Compositions and methods for generating antibodies based on the use of expression-enhanced loci

Abstract

To provide compositions and methods for making antibodies based on the use of an expression-enhancing locus.SOLUTION: This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antibodies including bispecific antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing an expression-enhancing locus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 325,385, filed April 20, 2016, the entire contents of which are incorporated herein by reference.

[0002] This disclosure relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, this disclosure relates to compositions and methods for improved expression of antigen-binding proteins, such as bispecific antibodies, in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by utilizing expression-enhancing sites. [Background technology]

[0003] Cellular expression systems aim to provide a reliable and efficient source for the production of a given protein, both for research and therapeutic applications. For example, mammalian expression systems are capable of appropriate post-translational modification of recombinant proteins, and therefore, for therapeutic protein production purposes, expression of recombinant proteins in mammalian cells is the preferred method.

[0004] Despite the availability of various expression systems, efficient gene transfer and integrated gene stability for recombinant protein expression remain challenges. One of the concerns for long-term expression of a target transgene is minimizing disruption of cellular genes to avoid altering the phenotype of the cell line.

[0005] Engineering stable cell lines to accommodate the expression of multiple genes, such as multiple antibody chains found in multispecific antibodies, is particularly challenging. Wide variations in expression levels of integrated genes can occur. Integration of additional genes can result in even greater expression variation and instability due to the local genetic environment (i.e., position effects). Expression systems for producing multispecific antigen-binding proteins often require the expression of two or more different immunoglobulin chains intended to pair into unique multimeric forms, and often result in the predominance of homodimers over the desired heterodimer or multimeric combinations. Thus, there is a need in the art for improved mammalian expression systems. Summary of the Invention [Means for solving the problem]

[0006] In one embodiment, a cell is provided that contains an exogenous nucleic acid integrated at a specific site within an expression-enhanced locus, wherein the exogenous nucleic acid sequence encodes a bispecific antigen-binding protein.

[0007] In some embodiments, the exogenous nucleic acid sequence comprises a first exogenous nucleic acid containing a nucleotide sequence encoding a first light chain fragment (LCF), a second exogenous nucleic acid containing a nucleotide sequence encoding a first heavy chain fragment (HCF), and a third exogenous nucleic acid containing a nucleotide sequence encoding a second HCF (or designated HCF* if the second HCF is different from the first HCF), wherein the first and second HCFs and the first LCF form a bispecific antigen-binding protein. In certain embodiments, the first and second HCFs and the first LCF contain at least two variable regions and two CH3 constant domains of the bispecific antigen-binding protein. In some embodiments, the two variable regions are different. In some embodiments, the two CH3 regions are different. In some embodiments, each exogenous nucleic acid sequence is simultaneously integrated into a specific site within the enhanced expression locus.

[0008] In some embodiments, the nucleotide sequence encoding the first HCF encodes amino acids from a first constant region (e.g., encoding one or more of the CH1, CH2, hinge, or CH3 domains), and the nucleotide sequence encoding the second HCF encodes amino acids from a second constant region. The amino acids from the first constant region may be the same as or different from the amino acids from the second constant region. In certain embodiments, the nucleotide sequence encoding the first HCF encodes a first CH3 domain, and the nucleotide sequence encoding the second HCF encodes a second CH3 domain, in which case the first and second CH3 domains may be the same or different. In some embodiments, the first and second CH3 domains differ at at least one amino acid position. For example, one of the two CH3 domains is a human IgG CH3 domain and the other is a modified human IgG CH3 domain, and the two CH3 domains have different protein A binding properties. In other embodiments, the nucleotide sequences encoding the first and second CH3 domains differ from each other in that one of the nucleotide sequences has been codon-modified.

[0009] In another specific embodiment, the nucleotide sequence encoding the first HCF encodes a first heavy chain variable (VH) region and the nucleotide sequence encoding the second HCF encodes a second VH region, wherein the first and second heavy chains may have the same or different VH regions. In another embodiment, the first and second VHs are linked to the same or different constant regions.

[0010] In some embodiments, the nucleotide sequence encoding the first LCF encodes a first light chain variable (VL) region.

[0011] In some embodiments, the exogenous nucleic acid sequence comprises an additional exogenous nucleic acid comprising a nucleotide sequence encoding a second LCF, e.g., a second light chain variable (VL) region. In some embodiments, the nucleotide sequence encoding the second VL region encodes a second light chain constant region.

[0012] The relative positions of the multiple exogenous nucleic acids at the locus can vary. In some embodiments, the nucleic acid encoding LCF is positioned upstream or downstream relative to both nucleic acids encoding HCF.

[0013] In some embodiments, each of the sequences encoding HCF or LCF is independently linked to a transcriptional regulatory sequence. In certain embodiments, the first exogenous nucleic acid further comprises a first promoter operably linked to the nucleotide sequence encoding the first LCF, the second exogenous nucleic acid further comprises a second promoter operably linked to the nucleotide sequence encoding the first HCF, and the third exogenous nucleic acid further comprises a third promoter operably linked to the nucleotide sequence encoding the second HCF, wherein the first, second, and third promoters may be the same or different, and / or the promoter is the same or different from the fourth promoter to which the fourth exogenous nucleic acid is operably linked. In some embodiments, the first, second, and third promoters are the same.

[0014] In some embodiments, the exogenous nucleic acid sequence further comprises a recombinase recognition site at the integration site, e.g., a first recombinase recognition site (RRS) positioned 5' to the first exogenous nucleic acid and a second recombinase recognition site (RRS) positioned 3' to both the second and third exogenous nucleic acids, where the first and second RRSs are different. In some embodiments, a third RRS is also included, where the third RRS is positioned 3' to the first exogenous nucleic acid and 5' to one or both of the second and third exogenous nucleic acids, where the third RRS is different from the first and second RRSs.

[0015] In some embodiments, the exogenous nucleic acid sequence may include a fourth exogenous nucleic acid containing a selectable marker gene. In certain embodiments, the fourth exogenous nucleic acid is located 3' to the first exogenous nucleic acid. In certain embodiments, the fourth exogenous nucleic acid is integrated as a split gene. In other embodiments, the fourth exogenous nucleic acid, i.e., the selectable marker, is located 3' to a third RRS, which is 3' to a fourth promoter operably linked to the fourth exogenous nucleic acid. In some embodiments, the selectable marker gene comprises an inserted third RRS, which is optionally inserted within an intron of the selectable marker gene, where the third RRS is different from the first and second RRSs.

[0016] In some embodiments, the order of exogenous nucleic acids at the locus can be as follows: from 5' to 3', a first exogenous nucleic acid (encoding an LCF), a fourth exogenous nucleic acid (encoding a selectable marker), a second exogenous nucleic acid (encoding a first HCF), and a third exogenous nucleic acid (encoding a second HCF). In some particular embodiments, the second exogenous nucleic acid contains a nucleotide sequence encoding a modified CH3 domain of human IgG, and the third exogenous nucleic acid comprises a nucleotide sequence encoding a native CH3 domain of human IgG.

[0017] In one embodiment, the order of the exogenous nucleic acids at the locus is, from 5' to 3', a first exogenous nucleic acid (encoding an LCF), a second exogenous nucleic acid (encoding a first HCF), a fourth exogenous nucleic acid (encoding a selectable marker), and a third exogenous nucleic acid (encoding a second HCF), wherein the second exogenous nucleic acid comprises a nucleotide sequence encoding a native CH3 domain of human IgG, and the third exogenous nucleic acid comprises a nucleotide sequence encoding a modified CH3 domain of human IgG.

[0018] In some embodiments, the promoters linked to the sequence encoding HCF or LCF are the same and different from the promoter to which the selectable marker gene is operably linked.

[0019] In some embodiments, the bispecific antigen binding protein specifically binds to a T cell antigen and a tumor cell antigen. Other suitable dual antigen specificities are also provided.

[0020] In some embodiments, the enhanced expression locus is selected from a locus with a nucleotide sequence at least 90% identical to SEQ ID NO:1, or a locus with a nucleotide sequence at least 90% identical to SEQ ID NO:2.

[0021] In various embodiments, the cells are CHO cells.

[0022] In another aspect, vectors designed for site-specific integration of multiple exogenous nucleic acids are provided.

[0023] In some embodiments, the disclosure provides a vector set comprising, from 5' to 3', a first nucleic acid containing a nucleotide sequence encoding a first RRS, a first LCF, and a first vector containing a third RRS; a second nucleic acid containing a nucleotide sequence encoding a third RRS, a first VH region, and a second vector containing a second RRS, wherein either the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF, and wherein the first and second HCFs and the first LCF form a bispecific antigen-binding protein.

[0024] In some embodiments, the nucleotide sequence encoding the second HCF is contained in a first nucleic acid and is optionally located downstream of the nucleotide sequence encoding the first LCF. In other embodiments, the nucleotide sequence encoding the second HCF is contained in a second nucleic acid.

[0025] In some embodiments, the nucleotide sequence encoding the first HCF encodes a first chimeric constant region (e.g., encoding one or more of the CH1, hinge, CH2, or CH3 domains, or fragments thereof, from any isotype), and the nucleotide sequence encoding the second HCF encodes a second chimeric constant region. Examples of chimeric constant regions are described in PCT International Application Publication No. WO2014 / 121087A1, published August 7, 2014, which is incorporated herein by reference. The amino acids from the first constant region may be the same as or different from the amino acids from the second chimeric constant region. In certain embodiments, the nucleotide sequence encoding the first HCF encodes a first CH3 domain, and the nucleotide sequence encoding the second HCF encodes a second CH3 domain, in which case the first and second CH3 domains may be the same or different. In some embodiments, the first and second CH3 domains differ at at least one amino acid position. For example, one of the two CH3 domains is a human IgG CH3 domain and the other is a modified human IgG CH3 domain, and the two CH3 domains have different Protein A binding properties. In other embodiments, the nucleotide sequences encoding the first and second CH3 domains differ from each other in that one of the nucleotide sequences has been codon modified.

[0026] In other specific embodiments, the nucleotide sequence encoding the first VH region encodes a first heavy chain and the nucleotide sequence encoding the second VH region encodes a second heavy chain, wherein the first and second heavy chains may have the same or different constant regions.

[0027] In some embodiments, the nucleotide sequence encoding the first LCF encodes a first light chain variable region (VL).

[0028] In some embodiments, each of the sequences encoding LCF or HCF is independently linked to a transcription control sequence, such as a promoter. In certain embodiments, the promoter linked to the sequence encoding the first HCF and the promoter linked to the sequence encoding the second HCF are the same promoter. In certain embodiments, the promoters linked to the LCF and HCF are all the same promoter.

[0029] In some embodiments, the first nucleic acid in the first vector further comprises a 5' portion of a selectable marker gene located 5' to the third RRS in the first vector. The second nucleic acid further comprises the remaining 3' portion of the selectable marker gene located 3' to the third RRS in the second vector. That is, the selectable marker gene is split between two vectors. In other embodiments, the selectable marker and the promoter to which the selectable marker is operably linked are split between two vectors. In other words, the promoter and the selectable marker gene are located on different vectors. In one embodiment, the promoter operably linked to the marker gene is located 5' to the third RRS in the first vector, and the marker gene is located 3' to the third RRS in the second vector, as well as 5' to a second promoter operably linked to the second nucleic acid and a third promoter operably linked to the third nucleic acid. In some embodiments, the third RRS in the first vector is present within the 5' portion of an intron of the selectable marker gene. The third RRS in the second vector is located within the 3' portion of the intron of the selectable marker gene.

[0030] In certain embodiments, the first vector comprises, from 5' to 3', a first RRS, a first nucleic acid, and a third RRS. The second vector comprises, from 5' to 3', a third RRS, a second nucleic acid containing a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HCF, and a second RRS. In other specific embodiments, the first vector comprises, from 5' to 3', a first RRS, a first nucleic acid, and a third RRS, wherein the first nucleic acid comprises a nucleotide sequence encoding a first HCF region and a nucleotide sequence encoding a second HCF region. The second vector comprises, from 5' to 3', a third RRS, a second nucleic acid, and a second RRS, wherein the second nucleic acid comprises a nucleotide sequence encoding the first HCF region. In any of these specific embodiments, the first nucleic acid may further comprise a 5' portion of a selectable marker gene located 5' to the third RRS in the first vector. and the second nucleic acid further comprises the remaining 3' portion of the selectable marker gene located 3' to the third RRS in the second vector, where, optionally, the third RRS in the first vector is within the 5' portion of an intron of the selectable marker gene and the third RRS in the second vector is within the 3' portion of an intron of the selectable marker gene.

[0031] In some embodiments, the vector set may include additional vectors, such as vectors containing nucleotide sequences encoding one or more RRSs and a second LCF, or vectors encoding one or more recombinases that recognize an RRS.

[0032] In other embodiments, the present disclosure provides vectors designed to achieve site-specific integration of multiple exogenous nucleic acids via homologous arm-based homologous recombination, hi some embodiments, the vectors contain exogenous nucleic acid sequences encoding bispecific antigen binding proteins and flanked by 5' and 3' homologous arms for integration into an expression-enhancing locus in a cell.

[0033] In a further aspect, the present disclosure provides a system that can be used to generate a cell comprising a combination of a cell and one or more vectors, wherein the exogenous nucleic acid is integrated into an expression-enhanced locus, wherein the exogenous nucleic acid together encodes a bispecific antigen-binding protein.

[0034] In one embodiment, a system is provided that includes a cell and a vector set, wherein the cell contains a set of RRSs integrated into expression-enhancing loci in its genome, the RRSs being distinct from each other and separated among one or more exogenous nucleic acids, such as selectable markers, for recombination exchange with genes of interest in the vector set, and wherein the RRSs in the vector set have the same configuration as the RRSs in the cell.

[0035] In some embodiments, a system is provided that includes a cell and a vector set, wherein the cell contains, in a 5' to 3' direction, the following integrated into an expression-enhancing locus in its genome: a first RRS, a first exogenous nucleic acid, a second RRS, a second exogenous nucleic acid, and a third RRS, wherein the three RRSs are different from one another. In this case, the vector set includes, in a 5' to 3' direction, a first vector containing the first RRS, a first nucleic acid containing a nucleotide sequence encoding a first immunoglobulin chain or a fragment thereof, and the second RRS; and a second vector containing the second RRS, a second nucleic acid containing a nucleotide sequence encoding a second immunoglobulin chain or a fragment thereof, and the third RRS, wherein either the first nucleic acid or the second nucleic acid further contains a nucleotide sequence encoding a third immunoglobulin chain or a fragment thereof. When the vector is introduced into a cell, the first and second nucleic acids in the vector integrate into the expression-enhancing locus via recombination mediated by the first, second, and third RRSs.

