Compositions and methods for making antibodies based on the use of expression enhancing loci

Abstract

The present invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the present invention includes compositions and methods for improving expression of antibodies, including bispecific antibodies, in eukaryotic cells, particularly Chinese hamster cell lines, by employing expression-enhancing loci.

Description

[0001] This application is a divisional application of CN201780024560.2.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 325,385, filed April 20, 2016, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This disclosure relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. Specifically, this disclosure relates to compositions and methods for improving the expression of antigen-binding proteins (such as bispecific antibodies) in eukaryotic cells (especially Chinese hamster (Cricetulus griseus) cell lines) by employing the expression of enhancing loci. Background Technology

[0005] Cellular expression systems are designed to provide a reliable and efficient source for the preparation of a given protein, whether for research or therapeutic use. Recombinant protein expression in mammalian cells is a preferred method for preparing therapeutic proteins due to the ability of, for example, mammalian expression systems to make appropriate post-translational modifications to recombinant proteins.

[0006] Despite the availability of numerous systems, challenges remain in achieving efficient gene transfer and stability of integrating genes for recombinant protein expression. For long-term expression of the target transgene, one consideration is minimizing disruption to cellular genes to avoid alterations in cell line phenotype.

[0007] Engineering stable cell lines to accommodate multiple genes for expression (e.g., multiple antibody chains in a multispecific antibody) is particularly challenging. Significant variations in the expression levels of integrated genes can occur. The integration of additional genes can lead to substantial variability and instability in expression due to local genetic conditions (i.e., position effects). Expression systems for producing multispecific antigen-binding proteins typically require the expression of two or more distinct immunoglobulin chains intended to pair as specific multimers and may generally favor the production of homodimers rather than the desired heterodimers or multimer combinations. Therefore, there is a need in the art for improved mammalian expression systems. Summary of the Invention

[0008] In one aspect, a cell containing a foreign nucleic acid sequence integrated at a specific site within an enhanced expression locus is provided, wherein the foreign nucleic acid sequence encodes a bispecific antigen-binding protein.

[0009] 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 denoted as HCF*, wherein the second HCF is different from the first HCF), wherein the first HCF, the second HCF, and the first LCF form a bispecific antigen-binding protein. In some embodiments, the first HCF, the second HCF, and the first LCF contain at least two variable regions and two CH3 constant domains of a 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 specificity site within an expression locus.

[0010] In some embodiments, the nucleotide sequence encoding the first HCF encodes an amino acid in 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 an amino acid in a second constant region. The amino acids in the first constant region may be the same as or different from those in the second constant region. In a particular embodiment, 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, wherein the first CH3 domain and the second CH3 domain may be the same as or different from each other. In some embodiments, the first CH3 domain and the second CH3 domain 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 characteristics. In other embodiments, the nucleotide sequences encoding the first CH3 domain and the second CH3 domain differ from each other in that one of the nucleotide sequences is codon-modified.

[0011] In other specific embodiments, 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 VH and the second VH may be linked to the same or different constant regions.

[0012] In some implementations, the nucleotide sequence encoding the first LCF encodes the first light chain variable (VL) region.

[0013] In some embodiments, the exogenous nucleic acid sequence contains additional exogenous nucleic acids, such as a second light chain variable (VL) region, including a nucleotide sequence encoding a second LCF. In some embodiments, the nucleotide sequence encoding the second VL region also encodes a second light chain constant region.

[0014] The relative positions of the multiple exogenous nucleic acids at the locus can vary. In some implementations, the nucleic acid encoding LCF is located upstream or downstream of the two nucleic acids encoding HCF.

[0015] In some embodiments, each of the sequences encoding HCF or LCF is independently linked to a transcriptional regulatory sequence. In a particular embodiment, the first exogenous nucleic acid further includes a first promoter operatively linked to a nucleotide sequence encoding the first LCF, the second exogenous nucleic acid further includes a second promoter operatively linked to a nucleotide sequence encoding the first HCF, and the third exogenous nucleic acid includes a third promoter operatively linked to a nucleotide sequence encoding the second HCF, wherein the first, second, and third promoters are the same or different, and / or the promoters are the same or different from a fourth promoter operatively linked to a fourth exogenous nucleic acid. In some embodiments, the first, second, and third promoters are the same.

[0016] In some embodiments, the exogenous nucleic acid sequence at the integration site also includes recombinase recognition sites, such as a first recombinase recognition site (RRS) located at 5' relative to the first exogenous nucleic acid, and a second recombinase recognition site (RRS) located at 3' relative to the second and third exogenous nucleic acids, wherein the first and second RRSs are different. In some embodiments, a third RRS is also included and is located at 3' relative to the first exogenous nucleic acid and at 5' relative to one or both of the second and third exogenous nucleic acids, wherein the third RRS is different from the first and second RRSs.

