comprising in 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain It is a deoxyribonucleic acid containing any one of the following:

[0025] One aspect of the invention is the use of deoxyribonucleic acid for the expression of a multivalent, bispecific antibody in a mammalian cell, wherein the deoxyribonucleic acid comprises, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain The present invention relates to the use of deoxyribonucleic acid, including any of the following:

[0026] In one embodiment of the use, the deoxyribonucleic acid is integrated into the genome of the mammalian cell.

[0027] In one embodiment of the use, exactly one copy of the deoxyribonucleic acid is stably integrated into a single site or locus in the genome of the mammalian cell.

[0028] One aspect of the present invention is a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated into the genome of the cell, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain A recombinant mammalian cell comprising either

[0029] In one embodiment, exactly one copy of the deoxyribonucleic acid is stably integrated into a single site or locus in the genome of the mammalian cell.

[0030] In one embodiment of all the preceding aspects, the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises: - a first recombination recognition sequence located 5' of the first (5'-most) expression cassette, - a second recombination recognition sequence located 3' to the sixth or eighth (3'-most) expression cassette, and - a third recombination recognition sequence, - between the first recombination recognition sequence and the second recombination recognition sequence, and - Between two expression cassettes a third recombination recognition sequence located at wherein all recombination recognition sequences are different.

[0031] In one embodiment, the third recombination recognition sequence is located between the third and fourth or fourth and fifth expression cassettes.

[0032] In one embodiment, the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises another expression cassette encoding a selectable marker, wherein the expression cassette encoding the selectable marker is partially located 5' and partially located 3' to a third recombination recognition sequence, wherein the portion of the expression cassette located 5' comprises a promoter and a start codon, and the portion of the expression cassette located 3' comprises a coding sequence without a start codon and a polyA signal, wherein the start codon is operably linked to the coding sequence.

[0033] One embodiment of the present invention is a composition comprising two deoxyribonucleic acids, wherein the two deoxyribonucleic acids comprise three different recombination recognition sequences and eight expression cassettes; - the first deoxyribonucleic acid is, in the 5' to 3' direction, (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding the first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, and - a first copy of a third recombination recognition sequence including any of the following: the second deoxyribonucleic acid is in the 5' to 3' direction (1) - a second copy of a third recombination recognition sequence, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding a first light chain, and - a second recombination recognition sequence, or (2) - a second copy of a third recombination recognition sequence, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, - an eighth expression cassette encoding a first light chain, and - a second recombination recognition sequence A composition comprising any one of the following:

[0034] In one embodiment, the first and second deoxyribonucleic acids both comprise the configuration of (1), or the first and second deoxyribonucleic acids both comprise the configuration of (2).

[0035] In one embodiment of all the preceding aspects, the deoxyribonucleic acid encoding the multivalent, bispecific antibody further comprises another expression cassette encoding a selectable marker.

[0036] In one embodiment, the expression cassette encoding the selectable marker comprises a third recombination recognition sequence: i) the 5' side, or ii) the 3' side, or iii) Partially on the 5' side and partially on the 3' side Located in.

[0037] In one embodiment, an expression cassette encoding a selectable marker is located partially 5' and partially 3' of a third recombination recognition sequence, and the 5' portion of the expression cassette includes a promoter and a start codon, while the 3' portion of the expression cassette includes a coding sequence without a start codon and a polyA signal.

[0038] In one embodiment, the 5'-located portion of the expression cassette encoding the selectable marker comprises a promoter sequence operably linked to a start codon, the promoter sequence being adjacent to the third or fourth expression cassette on the upstream side (i.e., located downstream relative to the third or fourth expression cassette), respectively, and the start codon being adjacent to the third recombination recognition sequence on the downstream side (i.e., located upstream relative to the third recombination recognition sequence); and the 3'-located portion of the expression cassette encoding the selectable marker comprises a nucleic acid encoding a selectable marker lacking a start codon, being adjacent to the third recombination recognition sequence on the upstream side and adjacent to the fourth or fifth expression cassette on the downstream side, respectively.

[0039] In one embodiment, the start codon is a translation initiation codon, hi one embodiment, the start codon is ATG.

[0040] One aspect of the present invention is a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated into the genome of the cell, The deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises the following elements: a first recombination recognition sequence, a second recombination recognition sequence, and a third recombination recognition sequence; a first selectable marker and a second selectable marker, and the first to sixth or first to eighth expression cassettes, and the sequence of the element comprises, in the 5' to 3' direction: RRS1-1 st EC-2 nd EC-3 rd EC-RRS3-SM1-4 th EC-5 th EC-6 th EC-RRS2 or RRS1-1 st EC-2 nd EC-3 rd EC-4 th EC-RRS3-SM1-5 th EC-6 th EC-7th EC-8 th EC-RRS2 where: RRS = recombination recognition sequence; EC = expression cassette; SM = selection marker is.

[0041] One embodiment of the present invention provides a method for producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding a multivalent bispecific antibody and that secretes the multivalent bispecific antibody, comprising: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus in the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and wherein the recombination recognition sequences are all different; b) introducing into the cells provided in a) two deoxyribonucleic acid compositions comprising three different recombination recognition sequences and six or eight expression cassettes, - the first deoxyribonucleic acid is in the 5' to 3' direction (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding the first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, and - a first copy of a third recombination recognition sequence including any of the following: a second deoxyribonucleic acid in the 5' to 3' direction, (1) - a second copy of a third recombination recognition sequence, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding a first light chain, and - a second recombination recognition sequence, or (2) - a second copy of a third recombination recognition sequence, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, - an eighth expression cassette encoding a first light chain, and - a second recombination recognition sequence including any of the following: the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are identical to the first to third recombination recognition sequences of the integrated exogenous nucleotide sequence; the 5'-end portion and the 3'-end portion of the expression cassette encoding said one second selection marker together form a functional expression cassette of said one second selection marker; introducing into a cell a composition comprising two deoxyribonucleic acids; c) one or more recombinases i) simultaneously with the first and second deoxyribonucleic acids of b), or ii) Then sequentially The step of introducing one or more recombinases recognize recombination recognition sequences in the first and second deoxyribonucleic acids (and optionally, the one or more recombinases perform two recombinase-mediated cassette exchanges); introducing one or more recombinases; and d) selecting cells that express a second selection marker and secrete a multivalent bispecific antibody. whereby a recombinant mammalian cell is produced that contains deoxyribonucleic acid encoding the multivalent bispecific antibody and that secretes the multivalent bispecific antibody.

[0042] In one embodiment, the first and second deoxyribonucleic acids both comprise the configuration of (1), or the first and second deoxyribonucleic acids both comprise the configuration of (2).

[0043] In one embodiment, an expression cassette encoding one of the second selection markers is located partially on the 5' side and partially on the 3' side of a third recombination recognition sequence, and the portion of the expression cassette located on the 5' side includes a promoter and a start codon, while the portion of the expression cassette located on the 3' side includes a sequence encoding the one of the second selection markers without a start codon and a polyA signal.

[0044] In one embodiment, the 5'-end portion of the expression cassette encoding the one second selection marker comprises a promoter sequence operably linked to a start codon, the promoter sequence being adjacent to the third or fourth expression cassette on the upstream side (i.e., located downstream relative to the third or fourth expression cassette), respectively, and the start codon being adjacent to the third recombination recognition sequence on the downstream side (i.e., located upstream relative to the third recombination recognition sequence); and the 3'-end portion of the expression cassette encoding the one second selection marker lacks a start codon and comprises a sequence encoding the one second selection marker, which is adjacent to the third recombination recognition sequence on the upstream side and adjacent to the fourth or fifth expression cassette on the downstream side, respectively.

[0045] In one embodiment, the start codon is a translation initiation codon, hi one embodiment, the start codon is ATG.

[0046] In one embodiment of all the preceding aspects and embodiments, the first deoxyribonucleic acid is incorporated into a first vector and the second deoxyribonucleic acid is incorporated into a second vector.

[0047] In one embodiment of all the preceding aspects and embodiments, - the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a first light chain variable domain; - the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a second heavy chain variable domain; - the first light chain comprises, from N-terminus to C-terminus, a second light chain variable domain and a CL domain; wherein the first heavy chain variable domain and the second light chain variable domain form a first binding site, and the second heavy chain variable domain and the first light chain variable domain form a second binding site.

[0048] In one embodiment of all the preceding aspects and embodiments, each expression cassette comprises, in the 5' to 3' direction, a promoter, a coding sequence, and a polyadenylation signal sequence, optionally followed by a terminator sequence.

[0049] In one embodiment of all the preceding aspects and embodiments, Each expression cassette of an antibody chain comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the antibody chain, and a polyadenylation signal sequence, and optionally a terminator sequence; and Each expression cassette encoding a selectable marker comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the selectable marker, and a polyadenylation signal sequence, and optionally, a terminator sequence.

[0050] In one embodiment of all the preceding aspects and embodiments, the promoter is the human CMV promoter with or without intron A, the polyadenylation signal sequence is the bGH polyA site, and the terminator is the hGT terminator.

[0051] Terminator sequences prevent the generation of very long RNA transcripts by RNA polymerase II, i.e., read-through into the next expression cassette in the deoxyribonucleic acid according to the invention, and are used in the method according to the invention, i.e., the expression of one structural gene of interest is controlled by its own promoter.

[0052] Thus, the combination of a polyadenylation signal and a terminator sequence achieves efficient transcription termination; readthrough of RNA polymerase II is prevented by the presence of a dual termination signal. The terminator sequence initiates complex degradation, facilitating dissociation of RNA polymerase from the DNA template.

[0053] In one embodiment of all the preceding aspects and embodiments, the promoter is the human CMV promoter including intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence, and the terminator sequence is the hGT terminator, except in the expression cassette for the selectable marker, where the promoter is the SV40 promoter, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence, and no terminator sequence is present.

[0054] In one embodiment of all the above aspects and embodiments, all expression cassettes are arranged in one direction.

[0055] In one embodiment of all the preceding aspects and embodiments, the mammalian cell is a CHO cell. In one embodiment, the CHO cell is a CHO-K1 cell.

[0056] In one embodiment of all aspects and embodiments, the multivalent bispecific antibody is an anti-FAP / Ox40 bispecific antibody. Such antibodies are reported in WO 2017 / 060144, which is incorporated herein by reference in its entirety.

[0057] In one embodiment of all the preceding aspects and embodiments, neither the first nor the second light chain of the multivalent bispecific antibody is a common or universal light chain.

[0058] In one preferred embodiment of all aspects and embodiments, exactly two deoxyribonucleic acids are included or introduced.

[0059] The individual expression cassettes in the deoxyribonucleic acid according to the invention are arranged sequentially, with the distance between the end of one expression cassette and the start of the next one being only a few nucleotides, which is what was necessary for the cloning procedure, i.e. what results.