[0036] In some embodiments, a first exogenous nucleic acid in a cell contains a first selectable marker gene and a second exogenous nucleic acid in a cell contains a second selectable marker gene, wherein the first and second selectable marker genes are different. The selectable marker replaces the exogenous nucleic acid that is integrated in the cell.

[0037] In some embodiments, the first vector contains, from 5' to 3', a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF, and a third RRS. The second vector contains, from 5' to 3', a third RRS, a second nucleic acid, and a second RRS, where the second nucleic acid contains both a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HC. In other embodiments, the first vector contains, from 5' to 3', a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF and a nucleotide sequence encoding a second HCF, and a third RRS. The second vector contains, from 5' to 3', a third RRS, a second nucleic acid containing a nucleotide sequence encoding a first HCF, and a second RRS.

[0038] In some embodiments, the first vector comprises, from 5' to 3', a first RRS, a first nucleic acid containing a nucleotide sequence encoding the first HCF, and a third RRS. The second vector comprises, from 5' to 3', a third RRS, a second nucleic acid, and a second RRS, where the second nucleic acid contains both a nucleotide sequence encoding the first LCF and a nucleotide sequence encoding the second HCF. In other embodiments, the first vector comprises, from 5' to 3', a first RRS, a first nucleic acid containing a nucleotide sequence encoding the first HCF and a nucleotide sequence encoding the second HCF, and a third RRS. The second vector comprises, from 5' to 3', a third RRS, a second nucleic acid containing a nucleotide sequence encoding the first LCF, and a second RRS. In any of these embodiments, the first nucleic acid in the first vector may further comprise a promoter located 5' to the third RRS, and the second nucleic acid in the second vector further comprises a selectable marker gene operably linked to the promoter and located 3' to the third RRS. In other embodiments, the first nucleic acid in the first vector may further comprise a 5' portion of a selectable marker gene located 5' to the third RRS, and the second nucleic acid in the second vector further comprises the remaining 3' portion of the selectable marker gene located 3' to the third RRS. In this case, optionally, the third RRS in the first vector is present within the 5' portion of an intron of the selectable marker gene, and the third RRS in the second vector is present within the 3' portion of an intron of the selectable marker gene.

[0039] In some embodiments, the nucleotide sequence encoding an LCF is operably linked to a first promoter, the nucleotide sequence encoding a first HCF is operably linked to a second promoter, and the nucleotide sequence encoding a second HCF is operably linked to a third promoter, in which case the first, second, and third promoters are the same promoter, which is different from the promoter to which the selectable marker gene, if present in one of the vectors, is operably linked.

[0040] In some embodiments, the nucleotide sequence encoding the first HCF encodes a first CH3 domain, and the nucleotide sequence encoding the second HCF encodes a second CH3 domain, where the first and second CH3 domains may be the same or different. In some embodiments, one of the two CH3 domains is a native CH3 domain of human IgG, and the other CH3 domain is a modified CH3 domain of human IgG. In certain embodiments, the nucleotide sequence encoding the modified CH3 domain is in a first vector (i.e., a vector encoding the first HCF) and is optionally downstream of the nucleotide sequence encoding the first LCF. In other specific embodiments, the nucleotide sequence encoding the modified CH3 domain is in a second vector and is upstream of the nucleotide sequence encoding the unmodified CH3 domain.

[0041] In another aspect, the disclosure provides methods for making bispecific antigen-binding proteins.

[0042] In some embodiments, the method comprises providing a system as described herein containing a cell having an RRS and a set of vectors containing a plurality of exogenous nucleic acids; introducing the vectors into the cells by transfection; selecting transfected cells in which the exogenous nucleic acids in the vectors have integrated into an expression-enhanced locus via RRS-mediated recombination; expressing the polypeptides encoded by the nucleic acids in the transformed cells; and obtaining the bispecific antigen binding protein from the transfected cells, wherein the plurality of exogenous nucleic acids together encode the bispecific antigen binding protein and an RRS that matches an RRS in the cell.

[0043] In some embodiments, the method may comprise a cell containing an exogenous nucleic acid sequence encoding the bispecific antigen binding protein integrated within an expression-enhanced locus, expressing the bispecific antigen binding protein from the exogenous nucleic acid, and obtaining the bispecific antigen binding protein from the cell. In certain embodiments, for example, the following items are provided: (Item 1) A cell comprising an exogenous nucleic acid integrated at a specific site within an expression-enhanced locus, said exogenous nucleic acid sequence encoding a bispecific antigen-binding protein. (Item 2) 2. The cell of item 1, wherein the exogenous nucleic acid sequences comprise a first exogenous nucleic acid having a nucleotide sequence encoding a first LCF, a second exogenous nucleic acid having a nucleotide sequence encoding a first HCF, and a third exogenous nucleic acid having a nucleotide sequence encoding a second HCF. (Item 3) The cell of item 2, wherein the nucleotide sequence encoding the first HCF encodes a first CH2 domain and a first CH3 domain, and the nucleotide sequence encoding the second HCF encodes a second CH2 domain and a second CH3 domain. (Item 4) 4. The cell of item 3, wherein the first and second CH3 domains differ at at least one amino acid position. (Item 5) 4. The cell of item 3, wherein the first and second CH3 domains are human IgG CH3 domains, and one of the two CH3 domains is modified at at least one amino acid position, thereby resulting in Protein A binding properties that differ from those of the unmodified CH3 domain. (Item 6) 4. The cell of item 3, wherein the nucleotide sequences encoding the first and second CH domains differ from each other in that one of the nucleotide sequences is codon modified. (Item 7) 3. The cell of item 2, wherein the nucleotide sequence encoding the first HCF encodes a first VH and the nucleotide sequence encoding the second HCF encodes a second VH. (Item 8) 3. The cell of item 2, wherein the nucleotide sequence encoding the first LCF encodes a first VL. (Item 9) 2. The cell of item 1, wherein the exogenous nucleic acid sequence further comprises an additional exogenous nucleic acid comprising a nucleotide sequence encoding a second LCF. (Item 10) 10. The cell of item 9, wherein the nucleotide sequence encoding the second LCF encodes a second VL. (Item 11) 3. The cell of claim 2, wherein the first exogenous nucleic acid further comprises a first promoter operably linked to the nucleotide sequence encoding a first HCF, the second exogenous nucleic acid further comprises a second promoter operably linked to the nucleotide sequence encoding a first HCF, and the third exogenous nucleic acid further comprises a third promoter operably linked to the nucleotide sequence encoding a second HCF, and the first, second, and third promoters are the same or different promoters. (Item 12) 3. The cell of item 2, wherein the first exogenous nucleic acid is positioned upstream relative to the second and third exogenous nucleic acids. (Item 13) 13. The cell of item 12, further comprising a first recombinase recognition site (RRS) positioned 5' to the first exogenous nucleic acid and a second recombinase recognition site (RRS) positioned 3' to both the second and third exogenous nucleic acids, wherein the first and second RRSs are different. (Item 14) 13. The cell of item 12, further comprising a third RRS positioned 3' to the first exogenous nucleic acid and 5' to one or both of the second and third exogenous nucleic acids, wherein the third RRS is different from the first and second RRSs. (Item 15) 14. The cell of item 13, further comprising a fourth exogenous nucleic acid comprising a selectable marker gene. (Item 16) 16. The cell of item 15, wherein the fourth exogenous nucleic acid further comprises a third RRS and is positioned 3' to the first exogenous nucleic acid. (Item 17) 3. The cell of item 2, wherein the second exogenous nucleic acid is positioned upstream relative to the first and third exogenous nucleic acids. (Item 18) 17. The cell of item 16, wherein the order of the exogenous nucleic acids at the locus is, from 5' to 3', the first exogenous nucleic acid, the fourth exogenous nucleic acid, the second exogenous nucleic acid, and the third exogenous nucleic acid, wherein the second exogenous nucleic acid comprises a nucleotide sequence encoding a modified CH3 domain of human IgG, and the third exogenous nucleic acid comprises a nucleotide sequence encoding a native CH3 domain of human IgG. (Item 19) 17. The cell of item 16, wherein the order of the exogenous nucleic acids at the locus is, from 5' to 3', the first exogenous nucleic acid, the second exogenous nucleic acid, the fourth exogenous nucleic acid, and the third exogenous nucleic acid, wherein the second exogenous nucleic acid comprises a nucleotide sequence encoding the native CH3 domain of human IgG, and the third exogenous nucleic acid comprises a nucleotide sequence encoding the modified CH3 domain of human IgG. (Item 20) 20. The cell of item 18 or 19, wherein the first, second, and third promoters are the same promoter and are different from the promoter to which the selectable marker gene is operably linked. (Item 21) 2. The cell of item 1, wherein the bispecific antigen-binding protein specifically binds to a T cell antigen and a tumor cell antigen. (Item 22) 2. The cell of item 1, wherein the enhanced expression locus is selected from the group consisting of a locus having a nucleotide sequence at least 90% identical to SEQ ID NO: 1 and a locus having a nucleotide sequence at least 90% identical to SEQ ID NO: 2. (Item 23) 23. The cell according to any one of items 1 to 22, wherein the cell is a CHO cell. (Item 24) a first vector comprising, in the 5' to 3' direction, a first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF, and a third RRS; a vector set comprising, in a 5' to 3' direction, the third RRS, a second nucleic acid comprising a nucleotide sequence encoding a first HCF, and a second vector comprising a second RRS, the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF, and A vector set, wherein the first and second HCFs and the first LCF encode a bispecific antigen-binding protein. (Item 25) 25. The vector set of item 24, wherein the nucleotide sequence encoding the second HCF is contained in the first nucleic acid. (Item 26) 26. The vector set of item 25, wherein the nucleotide sequence encoding the second HCF is located upstream or downstream of the nucleotide sequence encoding the first LCF. (Item 27) 25. The vector set of item 24, wherein the nucleotide sequence encoding the second HCF is contained in the second nucleic acid. (Item 28) 25. The vector set of item 24, wherein the nucleotide sequence encoding the first HCF encodes a first CH3 domain, and the nucleotide sequence encoding the second HCF encodes the second CH3 domain. (Item 29) 29. The vector set of item 28, wherein the first and second CH3 domains differ at at least one amino acid position. (Item 30) 29. The vector set of Item 28, wherein the first and second CH3 domains are human IgG CH3 domains, and one of the two CH3 domains is modified at at least one amino acid position, thereby resulting in Protein A binding properties that differ from those of the unmodified CH3 domain. (Item 31) 29. The vector set of item 28, wherein the nucleotide sequences encoding the first and second CH domains differ from each other in that one of the nucleotide sequences is codon-modified. (Item 32) 25. The vector set of item 24, wherein the nucleotide sequence encoding the first HCF encodes a first VH, and the nucleotide sequence encoding the second HCF encodes a second heavy chain VH. (Item 33) 25. The vector set of item 24, wherein the nucleotide sequence encoding the first LCF encodes a first VL. (Item 34) 25. The vector set of item 24, wherein the nucleotide sequence encoding a first light chain LCF is operably linked to a first promoter, the nucleotide sequence encoding a first HCF is operably linked to a second promoter, and the nucleotide sequence encoding a second HCF is operably linked to a third promoter. (Item 35) 35. The vector set according to Item 34, wherein the first, second, and third promoters are the same or different promoters. (Item 36) 25. The vector set of Item 24, wherein the first nucleic acid further comprises a second promoter positioned 5' to the third RRS in the first vector, and the second nucleic acid further comprises the selectable marker gene positioned 3' to the third RRS in the second vector. (Item 37) 25. The vector set of Item 24, wherein the first nucleic acid further comprises a second promoter in the first vector positioned 5' to a 5' portion of a selectable marker gene, and the 3' portion of the selectable marker in the second vector is present 3' to a homologous arm of the 5' portion of the selectable marker gene. (Item 38) the first vector comprises, in a 5' to 3' direction, the first RRS, the first nucleic acid, and the third RRS; 25. The vector set of Item 24, wherein the second vector comprises, in a 5' to 3' direction, the third RRS, the second nucleic acid, and the second nucleic acid comprises the nucleotide sequence encoding a first heavy chain or fragment thereof, and the nucleotide sequence encoding a second heavy chain or fragment thereof. (Item 39) the first vector comprises, in a 5' to 3' direction, the first RRS, the first nucleic acid, the first nucleic acid comprising the nucleotide sequence encoding a first LCF and the nucleotide sequence encoding a second HCF, and the third RRS; 25. The vector set of item 24, wherein the second vector comprises, in a 5' to 3' direction, the third RRS and the second nucleic acid, and the second nucleic acid comprises a nucleotide sequence encoding the first HCF. (Item 40) 40. The vector set of claim 38 or 39, wherein the first nucleic acid further comprises a 5' portion of a selectable marker gene positioned 5' to the third RRS in the first vector, and the second nucleic acid further comprises a remaining 3' portion of the selectable marker gene positioned 3' to the third RRS in the second vector, and optionally, the third RRS in the first vector is present within a 5' portion of an intron of the selectable marker gene, and the third RRS in the second vector is present within a 3' portion of an intron of the selectable marker gene. (Item 41) 25. The vector set of item 24, further comprising a third vector comprising nucleotides encoding one or more RRSs and a second LCF. (Item 42) 25. The vector set of item 24, further comprising a third vector encoding one or more recombinases that recognize the RRS. (Item 43) A vector comprising an exogenous nucleic acid encoding a bispecific antigen binding protein and flanked by 5' and 3' homology arms for integration into an expression-enhancing locus in a cell. (Item 44) A system comprising a cell and a vector set, the cell comprises, in a 5' to 3' orientation, a first RRS, a first exogenous nucleic acid, a third RRS, a second exogenous nucleic acid, and a second RRS integrated into an expression-enhancing locus of the cell's genome, wherein the three RRSs are different from one another; The vector set comprises: a first vector comprising, in a 5' to 3' direction, the first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF, and the third RRS; a second nucleic acid comprising a nucleotide sequence encoding the third RRS, the first HCF, and a second vector comprising the second RRS; Either the first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF; When the vector is introduced into the cell, the first and second nucleic acids in the vector are integrated into the expression-enhancing locus via recombination mediated by the first, second, and third RRSs. (Item 45) 45. The system of item 44, wherein the first exogenous nucleic acid comprises a first selectable marker gene and the second exogenous nucleic acid comprises a second selectable marker gene, and the first and second selectable marker genes are different. (Item 46) the first vector comprises, in a 5' to 3' direction, the first RRS, the first nucleic acid comprising the first LCF, and the third RRS; and 45. The system of item 44, wherein the second vector comprises, in a 5' to 3' direction, the third RRS, the second nucleic acid comprising both the nucleotide sequence encoding the first HCF and the nucleotide sequence encoding the second HCF, and a second RRS. (Item 47) the first vector comprises, in a 5' to 3' direction, the first RRS, the first nucleic acid comprising the nucleotide sequence encoding the first LCF and the nucleotide sequence encoding the second HCF, and the third RRS; 45. The system of item 44, wherein the second vector comprises, in a 5' to 3' direction, the third RRS, the second nucleic acid comprising the nucleotide sequence encoding the first HCF, and the second RRS. (Item 48) 48. The system of claim 46 or 47, wherein the first nucleic acid in the first vector further comprises a 5' portion of a selectable marker gene positioned 5' to the third RRS, and the second nucleic acid in the second vector further comprises a remaining 3' portion of the selectable marker gene positioned 3' to the third RRS. (Item 49) 49. The system of Item 48, wherein the third RRS in the first vector is present within a 5' portion of an intron of the selectable marker gene, and the third RRS in the second vector is present within a 3' portion of an intron of the selectable marker gene. (Item 50) 49. The system of claim 48, wherein the nucleotide sequence encoding the LCF is operably linked to a first promoter, the nucleotide sequence encoding the first HCF is operably linked to a second promoter, and the nucleotide sequence encoding the second HCF is operably linked to a third promoter, wherein the first, second, and third promoters are the same or different promoters, and / or the promoter is the same or different from the promoter to which the selectable marker gene is operably linked. (Item 51) 45. The system of item 44, wherein the nucleotide sequence encoding the first HCF encodes a first CH3 domain and the nucleotide sequence encoding the second HCF encodes a second CH3 domain. (Item 52) 52. The system of claim 51, wherein one of the first and second CH3 domains is the CH3 domain of human IgG, and the other is a modified CH3 domain of human IgG comprising a modification at at least one amino acid position. (Item 53) 53. The system of claim 52, wherein the nucleotide sequence encoding the modified CH3 domain is in the first vector. (Item 54) 53. The system of claim 52, wherein the nucleotide sequence encoding the modified CH3 domain is in the second vector and is upstream of the nucleotide sequence encoding the unmodified CH3 domain. (Item 55) 1. A method comprising: (i) providing a system according to any one of items 44 to 54; (ii) co-introducing the vectors into the cells by transfection; and (iii) selecting transfected cells in which the first and second nucleic acids in the vector have been integrated into the expression-enhancing locus of the cells via recombination mediated by the first, second, and third RRSs. (Item 56) (iv) expressing the first LCF, the first HCF, and the second HCF in the selected transfected cells; and (v) obtaining the bispecific antigen-binding protein comprising the first LCF, the first HCF, and the second HCF from the selected transfected cells. (Item 57) 1. A method of making a bispecific antigen-binding protein, comprising: (i) providing the cell according to any one of items 1 to 23; (ii) expressing said bispecific antigen-binding protein from said exogenous nucleic acid sequence; and (iii) obtaining the bispecific antigen-binding protein from the cell. [Brief explanation of the drawings]