[0017] In some embodiments, the exogenous nucleic acid sequence may include a fourth exogenous nucleic acid containing a selective marker gene. In a particular embodiment, the fourth exogenous nucleic acid is located at 3' relative to the first exogenous nucleic acid. In some embodiments, the fourth exogenous nucleic acid is integrated as a break gene. In other embodiments, the fourth exogenous nucleic acid or selective marker is located at 3' of the third RRS, i.e., operatively linked to the 3' of a fourth promoter of the fourth exogenous nucleic acid. In some embodiments, the selective marker gene contains a third RRS that has been inserted, optionally inserted within an intron of the selective marker gene, wherein the third RRS is different from the first and second RRSs.

[0018] In some embodiments, the sequence of the exogenous nucleic acids at the locus from 5' to 3' can be: a first exogenous nucleic acid (encoding LCF), a fourth exogenous nucleic acid (encoding a selective marker), a second exogenous nucleic acid (encoding a first HCF), and a third exogenous nucleic acid (encoding a second HCF); and in some specific embodiments, the second exogenous nucleic acid contains a nucleotide sequence encoding a modified CH3 domain of human IgG, and the third exogenous nucleic acid contains a nucleotide sequence encoding a native CH3 domain of human IgG.

[0019] In some implementations, the sequence of the exogenous nucleic acids at the locus from 5' to 3' is: first exogenous nucleic acid (encoding LCF), second exogenous nucleic acid (encoding first HCF), fourth exogenous nucleic acid (encoding a selective marker), and third exogenous nucleic acid (encoding second HCF), wherein the second exogenous nucleic acid contains a nucleotide sequence encoding the natural CH3 domain of human IgG, and the third exogenous nucleic acid contains a modified CH3 domain encoding human IgG.

[0020] In some implementations, the promoter linked to the sequence encoding HCF or the sequence encoding LCF is the same and is different from the promoter to which the selective marker gene is operatively linked.

[0021] In some implementations, the bispecific antigen-binding protein specifically binds to both T-cell antigens and tumor cell antigens. Other suitable bispecific antigens are also provided.

[0022] In some embodiments, the enhanced expression locus is selected from: a locus containing a nucleotide sequence having at least 90% identity with SEQ ID NO:1, or a locus containing a nucleotide sequence having at least 90% identity with SEQ ID NO:2.

[0023] In several implementations, the cells are CHO cells.

[0024] In another aspect, vectors designed for site-specific integration of multiple exogenous nucleic acids are provided.

[0025] In some embodiments, this disclosure provides a set of vectors comprising a first vector comprising, from 5' to 3': a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF, and a third RRS; and a second vector comprising, from 5' to 3': a third RRS, a second nucleic acid containing a nucleotide sequence encoding a first VH region, and a second RRS; wherein the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF; and wherein the first HCF, the second HCF, and the first LCF form a bispecific antigen-binding protein.

[0026] In some embodiments, the nucleotide sequence encoding the second HCF is included within the first nucleic acid, optionally downstream of the nucleotide sequence encoding the first LCF. In other embodiments, the nucleotide sequence encoding the second HCF is included within the second nucleic acid.

[0027] In some embodiments, the nucleotide sequence encoding the first HCF encodes a first chimeric constant region (e.g., encoding one or more of any isotype of CH1, hinge CH2, or CH3 domains, or fragments thereof), and the nucleotide sequence encoding the second HCF encodes a second chimeric constant region. Examples of chimeric constant regions are described in PCT International Publication No. WO 2014 / 121087 A1, published August 7, 2014, which is incorporated herein by reference. The amino acids of the first constant region may be the same as or different from those of the second chimeric constant region. In a particular embodiment, 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, wherein the first CH3 domain and the second CH3 domain may be the same as or different from each other. In some embodiments, the first CH3 domain and the second CH3 domain 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 characteristics. In other embodiments, the nucleotide sequences encoding the first CH3 domain and the second CH3 domain differ from each other in that one of the nucleotide sequences is codon-modified.

[0028] In other specific embodiments, the nucleotide sequence encoding the first VH region encodes the first heavy chain, and the nucleotide sequence encoding the second VH region encodes the second heavy chain, wherein the first and second heavy chains may have the same or different constant regions.

[0029] In some implementations, the nucleotide sequence encoding the first LCF encodes the first light chain variable region (VL).

[0030] In some embodiments, each of the sequences encoding LCF or HCF is independently linked to a transcriptional regulatory sequence, such as a promoter. In a particular embodiment, the promoter linked to the sequence encoding a first HCF and the promoter linked to the sequence encoding a second HCF are the same. In a particular embodiment, the promoters linked to one or more LCFs and one or more HCFs are all the same.