[0060] In one embodiment of all the preceding aspects and embodiments, the two immediately following expression cassettes are separated by a maximum of 100 bps (i.e., a maximum of 100 base pairs (bps) from the end of the polyA signal sequence or terminator sequence to the start of the next promoter element, respectively). In one embodiment, the two immediately following expression cassettes are separated by a maximum of 50 bps. In one preferred embodiment, the two immediately following expression cassettes are separated by a maximum of 25 bps. [The present invention 1001] 1. A method for producing a multivalent bispecific antibody, comprising: a) culturing mammalian cells containing deoxyribonucleic acid encoding said multivalent bispecific antibody, and b) recovering said multivalent, bispecific antibody from said cells or culture medium. Including, the deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of the mammalian cell and comprises, in a 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and - an eighth expression cassette encoding the first light chain 1. A method for producing a multivalent bispecific antibody, comprising any one of the following steps: [The present invention 1002] A deoxyribonucleic acid encoding a multivalent, bispecific antibody, comprising, in 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and - an eighth expression cassette encoding the first light chain Deoxyribonucleic acid, including any of the following: [The present invention 1003] 1. Use of deoxyribonucleic acid for the expression of a multivalent, bispecific antibody in a mammalian cell, the deoxyribonucleic acid comprising, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and - an eighth expression cassette encoding said first light chain, Use, including any of the following: [The present invention 1004] 1. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated into the genome of the cell, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises, in a 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and - an eighth expression cassette encoding the first light chain A recombinant mammalian cell comprising any one of the above. [The present invention 1005] A composition comprising two deoxyribonucleic acids, wherein the two deoxyribonucleic acids comprise three different recombination recognition sequences and six or eight expression cassettes; - the first deoxyribonucleic acid is in the 5' to 3' direction (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, and - a first copy of a third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, (1) - a second copy of said third recombination recognition sequence; - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding said first light chain, and - a second recombination recognition sequence, or (2) - a second copy of said third recombination recognition sequence; - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, - an eighth expression cassette encoding said first light chain, and - a second recombination recognition sequence A composition comprising any one of the following: [The present invention 1006] 1. A method for producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding a multivalent bispecific antibody and that secretes said multivalent bispecific antibody, comprising: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a genomic locus of said mammalian cell, said exogenous nucleotide sequence comprising first and second recombination recognition sequences flanking at least one first selectable marker, and a third recombination recognition sequence located between said first and second recombination recognition sequences, and wherein said recombination recognition sequences are all different; b) introducing into the cells provided in a) two deoxyribonucleic acid compositions comprising three different recombination recognition sequences and six or eight expression cassettes, - the first deoxyribonucleic acid is in the 5' to 3' direction (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, and - a first copy of a third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, (1) - a second copy of said third recombination recognition sequence; - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding said first light chain, and - a second recombination recognition sequence, or (2) - a second copy of said third recombination recognition sequence; - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, - an eighth expression cassette encoding said first light chain, and - a second recombination recognition sequence including any of the following: the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are identical to the first to third recombination recognition sequences of the integrated exogenous nucleotide sequence; the 5'-end portion and the 3'-end portion of an expression cassette encoding one second selection marker together form a functional expression cassette for said one second selection marker; introducing a composition of two deoxyribonucleic acids; c) adding one or more recombinases to i) simultaneously with said first and second deoxyribonucleic acids of b), or ii) Then sequentially A step of introducing the compound by any one of the following methods: the one or more recombinases recognize recombination recognition sequences in the first and second deoxyribonucleic acids (and optionally, the one or more recombinases perform two recombinase-mediated cassette exchanges); introducing one or more recombinases; and d) selecting cells that express said second selectable marker and secrete said multivalent, bispecific antibody. thereby producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding said multivalent bispecific antibody and that secretes said multivalent bispecific antibody. Methods for producing recombinant mammalian cells. [The present invention 1007] 1006. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001 to 1006, wherein said first heavy chain comprises the mutation T366W (Kabat numbering) in the CH3 domain and said second heavy chain comprises the mutations T366S, L368A, and Y407V (Kabat numbering) in the CH3 domain, or vice versa. [The present invention 1008] 1007. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to the invention, wherein one of said heavy chains further comprises the mutation S354C and each other heavy chain comprises the mutation Y349C (numbering according to Kabat). [The present invention 1009] 1008. A method for producing a multivalent bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001 to 1008, wherein said first light chain is a domain-swapped light chain VH-VL or CH1-CL. [The present invention 1010] - the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a first light chain variable domain; - the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a second heavy chain variable domain; and - the first light chain comprises, from N-terminus to C-terminus, a second light chain variable domain and a CL domain; the first heavy chain variable domain and the second light chain variable domain form a first binding site, and the second heavy chain variable domain and the first light chain variable domain form a second binding site; A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001 to 1009. [The present invention 1011] 1010. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001, 1003, 1004 and 1006 to 1010, wherein exactly one copy of said deoxyribonucleic acid is stably integrated into the genome of said mammalian cell at a single site or locus. [The present invention 1012] the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises a further expression cassette encoding a selection marker, the expression cassette encoding the selection marker being partially located on the 5' side and partially located on the 3' side of the third recombination recognition sequence, the portion of the expression cassette located on the 5' side comprising a promoter and a start codon, the portion of the expression cassette located on the 3' side comprising a coding sequence without a start codon and a polyA signal, the start codon being operably linked to the coding sequence, and the portion of the expression cassette located on the 5' side encoding the selection marker comprising a promoter sequence operably linked to a start codon, wherein the promoter sequence is located upstream the 3'-located portion of the expression cassette encoding the selectable marker comprises a nucleic acid encoding the selectable marker lacking a start codon and is adjacent to the third recombination recognition sequence on the upstream side and adjacent to the fourth or fifth expression cassette on the downstream side, respectively, wherein the start codon is operably linked to the coding sequence, or the deoxyribonucleic acid, or the use, or the recombinant mammalian cell, or the composition, or the method for producing a recombinant mammalian cell of any of claims 1001 to 1005 and 1007 to 1011. [The present invention 1013] each expression cassette of the antibody chain comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the antibody chain, and a polyadenylation signal sequence, and optionally a terminator sequence; and each expression cassette encoding the selectable marker comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the selectable marker, and a polyadenylation signal sequence, and optionally a terminator sequence; the promoter is a human CMV promoter containing intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence, and the terminator is an hGT terminator; except that in the expression cassette of the selectable marker, the promoter is an SV40 promoter, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, and no terminator is present. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001 to 1012. [The present invention 1014] 10. The method for producing a multivalent, bispecific antibody, or the deoxyribonucleic acid, or the use, or the recombinant mammalian cell, or the composition, or the method for producing a recombinant mammalian cell of any of claims 1001, 1003, 1004 and 1006 to 1013, wherein the mammalian cell is a CHO cell. [The present invention 1015] 10. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, of any of claims 1001 to 1014, wherein all expression cassettes are arranged in one direction. DETAILED DESCRIPTION OF THE INVENTION

[0061] Detailed Description of Embodiments of the Invention The present invention is based, at least in part, on the discovery that the use of predetermined and specific expression cassette configurations for the expression of multivalent bispecific antibodies, which are complex molecules comprising different polypeptides, i.e. heteromultimers, results in the efficient expression and production of multivalent bispecific antibodies in mammalian cells, such as CHO cells.

[0062] The present invention is based, at least in part, on the discovery that dual recombinase-mediated cassette exchange (RMCE) can be used to produce recombinant mammalian cells, such as recombinant CHO cells, whereby a predetermined and specific expression cassette sequence is integrated into the genome, resulting in efficient expression and production of multivalent bispecific antibodies. This integration occurs at a specific site in the genome of the mammalian cell by targeted integration. This makes it possible to control the expression ratio of the different polypeptides of the heteromultimeric multivalent bispecific antibody relative to each other. This results in efficient expression, accurate assembly, and successful secretion of correctly folded and assembled multivalent bispecific antibodies with high expression yields.

[0063] I. Definition Useful methods and techniques for practicing the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones, NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0064] The use of recombinant DNA technology makes it possible to produce derivatives of nucleic acids. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, replacement, deletion, or insertion. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).

[0065] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0066] The term "about" refers to a range of ±20% of the preceding numerical value. In one embodiment, the term "about" refers to a range of ±10% of the preceding numerical value. In one embodiment, the term "about" refers to a range of ±5% of the preceding numerical value.

[0067] The term "comprising" also includes the term "consisting of."

[0068] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acids have been introduced and which are intended to be the starting point for subsequent genetic modification, including the progeny of such cells. Thus, the term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells containing an exogenous nucleotide sequence integrated at a single site within a genomic locus of the mammalian cell, the exogenous nucleotide sequence comprising at least one first and at least one second recombinase recognition sequence (the recombinase recognition sequences are different) flanked by at least one first selectable marker. In one embodiment, a mammalian cell comprising an exogenous nucleotide sequence is a cell containing an exogenous nucleotide sequence integrated at a single site within a genomic locus of the host cell, the exogenous nucleotide sequence comprising first and second recombination recognition sequences flanking at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and the recombination recognition sequences are all different.

[0069] As used herein, the term "recombinant cell" refers to a cell after final genetic modification, such as a cell that expresses a polypeptide of interest and can be used for the production of the polypeptide of interest on any scale. For example, a "mammalian cell containing an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), thereby introducing the coding sequence of the polypeptide of interest into the genome of the host cell, is a "recombinant cell". Although this cell can still undergo further RMCE reactions, it is not intended to do so.

[0070] Both "mammalian cells containing an exogenous nucleotide sequence" and "recombinant cells" are "transformed cells." This term includes the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell, for example, in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are embraced.

[0071] An "isolated" composition is one that has been separated from a component of its natural environment. In some embodiments, the composition is purified to greater than 95% or 99% purity, as measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B848 (2007) 79-87.

[0072] An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0073] An "isolated" polypeptide or antibody means a polypeptide or antibody molecule that has been separated from a component of its natural environment.

[0074] The term "integration site" refers to a nucleic acid sequence within a cell's genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides in the cell's genome. In certain embodiments, the integration site comprises a stretch of nucleotide sequence. In certain embodiments, the integration site is located within a specific locus in the genome of a mammalian cell. In certain embodiments, the integration site is within an endogenous gene of a mammalian cell.

[0075] The terms "vector" or "plasmid" can be used interchangeably and, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0076] The term "bind" refers to the binding of a binding moiety to its target, e.g., the binding of an antibody binding moiety comprising an antibody heavy chain variable domain and an antibody light chain variable domain to a respective antigen. This binding can be measured, for example, using a BIAcore® assay (GE Healthcare, Uppsala, Sweden). That is, the term "bind (to an antigen)" refers to the binding of an antibody to its antigen in an in vitro assay. In one embodiment, binding is measured in a binding assay in which the antibody is bound to a surface and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). Binding refers to, for example, the binding of an antibody to a surface of a given antigen, e.g., 10 -8 M or less, in some embodiments 10 -13 ~10 -8 M, in some embodiments, 10 -13 ~10 -9 Binding affinity (K D The term "bind" also includes the term "specifically bind."

[0077] For example, in one possible embodiment of a BIAcore® assay, an antigen is bound to a surface and the binding of the antibody, i.e., its binding site, is measured by surface plasmon resonance (SPR). Binding affinity is measured using the term k a (association constant: rate constant for binding to form a complex), k d (dissociation constant; rate constant for dissociation of the complex), and K D (k d / ka Alternatively, the binding signal of an SPR sensorgram can be directly compared to the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0078] The term "binding site" refers to any proteinaceous entity that exhibits binding specificity for a target. It may be, for example, a receptor, a receptor ligand, anticalin, an affibody, an antibody, etc. Thus, as used herein, the term "binding site" refers to a polypeptide that is capable of specifically binding to a second polypeptide or being specifically bound by a second polypeptide.

[0079] As used herein, the term "selection marker" refers to a gene that allows cells carrying the gene to be specifically selected, either positively or negatively, in the presence of a corresponding selection agent. For example, but not limited to, a selection marker can allow host cells transformed with the selection marker gene to be positively selected in the presence of each selection agent (under selective culture conditions); untransformed host cells would not be able to grow or survive under selective culture conditions. A selection marker can be positive, negative, or bifunctional. A positive selection marker can allow for the selection of cells carrying the marker, while a negative selection marker can allow for the selective elimination of cells carrying the marker. A selection marker can confer drug resistance to a host cell or complement metabolic or catabolic defects. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D)), as well as genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0080] In addition to facilitating selection in the presence of a corresponding selection agent, a selection marker may alternatively be a molecule not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. For example, cells expressing such molecules can be distinguished from cells that do not harbor the gene based on the detection or absence, respectively, of fluorescence emitted by the encoded polypeptide.

[0081] As used herein, the term "operably linked" refers to the juxtaposition of two or more components in a relationship permitting them to function in a desired manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it functions to regulate the transcription of the coding sequence. In certain embodiments, DNA sequences that are "operably linked" are contiguous and adjacent on a single chromosome. In certain embodiments, where it is necessary to join two protein coding regions, such as a secretory leader and a polypeptide, these sequences are contiguous, adjacent, and in reading frame. In certain embodiments, an operably linked promoter may be located upstream of and adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates expression of a coding sequence, two components may be operably linked even though they are not contiguous. An enhancer is operably linked to a coding sequence if the enhancer increases the transcription of the coding sequence. An operably linked enhancer may be located upstream, inside, or downstream of a coding sequence, and may be located at a considerable distance from the promoter of the coding sequence. Operable linkage can be achieved by recombinant methods known in the art, for example, by using PCR methods and / or by ligation at a convenient restriction site. If a convenient restriction site does not exist, synthetic oligonucleotide adapters or linkers can be used according to conventional methods. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it allows translation to begin at an internal position of the ORF independently of the 5' end.

[0082] As used herein, the term "adjacent" means that a first nucleotide sequence is located at either the 5' or 3' end of a second nucleotide sequence, or at both ends.The adjacent nucleotide sequence may be located next to the second nucleotide sequence, or at a predetermined distance from it.The length of the adjacent nucleotide sequence is not particularly limited.For example, the adjacent sequence may be a few base pairs or several thousand base pairs.