[0044] [Figure 1]Figure 1. Example of a bispecific cloning strategy for integration into an expression-enhancing locus. A light chain (LC) vector, e.g., a common light chain, and a dual heavy chain (HC) vector (where "*" indicates that the two HCs are different, e.g., HC* contains modifications in the CH3 domain and / or is codon-modified) are generated by cloning the variable regions of the antibody of interest into the appropriate vectors. The 3' RRS site of the LC vector and the 5' RRS site of the dual HC vector are identical and are contained within the split intron of the hygromycin resistance gene. The split intron of the hygromycin resistance gene has been engineered to join and remove the intron, allowing expression of the protein encoded by the hygromycin resistance gene for efficient selection of recombinants. The arrows represent promoters.

[0045] [Figure 2] Figure 2. Example of a bispecific cloning strategy for integration into an expression-enhancing locus. Utilizing a universal light chain with a 5' RRS (RRS1) (see, for example, the light chain from the humanized Universal Light Chain (ULC) VelocImmune® mouse described in WO2013022782) allows for the efficient construction of novel bispecific antibodies by inserting one heavy chain (HC*) flanked by a third RRS (RRS3) into an existing plasmid containing an expression cassette for the universal light chain. The second heavy chain (HC) is cloned into a second plasmid with RRS2 and RRS3 sites.

[0046] [Figure 3]Figure 3. Example of a bispecific cloning strategy for integration into an expression-enhancing locus. The three separate antibody chains of a bispecific antibody (AbC1, AbC2, and AbC3) are first cloned into individual vectors. The AbC1 and AbC3 vectors each contain RRS sites flanking the antibody expression cassette. The expression cassette for AbC2 is excised from the AbC2 plasmid and then subcloned into the AbC3 expression plasmid, resulting in a plasmid containing, in 5' to 3' direction, an RRS3 site, an AbC2 expression cassette, an AbC3 expression cassette, and an RRS2 site. This plasmid, along with the AbC1 plasmid and recombinase, is introduced into host cells carrying RRS1 and RRS2 in the expression-enhancing locus. Bispecific antibody-expressing cell lines are isolated after recombinase-mediated cassette exchange.

[0047] [Figure 4] Figure 4. Example of a bispecific cloning strategy for integration into an expression-enhancing locus. The three separate antibody chains of a bispecific antibody (AbC1, AbC2, and AbC3) are first cloned into individual vectors. The AbC1 and AbC3 vectors each contain RRS sites flanking the antibody expression cassette. The expression cassette for AbC2 is excised from the AbC2 plasmid and then subcloned into the AbC1 expression plasmid, resulting in a plasmid containing, in 5' to 3' direction, an RRS1 site, an AbC1 expression cassette, an AbC2 expression cassette, and an RRS3 site. This plasmid, along with the AbC3 plasmid and recombinase, is introduced into host cells carrying RRS1 and RRS2 in the expression-enhancing locus. Bispecific antibody-expressing cell lines are isolated after recombinase-mediated cassette exchange.

[0048] [Figure 5]Figure 5. Expression of bispecific antibodies from expression cassettes integrated at a single genomic site (EESYR®). CHO cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 were generated by recombinase-mediated cassette exchange at the EESYR® locus. The arrangement of the expression cassettes for the three separate antibody chains (AbC1, AbC2, and AbC3) of the bispecific Ab at the EESYR® locus is shown on the left. The titer of each bispecific antibody in the conditioned medium of 4-day shake-flask cultures was determined by HPLC and is shown in the bar graph on the right. DETAILED DESCRIPTION OF THE INVENTION

[0049] Definition of Terms The term "antibody," as used herein, includes immunoglobulin molecules composed of four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interrupted by more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may also be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may also be abbreviated as LCDR1, LCDR2, and LCDR3).

[0050] The term "antigen binding protein" includes proteins having at least one CDR and capable of selectively recognizing an antigen, i.e., having a K of at least micromolar. D Therapeutic antigen-binding proteins (e.g., therapeutic antibodies) often have K in the nanomolar or picomolar range. D Typically, an antigen-binding protein comprises two or more CDRs, e.g., two, three, four, five, or six CDRs. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies, such as polypeptides containing the variable regions of the heavy and light chains of an antibody (e.g., Fab fragments (F(ab')2 fragments)), proteins containing the variable regions of the heavy and light chains of an antibody and additional amino acids from the constant regions of the heavy and / or light chains (e.g., one or more constant domains, i.e., one or more of the CL, CH1, CH2, and CH3 domains).

[0051] The term "bispecific antigen-binding protein" includes antigen-binding proteins that can selectively bind to, or have different specificities for, two or more epitopes, either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). The antigen-binding portion, or antigen-binding fragment portion (Fab) of such a protein, confers specificity for a particular antigen and is typically composed of immunoglobulin heavy and light chain variable regions. In some circumstances, the heavy and light chain variable regions may not be a cognate pair, or may have different binding specificities.

[0052] An example of a bispecific antigen-binding protein is a "bispecific antibody," which includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies often comprise two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antigen-binding protein can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the variable region of the first heavy chain for the first epitope is generally at least one to two orders of magnitude, or three or four orders of magnitude, lower than the affinity of the variable region of the first heavy chain for the second epitope, or vice versa. Bispecific antigen-binding proteins, such as bispecific antibodies, may comprise heavy chain variable regions that recognize different epitopes of the same antigen. A typical bispecific antibody has two heavy chains, each containing three heavy-chain CDRs followed (from N- to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A typical bispecific antibody also has an immunoglobulin light chain, which does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind one or more epitopes bound by the heavy-chain antigen-binding region, or can associate with each heavy chain and bind one or both epitopes of the heavy chain. In one embodiment, the Fc domain comprises at least a CH2 and a CH3. The Fc domain may comprise a hinge, a CH2 domain, and a CH3 domain.

[0053] One embodied bispecific format comprises a first heavy chain (HC), a second heavy chain with a modified CH3 (HC*), and a common light chain (LC) (two copies of the same light chain). In another embodiment, it comprises a first heavy chain (HC), a common LC, and an HC-ScFv fusion polypeptide (where the second HC is fused to the N-terminus of the ScFv). In another embodiment, it comprises a first heavy chain (HC), a cognate LC, and an HC-ScFv fusion polypeptide (where the second HC is fused to the N-terminus of the ScFv). In another embodiment, it comprises a first heavy chain (HC), an LC, and an Fc domain. In another embodiment, it comprises a first HC, an LC, and an ScFv-Fc fusion polypeptide (where the Fc is fused to the C-terminus of the ScFv). In another embodiment, it comprises a first HC, a common LC, and an Fc-ScFv fusion polypeptide (where the Fc is fused to the N-terminus of the ScFv). In another embodiment, it comprises a first HC, an LC, and an ScFv-HC (where the second HC is fused to the C-terminus of the ScFv).

[0054] In some embodiments, one heavy chain (HC) may be the native or "wild-type" sequence and the second heavy chain may be Fc domain modified. In other embodiments, one heavy chain (HC) may be the native or "wild-type" sequence and the second heavy chain may be codon modified.

[0055] The term "cell" includes any cell suitable for expression of a recombinant nucleic acid sequence and having a locus that allows for stable integration and enhanced expression of exogenous nucleic acid. Cells include mammalian cells, such as non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a mammalian cell selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cells are retinal cells expressing one or more viral genes, e.g., viral genes (e.g., PER.C6 TM cells).

[0056] "Cell density" refers to the number of cells per sample volume, e.g., total number of cells (live and dead) per mL. Cell counts may be performed manually or automatically, e.g., using a flow cytometer. Automated cell counters are adapted to count live or dead cells or both live and dead cells, e.g., using standard methods such as trypan blue uptake. The phrase "viable cell density" or "viable cell concentration" refers to the number of live cells per sample volume (also referred to as "viable cell count"). Many known manual or automated techniques may be used to determine cell density. Online biomass measurements of the culture may be taken, with capacitance or optical density correlating to the number of cells per volume. Final cell densities in cell cultures, e.g., production cultures, may range from, e.g., about 1.0 to 10 x 10, depending on the starting cell line. 6 In some embodiments, the final cell density varies between 1.0 and 10x10 cells / mL prior to harvesting the protein of interest from the production cell culture. 6 In other embodiments, the final cell density reaches at least 5.0 x 10 cells / mL. 6 cells / mL, at least 6x10 6 cells / mL, at least 7x10 6 cells / mL, at least 8x10 6 cells / mL, at least 9x10 6 cells / mL, or at least 10x10 6 cells / mL.

[0057] The term "codon-modified" means that a protein-encoding nucleotide sequence has been modified at one or more nucleotides, i.e., one or more codons, without changing the amino acid encoded by that codon, resulting in a codon-modified version of the nucleotide sequence. The codon modification of a nucleotide sequence can provide a convenient basis for distinguishing the nucleotide sequence from its codon-modified version in nucleic acid-based assays (e.g., hybridization-based assays, PCR, etc., among others). In some instances, codons of a nucleotide sequence are modified to improve or optimize expression of the encoded protein in a host cell using codon optimization techniques known in the art (Gustafsson, C., et al., 2004, Trends in Biotechnology, 22:346-353; Chung, BK-S., et al., 2013, Journal of Biotechnology, 167:326-333; Gustafsson, C., et al., 2012, Protein Expr Purif, 83(1):37-46). Sequence design software tools using such techniques are also known in the art, including, but not limited to, Codon optimizer (Fuglsang A. 2003, Protein Expr Purif, 31:247-249), Gene Designer (Villalobos A, et al., 2006, BMC Bioinforma, 7:285), and OPTIMIZER (Puigbo P, et al. 2007, Nucleic Acids Research, 35:W126-W131).

[0058] The term "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene of an organism, which amino acid sequence is normally (i.e., in wild-type animals) located between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T-cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, and can be encoded, for example, by naive B cells or mature B cells or T cells. Under some circumstances (e.g., with respect to CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in the unrearranged nucleic acid sequence) but are contiguous in the B-cell nucleic acid sequence as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form the heavy chain CDR3).

[0059] The term "enhanced expression locus" refers to a locus in a cellular genome that contains a sequence and exhibits increased expression levels relative to other regions or sequences in the genome when an appropriate gene or construct is exogenously added (i.e., integrated) into or near the sequence, or is "operably linked" to the sequence.

[0060] When used to describe enhanced expression, the term "enhanced" includes at least about 1.5-fold to at least about 3-fold enhanced expression over expression typically observed with random integration of an exogenous sequence into a genome or integration at another locus, e.g., compared to various random integrations of a single copy of the same expression construct. The fold enhanced expression observed using the sequences of the invention is compared to the expression level of the same gene, measured under substantially the same conditions in the absence of the sequences of the invention, e.g., when integrated at another locus in the homologous genome. Enhanced recombination efficiency includes enhanced recombination capacity of a locus (e.g., utilization of recombinase recognition sites (RRSs)). Enhancement refers to recombination efficiency over random recombination, typically 0.1% when no recombinase recognition sites or homologs are utilized. Preferred enhanced recombination efficiencies are about 10-fold over random or about 1%. Unless specified, the claimed inventions are not limited to a particular recombination efficiency. Enhanced expression loci often support higher production of a protein of interest by host cells. Enhanced expression therefore involves higher production of the protein of interest per cell (higher titer per gram of protein) rather than simply achieving higher titer through higher copy number of cells in culture. Specific productivity, Qp (pg / cell / day, or pcd), is considered a measure of sustainable productivity. Recombinant host cells that exhibit a Qp of greater than 5 pcd, greater than 10 pcd, or greater than 15 pcd, or greater than 20 pcd, or greater than 25 pcd, or even greater than 30 pcd are desirable. Host cells with inserted genes of interest at expression-enhancing loci or "hot spots" exhibit high specific productivity.