[0083] Deoxyribonucleic acid includes a coding strand and a non-coding strand. The terms "5'" and "3'" as used herein refer to positions on the coding strand.

[0084] As used herein, the term "exogenous" refers to a nucleotide sequence that is not native to a particular cell and is introduced into the cell by DNA delivery methods, such as transfection, electroporation, or transformation.Therefore, an exogenous nucleotide sequence is an artificial sequence, and this artifact can be, for example, the combination of subsequences of different origins (for example, the combination of a recombinase recognition sequence with an SV40 promoter and the coding sequence of green fluorescent protein is an artificial nucleic acid), or the partial deletion or mutation of nucleic acid bases of a sequence (for example, the sequence or cDNA that codes only for the extracellular domain of a membrane-bound receptor).The term "endogenous" refers to a nucleotide sequence that originates from a cell.An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but an "exogenous" sequence is introduced into a cell by, for example, recombinant DNA technology.

[0085] antibody General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0086] The term "heavy chain" is used herein in its original sense, i.e., to refer to the two larger of the four polypeptide chains that form an antibody (see, e.g., Edelman, GM and Gally JA, J. Exp. Med. 116 (1962) 207-227). The term "larger" in this context can refer to either molecular weight, length, or number of amino acids. The term "heavy chain" is independent of the sequence and number of individual antibody domains present therein. It is assigned solely on the basis of the molecular weight of the respective polypeptide.

[0087] The term "light chain" is used herein in its original sense, i.e., to refer to the smaller of the four polypeptide chains that form an antibody (see, e.g., Edelman, GM and Gally JA, J. Exp. Med. 116 (1962) 207-227). The term "smaller" in this context can refer to either molecular weight, length, or number of amino acids. The term "light chain" is independent of the sequence and number of individual antibody domains present therein. It is assigned solely based on the molecular weight of the respective polypeptide.

[0088] As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as set forth in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering." Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which are further clarified herein by referring to "Kabat EU index numbering" in this case).

[0089] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, full length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment-fusions and combinations thereof.

[0090] The term "native antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a heavy chain variable region (VH) followed by three heavy chain constant domains (CH1, CH2, and CH3), thereby positioning a hinge region between the first and second heavy chain constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a light chain variable region (VL) followed by a light chain constant domain (CL). Based on the amino acid sequence of its constant domain, the light chain of an antibody may be assigned to one of two types, called kappa (κ) or lambda (λ).

[0091] The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. A full-length antibody comprises two or more full-length antibody light chains, each comprising, from N to C terminus, a variable region and a constant domain, and two antibody heavy chains, each comprising, from N to C terminus, a variable region, a first constant domain, a hinge region, a second constant domain, and a third constant domain. In contrast to natural antibodies, a full-length antibody may comprise additional immunoglobulin domains, such as one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs, which are conjugated to one or more ends of different chains of a full-length antibody, but only one fragment at each end. These conjugates are also encompassed by the term full-length antibody.

[0092] The term "antibody binding site" refers to a pair of heavy-chain variable domains and light-chain variable domains. To ensure proper binding to an antigen, these variable domains are cognate, i.e., belong together. An antibody binding site comprises at least three HVRs (e.g., in the case of a VHH) or three to six HVRs (e.g., in the case of a naturally occurring, i.e., conventional antibody with a VH / VL pair). Generally, the amino acid residues of an antibody involved in antigen binding form the binding site. These residues are usually contained in a pair of antibody heavy-chain variable domains and corresponding antibody light-chain variable domains. An antibody antigen-binding site comprises amino acid residues from "hypervariable regions" or "HVRs." "Framework" or "FR" regions are variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N- to C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy chain variable domain is the region that contributes most to antigen binding and defines the binding specificity of the antibody. A "functional binding site" can specifically bind to its target. The term "specifically bind" refers to the binding of a binding site to a target in an in vitro assay, in one embodiment, in a binding assay. Such a binding assay can be any assay as long as a binding event can be detected. For example, a binding assay in which an antibody is bound to a surface and the binding of an antigen to the antibody is measured by surface plasmon resonance (SPR). Alternatively, bridging ELISA can be used.

[0093] The term "hypervariable region" or "HVR", as used herein, refers to each region of an antibody variable domain comprising stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs: three in the heavy chain variable domain VH (H1, H2, H3) and three in the light chain variable domain VL (L1, L2, L3).

[0094] HVR includes: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c) comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0095] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0096] The "class" of an antibody refers to the type of constant domain or constant region, preferably the Fc region, possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0097] The term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain that contains the constant domains, i.e., the CH1 domain, hinge region, CH2 domain, and CH3 domain in the case of a native immunoglobulin, or the first constant domain, hinge region, second constant domain, and third constant domain in the case of a full-length immunoglobulin. In one embodiment, a human IgG heavy chain constant region extends from Ala118 to the carboxyl terminus of the heavy chain (Kabat EU index numbering), although the C-terminal lysine (Lys447) of the constant region may or may not be present (Kabat EU index numbering). The term "constant region" refers to a dimer comprising two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0098] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the hinge region (middle and lower hinge regions), the second constant domain (e.g., CH2 domain), and the third constant domain (e.g., CH3 domain). In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (Kabat EU index numbering). Thus, the Fc region is smaller than the constant region, but the C-terminal portion is identical to it. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (Kabat EU index numbering). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0099] The constant region of an antibody, more precisely the Fc region (and similarly the constant region), is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. The effect of an antibody on the complement system depends on the specific conditions, but binding to C1q is triggered by a defined binding site in the Fc region. Such binding sites are known in the prior art and are described, for example, in Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324, and EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat). Antibodies of the subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. The term "Fc region of an antibody" is well known to those skilled in the art and is defined based on papain cleavage of an antibody.

[0100] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0101] The term "valency," as used herein, refers to the presence of a particular number of binding sites within an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, within an antibody.

[0102] "Monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2), or combinations thereof (e.g., full-length antibodies with additional scFv or Fab fragments). Monospecific antibodies need not be monovalent; that is, they may contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0103] A "multispecific antibody" refers to an antibody that has binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies), or combinations thereof (e.g., full-length antibodies with additional scFv or Fab fragments). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. Multispecific antibodies are also at least bispecific. Thus, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies with two, three, or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587).

[0104] In certain embodiments, the antibody is a multispecific antibody, for example, at least a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different antigens or epitopes. In certain embodiments, one of the binding specificities is for a first antigen, and the other is for a different second antigen. In certain embodiments, a multispecific antibody can bind to two different epitopes of the same antigen. A multispecific antibody can be used to localize a cytotoxic substance to cells that express the antigen.

[0105] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science, 229 (1985) 81-83), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553; bispecific antibody fragments). (See, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 6448-6444), and by using single-chain Fv (scFv) dimers (See, e.g., Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374, and by preparing triabodies as described, e.g., Tutt, A. et al., J. Immunol. 147: (1991) 60-69).

[0106] The antibody or fragment may also be a multispecific antibody as described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, or WO2010 / 145793.

[0107] The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO2012 / 163520.

[0108] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0109] The term "non-overlapping" in this context indicates that amino acid residues contained within the first paratope of a bispecific Fab are not contained within the second paratope, and that amino acids contained within the second paratope of a bispecific Fab are not contained within the first paratope.

[0110] The "knob-into-hole" dimerization module and its use in antibody engineering is described in Carter P.; Ridgway JBB; Presta LG: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

[0111] The CH3 domains of antibody heavy chains can be modified using the "knob-into-hole" technique. This technique is described in detail with some examples in, for example, WO96 / 027011, Ridgway, JB et al., Protein Eng. 9 (1996) 617-621, and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681. This method involves modifying the interaction surfaces of two CH3 domains to increase heterodimerization of these two CH3 domains, thereby increasing heterodimerization of polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) can be a "knob," and the other a "hole." The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0112] The mutation T366W in the CH3 domain (of an antibody heavy chain) is designated as a "knob mutation" or "mutated knob," and the mutations T366S, L368A, and Y407V in the CH3 domain (of an antibody heavy chain) are designated as "hole mutations" or "mutated hole" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a S354C mutation in the CH3 domain of a heavy chain with a "knob mutation" (designated as a "knob-cys-mutation" or "mutated knob-cys") and a Y349C mutation in the CH3 domain of a heavy chain with a "hole mutation" (designated as a "hole-cys-mutation" or "mutated hole-cys") (numbering according to the EU index of Kabat).

[0113] The term "domain crossover" as used herein refers to deviations in domain sequence from that of a native antibody in that in a pair of antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., antibody Fab (fragment-antigen binding), at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and the domain crossover in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain having a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and the domain crossover in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of an intact light chain (VL-CL) and an intact VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and the domain crossover results in a heavy chain fragment with a VL-CL domain sequence (all domain sequences listed above are in the N-terminal to C-terminal direction).

[0114] As used herein, the term "replaced with each other" with respect to corresponding heavy chain domains and light chain domains refers to the above-mentioned domain crossover. Thus, when CH1 and CL domains are "replaced with each other", the term refers to the domain crossover described under item (i) and the resulting heavy chain and light chain domain sequences. Thus, when VH and VL are "replaced with each other", the term refers to the domain crossover described under item (ii); and when CH1 and CL domains are "replaced with each other" and VH and VL domains are "replaced with each other", the term refers to the domain crossover described under item (iii). Bispecific antibodies comprising domain crossovers have been reported, for example, in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, and Schaefer, W. et al., Proc. Natl. Acad. Sci USA 108 (2011) 11187-11192. Such antibodies are generally referred to as CrossMabs.

[0115] In one embodiment, the multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in the above item (i), the domain crossover of the VH and VL domains described in the above item (ii), or the domain crossover of the VH-CH1 and VL-VL domains described in the above item (iii). In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen are constructed to have the same domain sequence. Therefore, when more than one Fab with domain crossover is included in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0116] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0117] The term "recombinant antibody," as used herein, refers to all antibodies (chimeric, humanized, and human) that are prepared, expressed, produced, or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, CHO cells, etc.

[0118] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabody, linear antibody, and single-chain antibody molecules (e.g., scFv or scFab).

[0119] II. Compositions and Methods Typically, recombinant mass production of a polypeptide of interest, such as a therapeutic polypeptide, requires cells that stably express and secrete the polypeptide. These cells are called "recombinant cells" or "recombinant production cells," and the method used to generate such cells is called "cell line development." In the first step of the cell line development method, a suitable host cell, such as a CHO cell, is transfected with a nucleic acid sequence suitable for expressing the polypeptide of interest. In the second step, cells that stably express the polypeptide of interest are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the polypeptide of interest.

[0120] A nucleic acid that encodes a polypeptide, i.e., a coding sequence, is called a structural gene. Such a structural gene is a simple message, and its expression requires additional control elements. Therefore, a structural gene is usually incorporated into an expression cassette. The minimum control elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream (i.e., 5') of the structural gene, and a polyadenylation signal sequence functional in mammalian cells located downstream (i.e., 3') of the structural gene. The promoter sequence, structural gene sequence, and polyadenylation signal sequence are arranged in an operably linked form.

[0121] If the polypeptide of interest is a heteromultimeric polypeptide composed of different (monomeric) polypeptides, not only one expression cassette but multiple expression cassettes containing different structural genes are required, i.e., at least one expression cassette for each of the different (monomeric) polypeptides of the heteromultimeric polypeptide. For example, a full-length antibody is a heteromultimeric polypeptide containing two copies of a light chain and two copies of a heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Therefore, two expression cassettes are required for the expression of a full-length antibody, one for the light chain and one for the heavy chain. For example, if a full-length antibody is a bispecific antibody, i.e., the antibody contains two different binding sites that specifically bind to two different antigens, the light chains are different from each other and the heavy chains are different from each other. Thus, such a bispecific full-length antibody is composed of four different polypeptides, and four expression cassettes are required.

[0122] The expression cassette for the polypeptide of interest is in turn incorporated into a so-called "expression vector." An "expression vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene in mammalian cells. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, which in the case of, for example, E. coli, contains an origin of replication and a prokaryotic or eukaryotic selection marker, as well as the expression cassette required for expression of the structural gene of interest. An "expression vector" is a delivery vehicle for introducing an expression cassette into mammalian cells.

[0123] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the greater the number of different expression cassettes required. Essentially, the larger the number of expression cassettes, the larger the size of the nucleic acid to be integrated into the genome of the host cell. At the same time, the size of the expression vector also increases. However, the practical upper limit of the vector size is in the range of about 15 kbp, beyond which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors. Thereby, the expression cassette can be shared between different expression vectors, each containing only a part of the expression cassette.

[0124] Conventional cell line development (CLD) relies on random integration (RI) of a vector carrying an expression cassette for a polypeptide of interest (SOI). Generally, when a vector is transfected by a random method, some vectors or fragments thereof are integrated into the cell genome. Therefore, the RI-based transfection process is unpredictable.

[0125] Thus, by addressing the size problem by sharing the expression cassettes among different expression vectors, a new problem arises - the uneven number of expression cassettes integrated and their spatial distribution.

[0126] Generally, the more expression cassettes for expressing structural genes are integrated into the cell genome, the greater the amount of each polypeptide that is expressed.In addition to the number of expression cassettes that are integrated, the integration site and locus also affect the expression yield.For example, if an expression cassette is integrated into a site with low transcriptional activity in the cell genome, only a small amount of the encoded polypeptide will be expressed.However, if the same expression cassette is integrated into a site with high transcriptional activity in the cell genome, the encoded polypeptide will be expressed in large amounts.

[0127] This difference in expression does not pose a problem as long as the expression cassettes for the different polypeptides of a heteromultimeric polypeptide are all integrated at the same frequency and into loci with similar transcriptional activity, under which circumstances all polypeptides contained in the multimeric polypeptide are expressed in equal amounts and the multimeric polypeptide is assembled correctly.

[0128] However, this scenario is highly unlikely and cannot be guaranteed for molecules composed of more than two polypeptides. For example, International Publication No. 2018 / 162517 discloses that significant differences in expression yield and product quality were observed by RI depending on i) the expression cassette sequence and ii) the distribution of the expression cassettes among different expression vectors. Without being bound by theory, this observation is attributed to the fact that different expression cassettes from different expression vectors are integrated into different loci at different frequencies in cells, resulting in the differential expression of various polypeptides contained in the heteromultimeric polypeptide, i.e., in various, improper ratios. As a result, some monomeric polypeptides are present in relatively high amounts, while others are present in relatively low amounts. This imbalance in the monomers contained in the heteromultimeric polypeptide leads to incomplete assembly, incorrect assembly, and reduced secretion rates. All of the above results in a reduced expression yield of correctly folded heteromultimeric polypeptides and an increased proportion of by-products associated with the product.

[0129] Unlike conventional RI CLD, targeted integration (TI) CLD introduces transgenes containing various expression cassettes into predetermined "hot spots" in the cell genome. Furthermore, this introduction uses a predetermined ratio of the expression cassettes. As a result, without being bound by this theory, all of the various polypeptides contained in the heteromultimeric polypeptide are expressed at the same (or at least similar, with only minor differences) speed and in the appropriate ratio. This should result in an increased amount of correctly assembled heteromultimeric polypeptide, and a decreased rate of by-products associated with the product.

[0130] Also, given a given copy number and a given integration site, recombinant cells obtained by TI should have superior stability compared to cells obtained by RI. Furthermore, because the selection marker is used only to select cells with the appropriate TI and not to select cells showing high levels of transgene expression, a less mutagenic marker may be applied to minimize the possibility of sequence variants (SVs) arising in part due to the mutagenicity of selection agents such as methotrexate (MTX) or methionine sulfoximine (MSX).

[0131] However, it is now known that the sequence of the expression cassette in the transgene used in TI, i.e., the expression cassette configuration, has a strong influence on the expression of multivalent bispecific antibodies.

[0132] The present invention uses a specific expression cassette configuration with a predetermined number and sequence of individual expression cassettes, which results in high expression yields and high product quality for multivalent, bispecific antibodies expressed in mammalian cells.

[0133] The TI method is used for the directed integration of a transgene carrying an expression cassette sequence according to the present invention. The present invention provides a novel method for generating recombinant mammalian cells expressing multivalent bispecific antibodies using a two-plasmid recombinase-mediated cassette exchange (RMCE) reaction. The improvement is particularly the directed integration at the same locus in the directed sequence, thereby resulting in high expression of the multivalent bispecific antibody and reduced formation of product-related by-products.

[0134] The presently disclosed subject matter provides not only methods for producing recombinant mammalian cells for stable large-scale production of multivalent, bispecific antibodies, but also recombinant mammalian cells with high productivity of multivalent, bispecific antibodies with advantageous by-product profiles.

[0135] The two-plasmid RMCE strategy used here allows for the insertion of multiple expression cassettes into the same TI locus.

[0136] II.a Transgenes and Methods According to the Invention Recombinant mammalian cells expressing multivalent bispecific antibodies are reported herein. Multivalent bispecific antibodies are heteromultimeric polypeptides that are not naturally expressed by said mammalian cells. More specifically, multivalent bispecific antibodies are heterodimeric proteins consisting of three polypeptides: an antibody light chain and first and second antibody heavy chains. To achieve expression of the multivalent bispecific antibodies, recombinant nucleic acids containing multiple different expression cassettes in specific and predetermined sequences were integrated into the genome of the mammalian cells.

[0137] Also reported herein are methods for making recombinant mammalian cells that express multivalent, bispecific antibodies and methods for producing multivalent, bispecific antibodies using the recombinant mammalian cells.

[0138] The present invention is based, at least in part, on the discovery that the sequences of the different expression cassettes required for expression of heteromultimeric multivalent bispecific antibodies, i.e., the expression cassette configuration, influence the expression yield of the multivalent bispecific antibody when integrated into the genome of a mammalian cell.

[0139] The present invention is based, at least in part, on the discovery that dual recombinase-mediated cassette exchange (RMCE) can be used to produce recombinant mammalian cells, such as recombinant CHO cells, whereby a predetermined and specific expression cassette sequence is integrated into the genome, resulting in efficient expression and production of multivalent, bispecific antibodies. This integration occurs at a specific site in the genome of the mammalian cell by targeted integration. This allows for control of the expression ratio of different polypeptides of the heteromultimeric antibody relative to each other. This results in efficient expression, accurate assembly, and successful secretion of correctly folded and assembled multivalent, bispecific antibodies with high expression yields.

[0140] Because multivalent bispecific antibodies are heterotrimers, their expression requires at least three different expression cassettes: one for the expression of the antibody light chain, a second for the expression of the first antibody heavy chain, and a third for the expression of the second antibody heavy chain. In addition, an additional expression cassette for a positive selection marker may also be included.

[0141] To investigate the effect of expression cassette configuration on productivity in TI hosts, RMCE pools were generated by transfecting two plasmids (front and back vectors) containing different numbers and configurations of individual chains of a multivalent bispecific antibody. After selection, recovery, and validation of RMCE by flow cytometry, the productivity of the pools was assessed in a 14-day fed-batch production assay.

[0142] The effect of antibody chain expression cassette organization on the expression of multivalent bispecific antibodies was evaluated. For multivalent bispecific antibodies, the following results were obtained: TIFF2025134010000002.tif50145

[0143] The present invention is summarized below. An independent aspect according to the invention is a method for producing a multivalent, bispecific antibody, comprising: a) culturing mammalian cells containing deoxyribonucleic acid encoding a multivalent bispecific antibody, and b) recovering the multivalent bispecific antibody from the cells or culture medium; wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of a mammalian cell and comprises, in a 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain A method comprising:

[0144] Stable integration of the deoxyribonucleic acid encoding the multivalent, bispecific antibody can be performed by any method known to those skilled in the art, as long as it is stably integrated into the genome of the mammalian cell and the specific sequence of the expression cassette is maintained.

[0145] The individual expression cassettes in the deoxyribonucleic acid according to the invention are arranged sequentially, with the distance between the end of one expression cassette and the start of the next one being only a few nucleotides, which is what was necessary for the cloning procedure, i.e. what results.

[0146] An independent embodiment according to the invention is a deoxyribonucleic acid encoding a multivalent, bispecific antibody, comprising, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain It is a deoxyribonucleic acid containing any one of the following:

[0147] An independent aspect according to the invention is the use of deoxyribonucleic acid for the expression of a multivalent, bispecific antibody in a mammalian cell, the deoxyribonucleic acid comprising, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding a first light chain, The present invention relates to the use of deoxyribonucleic acid, including any of the following:

[0148] An independent aspect according to the invention is a recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated into the genome of the cell, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises, in the 5' to 3' direction: (1) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, and - a sixth expression cassette encoding a first light chain, or (2) - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, and - an eighth expression cassette encoding the first light chain A recombinant mammalian cell comprising either

[0149] An independent aspect of the present invention is a composition comprising two deoxyribonucleic acids, the two deoxyribonucleic acids comprising three different recombination recognition sequences and six or eight expression cassettes; - the first deoxyribonucleic acid is in the 5' to 3' direction (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding the first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, and - a first copy of a third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, (1) - a second copy of a third recombination recognition sequence, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding a first light chain, and - a second recombination recognition sequence, or (2) - a second copy of a third recombination recognition sequence, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, - an eighth expression cassette encoding a first light chain, and - a second recombination recognition sequence A composition comprising any one of the following:

[0150] In one embodiment, the first and second deoxyribonucleic acids both comprise the configuration of (1), or the first and second deoxyribonucleic acids both comprise the configuration of (2).

[0151] An independent aspect according to the invention is a method for producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding a multivalent bispecific antibody and that secretes the multivalent bispecific antibody, comprising: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a locus in the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and wherein the recombination recognition sequences are all different; b) introducing into the cells provided in a) two deoxyribonucleic acid compositions comprising three different recombination recognition sequences and six or eight expression cassettes, - the first deoxyribonucleic acid is in the 5' to 3' direction (1) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding the first light chain, and - a first copy of a third recombination recognition sequence or (2) - a first recombination recognition sequence, - a first expression cassette encoding a first heavy chain, - a second expression cassette encoding a first light chain, - a third expression cassette encoding a first light chain, - a fourth expression cassette encoding a first light chain, and - a first copy of a third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, (1) - a second copy of a third recombination recognition sequence, - a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding a first light chain, - a sixth expression cassette encoding a first light chain, and - a second recombination recognition sequence, or (2) - a second copy of a third recombination recognition sequence, - a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding a first light chain, - a seventh expression cassette encoding a first light chain, - an eighth expression cassette encoding a first light chain, and - a second recombination recognition sequence, including any of the following: the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are identical to the first to third recombination recognition sequences of the integrated exogenous nucleotide sequence; the 5'-end portion and the 3'-end portion of the expression cassette encoding said one second selection marker together form a functional expression cassette of said one second selection marker; introducing into a cell a composition comprising two deoxyribonucleic acids; c) one or more recombinases i) simultaneously with the first and second deoxyribonucleic acids of b), or ii) then sequentially, introducing, wherein one or more recombinases recognize recombination recognition sequences of the first and second deoxyribonucleic acids; (and optionally, the one or more recombinases effect two recombinase-mediated cassette exchanges); introducing one or more recombinases; and d) selecting cells that express a second selection marker and secrete a multivalent bispecific antibody. whereby a recombinant mammalian cell is produced that contains deoxyribonucleic acid encoding the multivalent bispecific antibody and that secretes the multivalent bispecific antibody.

[0152] In one embodiment, the first and second deoxyribonucleic acids both comprise the configuration of (1), or the first and second deoxyribonucleic acids both comprise the configuration of (2).

[0153] In one embodiment of all independent aspects and all dependent embodiments of the invention, exactly one copy of a deoxyribonucleic acid encoding a multivalent, bispecific antibody is stably integrated into a single locus in the genome of a mammalian cell by targeted integration.

[0154] In one embodiment of all independent aspects and all dependent embodiments of the invention, exactly one copy of a deoxyribonucleic acid encoding a multivalent, bispecific antibody is stably integrated into a single locus in the genome of a mammalian cell by a single or double recombinase-mediated cassette exchange reaction.

[0155] In one embodiment of every independent aspect and every dependent embodiment of the invention, the first heavy chain comprises the mutation T366W (Kabat numbering) in the CH3 domain and the second heavy chain comprises the mutations T366S, L368A, and Y407V (Kabat numbering) in the CH3 domain, or vice versa.

[0156] In one embodiment of every independent aspect and every dependent embodiment of the invention, one of the heavy chains further comprises the mutation S354C and each other heavy chain comprises the mutation Y349C (numbering according to Kabat).

[0157] In one embodiment of every independent aspect and every dependent embodiment of the invention, the first heavy chain is an extended heavy chain comprising an additional domain-swapped Fab fragment.

[0158] In one embodiment of every independent aspect and every dependent embodiment of the invention, the first light chain is a domain-swapped light chain VH-VL or CH1-CL.