[0061] The terms "exogenously added gene," "exogenously added nucleic acid," or simply "exogenous nucleic acid," when used in reference to a locus of interest, refer to any DNA sequence or gene that is not present within the locus of interest when the locus exists in nature. For example, an "exogenous nucleic acid" within a CHO locus (e.g., a locus having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2) can be a hamster gene that is not present within the particular CHO locus in nature (i.e., a hamster gene from another locus in the hamster genome), a gene from any other species (e.g., a human gene), a chimeric gene (e.g., human / mouse), or any other gene not known to be present within the CHO locus in nature.

[0062] The terms "heavy chain" or "immunoglobulin heavy chain" include immunoglobulin heavy chain constant region sequences from any organism, and, unless otherwise specified, include a heavy chain variable domain. Unless otherwise specified, a heavy chain variable domain includes three heavy chain CDRs and four FR regions. A typical heavy chain comprises (from N- to C-terminus) the variable domain followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. The term "heavy chain fragment" or "heavy chain fragment" (also referred to herein as "HCF") includes a peptide of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids of a heavy chain, and may include one or more CDRs, one or more CDRs combined with one or more FRs, a CH1, a hinge, a CH2, or a CH3, a variable region, a constant region, a fragment of a constant region (e.g., a CH1, a CH2, a CH3), or a combination thereof. Examples of HCFs include VHs and all or part of the Fc region. The phrase "nucleotide sequence encoding an HCF" includes nucleotide sequences encoding a polypeptide consisting of an HCF and nucleotide sequences encoding a polypeptide containing an HCF, including, for example, polypeptides that may contain additional amino acids in addition to a specific HCF. For example, a nucleotide sequence encoding an HCF specifically includes nucleotide sequences encoding a polypeptide consisting of a VH, a polypeptide consisting of a VH linked to a CH3, and a polypeptide consisting of a full-length heavy chain.

[0063] "Homologous sequence," in the context of nucleic acid sequences, refers to a sequence that is substantially homologous to a reference nucleic acid sequence. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding nucleotides are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete (i.e., full-length) sequence.

[0064] The term "light chain" includes immunoglobulin light chain constant region sequences from any organism, including human kappa light chains and human lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically contains three light chain CDRs and four framework (FR) regions, unless otherwise specified. A full-length light chain generally contains, from the amino terminus to the carboxyl terminus, a VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a light chain constant domain. Light chains that can be used in the present invention include, for example, light chains that do not selectively bind to either the first or second epitope selectively bound by a bispecific antibody. Suitable light chains also include light chains that can bind to or contribute to binding to one or both epitopes bound by the antigen-binding region of an antibody. The terms "light chain fragment" or "light chain fragment" (or "LCF") include peptides of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids of a light chain, and may include one or more CDRs combined with one or more CDRs, one or more FRs, a variable region, a constant region, a fragment of a constant region, or a combination thereof. Examples of LCFs include all or a portion of VLs and light chain constant regions (CLs). The term "nucleotide sequence encoding an LCF" includes nucleotide sequences that encode polypeptides consisting of an LCF as well as nucleotide sequences that encode polypeptides containing an LCF, including, for example, polypeptides that may contain additional amino acids in addition to the particular LCF. For example, a nucleotide sequence encoding an LCF specifically includes a nucleotide sequence that encodes a polypeptide consisting of a VL or a polypeptide consisting of a full-length light chain.

[0065] The phrase "operably linked" refers to the linkage of nucleic acids or proteins in a manner that allows the linked molecules to function as intended. DNA regions are operably linked when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it is capable of mediating transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Generally, operably linked can include, but does not require, contiguity. In the case of sequences such as secretory leaders, contiguity and proper placement in reading frame are typical characteristics. An expression-enhancing sequence of a locus of interest is operably linked to a gene of interest (GOI) if it is functionally associated with the GOI, e.g., if its presence results in enhanced expression of the GOI.

[0066] "Percent identity," when describing a subject locus, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof, is meant to include homologous sequences that show identity along the contiguous region of homology, but non-homologous gaps, deletions, or insertions in the comparison sequence are not taken into account in calculating percent identity.

[0067] As used herein, the determination of "percent identity" between, for example, SEQ ID NO: 1 or a fragment thereof and a species homolog does not include comparisons of sequences where the species homolog does not have a homologous sequence to compare in an alignment (i.e., it does not include comparisons of sequences where SEQ ID NO: 1 or a fragment thereof has an insertion at that point, or where the species homolog possibly has a gap or deletion). Thus, "percent identity" does not include penalties for gaps, deletions, and insertions.

[0068] A "recognition site" or "recognition sequence" is a specific DNA sequence recognized by a nuclease or other enzyme that binds to the DNA backbone and directs site-specific cleavage. Endonucleases cleave DNA within a DNA molecule. Recognition sites are also referred to in the art as recognition target sites.

[0069] A "recombinase recognition site" ("RRS") is a specific DNA sequence recognized by a recombinase, such as Cre recombinase (Cre) or flippase (flp). Site-specific recombinases can carry out DNA rearrangements, including deletions, rearrangements, and translocations, when one or more of their target recognition sequences are strategically placed within an organism's genome. In one example, Cre specifically mediates recombination events with its DNA target recognition site, loxP, which consists of two 13-bp inverted repeats separated by an 8-bp spacer. Two or more recombinase recognition sites can be used to facilitate, for example, recombination-mediated DNA exchange. Variants or mutants of recombinase recognition sites, such as lox sites, can also be used (Araki, N. et al., 2002, Nucleic Acids Research, 30:19, e103).

[0070] "Recombinase-mediated cassette exchange" or "RMCE" refers to the process of precisely replacing a genomic target cassette with a donor cassette. The molecular configuration typically provided to carry out this process includes: 1) a genomic target cassette flanked on both the 5' and 3' ends by recognition target sites specific to a particular recombinase; 2) a donor cassette flanked by matching recognition target sites; and 3) a site-specific recombinase. Recombinase proteins are known in the art (Turan, S. and Bode J., 2011, FASEB J., 25, pp. 4088-4107) and are capable of precisely cleaving DNA within a specific recognition target site (DNA sequence) without adding or deleting nucleotides. Common recombinase / site combinations include, but are not limited to, Cre / lox and Flp / frt. Vectors containing the R4-attP site and encoding the phiC31 integrase for RMCE are also provided in commercially available kits (see, e.g., U.S. Patent Application Publication No. US20130004946).

[0071] "Site-specific integration" or "targeted insertion" refers to a gene targeting method used to direct the insertion or integration of a gene or nucleic acid sequence into a specific location in the genome, i.e., to move DNA to a specific site between two nucleotides in a continuous polynucleotide chain. Site-specific integration or targeted insertion may be performed on a particular nucleic acid that contains multiple expression units or cassettes, for example, multiple genes, each with its own regulatory elements (e.g., promoters, enhancers, and / or transcription termination sequences). "Insertion" and "integration" are used interchangeably. It is understood that insertion of a gene or nucleic acid sequence (e.g., a nucleic acid sequence with an expression cassette) may result in (or be engineered to result in) the replacement or deletion of one or more nucleic acids depending on the gene editing technique used.

[0072] "Stable integration" means that the exogenous nucleic acid integrated into the host cell genome remains integrated for an extended period of time in cell culture, such as at least 7 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, or longer. It is understood that producing bispecific antigen-binding proteins for large-scale production and purification is challenging. Stability and clonality are essential for the reproducibility of any biomolecule, particularly a biomolecule used for therapeutic purposes. The stable clones expressing bispecific antibodies generated by the disclosed methods provide a consistent and reproducible means for generating therapeutic biomolecules.

[0073] overview The present disclosure provides compositions and methods for improving the expression of multiple polypeptides in host cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by using an expression-enhancing locus in the host cell. More specifically, the present disclosure provides compositions and methods designed to integrate multiple exogenous nucleic acids, which together encode a bispecific antigen-binding protein, into an expression-enhancing locus in a host cell, such as a CHO cell. In particular, the present disclosure provides cells containing multiple exogenous nucleic acids integrated at specific sites within the expression-enhancing locus, where the multiple exogenous nucleic acids together encode a bispecific antigen-binding protein. The present disclosure also provides nucleic acid vectors designed for site-specific integration of multiple exogenous nucleic acids into an expression-enhancing locus. The present disclosure further provides a system for site-specific integration of the multiple exogenous nucleic acids from the vectors into an expression-enhancing locus, comprising a host cell containing two or more recombinase recognition sites (RRSs) and a set of vectors containing matching RRSs and multiple exogenous nucleic acids. Additionally, the present disclosure provides methods of making bispecific antigen-binding proteins using the cells, vectors, and systems disclosed herein.

[0074] Cells with multiple exogenous nucleic acids integrated at specific sites within expression-enhanced loci In one aspect, the disclosure provides a cell containing an exogenous nucleic acid integrated at a specific site within an expression-enhanced locus, wherein the exogenous nucleic acid sequence encodes a bispecific antigen-binding protein.

[0075] The cells provided herein are capable of producing bispecific antigen-binding proteins (e.g., bispecific antibodies) at high titers and / or high specific productivity (pg / cell / day). In some embodiments, the cells produce the bispecific antigen-binding proteins at a titer of at least 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L or more. In some embodiments, the cells produce the bispecific antigen-binding proteins with a ratio of bispecific antigen-binding protein titer to total antigen-binding protein titer of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more. In some embodiments, cells producing the bispecific antigen binding protein have a specific productivity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 picograms / cell / day (pcd) or more, as determined based on total antigen binding protein (pg) produced per cell per day.

[0076] Host cells with an exogenous nucleic acid sequence encoding a bispecific antigen binding protein integrated into a specific site within an expression-enhancing locus can be used to express, for example, 1-10x10 6 In other embodiments, the host cells encoding the bispecific antigen-binding protein exhibit a high cell density in the production culture, such as at least 5 x 10 cells / mL. 6 cells / mL, 6x10 6 cells / mL, 7x10 6 cells / mL, 8x10 6 cells / mL, 9x10 6 cells / mL, or 10x10 6 with a final cell density of 1000 cells / mL.

[0077] In some embodiments, the bispecific antigen-binding protein contains two HC fragments (HCFs) with different antigen specificities and two LCFs. When two VL regions are used, the two VL regions may be the same or different. In certain embodiments, the two VL regions are the same, e.g., a common light chain.

[0078] In some embodiments, each of the two HCFs comprises amino acids from a heavy chain constant region, such as CH1, CH2, or CH3. In certain embodiments, each of the two HCFs comprises a CH3 domain. In certain embodiments, each of the two HCFs comprises a constant region, i.e., a full-length constant region.

[0079] In some embodiments, each of the two HCFs comprises a VH, and the two VHs may be the same or different.

[0080] In some embodiments, the bispecific antigen-binding protein comprises two heavy chains (ie, two full-length heavy chains).

[0081] In some embodiments, each of the two LCFs comprises a VL. In certain embodiments, each LCF consists of a VL region, and the VL region is operably linked to an amino acid sequence comprising amino acids from a light chain constant region. In certain embodiments, each VL region is operably linked to a CL region. That is, the bispecific antigen-binding protein comprises a light chain (i.e., a full-length light chain).

[0082] In some embodiments, the exogenous nucleic acid sequence integrated into the expression-enhanced locus comprises a first exogenous nucleic acid containing a nucleotide sequence encoding a first LCF, a second exogenous nucleic acid containing a nucleotide sequence encoding a first HCF, and a third exogenous nucleic acid containing a nucleotide sequence encoding a second HCF.

[0083] In some embodiments, the nucleotide sequence encoding the first LCF may encode a light chain variable (VL) region sequence. In certain embodiments, the nucleotide sequence encoding the first VL region encodes a first light chain.

[0084] In some embodiments, the nucleotide sequence encoding a first HCF encodes amino acids from a first heavy chain constant region (e.g., one or more of the CH1, hinge, CH2, or CH3 domains), and the nucleotide sequence encoding a second HCF encodes amino acids from a second heavy chain constant region. The amino acids from the first heavy chain constant region may be the same as or different from the amino acids from the second heavy chain constant region. For example, the nucleotide sequence encoding a first HCF encodes a first CH3 domain, and the nucleotide sequence encoding a second HCF encodes a second CH3 domain, where the first and second CH3 domains may be the same or may differ at one or more amino acid positions, as described herein below for bispecific antigen-binding proteins.

[0085] In some embodiments, the nucleotide sequence encoding the first HCF encodes a first VH, and the nucleotide sequence encoding the second HCF encodes a second VH.

[0086] In some embodiments, the nucleotide sequence encoding the first HCF encodes a first heavy chain, and the nucleotide sequence encoding the second HCF encodes a second heavy chain. The first and second heavy chains may have the same constant region or may differ in one or more amino acids. Various examples of bispecific antigen-binding proteins having different heavy chain constant domains (e.g., different CH3 domains) are further described herein below. Regardless of the encoded amino acid sequence, the nucleotide sequences encoding amino acids from the two heavy chain constant regions may differ in that one of the two encoding nucleotide sequences may be codon-modified, thereby providing a convenient basis for distinguishing the two nucleotide sequences in nucleic acid-based detection assays.

[0087] In some embodiments, the nucleotide sequences encoding each HCF or LCF are independently operably linked to a transcription control sequence, including a promoter. "Independently" means that each coding sequence is operably linked to a separate transcription control sequence, e.g., a promoter, such that transcription of the coding sequence is under separate control and management. In some embodiments, the promoters directing transcription of the two HCF-containing polypeptides are the same. In some embodiments, the promoters directing transcription of the two HCF-containing polypeptides and the promoter directing transcription of the LCF-containing polypeptide are all the same promoter, e.g., a CMV promoter. In some embodiments, the nucleotide sequences encoding each HCF or LCF are independently operably linked to an inducible or repressible promoter. Inducible and repressible promoters allow, for example, production to occur only during the production phase (fed-batch culture) and not during the growth phase (seed train culture). Better control of the production (expression) of each gene product can be achieved by using different promoters.