[0159] In one embodiment of all independent aspects and all dependent embodiments of the invention: - the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a first light chain variable domain; - the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain and a second heavy chain variable domain; - the first light chain comprises, from N-terminus to C-terminus, a second light chain variable domain and a CL domain; wherein the first heavy chain variable domain and the second light chain variable domain form a first binding site, and the second heavy chain variable domain and the first light chain variable domain form a second binding site.

[0160] In one embodiment of all independent aspects and all dependent embodiments of the invention, exactly one copy of the deoxyribonucleic acid is stably integrated into a single site or locus in the genome of the mammalian cell.

[0161] In one embodiment of every independent aspect and every dependent embodiment of the invention, the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises an additional expression cassette encoding a selectable marker.

[0162] In one embodiment of all independent aspects and all dependent embodiments of the invention, an expression cassette encoding a selectable marker is located partially 5' and partially 3' to a third recombination recognition sequence, the 5' portion of the expression cassette comprising a promoter and a start codon, and the 3' portion of the expression cassette comprising a coding sequence without a start codon and a polyA signal, the start codon being operably linked to the coding sequence.

[0163] In one embodiment of all independent aspects and all dependent embodiments of the invention, the 5'-located portion of the expression cassette encoding the selectable marker comprises a promoter sequence operably linked to a start codon, the promoter sequence flanked upstream by a third or fourth expression cassette, respectively, and the start codon flanked downstream by a third recombination recognition sequence; and the 3'-located portion of the expression cassette encoding the selectable marker comprises a nucleic acid encoding a selectable marker lacking a start codon, flanked upstream by a third recombination recognition sequence and downstream by a fourth or fifth expression cassette, respectively, and the start codon operably linked to a coding sequence.

[0164] In one embodiment of all independent aspects and all dependent embodiments of the invention: Each expression cassette of an antibody chain comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the antibody chain, and a polyadenylation signal sequence, and optionally a terminator sequence; and Each expression cassette encoding a selectable marker comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the selectable marker, and a polyadenylation signal sequence, and optionally, a terminator sequence.

[0165] Terminator sequences prevent the generation of very long RNA transcripts by RNA polymerase II, i.e., read-through into the next expression cassette in the deoxyribonucleic acid according to the invention, and are used in the method according to the invention, i.e., the expression of one structural gene of interest is controlled by its own promoter.

[0166] Thus, the combination of a polyadenylation signal and a terminator sequence achieves efficient transcription termination; readthrough of RNA polymerase II is prevented by the presence of a dual termination signal. The terminator sequence initiates complex degradation, facilitating dissociation of RNA polymerase from the DNA template.

[0167] In one embodiment of all independent aspects and all dependent embodiments of the invention, the promoter is the human CMV promoter including intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence, and the terminator is the hGT terminator, except in the expression cassette for the selectable marker, where the promoter is the SV40 promoter, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence, and the terminator is absent.

[0168] In one embodiment of all independent aspects and all dependent embodiments of the invention, the mammalian cells are CHO cells.

[0169] In one embodiment of all independent aspects and all dependent embodiments of the invention, all expression cassettes are unidirectionally arranged.

[0170] In one embodiment of all independent aspects and all dependent embodiments of the invention, an expression cassette encoding a selectable marker is located partially 5' and partially 3' of a third recombination recognition sequence, the 5' portion of the expression cassette comprising a promoter and a start codon, and the 3' portion of the expression cassette comprising a coding sequence without a start codon and a polyA signal.

[0171] In one embodiment of all independent aspects and all dependent embodiments of the invention, the 5'-located portion of the expression cassette encoding the selectable marker comprises a promoter sequence operably linked to a start codon, which is adjacent on the upstream side to the third or fourth expression cassette, respectively (i.e., located downstream relative to the third or fourth expression cassette), and the start codon is adjacent on the downstream side to the third recombination recognition sequence (i.e., located upstream relative to the third recombination recognition sequence); and the 3'-located portion of the expression cassette encoding the selectable marker comprises a nucleic acid encoding a selectable marker lacking a start codon operably linked to a polyadenylation sequence, which is adjacent on the upstream side to the third recombination recognition sequence and adjacent on the downstream side to the fourth or fifth expression cassette, respectively.

[0172] In one embodiment of every independent aspect and every dependent embodiment of the invention, the start codon is a translation initiation codon, hi one embodiment, the start codon is ATG.

[0173] In one embodiment of every independent aspect and every dependent embodiment of the invention, the first deoxyribonucleic acid is incorporated into a first vector and the second deoxyribonucleic acid is incorporated into a second vector.

[0174] In one embodiment of all independent aspects and all dependent embodiments of the invention, each expression cassette comprises, in the 5' to 3' direction, a promoter, a coding sequence, and a polyadenylation signal sequence, optionally followed by a terminator sequence, all operably linked to each other.

[0175] In one embodiment of all independent aspects and all dependent embodiments of the invention, the mammalian cell is a CHO cell, hi one embodiment, the CHO cell is a CHO-K1 cell.

[0176] In one embodiment of every independent aspect and every dependent embodiment of the invention, the recombinase recognition sequences are L3, 2L, and LoxFas. In one embodiment, L3 has the sequence of SEQ ID NO: 01, 2L has the sequence of SEQ ID NO: 02, and LoxFas has the sequence of SEQ ID NO: 03. In one embodiment, the first recombinase recognition sequence is L3, the second recombinase recognition sequence is 2L, and the third recombinase recognition sequence is LoxFas.

[0177] In one embodiment of all independent aspects and all dependent embodiments of the invention, the promoter is a human CMV promoter including intron A, the polyadenylation signal sequence is a bGH polyA site, and the terminator sequence is an hGT terminator.

[0178] In one embodiment of all independent aspects and all dependent embodiments of the invention, the promoter is the human CMV promoter including intron A, the polyadenylation signal sequence is the bGH polyA site, and the terminator sequence is the hGT terminator, except in the expression cassette for the selectable marker, where the promoter is the SV40 promoter, the polyadenylation signal sequence is the SV40 polyA site, and the terminator sequence is absent.

[0179] In one embodiment of every independent aspect and every dependent embodiment of the invention, the human CMV promoter has the sequence of SEQ ID NO: 04. In one embodiment, the human CMV promoter has the sequence of SEQ ID NO: 06.

[0180] In one embodiment of all independent aspects and all dependent embodiments of the invention, the bGH polyadenylation signal sequence is SEQ ID NO:08.

[0181] In one embodiment of every independent aspect and every dependent embodiment of the invention, the hGT terminator has the sequence of SEQ ID NO:09.

[0182] In one embodiment of all independent aspects and all dependent embodiments of the invention, the SV40 promoter has the sequence of SEQ ID NO:10.

[0183] In one embodiment of all independent aspects and all dependent embodiments of the invention, the SV40 polyadenylation signal sequence is SEQ ID NO:07.

[0184] In one embodiment of all aspects and embodiments, the multivalent bispecific antibody is an anti-FAP / Ox40 bispecific antibody. Such antibodies are reported in WO 2017 / 060144, which is incorporated herein by reference in its entirety.

[0185] II.b Recombinase-mediated cassette exchange (RMCE) Targeted integration allows exogenous nucleotide sequence to be integrated into predetermined site of mammalian cell genome.In certain embodiments, targeted integration is mediated by recombinase that recognizes one or more recombination recognition sequences (RRS).In certain embodiments, targeted integration is mediated by homologous recombination.

[0186] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. RRSs can be used to define locations in a nucleotide sequence where recombination events are expected to occur.

[0187] In certain embodiments, the RRS is selected from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence. When multiple RRSs must be present, the selection of each sequence is dependent on the other, provided that non-identical RRSs are selected.

[0188] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0189] In certain embodiments, where the RRS is a LoxP site, the cells require Cre recombinase to effect recombination. In certain embodiments, where the RRS is an FRT site, the cells require FLP recombinase to effect recombination. In certain embodiments, where the RRS is a Bxb1 attP site or a Bxb1 attB site, the cells require Bxb1 integrase to effect recombination. In certain embodiments, where the RRS is a φC31 attP site or a φC31 attB site, the cells require φC31 integrase to effect recombination. These recombinases can be introduced into cells using expression vectors containing the coding sequences for these enzymes.

[0190] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre is a 38-kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp nonpalindromic core region flanked by two 13-bp inverted repeats. Cre recombinase binds to the 13-bp repeats, thereby mediating recombination within the 8-bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. When two LoxP sequences are located in the same nucleotide sequence and in the same orientation, Cre-mediated recombination excises the DNA sequence located between the two LoxP sequences into a covalently closed circle. If two LoxP sequences are located in opposite positions on the same nucleotide sequence, Cre-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are located on two different DNA molecules and one of the DNA molecules is circular, Cre-mediated recombination will result in the integration of a circular DNA sequence.

[0191] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. The mutant LoxP sequence was developed to increase the efficiency of Cre-mediated integration or replacement. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, the Lox71 sequence has a 5-bp mutation in the left 13-bp repeat. The Lox66 sequence has a 5-bp mutation in the right 13-bp repeat. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0192] The term "matched RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matched RRSs are different sequences from each other but can be recognized by the same recombinase. In certain embodiments, the first matched RRS is a Bxb1 attP sequence and the second matched RRS is a Bxb1 attB sequence. In certain embodiments, the first matched RRS is a φC31 attB sequence and the second matched RRS is a φC31 attB sequence.

[0193] II.c Exemplary mammalian cells suitable for TI Any known or future mammalian cell suitable for TI containing exogenous nucleic acid (a "landing site"), as described above, can be used in the present invention.

[0194] The present invention is exemplified using CHO cells containing exogenous nucleic acid (landing site) based on the previous section. This is presented solely to illustrate the invention and should not be construed as limiting in any way. The true scope of the invention is set forth in the claims.

[0195] In one preferred embodiment, the mammalian cell comprising the exogenous nucleotide sequence integrated at a single site within a locus in the genome of the mammalian cell is a CHO cell.

[0196] An exemplary mammalian cell suitable for use in the present invention, which contains an exogenous nucleotide sequence integrated at a single site within its genomic locus, is a CHO cell harboring a landing site (= an exogenous nucleotide sequence integrated at a single site within a mammalian cell's genomic locus) containing three heterospecific loxP sites for Cre recombinase-mediated DNA recombination. These heterospecific loxP sites are L3, LoxFas, and 2L (see, e.g., Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further comprises a bicistronic unit that connects the expression of the selection marker via IRES to the expression of fluorescent GFP protein, allowing for the stabilization of the landing site by positive selection and the selection of the absence of the site after transfection and Cre recombination (negative selection). The green fluorescent protein (GFP) is useful for monitoring the RMCE reaction. An exemplary GFP has the sequence of SEQ ID NO: 11.

[0197] This organization of the landing sites, as outlined in the previous paragraph, allows the simultaneous integration of two vectors, the so-called front vector with the L3 and LoxFas sites and the back vector with the internal LoxFas and 2L sites. The functional elements of the selectable marker gene, which are different from those present in the landing sites, are distributed between both vectors: the promoter and start codon are located on the front vector, whereas the coding region and polyA signal are located on the back vector. Resistance to each selection agent is induced only upon correct Cre-mediated integration of the nucleic acids from both vectors.

[0198] Typically, a mammalian cell suitable for TI is a mammalian cell that contains an exogenous nucleotide sequence integrated at a single site within a locus in the genome of the mammalian cell, the exogenous nucleotide sequence comprising first and second recombination recognition sequences flanking at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and the recombination recognition sequences are all different. The exogenous nucleotide sequence is referred to as a "landing site."

[0199] The subject matter disclosed in the present invention uses mammalian cells suitable for TI of exogenous nucleotide sequences. In certain embodiments, the mammalian cells suitable for TI contain exogenous nucleotide sequences integrated into an integration site in the genome of the mammalian cells. Such mammalian cells suitable for TI can also be referred to as TI host cells.

[0200] In certain embodiments, the mammalian cells suitable for TI are hamster, human, rat, or mouse cells containing a landing site. In certain embodiments, the mammalian cells suitable for TI are Chinese hamster ovary (CHO) cells, CHO K1 cells, CHO K1SV cells, CHO DG44 cells, CHO DUKXB-11 cells, CHO K1S cells, or CHO K1M cells containing a landing site.

[0201] In certain embodiments, a mammalian cell suitable for TI comprises an integrated exogenous nucleotide sequence, the exogenous nucleotide sequence comprising one or more recombination recognition sequences (RRSs). In certain embodiments, the exogenous nucleotide sequence comprises at least two RRSs. The RRSs can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRSs can be selected from the group consisting of LoxP, LoxP L3, LoxP 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, Lox66, FRT, Bxb1 attP, Bxb1 attB, φC31 attP, and φC31 attB sequences.