[0088] In one such example, cells are first engineered to express the tetracycline repressor protein (TetR), and each HCF-encoding nucleotide sequence and each LCF-encoding nucleotide sequence are placed under the transcriptional control of a promoter whose activity is controlled by TetR. Two tandem TetR operators (tetO) are positioned immediately downstream of a CMV promoter. In some embodiments, each HCF- and / or LCF-encoding nucleotide sequence is independently operably linked to a promoter upstream of at least one TetR operator (TetO) or Arc operator (ArcO). In other embodiments, each HCF- and / or LCF-encoding nucleotide sequence is independently operably linked to a CMV / TetO or CMV / ArcO hybrid promoter. Additional suitable promoters are described herein below.

[0089] The relative positions of multiple exogenous nucleic acids within a locus can vary. While not intending to be bound by any theory, it is believed that achieving balanced (i.e., equivalent) expression levels of the two HCF-containing polypeptides is important. In some embodiments, the nucleic acid encoding LCF is positioned upstream of both HCF-encoding nucleic acids. When the three promoters controlling the expression of the LCF-containing polypeptides and the two HCF-containing polypeptides are identical, a suitable arrangement includes, from 5' to 3', a nucleotide sequence encoding LCF, a nucleotide sequence encoding the first HCF, an additional, different promoter operably linked to the nucleotide sequence (e.g., a selectable marker gene), and a nucleotide sequence encoding the second HCF. Another suitable arrangement includes, from 5' to 3', a nucleotide sequence encoding LCF, an additional, different promoter operably linked to the nucleotide sequence (e.g., a selectable marker gene), a nucleotide sequence encoding the first HCF, and a nucleotide sequence encoding the second HCF. When the nucleotide sequence encoding HCF encodes a constant region sequence, each upstream nucleotide sequence may encode a modified constant region sequence (e.g., a modified CH3), or one upstream nucleotide sequence may encode a modified constant region sequence and the other may encode an unmodified constant region sequence.

[0090] In some embodiments, the cell further contains one or more RRSs integrated into the locus. In some embodiments, the cell contains a first RRS and a second RRS, which are different from each other and adjacent to the exogenous nucleic acid sequence. In this case, the exogenous nucleic acid sequence contains, in order, a nucleic acid encoding a first LCF, a nucleic acid encoding a first HCF, and a nucleic acid encoding a second HCF. In certain embodiments, the nucleic acid encoding an LCF is located upstream from both of the nucleic acids encoding the HCF, and the cell contains a third RRS located 3' to the nucleic acid encoding the first LCF and 5' to one or both of the exogenous nucleic acids encoding the HCF, where the third RRS is different from the first RRS and the second RRS. The third RRS may be engineered to be contained in an intron of a gene, which may be located between any two of the sequences encoding the HCF or the LCF.

[0091] Bispecific antigen-binding proteins Bispecific antigen-binding proteins, e.g., bispecific antibodies, suitable for cloning and production in the cells, vectors and systems described in this disclosure are not limited to any particular format of the bispecific antigen-binding protein.

[0092] In various embodiments, a bispecific antigen-binding protein comprises two polypeptides, each polypeptide containing an antigen-binding portion (e.g., a VH region) and a CH3 domain, wherein the antigen-binding portions of the two polypeptides have different antigen specificities, and wherein the two CH3 domains are heterodimeric with respect to each other in that one of the CH3 domains is modified at at least one amino acid position to result in different Protein A binding properties between the two polypeptides. See, for example, the bispecific antibodies described in U.S. Patent No. 8,586,713. In this manner, different Protein A isolation schemes can be used to readily isolate heterodimeric bispecific antigen-binding proteins from homodimers.

[0093] In some embodiments, the bispecific antigen binding protein comprises two heavy chains that have different antigen specificities and differ at least one amino acid position in the CH3 domain, resulting in different Protein A binding properties between the two heavy chains.

[0094] In some embodiments, the two polypeptides contain a CH3 domain of a human IgG, wherein one of the two polypeptides contains a CH3 domain of a human IgG selected from IgG1, IgG2, and IgG4, and the other of the two polypeptides contains a modified CH3 domain of a human IgG selected from IgG1, IgG2, and IgG4, wherein the modification reduces or abolishes binding of the modified CH3 region to Protein A. In particular embodiments, one of the two polypeptides contains a CH3 domain of a human IgG1, and the other of the two polypeptides contains a modified CH3 domain of a human IgG1, wherein the modification is selected from the group consisting of (i) 95R and (ii) 95R and 96F according to the IMGT exon numbering system. In other particular embodiments, the modified CH3 domain comprises 1 to 5 additional modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I according to the IMGT exon numbering system.

[0095] In various other embodiments, the two polypeptides contain CH3 domains of mouse IgG, wherein one of the two polypeptides contains an unmodified CH3 domain of mouse IgG and the other of the two polypeptides contains a modified CH3 domain of mouse IgG, wherein the modification reduces or eliminates binding of the modified CH3 region to Protein A. In various embodiments, the mouse IgG CH3 domain is modified to comprise specific amino acids at specific positions (EU numbering) selected from the group consisting of: 252T, 254T, and 256T; 252T, 254T, 256T, and 258K; 247P, 252T, 254T, 256T, and 258K; 435R and 436F; 252T, 254T, 256T, 435R, and 436F; 252T, 254T, 256T, 258K, 435R, and 436F; 24tP, 252T, 254T, 256T, 258K, 435R, and 436F; and 435R. In certain embodiments, a particular group of modifications selected from the group consisting of: M252T, S254T, S256T; M252T, S254T, S256T, I258K; I247P, M252T, S254T, S256T, I258K; H435R, H436F; M252T, S254T, S256T, H435R, H436F; M252T, S254T, S256T, I258K, H435R, H436F; I247P, M252T, S254T, S256T, I258K, H435R, H436F; and H435R.

[0096] In various embodiments, the bispecific antigen-binding protein is a hybrid of mouse and rat monoclonal antibodies or antigen-binding proteins, such as a hybrid of mouse IgG2a and rat IgG2b. According to these embodiments, the bispecific antibody is composed of a heterodimer of two antibodies, each with one heavy / light chain pair, linked via their Fc portions. The described heterodimer can be easily purified from a mixture of the two original antibody homodimers and bispecific heterodimers because the binding properties of the bispecific antibody for Protein A are different from those of the original antibodies. Rat IgG2b does not bind to Protein A, whereas mouse IgG2a does. Consequently, the mouse-rat heterodimer binds to Protein A but elutes at a higher pH than the mouse IgG2a homodimer. This allows for selective purification of the bispecific heterodimer.

[0097] In various other embodiments, the bispecific antigen-binding proteins are of a type referred to in the art as "knobs-into-holes" (see, e.g., U.S. Pat. No. 7,183,076). In these embodiments, the Fc portions of two antibodies are engineered to create a protruding "knob" in one and a complementary "hole" in the other. When produced in the same cell, the combination of the engineered "knob" and the engineered "hole" is said to cause the heavy chains to preferentially form heterodimers over homodimers.

[0098] In another embodiment, the first heavy chain and the second heavy chain comprise one or more amino acid modifications in the CH3 domain that allow interaction between the two heavy chains. Amino acid residues at the CH3-CH3 interface are substituted with charged amino acids, thereby electrostatically disfavoring homodimer formation. (See, e.g., PCT International Publication No. WO2009089004 and European Publication No. EP1870459.)

[0099] In other embodiments, the first heavy chain comprises a CH3 domain of isotype IgA and the second heavy chain comprises a CH3 domain of IgG (or vice versa), promoting preferential formation of heterodimers (see, e.g., PCT International Patent Application Publication No. WO2007110205).

[0100] In other embodiments, various formats can be incorporated into immunoglobulin chains by engineering methods that promote heterodimer formation, such as Fab-arm exchange (PCT International Patent Application Publication No. WO2008119353; PCT International Patent Application Publication No. WO2011131746), coiled-coil domain interactions (PCT International Patent Application Publication No. WO2011034605), or leucine zipper peptides (Kostelny, et al. J. Immunol. 1992, 148(5):1547-1553).

[0101] Immunoglobulin heavy chain variable regions that can be used to create bispecific antigen-binding proteins can be produced using any method known in the art. For example, the first heavy chain comprises a variable region encoded by a nucleic acid derived from the genome of a mature B cell of a first animal, the first animal being immunized with a first antigen, and the first heavy chain specifically recognizes the first antigen. The second heavy chain comprises a variable region encoded by a nucleic acid derived from the genome of a mature B cell of a second animal, the second animal being immunized with a second antigen, and the second heavy chain specifically recognizes the second antigen. Immunoglobulin heavy chain variable region sequences can also be obtained by any other method known in the art, such as phage display. In other examples, nucleic acids encoding heavy chain variable regions include those of antibodies reported in the art or available by other means. In some embodiments, one of the two heavy chain coding sequences is codon-modified, thereby providing a convenient basis for distinguishing between the two coding sequences in nucleic acid-based assays.

[0102] Bispecific antibodies, with two heavy chains that recognize two different epitopes (or two different antigens), are more easily isolated if they can pair with the same light chain (i.e., the light chains have identical variable and constant domains). Various methods are known in the art for generating light chains that can pair with two heavy chains of different specificities without interfering or substantially interfering with the heavy chain variable domains and their selectivity and / or affinity for their target antigens, such as the techniques described and disclosed in U.S. Pat. No. 8,586,713.

[0103] Bispecific antigen-binding proteins may have a variety of dual antigen specificities and associated useful applications.

[0104] In some examples, bispecific antigen-binding proteins can be generated that have binding specificities for a tumor antigen and a T cell antigen, targeting an antigen on a cell, e.g., CD20, and also targeting an antigen on a T cell, e.g., a T cell receptor such as CD3. In this case, the bispecific antigen-binding protein targets both the target cells in the patient (e.g., B cells in a lymphoma patient via CD20 binding) as well as the patient's T cells. In various embodiments, the bispecific antigen-binding protein is designed to activate T cells by binding to them, e.g., by binding to CD3, thereby coupling T cell activation to specific selected tumor cells.

[0105] In the context of bispecific antigen-binding proteins, where one moiety binds to CD3 and the other moiety binds to the target antigen, the target antigen is a tumor-associated antigen. Non-limiting examples of specific tumor-associated antigens include, for example, AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BCMA, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CLEC-12, CTL A4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MA GE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PA X3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0106] In some embodiments, the bispecific antigen binding protein is selected from the group consisting of an anti-CD3x anti-CD20 bispecific antibody (described in U.S. Patent Application Publications US2014 / 0088295A1 and US20150266966A1, which are incorporated herein by reference), an anti-CD3x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3x anti-Muc16 bispecific antibody), and an anti-CD3x anti-prostate specific membrane antigen bispecific antibody (e.g., an anti-CD3x anti-PSMA bispecific antibody). In other embodiments, the bispecific antigen binding protein comprises one moiety that binds to CD3. Examples of anti-CD3 antibody moieties are described in U.S. Patent Application Publications US2014 / 0088295A1 and US20150266966A1, and International Patent Application Publication WO2017 / 053856, published March 30, 2017, all of which are incorporated herein by reference). In yet other embodiments, the bispecific antigen binding protein comprises one moiety that binds CD3 and one moiety that binds BCMA, CD19, CD20, CD28, CLEC-12, Her2, an HLA protein, a MAGE protein, Mucl6, PSMA, or Steap-2.

[0107] In the context of a bispecific antigen-binding protein, where one portion binds to a T cell receptor, e.g., binds to CD3, and the other portion binds to the target antigen, the target antigen may be an infectious disease-associated antigen. Non-limiting examples of infectious disease-associated antigens include, for example, antigens expressed on the surface of a viral particle or preferentially expressed on a cell infected with a virus, where the virus is selected from the group consisting of HIV, hepatitis virus (A, B, or C), herpesvirus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, or Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Alternatively, the target antigen may be an antigen expressed on the surface of a bacterium, or may be an antigen preferentially expressed on a cell infected with a bacterium, wherein the bacterium is selected from the group consisting of Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthrax, Yersinia pestis, Leptospira, and Lyme disease bacteria.In certain embodiments, the target antigen is an antigen expressed on the surface of a fungus or is an antigen preferentially expressed on cells infected with a fungus, wherein the fungus is selected from the group consisting of Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus), Mucorales (e.g., Mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and the like. In certain embodiments, the target antigen is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, and Babesia murin. The antigens are selected from the group consisting of Trypanosoma microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps, and Brugia malayi. Non-limiting examples of specific parasite-associated antigens include, for example, HIV gp120, HIV CD4, Hepatitis B virus glycoprotein L, Hepatitis B virus glycoprotein M, Hepatitis B virus glycoprotein S, Hepatitis C virus E1, Hepatitis C virus E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope protein.

[0108] Bispecific binding proteins can be created with two binding moieties, each directed to a binding partner on the same cell surface (i.e., each directed to a different target). This design is particularly suitable for targeting specific cells or cell types that express both targets on the same cell surface. Although the targets may also appear individually on other cells, the binding moieties of these binding proteins are selected so that each binding moiety binds to its target with relatively low affinity (e.g., low micromolar or high nanomolar, e.g., greater than 100 nanomolar KD, e.g., 500, 600, 700, 800 nanomolar). In this case, long-term target binding is preferred only when the two targets are in close proximity on the same cell.

[0109] Bispecific binding proteins can be created with two binding moieties that bind to the same target at different epitopes on the same target. This design is particularly suitable for maximizing the success rate of blocking a target using the binding protein. For example, multiple extracellular loops of a transmembrane channel or cell surface receptor can be targeted by the same bispecific binding molecule.

[0110] Bispecific binding proteins may be engineered with two binding molecules that cluster and activate negative regulators of immune signaling, resulting in immune suppression. In cis suppression can be achieved when the targets are on the same cell. In trans suppression can be achieved when the targets are on different cells. In cis suppression can be achieved, for example, using a bispecific binding protein with an anti-IgGRIIb binding moiety and an anti-FelD1 binding moiety, whereby IgGRIIb clusters only in the presence of FelD1, downregulating the immune response to FelD1. In trans suppression can be achieved, for example, using a bispecific binding protein with an anti-BTLA binding moiety and a binding moiety that specifically binds to a tissue-specific antigen of interest, whereby clustering of inhibitory BTLA molecules occurs only in selected target tissues, potentially providing a strategy for addressing autoimmune diseases.