[0202] In certain embodiments, the exogenous nucleotide sequence comprises a first, second, and third RRS, and at least one selectable marker located between the first and second RRSs, and the third RRS is different from the first and / or second RRS. In certain embodiments, the exogenous nucleotide sequence further comprises a second selectable marker, and the first and second selectable markers are different from each other. In certain embodiments, the exogenous nucleotide sequence further comprises a third selectable marker and an internal ribosome entry site (IRES), and the IRES is operably linked to the third selectable marker. The third selectable marker may be different from both the first and second selectable markers.

[0203] The selectable marker may be selected from the group consisting of aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. The selectable marker may also be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0204] In certain embodiments, the exogenous nucleotide sequence comprises a first, second, and third RRS, and at least one selectable marker located between the first and third RRS.

[0205] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell but can be introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, a mammalian cell suitable for TI comprises at least one exogenous nucleotide sequence integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the exogenous nucleotide sequence is integrated into one or more integration sites within a specific locus in the genome of the mammalian cell.

[0206] In certain embodiments, the exogenous nucleotide sequence being integrated comprises one or more recombination recognition sequences (RRSs), which can be recognized by a recombinase. In certain embodiments, the exogenous nucleotide sequence being integrated comprises at least two RRSs. In certain embodiments, the exogenous nucleotide sequence being integrated comprises three RRSs, with the third RRS being located between the first and second RRSs. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from both the first and second RRSs. In certain preferred embodiments, all three RRSs are different from each other. In certain embodiments, the RRSs are independently selected from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence.

[0207] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a first, second, and third RRS and at least one selectable marker. In certain embodiments, the selectable marker is located between the first and second RRS. In certain embodiments, two RRSs are adjacent to at least one selectable marker. That is, the first RRS is located 5' (upstream) of the selectable marker, and the second RRS is located 3' (downstream) of the selectable marker. In certain embodiments, the first RRS is located adjacent to the 5' end of the selectable marker, and the second RRS is located adjacent to the 3' end of the selectable marker.

[0208] In certain embodiments, the selection marker is located between the first and second RRSs, and these two flanking RRSs are different from each other. In certain preferred embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 5' to the selection marker, and the LoxP 2L sequence is located 3' to the selection marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In certain embodiments, the first flanking RRS is a φC31 attP sequence, and the second flanking RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are oriented in the same direction. In certain embodiments, the two RRSs are both forward or reverse oriented. In certain embodiments, the two RRSs are oriented in opposite directions.

[0209] In certain embodiments, the integrated exogenous nucleotide sequence comprises a first and a second selection marker flanked by two RRSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both of the two selection markers are, independently of each other, selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418). APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid; and the second selection marker is selected from the group consisting of GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent protein. In certain embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein, hi certain embodiments, the two RRSs flanking both selection markers are different from each other.

[0210] In certain embodiments, the selectable marker is operably linked to a promoter sequence. In certain embodiments, the selectable marker is operably linked to an SV40 promoter. In certain embodiments, the selectable marker is operably linked to a human cytomegalovirus (CMV) promoter.

[0211] In certain embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs. In certain embodiments, the third RRS is located between the first and second RRSs. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from both the first and second RRSs. In certain preferred embodiments, all three RRSs are different from each other.

[0212] II.d Exemplary Vectors Suitable for Implementing the Invention In addition to the "single-vector RMCE" outlined above, a novel "two-vector RMCE" can be performed for the targeted integration of two nucleic acids simultaneously.

[0213] A "two-vector RMCE" strategy is implemented in the method of the present invention using a combination of vectors according to the present invention. For example, but not limited to, the exogenous nucleotide sequence to be integrated can include three RRSs, e.g., a sequence in which the third RRS ("RRS3") is located between the first RRS ("RRS1") and the second RRS ("RRS2"), where the first vector includes two RRSs that match the first and third RRSs on the exogenous nucleotide sequence to be integrated, and the second vector includes two RRSs that match the third and second RRSs on the exogenous nucleotide sequence to be integrated. An example of a two-vector RMCE strategy is illustrated in FIG. 1. Such a two-vector RMCE strategy allows for the introduction of multiple SOIs by incorporating an appropriate number of SOIs between each pair of RRSs in each sequence, so that an expression cassette configuration according to the present invention can be obtained after TI in the genome of a mammalian cell suitable for TI.

[0214] The two-plasmid RMCE strategy involves using three RRS sites to simultaneously perform two independent RMCEs (Figure 1). Therefore, a mammalian cell landing site suitable for TI using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross-reactivity with either the first RRS site (RRS1) or the second RRS site (RRS2). The two expression plasmids to be targeted require identical flanking RRS sites for efficient targeting: one expression plasmid (front) is flanked by RRS1 and RRS3, and the other (back) is flanked by RRS3 and RRS2. Two selectable markers are also required in two-plasmid RMCE. A single selectable marker expression cassette is split into two parts. The front plasmid contains a promoter followed by a start codon and the RRS3 sequence. The back plasmid lacks the start codon (ATG) and has the RRS3 sequence fused to the N-terminus of the selectable marker coding region. To ensure in-frame translation of the fusion protein, i.e., operable linkage, additional nucleotides may need to be inserted between the RRS3 site and the selectable marker sequence. Only when both plasmids are correctly inserted can the complete expression cassette for the selectable marker be assembled, thus conferring resistance to each selection agent to the cells. Figure 1 is a schematic diagram illustrating the two-plasmid RMCE strategy.

[0215] Both single-vector and two-vector RMCE allow for the unidirectional integration of one or more donor DNA molecules into a predetermined site in the mammalian cell genome by precisely exchanging DNA sequences present on the donor DNA with DNA sequences in the mammalian cell genome where the integration site resides. These DNA sequences feature two heterospecific RRSs flanked by i) at least one selectable marker, or, in the case of certain two-vector RMCEs, a "split selectable marker," and / or ii) at least one exogenous SOI.

[0216] RMCE involves a double recombination crossover event, catalyzed by a recombinase, between two heterospecific RRSs and a donor DNA molecule within a target genomic locus. RMCE is designed to introduce copies of combined DNA sequences from a front vector and a back vector into a predetermined locus in a mammalian cell genome. Unlike recombination involving a single crossover event, RMCE can be performed such that the sequences of the prokaryotic vector are not introduced into the mammalian cell genome, thus reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0217] In certain embodiments, targeted integration is achieved by two rounds of RMCE, in which two different DNA sequences are both integrated into a predetermined site in the genome of a mammalian cell suitable for TI, each DNA sequence containing at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, targeted integration is achieved by multiple rounds of RMCE, in which DNA sequences from multiple vectors are all integrated into a predetermined site in the genome of a mammalian cell suitable for TI, each DNA sequence containing at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selectable marker may be partially encoded in a first vector and partially encoded in a second vector, such that expression of the selectable marker is possible only when both are correctly integrated by double RMCE. An example of such a system is shown in Figure 1.

[0218] In certain embodiments, targeted integration by recombinase-mediated recombination integrates various expression cassettes for selectable markers and / or multimeric polypeptides into one or more predetermined integration sites of the host cell genome, without including sequences derived from the prokaryotic vector.

[0219] In addition to the various embodiments depicted and claimed, the presently disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein may be combined with each other in other ways within the scope of the presently disclosed subject matter, such that the presently disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the presently disclosed subject matter have been presented for purposes of illustration and description and are not intended to be exhaustive or to limit the presently disclosed subject matter to the disclosed embodiments.

[0220] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions and methods of the presently disclosed subject matter without departing from the spirit or scope of the presently disclosed subject matter. Thus, it is intended that the presently disclosed subject matter cover modifications and variations that come within the scope of the appended claims and their equivalents.

[0221] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by reference in their entireties.

[0222] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. [Brief explanation of the drawings]

[0223] [Figure 1] Scheme of the two-plasmid RMCE strategy involving the use of three RRS sites to simultaneously perform two independent RMCEs. [Example]

[0224] Array Description SEQ ID NO: 01: Exemplary sequence of an L3 recombinase recognition sequence SEQ ID NO: 02: Exemplary sequence of 2L recombinase recognition sequence SEQ ID NO: 03: Exemplary sequence of a LoxFas recombinase recognition sequence SEQ ID NOs: 04-06: Exemplary variants of the human CMV promoter SEQ ID NO: 07: Exemplary SV40 polyadenylation signal sequence SEQ ID NO: 08: Exemplary bGH polyadenylation signal sequence SEQ ID NO: 09: Exemplary hGT terminator sequence SEQ ID NO: 10: Exemplary SV40 promoter sequence SEQ ID NO: 11: Exemplary GFP nucleic acid sequence

[0225] Working Example: Example 1 General Technology 1) Recombinant DNA Technology DNA was manipulated using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Molecular biological reagents were used according to the manufacturer's instructions.

[0226] 2) DNA sequencing DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany).

[0227] 3) DNA and protein sequence analysis and sequence data management The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 were used for sequence generation, mapping, analysis, annotation, and illustration.

[0228] 4) Gene and oligonucleotide synthesis The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0229] 5) Reagents Unless otherwise stated, all commercially available chemicals, antibodies, and kits were used as provided according to the manufacturer's protocols.

[0230] 6) Cultivation of TI host cell lines TI CHO host cells were cultured at 37°C in a humidified incubator with 85% humidity and 5% CO2. They were cultured in a proprietary DMEM / F12-based medium containing 300 μg / ml hygromycin B and 4 μg / ml of a second selection marker. Cells were split every 3 or 4 days at a concentration of 0.3 x 10E6 cells / ml into a total volume of 30 ml. Cultures were performed in 125 ml non-baffled Erlenmeyer flasks. Cells were shaken at 150 rpm with a 5 cm shaking amplitude. Cell numbers were determined using a Cedex HiRes Cell Counter (Roche). Cells were cultured until they reached 60 days of age.

[0231] 7) Cloning General Cloning at the R site relies on the DNA sequence adjacent to the gene of interest (GOI), which is identical to the sequence in the next fragment. Thus, assembly of the fragments is possible through overlapping of identical sequences followed by sealing of the nicks in the assembled DNA with DNA ligase. Therefore, cloning of a single gene, specifically a pre-vector containing an appropriate R site, is necessary. After successful cloning of these pre-vectors, the gene of interest, flanked by the R sites, is excised via restriction digestion with an enzyme that cuts immediately adjacent to the R site. The final step is to assemble all DNA fragments in one step. More specifically, 5'-exonuclease removes the 5' end of the overlapping region (R site). Then, R sites can be annealed, and DNA polymerase extends the 3' end to fill the gap in the sequence. Finally, DNA ligase seals the nicks between the nucleotides. The single fragments are assembled into a single plasmid by adding an assembly master mix containing various enzymes, such as exonucleases, DNA polymerases, and ligases, and then incubating the reaction mix at 50°C. Competent E. coli cells are then transformed with the plasmids.

[0232] Some vectors use a restriction enzyme-mediated cloning strategy. By selecting the appropriate restriction enzyme, the desired gene of interest can be excised and then inserted into another vector by ligation. Therefore, it is preferable to use an enzyme that cuts at the multiple cloning site (MCS) and select it in an intelligent way to ensure ligation of the fragments in the correct array. If the vector and fragment have previously been cut with the same restriction enzyme, the sticky ends of the fragment and vector will be perfectly compatible and can then be ligated with DNA ligase. After ligation, competent E. coli cells are transformed with the newly constructed plasmid.

[0233] Cloning by restriction digestion For digestion of the plasmid with restriction enzymes, the following components were pipetted together on ice:

[0234] Table: Restriction digestion reaction mix TIFF2025134010000003.tif32128

[0235] If more enzymes were used per digestion, 1 μl of each enzyme was used, adjusting the volume by adding more or less PCR-grade water. All enzymes were selected with the prerequisites that they be qualified for use in New England Biolabs' CutSmart buffer (100% activity) and at the same incubation temperature (all 37°C).

[0236] Incubation was performed using a thermomixer or thermal cycler, allowing the samples to incubate at a constant temperature (37°C). Samples were not stirred during incubation. The incubation time was set to 60 min. Afterwards, the samples were directly mixed with loading dye and loaded onto an agarose electrophoresis gel or stored at 4°C / on ice for further use.