[0111] Bispecific binding proteins may be engineered to activate multicomponent receptors. In this design, two binding moieties directed to two components of the receptor bind to and cross-link the receptor, activating signaling from the receptor. This can be achieved by using a bispecific binding protein with a binding moiety that binds to IFNAR1 and a binding moiety that binds to IFNAR2, where binding cross-links the receptors. Such bispecific binding proteins may provide an alternative to interferon therapy.

[0112] Bispecific binding proteins may be engineered to transport binding moieties across semipermeable barriers, such as the blood-brain barrier. In this design, one binding moiety binds to a target that can cross a particular selected barrier, while the other binding moiety targets a therapeutically active molecule, where the therapeutically active target molecule typically cannot cross the barrier. Bispecific binding proteins of this type are useful for delivering therapeutic agents to tissues that would otherwise be inaccessible to the therapeutic agent. Some examples include targeting the pIGR receptor to transport therapeutic agents to the gastrointestinal tract or lungs, or targeting the transferrin receptor to transport therapeutic agents across the blood-brain barrier.

[0113] Bispecific binding proteins can be generated that deliver binding moieties to specific cells or cell types. In this design, one binding moiety targets a cell surface protein (e.g., a receptor) that is readily internalized within the cell. The other binding moiety targets an intracellular protein, the binding of which produces a therapeutic effect.

[0114] Bispecific binding proteins that bind to surface receptors on phagocytic immune cells and to surface molecules on infectious pathogens (e.g., yeast or bacteria) deliver the infectious pathogen to the vicinity of the phagocytic immune cell, facilitating phagocytosis of the pathogen. An example of such a design is a bispecific antibody that targets the CD64 or CD89 molecule as well as the pathogen.

[0115] A bispecific binding protein has an antibody variable region as one binding moiety and a non-Ig moiety as the second binding moiety. The antibody variable region performs targeting, while the non-Ig moiety is an effector or toxin linked to Fc. In this case, a ligand (e.g., an effector or toxin) is delivered to the target bound to the antibody variable region.

[0116] Bispecific binding proteins with two moieties, each bound to an Ig region (e.g., an Ig sequence containing a CH2 and a CH3 region), can bring any two protein moieties near each other in an Fc environment. Examples of this design include traps, such as homodimeric or heterodimeric trap molecules.

[0117] Enhanced expression loci Expression-enhanced loci suitable for use in the present invention include, for example, loci with a nucleotide sequence having substantial homology to SEQ ID NO:1 described in U.S. Patent No. 8,389,239 (also referred to herein as "EESYR® loci"), loci with a nucleotide sequence having substantial homology to SEQ ID NO:2 or SEQ ID NO:3 described in U.S. Patent Application No. 14 / 919,300 (also referred to herein as "YARS loci"), and other expression-enhanced loci and sequences reported in the art (e.g., US20150167020A1 and U.S. Patent No. 6,800,457).

[0118] In some embodiments, the two expression-enhancing loci used in the present invention are selected from loci comprising nucleotide sequences with substantial homology to SEQ ID NO: 1, or loci comprising nucleotide sequences with substantial homology to SEQ ID NO: 2 or SEQ ID NO: 3. These loci contain sequences that not only confer enhanced expression of genes operably linked to and integrated into (i.e., within or adjacent to) the sequences, but also exhibit high recombination efficiency and improved integration stability compared to other sequences in the genome.

[0119] SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were identified in CHO cells. Other mammalian species (e.g., humans or mice) have been found to have limited homology to the identified expression-enhancing regions. However, sequences may be found in cell lines derived from other tissue types of Chinese hamsters (Cricetulus griseus) or other isogenic species and can be isolated using techniques known in the art. For example, cross-species hybridization or PCR-based techniques may identify other homologous sequences. Furthermore, variations can be generated in the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 using site-directed or random mutagenesis techniques known in the art. The resulting sequence variants may then be tested for expression-enhancing activity. DNAs at least about 90% identical in nucleic acid identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 that have expression-enhancing activity are expected to be isolated by routine experimentation and exhibit expression-enhancing activity.

[0120] The integration site, site, or nucleotide position of the insert of one or more exogenous nucleic acids may be any location within or adjacent to any of the expression-enhancing sequences (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3). Whether a particular chromosomal location within or adjacent to a locus of interest supports stable integration and efficient transcription of an integrated exogenous gene can be determined according to standard methods known in the art, such as those described in U.S. Pat. No. 8,389,239 and U.S. Patent Application Publication No. 14,919,300.

[0121] The integration sites contemplated herein are located within or within the vicinity of the expression-enhancing sequence, e.g., less than about 1 kb, 500 base pairs (bp), 250 bp, 100 bp, 50 bp, 25 bp, 10 bp, or less than about 5 bp upstream (5') or downstream (3') of the location of the expression-enhancing sequence on the chromosomal DNA. In yet some other embodiments, the integration sites used are located about 1000 base pairs, 2500 base pairs, 5000 base pairs, or more upstream (5') or downstream (3') of the location of the expression-enhancing sequence on the chromosomal DNA.

[0122] It is understood in the art that large genomic regions, such as scaffold / matrix attachment regions (S / MARs), also known as scaffold-attachment regions (SARs) or matrix-associated or matrix attachment regions (MARs), are regions of genomic DNA in eukaryotic cells to which the nuclear matrix attaches. Without being bound by any one theory, S / MARs often map to non-coding regions, separating a given transcriptional region (e.g., chromatin domain) from its neighbors and providing platforms for the structure and / or binding of factors that enable transcription, such as recognition sites for deoxyribonucleases or polymerases. Some S / MARs have been characterized at lengths of approximately 14-20 kb (Klar, et al. 2005, Gene 364:79-89). Therefore, integration of a gene at an enhanced expression locus (e.g., within or near SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) is predicted to result in enhanced expression. In some embodiments, host cells with an exogenous nucleic acid sequence encoding a bispecific antigen-binding protein integrated at a specific site within an enhanced expression locus exhibit high specific productivity. In other embodiments, host cells encoding the bispecific antigen-binding protein have a specific productivity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 picograms / cell / day (pcd).

[0123] In some embodiments, the integration site is within a locus comprising the nucleotide sequence of SEQ ID NO: 1. In particular embodiments, the integration site is within or near the nucleotide sequence of SEQ ID NO: 1. In particular embodiments, the integration site is at a position within SEQ ID NO: 1 selected from nucleotides spanning positions numbered 10-13,515; 20-12,020; 1,020-11,020; 2,020-10,020; 3,020-9,020; 4,020-8,020; 5,020-7,020; 6,020-6,920; 6,120-6,820; 6,220-6,720; 6,320-6,620; 6,420-6,520; 6,460-6,500; 6,470-6,490; and 6,475-6,485. In other embodiments, the integration site is in a sequence selected from the group consisting of nucleotides 5,000-7,400, 5,000-6,500, 6,400-7,400 of SEQ ID NO: 1, and nucleotides 6,400-6,500 of SEQ ID NO: 1. In certain embodiments, the integration site is before, after, or within the "turn on" triplet nucleotides 6471-6473 of SEQ ID NO: 1.

[0124] In some embodiments, the integration site is within a locus comprising the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3. In particular embodiments, the integration site is within or near the nucleotide sequence of SEQ ID NO:2. In particular embodiments, the integration site is within or near the nucleotide sequence of SEQ ID NO:3. In some embodiments, the integration site is within or near nucleotides 1990-1991, 1991-1992, 1992-1993, 1993-1994, 1995-1996, 1996-1997, 1997-1998, 1999-2000, 2001-2002, 2002-2003, 2003-2004, 2004-2005, 2005-2006, 2006-2008, 2009-3010, 2010-2011, 2011-2012, 2012-2013, 2013-2014, 2014-2015, 2015-2016, 2016-2017, 2017-2018, 2018-2019, 2019-3020, 2019-3021, 2019-3022, 2019-3023, 2019-3024, 2019-3025, 2019-3026, 2020-3021, 2020-3022, 2020-3024, 2020-3025, 2020-3026, 2020-3027, 2020-3028, In certain embodiments, the integration is at or within nucleotides 2001-2022 of SEQ ID NO:3. In some embodiments, the exogenous nucleic acid is inserted at or within nucleotides 2001-2002 or nucleotides 2021-2022 of SEQ ID NO:3, and as a result of the insertion, nucleotides 2002-2021 of SEQ ID NO:3 are deleted.

[0125] Site-specific integration into expression-enhancing loci Integration of multiple exogenous nucleic acids into an expression-enhancing locus in a site-specific manner, i.e., into one specific site within an expression-enhancing locus disclosed herein, can be achieved in several ways, including methods such as homologous recombination and recombinase-mediated cassette exchange, as described in the art (see, e.g., U.S. Pat. No. 8,389,239 and the techniques disclosed therein).

[0126] In some embodiments, cells are provided that contain at least two, i.e., two or more, different recombinase recognition sequences (RRS) within an expression-enhancing locus convenient for integration of a nucleic acid sequence containing multiple exogenous nucleic acids or genes of interest. Such cells can be obtained by introducing an exogenous nucleic acid sequence containing two or more RRSs into a desired locus using a variety of means, including homologous recombination, as described herein below and in the art, e.g., U.S. Patent No. 8,389,239 and the techniques disclosed therein.

[0127] In certain embodiments, cells are provided that contain three or more different recombinase recognition sequences (RRS) within an expression-enhancing locus that is convenient for the integration of multiple exogenous nucleic acids. In certain embodiments, cells are provided that contain three different recombinase recognition sequences (RRS) within an expression-enhancing locus that can mediate the integration of two separate exogenous nucleic acids, for example, where the 5' and middle RRS in the genome match the 5' and 3' RRS flanking a first exogenous nucleic acid to be integrated, and the middle and 3' RRS in the genome match the 5' and 3' RRS flanking a second exogenous nucleic acid to be integrated.

[0128] A suitable RRS may be selected from the group containing LoxP, Lox511, Lox5171, Lox2272, Lox2372, Loxm2, Lox-FAS, Lox71, Lox66, and mutants thereof, in which case the site-specific recombinase is Cre recombinase or a derivative thereof, and recombinase-mediated cassette exchange (RMCE) is performed using it. In another example, a suitable RRS may be selected from the group containing FRT, F3, F5, FRT mutant-10, FRT mutant+10, and mutants thereof, in which case the site-specific recombinase Flp recombinase or a derivative thereof is used, and RMCE is performed using it. In yet another example, the RRS may be selected from the group containing attB, attP, and mutants thereof, in which case the site-specific recombinase is phiC31 integrase or a derivative thereof, and RMCE is performed using it.

[0129] In other embodiments, the native cell is modified by homologous recombination techniques, whereby a nucleic acid sequence containing multiple exogenous nucleic acids is integrated into a specific site within the expression-enhancing locus.

[0130] With homologous recombination, homologous polynucleotide molecules (i.e., homologous arms) line up and exchange their sequence sections. During this exchange, a transgene can be introduced if it is flanked by homologous genomic sequences. In one example, a recombinase recognition site can be introduced into the host cell genome at the integration site via homologous recombination. In another example, a nucleic acid sequence containing multiple exogenous nucleic acids, such as multiple nucleic acids that together encode a bispecific antigen-binding protein, is inserted into the host genome, where the nucleic acid sequences are flanked by sequences homologous to the sequence of the target locus (homologous arms).

[0131] Homologous recombination in eukaryotic cells can be facilitated by introducing a break at the integration site in the chromosomal DNA. This can be accomplished by targeting a specific nucleus to a specific integration site. DNA-binding proteins that recognize DNA sequences at the target locus are known in the art. Gene targeting vectors can also be used to facilitate homologous recombination.

[0132] Gene targeting vector construction and nuclease selection for homologous recombination are within the skill of those skilled in the art to which the present invention pertains. In some instances, zinc finger nucleases (ZFNs), which have a modular structure and contain individual zinc finger domains, identify specific 3-nucleotide sequences within a target sequence (e.g., the site of targeted integration). Some embodiments can utilize ZFNs with a combination of individual zinc finger domains to target multiple target sequences. Transcription activator-like (TAL) effector nucleases (TALENs) can also be employed for site-specific genome editing. TAL effector protein DNA binding domains are typically used in combination with the non-specific cleavage domain of a restriction nuclease such as FokI. In some embodiments, a fusion protein containing a TAL effector protein DNA binding domain and a restriction nuclease cleavage domain is employed to identify and cleave DNA at target sequences within the locus of the present invention (Boch J et al., 2009 Science 326:1509-1512). RNA-guided endonucleases (RGENs) are programmable genome engineering tools developed from bacterial adaptive immune mechanisms. In this system, i.e., clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) immune response, the protein Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection. RGENs consist of components (Cas9 and tracrRNA) and target-specific CRISPRRNA (crRNA). Both the efficiency of DNA target cleavage and the location of the cleavage site vary based on the location of the protospacer adjacent motif (PAM), an additional requirement for target recognition (Chen, H. et al., J. Biol. Chem. Published online March 14, 2014, manuscript M113.539726).Sequences unique to specific targeting of loci (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) can be identified by aligning many of these sequences to the CHO genome, revealing potential off-target sites with 16-17 base pair matches.

[0133] In some embodiments, a targeting vector carrying a nucleic acid of interest flanked by 5' and 3' homology arms (e.g., a nucleic acid containing one or more RRSs optionally flanking one or more selectable marker genes, or a nucleic acid containing multiple exogenous nucleic acids that together encode a bispecific antigen-binding protein) is introduced into a cell along with one or more additional vectors or mRNAs. In one embodiment, the one or more additional vectors or mRNAs contain nucleotide sequences encoding site-specific nucleases, including, but not limited to, zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, and RNA-guided DNA endonucleases. In certain embodiments, the one or more vectors or mRNAs include a first vector comprising nucleotide sequences encoding a guide RNA, a tracrRNA, and a Cas enzyme, and a second vector comprising a donor (exogenous) nucleotide sequence. Such donor sequences contain nucleotide sequences encoding a gene of interest, or a recognition sequence, or a gene cassette with any one of these exogenous elements intended for targeted insertion. When mRNA is used, the mRNA can be transfected into cells using common transfection methods known to those skilled in the art and may encode enzymes such as transposases or endonucleases. The mRNA introduced into cells may be transient and not integrated into the genome, but the mRNA may carry exogenous nucleic acids necessary or beneficial for integration to occur. In some cases, mRNA is selected to eliminate any risk of long-lasting side effects of the accompanying polynucleotide, in which case only short-term expression is required to achieve the desired integration of the nucleic acid.