[0237] A 1% agarose gel was prepared for gel electrophoresis. 1.5 g of all-purpose agarose was weighed into a 125 Erlenmeyer flask and filled with 150 ml of TAE buffer. The mixture was heated in a microwave oven until the agarose was completely dissolved. 0.5 μg / ml of ethidium bromide was added to the agarose solution. The gel was then poured into a mold. After the agarose solidified, the mold was placed in the electrophoresis chamber, which was filled with TAE buffer. The sample was then loaded. The appropriate DNA molecular weight marker was loaded into the first pocket (from the left), followed by the sample. The gel was run at <130 V for approximately 60 minutes. After electrophoresis, the gel was removed from the chamber and analyzed using a UV-Imager.

[0238] The target band was excised and transferred to a 1.5 ml Eppendorf tube. Gel purification was performed using Qiagen's QIAquick Gel Extraction Kit according to the manufacturer's instructions. DNA fragments were stored at -20°C for further use.

[0239] The fragments for ligation were pipetted together at a vector-to-insert molar ratio of 1:2, 1:3, or 1:5, depending on the length of the insert and vector fragments and their relationship to each other. A 1:5 ratio was used when the fragment that needed to be inserted into the vector was short. The longer the insert, the less insert amount used relative to the vector. A vector amount of 50 ng was used for each ligation, and the specific insert amount was calculated using the NEBioCalculator. The T4 DNA Ligation Kit from NEB was used for the ligations. An example ligation mixture is shown in the following table:

[0240] Table: Ligation reaction mix TIFF2025134010000004.tif38154

[0241] Starting with the combination of DNA and water, followed by the addition of buffer, and finally the addition of enzyme, all components were pipetted together on ice. The reaction was gently mixed by pipetting up and down, briefly microcentrifuged, and then incubated at room temperature for 10 minutes. After incubation, the T4 ligase was heat-inactivated at 65°C for 10 minutes. The sample was then cooled on ice. In the final step, 10 beta-competent E. coli cells were transformed with 2 μl of the ligated plasmid (see below).

[0242] Cloning by R-site assembly For assembly, all DNA fragments with R sites at both ends were pipetted together on ice. When assembling more than four fragments, equimolar ratios of all fragments (0.05 ng) were used according to the manufacturer's recommendations. Half of the reaction mix was performed with NEBuilder HiFi DNA Assembly Master Mix. The total reaction volume was 40 μl, which was reached by filling with PCR-clean water. The following table shows an exemplary pipetting scheme.

[0243] Table: Assembly reaction mix TIFF2025134010000005.tif57167

[0244] After setting up the reaction mixture, the tubes were incubated in a thermocycler at 50 °C for 60 min at a constant temperature. After successful assembly, 10 beta-competent E. coli were transformed with 2 µl of assembled plasmid DNA (see below).

[0245] Transform 10 beta-competent E. coli cells For transformation, 10 beta-competent E. coli cells were thawed on ice. 2 μl of plasmid DNA was then pipetted directly into the cell suspension. The tube was flicked and placed on ice for 30 minutes. The cells were then placed in a 42°C warm thermal block for a heat shock of exactly 30 seconds. Immediately afterwards, the cells were cooled on ice for 2 minutes. 950 μl of NEB10 beta growth medium was added to the cell suspension. The cells were incubated at 37°C for 1 hour with shaking. Next, 50–100 μl was pipetted onto a prewarmed (37°C) LB-Amp agar plate and spread with a disposable spatula. The plate was incubated overnight at 37°C. Only bacteria that had successfully taken up the plasmid and possessed the ampicillin resistance gene were able to grow on this plate. The next day, single colonies were picked and cultured in LB-Amp medium for subsequent plasmid preparation.

[0246] bacterial culture E. coli was cultured in Luria Bertani LB medium, supplemented with 1 ml / L of 100 mg / ml ampicillin to achieve an ampicillin concentration of 0.1 mg / ml. For the different plasmid preparations, the following volumes were inoculated with a single bacterial colony:

[0247] Table: Volumes for E. coli culture TIFF2025134010000006.tif26154

[0248] For minipreps, 96-well 2 ml deep-well plates were filled with 1.5 ml of LB-Amp medium per well. Colonies were picked by pressing a toothpick into the medium. When all colonies were picked, the plate was closed with an adhesive air-porous membrane. The plate was incubated at 37°C in an incubator with a shaking speed of 200 rpm for 23 hours.

[0249] For minipreps, 15 ml tubes (with ventilated lids) were filled with 3.6 ml of LB-Amp medium and inoculated evenly with bacterial colonies. The toothpicks were left in the tubes during incubation. Similar to the 96-well plates, the tubes were incubated at 37°C and 200 rpm for 23 hours.

[0250] For maxipreps, 200 ml of LB-Amp medium was filled into an autoclaved 1 L glass Erlenmeyer flask and inoculated with 1 ml of a 5-hour-old bacterial day culture. The Erlenmeyer flask was closed with a paper stopper and incubated at 37°C and 200 rpm for 16 hours.

[0251] Plasmid preparation For minipreps, 50 μl of bacterial suspension was transferred to a 1 ml deep-well plate. The bacterial cells were then centrifuged in the plate at 3000 rpm for 5 minutes at 4°C. The supernatant was removed, and the plate containing the bacterial pellet was placed in an EpMotion. After approximately 90 minutes, analysis was performed, and the eluted plasmid DNA could be removed from the EpMotion for further use.

[0252] For minipreps, the 15 ml tubes were removed from the incubator and 3.6 ml of bacterial culture was divided into two 2 ml Eppendorf tubes. The tubes were centrifuged at 6,800 x g for 3 minutes at room temperature in a tabletop microcentrifuge. Minipreps were then performed using the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Plasmid DNA concentrations were measured using a Nanodrop™.

[0253] Maxipreps were performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF kit according to the manufacturer's instructions. DNA concentrations were measured with a Nanodrop.

[0254] Ethanol precipitation The volume of the DNA solution was mixed with 2.5 volumes of 100% ethanol. The mixture was incubated at -20°C for 10 minutes. The DNA was then centrifuged at 14,000 rpm at 4°C for 30 minutes. The supernatant was carefully removed, and the pellet was washed with 70% ethanol. Again, the tube was centrifuged at 14,000 rpm at 4°C for 5 minutes. The supernatant was carefully removed by pipetting, and the pellet was allowed to dry. Once the ethanol had evaporated, an appropriate amount of endotoxin-free water was added. The DNA was allowed to redissolve in water overnight at 4°C. A small aliquot was taken, and the DNA concentration was measured using a Nanodrop device.

[0255] Example 2 Plasmid preparation Expression cassette composition For expression of antibody chains, transcription units containing the following functional elements were used: - the immediate early enhancer and promoter from human cytomegalovirus, including intron A; - human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, - bovine growth hormone polyadenylation sequence (BGH pA), and - optionally, a human gastrin terminator (hGT).

[0256] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains: - the origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and - The beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0257] Front and back vector cloning To construct the two-plasmid antibody constructs, the antibody HC and LC fragments were cloned into a forward vector backbone containing the L3 and LoxFAS sequences, and a reverse vector containing the LoxFAS and 2L sequences and the pac selection marker. The Cre recombinase plasmid pOG231 (Wong, E.T. et al., Nuc. Acids Res. 33 (2005) e147; O'Gorman, S. et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) was used for all RMCE processes.

[0258] cDNA encoding each antibody chain was generated by gene synthesis (Geneart, Life Technologies Inc.). The gene synthesis and backbone vectors were digested with HindIII-HF and EcoRI-HF (NEB) at 37°C for 1 hour and separated by agarose gel electrophoresis. The insert and backbone DNA fragments were excised from the agarose gel and extracted using a QIAquick Gel Extraction Kit (Qiagen). The purified insert and backbone fragments were ligated at a 3:1 insert / backbone ratio using a rapid ligation kit (Roche) according to the manufacturer's protocol. The ligation approach was then transformed into competent E. coli DH5α by heat shock at 42°C for 30 seconds, incubated at 37°C for 1 hour, and plated onto agar plates containing selective ampicillin. The plates were incubated at 37°C overnight.

[0259] The next day, clones were picked and incubated overnight at 37°C with shaking for minipreps or maxipreps, which were performed using an EpMotion® 5075 (Eppendorf) or a QIAprep Spin Miniprep Kit (Qiagen) / NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure the absence of any unwanted mutations (SequiServe GmbH).

[0260] In the second cloning step, the previously cloned vector was digested with KpnI-HF / SalI-HF and SalI-HF / MfeI-HF using the same conditions as in the first cloning. The TI backbone vector was digested with KpnI-HF and MfeI-HF. Isolation and extraction were performed as previously described. Ligation of the purified insert and backbone at a 1:1:1 insert / insert / backbone ratio using T4 DNA ligase (NEB) according to the manufacturer's protocol was performed overnight at 4°C and inactivated at 65°C for 10 minutes. The following cloning steps were performed as previously described.

[0261] The cloned plasmids were used for TI transfection and pool generation.

[0262] Example 3 Culture, transfection, selection and pool generation TI host cells were grown in a proprietary DMEM / F12-based medium in disposable 125 ml vented shake flasks under standard humidified conditions (95% rH, 37°C, and 5% CO) with a constant agitation rate of 150 rpm. Every 3–4 days, cells were seeded at a concentration of 3 x 10E5 cells / ml in synthetic medium containing effective concentrations of selectable marker 1 and selectable marker 2. Culture density and viability were measured using a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[0263] For stable transfection, equimolar amounts of the front and back vectors were mixed. 1 μg of Cre expression plasmid was added to the 5 μg mixture.

[0264] Two days before transfection, TI host cells were seeded in fresh medium at a density of 4 x 10E5 cells / ml. Transfection was performed in a Nucleofector device using the Nucleofector Kit V (Lonza, Switzerland) according to the manufacturer's protocol. 3 x 10E7 cells were transfected with 30 μg of plasmid. After transfection, cells were seeded in 30 ml of medium without selection agent.

[0265] Five days after seeding, the cells were centrifuged and transferred to 80 mL of synthetic medium containing puromycin (selection agent 1) and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) at an effective concentration of 6 x 10E5 cells / mL for selection of recombinant cells. The cells were incubated at 37°C, 150 rpm, and maintained at 5% CO2 and 85% humidity. From this day on, the cells were not split. The cell density and viability of the cultures were monitored periodically. When the viability of the cultures began to increase again, the concentrations of selection agents 1 and 2 were reduced to approximately half of the amounts previously used. More specifically, to facilitate cell recovery, the selection pressure was reduced when the viability was greater than 40% and the viable cell density (VCD) was greater than 0.5 x 10E6 cells / mL. Therefore, 4 x 10E5 cells / ml were centrifuged and resuspended in 40 ml of selection medium II (synthetic medium, 1 / 2 selection markers 1 and 2). These cells were incubated under the same conditions as before, again without splitting.

[0266] Ten days after initiating selection, the success of Cre-mediated cassette exchange was confirmed by flow cytometry, measuring the expression of intracellular GFP and extracellular multivalent bispecific antibodies bound to the cell surface. APC antibodies (allophycocyanin-conjugated F(ab')2 fragment goat anti-human IgG) against the light and heavy chains of human antibodies were used for FACS staining. Flow cytometry was performed using a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). 10,000 events were measured per sample. Live cells were gated on a plot of forward scatter (FSC) versus side scatter (SSC). A live cell gate was defined on untransfected TI host cells and applied to all samples using FlowJo 7.6.5 EN software (TreeStar, Olten, Switzerland). GFP fluorescence was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). Multivalent bispecific antibodies were measured in the APC channel (excitation at 645 nm, detection at 660 nm). CHO parental cells, i.e., the cells used to generate the TI host cells, were used as a negative control for GFP and multivalent bispecific antibody expression. 14 days after the start of selection, viability was greater than 90%, and selection was considered complete.

[0267] Example 4 FACS screening FACS analysis was performed to examine the transfection efficiency and RMCE efficiency of the transfection. 4 x 10E5 cells of the transfected approach were centrifuged (1200 rpm, 4 min) and washed twice with 1 mL of PBS. After a washing step with PBS, the pellet was resuspended in 400 μL of PBS and transferred to a FACS tube (Falcon® round-bottom tube with cell strainer cap, Corning). Measurements were performed using FACS Canto II, and data were analyzed using the software FlowJo. was analyzed.

[0268] Example 5 Fed-batch culture Fed-batch production cultures were performed in shake flasks or Ambr15 vessels (Sartorius Stedim) containing proprietary synthetic medium. Cells were seeded at 1x10E6 cells / ml on day 0 and temperature shifted on day 3. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell counts (VCC) and percent viability of cells in the cultures were measured on days 0, 3, 7, 10, and 14 using a Vi-Cell™ XR instrument (Beckman Coulter). Glucose and lactate concentrations were measured on days 7, 10, and 14 using a Bioprofile 400 Analyzer (Nova Biomedical). On day 14 after the start of the feed, supernatants were harvested by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and clarified by filtration (0.22 μm). Titers on day 14 were measured using protein A affinity chromatography with UV detection. Product quality was determined by Caliper's LabChip (Caliper Life Sciences).