[0134] Vectors for site-specific integration Provided herein are nucleic acid vectors for introducing an exogenous nucleic acid into an expression-enhancing locus via site-specific integration. Suitable vectors include vectors designed to contain an exogenous nucleic acid sequence flanked by RRSs for integration via RMCE, and vectors designed to contain an exogenous nucleic acid sequence of interest flanked by homology arms for integration via homologous recombination.

[0135] In various embodiments, vectors are provided for performing site-specific integration via RMCE. In some embodiments, the vectors are designed to achieve simultaneous integration of multiple nucleic acids into target loci. In contrast to sequential integration, simultaneous integration allows for efficient and rapid isolation of desired clones that produce bispecific antigen-binding proteins.

[0136] In some embodiments, a vector set is provided, the set comprising two or more vectors, each vector containing at least two RRSs flanking one or more nucleic acids, wherein the nucleic acids in the vector set together encode a bispecific antigen binding protein.

[0137] In one embodiment, the vector set includes a first nucleic acid comprising, from 5' to 3', a nucleotide sequence encoding a first RRS, a first LCF, and a first vector comprising a third RRS; a second nucleic acid comprising, from 5' to 3', a nucleotide sequence encoding a third RRS, a first HCF, and a second vector comprising a second RRS. In this case, either the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF, and in this case, the first and second HCFs and the first LCF encode regions (e.g., variable regions) of a bispecific antigen-binding protein. In some embodiments, the nucleotide sequence encoding the second HCF is contained in the first nucleic acid on the first vector (i.e., the first LCF and the second HCF on one vector), optionally positioned, for example, downstream of the nucleotide sequence encoding the first LCF. In other embodiments, the nucleotide sequence encoding the second HCF is contained in the second nucleic acid on the second vector (i.e., the first HCF and the second HCF on one vector).

[0138] Nucleotide sequences encoding HCF may encode amino acids, such as amino acids or domains from the constant region, or may encode the entire constant region. In certain embodiments, nucleotide sequences encoding HCF or LCF may encode one or more constant domains, such as CL, CH1, hinge, CH2, CH3, or a combination thereof. In some embodiments, nucleotide sequences encoding HCF may encode a CH3 domain. For example, a nucleotide sequence encoding a first HCF may encode a first CH3 domain, and a nucleotide sequence encoding a second HCF may encode a second CH3 domain. The first and second CH3 domains may be identical or may differ by at least one amino acid. Differences in the CH3 domain or in the constant region can take any of the forms for the bispecific antigen-binding proteins described herein, such as differences that result in different protein A binding properties, electrostatic steering, or a "knob-and-hole" configuration. Regardless of any amino acid sequence differences, two HCF-encoding nucleotide sequences may differ in that one of the two nucleotide sequences has been codon-modified.

[0139] In some embodiments, the nucleotide sequence encoding each HCF is independently operably linked to a transcriptional control sequence, including, for example, a promoter. In some embodiments, the promoter directing transcription of the two HCF-containing polypeptides is the same. In some embodiments, the promoter directing transcription of the two HCF-containing polypeptides and the promoter directing transcription of the LCF-containing polypeptide are all the same promoter (e.g., a CMV promoter). In some embodiments, the nucleotide sequence encoding each HCF or LCF is independently operably linked to an inducible or repressible promoter. Inducible and repressible promoters allow, for example, production to occur only during the production phase (fed-batch culture) and not during the growth phase (seed train culture). Inducible or repressible promoters also allow for specific expression of one or more genes of interest. In some embodiments, the nucleotide sequence encoding each HCF and / or LCF is independently operably linked to a promoter upstream of at least one TetR operator (TetO) or Arc operator (ArcO). In yet other embodiments, the nucleotide sequences encoding each HCF and / or LCF are independently operably linked to a CMV / TetO or CMV / ArcO hybrid promoter. Examples of hybrid promoters (also referred to as regulatory fusion proteins) are found in International Patent Application Publication No. WO03101189A1, published December 11, 2003, which is incorporated herein by reference.

[0140] In some embodiments, the first nucleic acid in the first vector further comprises a 5' portion of a selectable marker gene located 5' to the third RRS in the first vector. The second nucleic acid further comprises the remaining 3' portion of the selectable marker gene located 3' to the third RRS in the second vector. In these embodiments, the first, second, and third RRSs mediate site-specific integration of the first and second nucleic acids, thereby properly linking the 5' and 3' portions of the selectable marker gene and simultaneously creating an integrated clone for convenient selection. In certain embodiments, the third RRS in the first vector is designed to be within the 5' portion of an intron of the selectable marker gene. The third RRS in the second vector is designed to be within the 3' portion of an intron of the selectable marker gene. In yet other embodiments, the third RRS in the first vector is designed to be between the operably linked (but separated on the other vector) promoter and the selectable marker gene. The third RRS in the first vector is designed to be 3' to the promoter. And the third RRS in the second vector is designed to be 5' of the selectable marker gene.

[0141] The vector set described above may include three or more vectors. For example, in addition to the two vectors described above, the vector set may include a third vector comprising at least two RRSs flanking a nucleotide sequence encoding a second LCF. The vector set may further include a vector encoding one or more recombinases that recognize the RRSs.

[0142] In other embodiments, vectors for achieving site-specific integration via homologous recombination are provided. In some examples, the polynucleotide sequence integrated into the host genome may be a DNA sequence, e.g., an RRS or multiple RRSs, intended to generate cells with one or more RRSs integrated into a desired locus for subsequent integration of a nucleic acid encoding a bispecific antigen-binding protein, where the RRSs are flanked by one or more selectable marker genes. In other examples, the polynucleotide sequence integrated into the host genome comprises multiple nucleic acids that together encode the bispecific antigen-binding protein. For example, the polynucleotide sequence comprises nucleic acids encoding two different heavy chains and a common light chain of a bispecific antibody. In some embodiments, the multiple nucleic acids that together encode the bispecific antigen-binding protein are each independently (i.e., separately) operably linked to a regulatory sequence (e.g., a promoter, enhancer, transcription termination sequence, or a combination thereof). That is, the regulatory sequences (e.g., promoters) for each of the multiple nucleic acids are distinct and may be the same or different (i.e., containing the same or different nucleotide sequences). When a nucleic acid in the plurality of nucleic acids comprises multiple coding sequences, each coding sequence, or each nucleotide sequence encoding the N-terminal portion of a polypeptide, is independently operably linked to its own regulatory sequence (e.g., a promoter).

[0143] It is within the skill of one in the art to select a sequence homologous to a sequence within an expression-enhancing locus and include the selected sequence as a homology arm in a targeting vector. In some embodiments, the vector or construct comprises a first homology arm and a second homology arm, wherein the combined first and second homology arms comprise a target sequence that replaces an endogenous sequence within the locus. In other embodiments, the first and second homology arms comprise a target sequence that is to be integrated or inserted into an endogenous sequence within the locus. In some embodiments, the homology arms contain nucleotide sequences homologous to nucleotide sequences present in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain embodiments, the vector contains a 5' homology arm having a nucleotide sequence corresponding to nucleotides 1001-2001 of SEQ ID NO:3 and a 3' homology arm having nucleotides homologous to nucleotides 2022-3022 of SEQ ID NO:3. The homologous arms, e.g., a first homologous arm (also referred to as a 5' homologous arm) and a second homologous arm (also referred to as a 3' homologous arm), are homologous to a target sequence within the locus. In the 5' to 3' direction, the homologous arms may extend a region or target sequence within the locus comprising at least 1 kb, or at least 2 kb, or at least 3 kb, or at least 4 kb, or at least 5 kb, or at least 10 kb. In other embodiments, the total number of nucleotides of the target sequence selected for the first and second homologous arms contains at least 1 kb, or at least 2 kb, or at least 3 kb, or at least 4 kb, or at least 5 kb, or at least 10 kb. In some examples, the distance between the 5' homology arm and the 3' homology arm (homologous to the target sequence) contains at least 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or at least 1 kb, or at least about 2 kb, or at least about 3 kb, or at least about 4 kb, or at least 5 kb, or at least about 10 kb.For example, when nucleotides 1001-2001 and 2022-3022 of SEQ ID NO: 3 are selected for the 5' and 3' homologous arms, the distance between the two homologous arms may be 20 nucleotides (corresponding to nucleotides 2002-2021 of SEQ ID NO: 3). Such homologous arms can mediate integration of an exogenous nucleic acid sequence into a locus containing SEQ ID NO: 3, e.g., within nucleotides 1990-2021 or 2002-2021 of SEQ ID NO: 3, and the simultaneous deletion of nucleotides 2002-2021 of SEQ ID NO: 3.

[0144] The vectors disclosed herein for introducing an exogenous nucleic acid for site-specific integration into an expression-enhancing locus may contain additional genes and sequences for directing expression of the exogenous nucleic acid of interest and the encoded polypeptide, and for selection and identification of cells into which the exogenous nucleic acid of interest has been successfully integrated. Such additional sequences include, for example, transcriptional and translational regulatory sequences, selectable marker genes, and the like, and are also described herein below.

[0145] Control arrays The vectors disclosed herein for introducing exogenous nucleic acids into expression-enhancing loci in a site-specific manner, and the cells resulting from site-specific integration, may contain control sequences for directing expression of the exogenous nucleic acid of interest and the encoded polypeptide. Control sequences include transcriptional promoters, enhancers, sequences encoding appropriate mRNA ribosomal binding sites, and sequences controlling transcription and translation termination. Transcriptional and translational control sequences may be provided by viral sources. For example, commonly used promoters and enhancers are derived from viruses such as polyoma, adenovirus 2, simian virus 40 (SV40), murine or human cytomegalovirus (CMV), the CMV immediate-early (CMV-IE) or CMV major IE (CMV-MIE) promoters, as well as RSV, the SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and the HIV LTR promoter. Viral genomic promoters, control sequences, and / or signal sequences may be utilized to drive expression, provided such control sequences are compatible with the selected host cell. Depending on the cell type in which the protein of interest is to be expressed, non-viral cellular promoters (e.g., β-globin and EF-1α promoters) can also be used. DNA sequences derived from the SV40 viral genome, such as the early and late promoters, enhancers, splice, and polyadenylation sites of SV40 origin, may be used to provide other genetic elements useful for expressing exogenous DNA sequences. The early and late promoters are particularly useful because both promoters are readily obtained as fragments from the SV40 virus and also contain the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used. Typically, an approximately 250-bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 origin of replication is included.Inducible promoters (e.g., induced by compounds, cofactors, or regulatory proteins) can be used and are particularly useful when production of the antigen-binding protein occurs only during the production phase (fed-batch culture) and not during the growth phase (seed train culture). Examples of inducible or repressible promoters include the alcohol dehydrogenase I gene promoter, tetracycline-responsive promoter systems, glucocorticoid receptor promoters, estrogen receptor promoters, ecdysone receptor promoters, metallothionein-based promoters, and T7 polymerase-based promoters. Sequences suitable for expression of multiple transcripts via bicistronic vectors have been previously reported (Kim SK and Wold BJ, Cell 42:129, 1985) and can be used in the present invention. Examples of suitable strategies for multicistronic expression of proteins include the use of 2A peptides (Szymczak et al., Expert Opin Biol Ther 5:627-638 (2005)) and the use of internal ribosome entry sites ("IRES"), both of which are known in the art. Other types of expression vectors are also useful, such as those described in U.S. Pat. No. 4,634,665 (Axel et al.) and U.S. Pat. No. 4,656,134 (Ringold et al.).

[0146] Selection Marker The vectors disclosed herein for introducing exogenous nucleic acids into expression-enhancing loci in a site-specific manner, and the cells resulting from site-specific integration, may contain one or more selectable marker genes.

[0147] In some embodiments, the selectable marker gene confers drug resistance, such as those listed in Table 1 of Kaufman, RJ (1988) Meth. Enzymology 185:537, including DHFR-MTX resistance, P-glycoprotein and multidrug resistance (MDR)-various lipophilic cytotoxic drugs (e.g., adriamycin, colchicine, vincristine), and adenosine deaminase (ADA)-Xyl-A, or adenosine and 2'-deoxycoformycin resistance. Other primary selectable markers include microbial antibiotic resistance genes, such as neomycin resistance, kanamycin resistance, or hygromycin resistance. Several suitable selection systems exist for mammalian hosts (Sambrook, supra, pp. 16.9-16.15). Protocols for cotransfection using two primary selectable markers have also been reported (Okayama and Berg, Mol. Cell Biol. 5:1136, 1985).

[0148] In other embodiments, the selectable marker gene encodes a polypeptide that provides a detectable signal for recognition of successful or unsuccessful insertion and / or replacement of the gene cassette, or that can generate a detectable signal. Suitable examples include, inter alia, fluorescent markers or proteins, and enzymes that catalyze chemical reactions that generate detectable signals. Examples of fluorescent markers are known in the art and include, but are not limited to, Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), and near-infrared fluorescent proteins (e.g., mKate, mKate2, mPlum, mRaspberry, or E2-Crimson). See, for example, Nagai, T., et al. 2002 Nature Biotechnology 20:87-90; Heim, R. et al. 1995 February 23 Nature 373:663-664; and Strack, R. et al. 2009 Biochemistry 48:8279-81.

[0149] System for producing bispecific antigen-binding proteins In a further aspect, the present disclosure provides a system that can be used to generate a cell comprising a combination of a cell and one or more vectors and an exogenous nucleic acid integrated into an expression-enhancing locus, wherein the exogenous nucleic acid together encodes a bispecific antigen-binding protein. The system may be provided, for example, in the form of a kit.

[0150] In some embodiments, the system is designed to allow efficient vector construction and co-integration of multiple exogenous nucleic acids via RMCE into specific sites within the expression-enhancing locus. Co-integration allows for rapid isolation of desired clones, and the use of a single expression-enhancing locus is also important for the generation of stable cell lines.