[0269] Example 6 Vector design effects To investigate the effect of expression cassette configuration on productivity in TI hosts, RMCE pools were generated by transfecting two plasmids (front and back vectors) containing different numbers and configurations of individual chains of a multivalent bispecific antibody. After selection, recovery, and validation of RMCEs by flow cytometry, the productivity of the pools was assessed in a 14-day fed-batch production assay.

[0270] The effect of antibody chain expression cassette organization on the expression of multivalent bispecific antibodies was evaluated. For multivalent bispecific antibodies, the following results were obtained: TIFF2025134010000007.tif50145

[0271] Sequence information SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Method for the generation of a multivalent, bispecific antibody expressing cell by targeted integration of multiple expression cassettes in a defined organization <150> EP 19181098.5 <151> 2019-06-19 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> L3 <400> 1 ataacttcgt ataaagtctc ctatacgaag ttat 34 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 2L <400> 2 ataacttcgt atagcataca ttatacgaag ttat 34 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> loxFas <400> 3 acaacttcgt atataccttt ctatacgaag ttgt 34 <210> 4 <211> 608 <212> DNA <213> Human cytomegalovirus <400> 4 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattagc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctccg tttagtgaac 600 gtcagatc 608 <210> 5 <211> 696 <212> DNA <213> Human cytomegalovirus <400> 5 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattagc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctccg tttagtgaac 600 gtcagatcta gctctgggag aggagcccag cactagaagt cggcggtgtt tccattcggt 660 gatcagcact gaacacagag gaagcttgcc gccacc 696 <210> 6 <211> 2125 <212> DNA <213> Human cytomegalovirus <400> 6 ctgcagtgaa taataaaatg tgtgtttgtc cgaaatacgc gttttgagat ttctgtcgcc 60 gactaaattc atgtcgcgcg atagtggtgt ttatcgccga tagagatggc gatattggaa 120 aaatcgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt tctgtgtaac 180 tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg cgatagcgct 240 tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat tctgtgtgtc 300 gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg ccgatagagg 360 cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc aatattggcc 420 attagccata ttattcattg gttatatagc ataaatcaat attggctatt ggccattgca 480 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc 540 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 600 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 660 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 720 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 780 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 840 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 900 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 960 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1020 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 1080 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1140 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 1200 ccgatccagc ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag 1260 tgacgtaagt accgcctata gagctatag gcccaccccc ttggcttctt atgcatgcta 1320 tactgttttt ggcttggggt ctatacaccc ccgcttcctc atgttatagg tgatggtata 1380 gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt ggtgacgata 1440 ctttccatta ctaatccata acatggctct ttgccacaac tctctttatt ggctatatgc 1500 caatacactg tccttcagag actgacacgg actctgtatt tttacaggat ggggtctcat 1560 ttattattta caaattcaca tatacaacac caccgtcccc agtgcccgca gtttttatta 1620 aacataacgt gggatctcca cgcgaatctc gggtacgtgt tccggacatg ggctcttc 1680 cggtagcggc ggagcttcta catccgagcc ctgctcccat gcctccagcg actcatggtc 1740 gctcggcagc tccttgctcc taacagtgga ggccagactt aggcacagca cgatgcccac 1800 caccaccagt gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa atgagctcgg 1860 ggagcgggct tgcaccgctg acgcatttgg aagacttaag gcagcggcag aagaagatgc 1920 aggcagctga gttgttgtgt tctgataaga gtcagaggta actcccgttg cggtgctgtt 1980 aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg ccaccagaca 2040 tatagctga cagactaaca gactgttcct ttccatgggt cttttctgca gtcaccgtcc 2100 ttgacacggt ttaaacgccg ccacc 2125 <210> 7 <211> 129 <212> DNA <213> Simian virus 40 <400> 7 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcacc attctagttg tggtttgtcc aaactcatca atgtatctta 120 tcatgtctg 129 <210> 8 <211> 225 <212> DNA <213> Taurus boss <400> 8 ctgtgccttc tagttgccag ccatctgttg tttgccccctc cccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtccttttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225 <210> 9 <211> 73 <212> DNA <213> Homo sapiens <400> 9 caggataata tatggtaggg ttcatagcca gagtaacctt tttttttaat ttttatttta 60 ttttattttt gag 73 <210> 10 <211> 288 <212> DNA <213> Simian virus 40 <400> 10 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 60 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 120 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 180 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 240 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcg 288 <210> 11 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> green fluorescent protein encoding nucleic acid <400> 11 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga 780 tccaccggat ctagatga 798

Claims

1. 1. A method for producing a multivalent bispecific antibody, comprising: a) culturing mammalian cells containing deoxyribonucleic acid encoding said multivalent bispecific antibody, and b) recovering the multivalent, bispecific antibody from the cells or culture medium Including, the deoxyribonucleic acid encoding the multivalent, bispecific antibody is stably integrated into the genome of the mammalian cell and comprises, in a 5' to 3' direction: （１） a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and an eighth expression cassette encoding said first light chain 1. A method for producing a multivalent bispecific antibody, comprising any one of the following steps:

2. A deoxyribonucleic acid encoding a multivalent, bispecific antibody, comprising, in 5' to 3' direction: （１） a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and an eighth expression cassette encoding said first light chain Deoxyribonucleic acid, including any of the following:

3. 1. Use of deoxyribonucleic acid for the expression of a multivalent, bispecific antibody in a mammalian cell, the deoxyribonucleic acid comprising, in the 5′ to 3′ direction: （１） a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and - an eighth expression cassette encoding said first light chain, Use, including any of the following:

4. 1. A recombinant mammalian cell comprising a deoxyribonucleic acid encoding a multivalent, bispecific antibody integrated into the genome of the cell, wherein the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises, in a 5′ to 3′ direction: （１） a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding a second heavy chain, - a fifth expression cassette encoding said first light chain, and - a sixth expression cassette encoding said first light chain, or (2) a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, - a fourth expression cassette encoding said first light chain, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, and an eighth expression cassette encoding said first light chain A recombinant mammalian cell comprising any one of the above.

5. A composition comprising two deoxyribonucleic acids, wherein the two deoxyribonucleic acids comprise three different recombination recognition sequences and six or eight expression cassettes; - the first deoxyribonucleic acid is in the 5' to 3' direction （１） a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, and - a first copy of the third recombination recognition sequence or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding said first light chain, and - a first copy of the third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, （１） - a second copy of said third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, - a sixth expression cassette encoding said first light chain, and - a second recombination recognition sequence, or (2) - a second copy of said third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, an eighth expression cassette encoding said first light chain, and - a second recombination recognition sequence A composition comprising any one of the following:

6. 1. A method for producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding a multivalent bispecific antibody and that secretes said multivalent bispecific antibody, comprising: a) providing a mammalian cell comprising an exogenous nucleotide sequence integrated at a single site within a genomic locus of said mammalian cell, said exogenous nucleotide sequence comprising first and second recombination recognition sequences flanking at least one first selectable marker, and a third recombination recognition sequence located between said first and second recombination recognition sequences, and wherein said recombination recognition sequences are all different; b) introducing into the cells provided in a) two deoxyribonucleic acid compositions comprising three different recombination recognition sequences and six or eight expression cassettes, - the first deoxyribonucleic acid is in the 5' to 3' direction （１） a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, and - a first copy of the third recombination recognition sequence or (2) a first recombination recognition sequence, a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first light chain, - a third expression cassette encoding said first light chain, a fourth expression cassette encoding said first light chain, and - a first copy of the third recombination recognition sequence and a second deoxyribonucleic acid in the 5' to 3' direction, （１） - a second copy of said third recombination recognition sequence, a fourth expression cassette encoding a second heavy chain, a fifth expression cassette encoding the first light chain, - a sixth expression cassette encoding said first light chain, and - a second recombination recognition sequence, or (2) - a second copy of said third recombination recognition sequence, a fifth expression cassette encoding a second heavy chain, - a sixth expression cassette encoding said first light chain, - a seventh expression cassette encoding said first light chain, an eighth expression cassette encoding said first light chain, and - a second recombination recognition sequence including any of the following: the first to third recombination recognition sequences of the first and second deoxyribonucleic acids are identical to the first to third recombination recognition sequences of the integrated exogenous nucleotide sequence; the 5'-end portion and the 3'-end portion of an expression cassette encoding one second selection marker together form a functional expression cassette of said one second selection marker; introducing a composition of two deoxyribonucleic acids; c) one or more recombinases, i) simultaneously with said first and second deoxyribonucleic acids of b), or ii) then sequentially A step of introducing the compound by any one of the following methods: said one or more recombinases recognize recombination recognition sequences in said first and said second deoxyribonucleic acids (and optionally said one or more recombinases effect two recombinase-mediated cassette exchanges); introducing one or more recombinases; and d) selecting cells that express said second selection marker and secrete said multivalent, bispecific antibody thereby producing a recombinant mammalian cell that contains deoxyribonucleic acid encoding said multivalent bispecific antibody and that secretes said multivalent bispecific antibody. Methods for producing recombinant mammalian cells.

7. 7. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to any one of claims 1 to 6, wherein the first heavy chain comprises the mutation T366W (numbering according to Kabat) in the CH3 domain and the second heavy chain comprises the mutations T366S, L368A, and Y407V (numbering according to Kabat) in the CH3 domain, or vice versa.

8. 8. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to claim 7, wherein one of the heavy chains further comprises the mutation S354C and each other heavy chain comprises the mutation Y349C (numbering according to Kabat).

9. 9. A method for producing a multivalent bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell according to any one of claims 1 to 8, wherein said first light chain is a domain-swapped light chain VH-VL or CH1-CL.

10. the first heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a first light chain variable domain; the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a second heavy chain variable domain; and - the first light chain comprises, from N- to C-terminus, a second light chain variable domain and a CL domain; the first heavy chain variable domain and the second light chain variable domain form a first binding site, and the second heavy chain variable domain and the first light chain variable domain form a second binding site; 10. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to any one of claims 1 to 9.

11. 11. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell according to any one of claims 1, 3, 4 and 6 to 10, wherein exactly one copy of said deoxyribonucleic acid is stably integrated into the genome of said mammalian cell at a single site or locus.

12. the deoxyribonucleic acid encoding the multivalent, bispecific antibody comprises a further expression cassette encoding a selection marker, the expression cassette encoding the selection marker being partially located 5' and partially located 3' to the third recombination recognition sequence, the portion of the expression cassette located 5' comprises a promoter and a start codon, the portion of the expression cassette located 3' comprises a coding sequence without a start codon and a polyA signal, the start codon is operably linked to the coding sequence, and the portion of the expression cassette located 5' to the selection marker comprises a promoter sequence operably linked to a start codon, is flanked on the upstream side by the third or fourth expression cassette, respectively, and the start codon is flanked on the downstream side by the third recombination recognition sequence; and the 3'-located portion of the expression cassette encoding the selectable marker comprises a nucleic acid encoding the selectable marker lacking a start codon and is flanked on the upstream side by the third recombination recognition sequence and on the downstream side by the fourth or fifth expression cassette, respectively, and the start codon is operably linked to the coding sequence.

13. each expression cassette of the antibody chain comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding the antibody chain, and a polyadenylation signal sequence, and optionally a terminator sequence; and each expression cassette encoding said selectable marker comprises, in the 5' to 3' direction, a promoter, a nucleic acid encoding said selectable marker, and a polyadenylation signal sequence, and optionally a terminator sequence; the promoter is a human CMV promoter containing intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence, and the terminator is an hGT terminator; except that in the expression cassette of the selectable marker, the promoter is an SV40 promoter, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, and no terminator is present.

13. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to any one of claims 1 to 12.

14. 14. The method for producing a multivalent, bispecific antibody, or the deoxyribonucleic acid, or the use, or the recombinant mammalian cell, or the composition, or the method for producing a recombinant mammalian cell, according to any one of claims 1, 3, 4 and 6 to 13, wherein the mammalian cell is a CHO cell.

15. 15. A method for producing a multivalent, bispecific antibody, or a deoxyribonucleic acid, or a use, or a recombinant mammalian cell, or a composition, or a method for producing a recombinant mammalian cell, according to any one of claims 1 to 14, wherein all expression cassettes are arranged in one direction.

Images