[0151] In some embodiments, a system is provided that includes any one of the above-described vector sets designed to integrate multiple exogenous nucleic acids via RMCE and a cell containing an RRS integrated at a specific site within an expression-enhancing locus that matches an RRS in the vector set. For example, the system includes a cell and a vector set, wherein the cell contains, in a 5' to 3' direction, the following integrated into an expression-enhancing locus of its genome: a first RRS, a first exogenous nucleic acid, a second RRS, a second exogenous nucleic acid, and a third RRS, wherein the three RRSs are different from one another. In this case, the vector set includes, in a 5' to 3' direction, a first vector comprising a first nucleic acid comprising a nucleotide sequence encoding a first LCF (e.g., a first VL), and a second RRS; and a second vector comprising a second RRS, a first HCF (e.g., a first VH), and a third RRS, wherein either the first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF (e.g., a second VH). When the vector is introduced into a cell, the first and second nucleic acids in the vector integrate into the expression-enhancing locus through recombination mediated by the first, second, and third RRSs. To facilitate screening of transfectants with nucleic acid from the vector properly integrated into the locus, the first exogenous nucleic acid in the cells of the system may contain a first selectable marker gene, and the second exogenous nucleic acid in the cells may contain a second selectable marker gene, where the first and second selectable marker genes are different from each other and from any selectable marker genes provided by the vector. In certain embodiments, the first and second selectable marker genes encode fluorescent proteins (which can provide negative selection), and the first and second nucleic acids on the vector provide an additional selectable marker gene in a split format to provide positive selection. While negative selection alone allows for rapid clonal isolation, the efficiency of isolating clones with the intended recombination may be limited (approximately 1%).By combining positive selection (new fluorescence, or resistance to drugs or antibiotics) with negative selection, the efficiency of isolating positive clones can be greatly improved (up to about 80%).

[0152] The system may include additional components, reagents, or information, such as protocols for introducing the vectors of the system into the cells of the system by transfection. Non-limiting transfection methods include chemical transfection methods, such as the use of liposomes, nanoparticles, calcium phosphate (Graham et al. (1973) Virology 52(2):456-67, Bacchetti et al. (1977) Proc Natl Acad Sci USA 74(4):1590-4 and, Kriegler, M (1991) Transfer and Expression: A Laboratory Manual. New York: W.H. Freeman and Company. pp.96-97), dendrimers, or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and optical transfection. Particle-based transfection methods include the use of gene guns and magnetically assisted transfection (Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods can also be used for transfection. mRNA delivery includes methods using TransMessenger™ and TransIT® (Bire et al. BMC Biotechnology 2013, 13:75). One commonly used method for introducing heterologous DNA into cells is the calcium phosphate precipitation method, as described, for example, in Wigler et al. (Proc. Natl. Acad. Sci. USA 77:3567, 1980). Polyethylene-induced fusion of bacterial protoplasts with mammalian cells (Schaffner et al., (1980) Proc. Natl. Acad. Sci. USA 77:2163) is another useful method for introducing heterologous DNA.Electroporation can also be used to directly introduce DNA into the cytoplasm of host cells, as described, for example, by Potter et al. (Proc. Natl. Acad. Sci. USA 81:7161, 1988) or Shigekawa et al. (BioTechniques 6:742, 1988). Other reagents useful for introducing heterologous DNA into mammalian cells have been reported, such as Lipofectin™ reagent and Lipofectamine™ reagent (Gibco BRL, Gaithersburg, Maryland, USA). Both of these commercially available reagents are used to form lipid-nucleic acid complexes (or liposomes), which, when applied to cultured cells, facilitate the uptake of nucleic acids into the cells.

[0153] Methods for producing bispecific antigen-binding proteins The present disclosure further provides methods for producing bispecific antigen-binding proteins. Using the methods, bispecific antigen-binding proteins (e.g., bispecific antibodies) can be produced at high titers and / or high specific productivity (pg / cell / day). In some embodiments, the bispecific antigen-binding proteins are produced at titers of at least 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, or more. In some embodiments, the bispecific antigen-binding proteins are produced with a ratio of bispecific antigen-binding protein titer to total antigen-binding protein titer of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or more. In some embodiments, the bispecific antigen binding protein is produced at a specific productivity determined based on total antigen binding protein (pg) produced per cell per day of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 picograms / cell / day or more.

[0154] In one embodiment, the method utilizes the system disclosed herein and introduces a vector of the system into cells of the system by transfection. Transfected cells that have properly integrated the exogenous nucleic acid into a targeted enhanced expression locus of the cell via RMCE may be screened and identified. Two HCF-containing polypeptides and at least one LCF-containing polypeptide may be expressed from the integrated nucleic acid, and a bispecific antigen-binding protein containing all three polypeptides may be obtained from the identified transfected cells and purified using known methods.

[0155] In some embodiments, the method includes (i) providing a system comprising a cell and a vector set, wherein the cell contains, in 5' to 3' direction, a first RRS, a first exogenous nucleic acid, a second RRS, a second exogenous nucleic acid, and a third RRS integrated into an expression-enhancing locus in the cell's genome, wherein the three RRSs are different from one another; and the vector set includes a first vector comprising, in 5' to 3' direction, the first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF (e.g., a first VL), and the second RRS; a second nucleic acid comprising a nucleotide sequence encoding a first HCF (e.g., a first VH), and a second vector comprising a third RRS, wherein either the first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF (e.g., a second VH); (ii) simultaneously introducing the vectors into the cell; and (iii) screening for transformed cells in which the first nucleic acid and the second nucleic acid in the vector have simultaneously integrated into the enhanced expression locus via recombination mediated by the first, second, and third RRSs.

[0156] In certain embodiments of the method, to facilitate screening for transformants having nucleic acid properly integrated into the locus from the vector, a first exogenous nucleic acid in the cells of the system may contain a first selectable marker gene, and a second exogenous nucleic acid in the cells may contain a second selectable marker gene, where the first and second selectable marker genes are different from each other, and the first and second nucleic acids on the vector together encode the additional selectable marker in a split format that, after co-integration, provides the complete sequence encoding the additional selectable marker gene. Screening for transformants may be performed by selecting for the first and second selectable markers (negative selection), and for the additional selectable marker (positive selection).

[0157] In another embodiment, the method simply utilizes a cell having an exogenous nucleic acid sequence integrated at a specific site within an expression-enhanced locus of the cell, wherein the exogenous nucleic acid sequence encodes a bispecific antigen binding protein and expresses the bispecific antigen binding protein from the cell. Consecutively cloned expression cassettes within the specific integration site.

[0158] The present specification is further illustrated by the following examples, which should not be construed as limiting. All cited references (including literature references, issued patents, and published patent applications cited throughout this application) are hereby expressly incorporated by reference. [Example]

[0159] Example 1: Cloning of bispecific antibody expression plasmids: The heavy and light chain components of bispecific antibodies may be cloned from hybridoma cells, B cells, plasma cells, or recombinant antibody gene libraries using methods known in the art. For example, antibodies may be cloned from hybridomas or B cells by five-prime RACE PCR or PCR using primers for the leader peptide, framework 1 sequence, framework 4 sequence, or constant region sequence. Alternatively, antibody genes or mRNA in antibody-expressing cells may be sequenced by next-generation sequencing and then identified through bioinformatics. It is also feasible to sequence antibody proteins and clone the corresponding antibody genes using synthetic DNA technology. Recombinant antibody libraries, such as yeast libraries or phage libraries, are also sources of antibody genes.

[0160] The CHO expression cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 each produce a bispecific antibody composed of three distinct polypeptides: AbC1, AbC2, and AbC3 (Figure 5). To generate the plasmid for RSX4189-1 construction, the AbC1 plasmid was linearized by digestion with MfeI, which was 3' to the AbC1 gene. The AbC2 expression cassette, excised from the AbC2 plasmid by MfeI digestion, was ligated into the MfeI site of the linearized AbC1 plasmid. The ligation product was transformed into DH10B Escherichia coli (E. coli). After transformation and growth in ampicillin-containing LB medium, individual E. coli colonies were analyzed for the presence of the desired plasmid containing the AbC1 and AbC2 genes. The sequences of maxi-prep DNA for the AbC3 plasmid and the AbC1-AbC2 dual expression plasmid were confirmed by Sanger sequencing. These two plasmids, along with the Cre expression plasmid pRG858, were transfected into EESYR® host cells carrying the RRS1 and RRS3 loci at the EESYR® locus using Lipofectamine. Transfected cells were selected using antibiotics for 12 days, after which the recombinant cells were stocked as RSX4189-1.

[0161] To generate the plasmid for constructing RSX4187-1, the AbC3 expression cassette, flanked by Mlu I and Nhe I sites, was cloned into the Mlu I and Spe I sites of the AbC2 plasmid. The Mlu I and Spe I sites were 3' to the AbC2 gene. The AbC2-AbC3 plasmid, AbC1 plasmid, and Cre plasmid pRG858 were combined and co-transfected into EESYR® host cells using Lipofectamine. The cells that underwent RMCE were stored as RSX4187-1.

[0162] To generate the plasmid for constructing RSX4191-1, the AbC3 expression cassette was cloned into the MfeI site in the AbC1 plasmid. The MfeI site was 3' to the AbC1 gene. The AbC1-AbC3 plasmid, the AbC2 plasmid, and the Cre plasmid pRG858 were combined and co-transfected into EESYR® host cells using Lipofectamine. The cells that underwent RMCE were stored as RSX4191-1.

[0163] To generate the plasmid for constructing RSX4188-1, the AbC2 expression cassette, flanked by Mlu I and Nhe I sites, was cloned into the Mlu I and Spe I sites of the AbC3 plasmid. The Mlu I and Spe I sites were 3' to the AbC3 gene. The AbC3-AbC2, AbC1, and Cre plasmids, pRG858, were combined and transfected into the EESYR using lipofectamine. ( The cells were co-transfected into RSX4188 (registered trademark) host cells. The cells that underwent RMCE were stored as RSX4188-1.

[0164] Example 2: Expression of bispecific antibodies from the EESYR® locus The bispecific antibody-expressing cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 were cultured in suspension in serum-free medium. To quantify the expression levels of the bispecific antibodies, the cell numbers of the cultures were counted on a Guava flow cytometer, and new shake flask cultures containing 2 million cells per ml of medium were initiated. After 4 days, the conditioned medium was collected by centrifugation to remove the cells. The bispecific antibody titers were determined using a protein A HPLC assay specific for the bispecific antibody. The titers of the bispecific antibody proteins expressed from RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 were 37.8 mg / L, 40.5 mg / L, 48.3 mg / L, and 21.8 mg / L, respectively. The total titers of all antibody proteins (including bispecific and monospecific antibody proteins) expressed from these cell lines and the ratio of bispecific antibody protein titer to total antibody protein titer are shown in the table below. [Table 1]

[0165] Sequence Listing [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 2-9 Table 2-10 Table 2-11 Table 2-12

Claims

1. A system for producing an antigen-binding protein, comprising: (i) a vector set, a first vector containing, from 5' to 3', a first recombinase recognition site (RRS), a first nucleic acid, a 5' portion of a selectable marker gene including a 5' portion of an intron, and a third RRS; a second vector containing, from 5' to 3', the third RRS, the remaining 3' portion of the selectable marker gene including the remaining 3' portion of the intron, a second nucleic acid, and a second RRS. Including, one of the first nucleic acid and the second nucleic acid contains a nucleotide sequence encoding a first light chain fragment (LCF), and the other of the first nucleic acid and the second nucleic acid contains a nucleotide sequence encoding a first heavy chain fragment (HCF); one of the first or second nucleic acids contains a nucleotide sequence encoding a second HCF; and a vector set, wherein the first and second HCFs and the first LCF each comprise a variable domain and are fragments of the antigen-binding protein; (ii) integrated 5' to 3' within an expression-enhancing locus in the genome; the first RRS, a first exogenous nucleic acid, the third RRS, a second exogenous nucleic acid, and The second RRS 1. A mammalian cell comprising: the first RRS, the second RRS, and the third RRS are different from one another; a mammalian cell, wherein the expression-enhanced locus comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 3; Including, the system.

2. The system of claim 1 , wherein the first nucleic acid comprises a nucleotide sequence encoding the first LCF and the second nucleic acid contains a nucleotide sequence encoding the first HCF.

3. The system of claim 1 , wherein the first nucleic acid comprises a nucleotide sequence encoding the first HCF and the second nucleic acid comprises a nucleotide sequence encoding the first LCF.

4. The system according to any one of claims 1 to 3, wherein the selectable marker gene confers resistance to an antibiotic.

5. The system of claim 4 , wherein the antibiotic is hygromycin, neomycin, or kanamycin.

6. 6. The system of claim 1, wherein the first HCF comprises a variable domain that is different from the variable domain of the second HCF, and the first HCF, the second HCF, and the first LCF are fragments of a bispecific antibody.

7. The system of any one of claims 1 to 5, wherein the first and second nucleic acids together encode a full-length antibody.

8. The system of claim 7 , wherein the antibody is a bispecific antibody.

9. A system described in any one of claims 1 to 8, wherein the mammalian cells are CHO cells.

10. Integrated within an expression-enhancing locus, from 5' to 3', a first recombinase recognition site (RRS), a first nucleic acid, a selectable marker gene containing an intron containing a third RRS; a second nucleic acid, and Second RRS 1. A mammalian cell for producing an antigen-binding protein, comprising: one of the first nucleic acid and the second nucleic acid contains a nucleotide sequence encoding a first light chain fragment (LCF), and the other of the first nucleic acid and the second nucleic acid contains a nucleotide sequence encoding a first heavy chain fragment (HCF); one of the first or second nucleic acids contains a nucleotide sequence encoding a second HCF; the first HCF, the second HCF, and the first LCF each comprise a variable domain and are fragments of the antigen-binding protein; the expression-enhanced locus comprises the nucleotide sequence set forth in SEQ ID NO:3; The mammalian cell is a CHO cell. cell.

11. The cell of claim 10, wherein the first nucleic acid comprises a nucleotide sequence encoding the first LCF and the second nucleic acid comprises a nucleotide sequence encoding the first HCF.

12. The cell of claim 10, wherein the first nucleic acid comprises a nucleotide sequence encoding the first HCF and the second nucleic acid comprises a nucleotide sequence encoding the first LCF.

13. The cell of any one of claims 10 to 12, wherein the selectable marker gene confers resistance to an antibiotic.

14. The cell of claim 13 , wherein the antibiotic is hygromycin, neomycin, or kanamycin.

15. The cell of any one of claims 10 to 14, wherein the first HCF comprises a variable domain that is different from the variable domain of the second HCF, and the first HCF, the second HCF, and the first LCF are fragments of a bispecific antibody.

16. The cell of any one of claims 10 to 14, wherein the first and second nucleic acids together encode a full-length antibody.

17. The cell of claim 16 , wherein the antibody is a bispecific antibody.

18. 1. A method of producing an antigen binding protein, comprising: Culturing the mammalian cell according to any one of claims 10 to 17, and producing said antigen binding protein from said cells. A method comprising:

19. 1. A method of producing an antigen binding protein, comprising: (a) obtaining a system according to any one of claims 1 to 9; (b) introducing the vector set of the system into the mammalian cell of the system; (c) culturing the mammalian cells obtained from (b); and (d) producing the antigen binding protein from the cultured mammalian cells. A method comprising:

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