[0031] In some embodiments, the first nucleic acid in the first vector further contains the 5' portion of a selective marker gene located at the 5' of the third RRS in the first vector; and the second nucleic acid further contains the remaining 3' portion of a selective marker gene located at the 3' of the third RRS in the second vector, i.e., the selective marker gene is broken into the two vectors. In other embodiments, the selective marker and its operatively linked promoter are separated into two vectors; in other words, the promoter and the selective marker gene are located in different vectors. In some embodiments, the promoter operatively linked to the marker gene is located at the 5' of the third RRS in the first vector, the marker gene is located at the 3' of the third RRS in the second vector, and there is a second promoter operatively linked to the second nucleic acid and a third promoter operatively linked to the third nucleic acid at the 5' of the third promoter. In some embodiments, the third RRS in the first vector is located within the 5' portion of the intron of the selective marker gene; and the third RRS in the second vector is located within the 3' portion of the intron of the selective marker gene.

[0032] In a particular embodiment, the first vector from 5' to 3' includes a first RRS, a first nucleic acid, and a third RRS; the second vector from 5' to 3' includes a third RRS, a second nucleic acid (wherein the second nucleic acid contains a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HCF), and a second RRS. In other particular embodiments, the first vector from 5' to 3' includes a first RRS, a first nucleic acid, wherein the first nucleic acid contains a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HCF region, and a third RRS; the second vector from 5' to 3' includes a third RRS, a second nucleic acid (wherein the second nucleic acid contains a nucleotide sequence encoding a first HCF region), and a second RRS. In any of these particular embodiments, the first nucleic acid may further include a 5' portion of a selective marker gene located at 5' of the third RRS in the first vector; and the second nucleic acid further includes the remaining 3' portion of a selective marker gene located at 3' of the third RRS in the second vector; wherein optionally the third RRS in the first vector is located within the 5' portion of an intron of the selective marker gene, and the third RRS in the second vector is located within the 3' portion of an intron of the selective marker gene.

[0033] In some embodiments, the set of vectors may include one or more additional vectors; for example, a vector containing one or more RRS and a nucleotide sequence encoding a second LCF, or a vector encoding one or more recombinases that recognize the RRS.

[0034] In other embodiments, this disclosure provides vectors designed to achieve site-specific integration of multiple exogenous nucleic acids via homologous recombination based on homologous arms. In some embodiments, the vector contains an exogenous nucleic acid sequence encoding a bispecific antigen-binding protein, said exogenous nucleic acid sequence being side-joined with 5' and 3' homologous arms for integration into a cell's expression-enhancing locus.

[0035] In another aspect, this disclosure provides a system comprising a combination of cells and one or more vectors, and which can be used to prepare cells in which exogenous nucleic acids are integrated into an expression-enhancing locus to co-encode a bispecific antigen-binding protein.

[0036] In some embodiments, a system is provided comprising a cell and a set of vectors, wherein the cell contains a set of RRSs integrated into its genome at an enhanced expression locus, the set of RRSs being distinct from one another and separated between one or more exogenous nucleic acids (such as selectable markers) for recombination exchange with the gene of interest in the set of vectors; and wherein the RRSs in the set of vectors contain the same arrangement as the RRSs in the cell.

[0037] In some embodiments, a system comprising cells and a set of vectors is provided, wherein the cells contain, from 5' to 3', an enhanced expression locus integrated into their 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 distinct from each other; wherein the set of vectors comprises a first vector containing, from 5' to 3', the first RRS, a first nucleic acid containing a nucleotide sequence encoding a first immunoglobulin chain or a fragment thereof, and a second RRS; 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 a third RRS; and wherein the first or second nucleic acid further includes a nucleotide sequence encoding a third immunoglobulin chain or a fragment thereof. Upon introduction of the vector into the cells, the first and second nucleic acids in the vectors are integrated into the enhanced expression locus via recombination mediated by the first, second, and third RRSs.

[0038] In some implementations, a first exogenous nucleic acid in the cell contains a first selective marker gene, and a second exogenous nucleic acid in the cell contains a second selective marker gene, wherein the first and second selective marker genes are different. The selective markers are exchanged with the integrated exogenous nucleic acid in the cell.

[0039] In some embodiments, the first vector from 5' to 3' includes a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF, and a third RRS; and the second vector from 5' to 3' includes a third RRS, a second nucleic acid (wherein the second nucleic acid contains a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HCF), and a second RRS. In other embodiments, the first vector from 5' to 3' includes 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; and the second vector from 5' to 3' includes a third RRS, a second nucleic acid containing a nucleotide sequence encoding a first HCF, and a second RRS.

[0040] In some embodiments, the first vector from 5' to 3' includes a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first HCF, and a third RRS; and the second vector from 5' to 3' includes a third RRS, a second nucleic acid (wherein the second nucleic acid contains a nucleotide sequence encoding a first LCF and a nucleotide sequence encoding a second HCF), and a second RRS. In other embodiments, the first vector from 5' to 3' includes a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first HCF and a nucleotide sequence encoding a second HCF, and a third RRS; and the second vector from 5' to 3' includes a third RRS, a second nucleic acid containing a nucleotide sequence encoding a first LCF, and a second RRS. In any of these embodiments, the first nucleic acid in the first vector may further include a promoter located at 5' of the third RRS, and the second nucleic acid in the second vector may further include a promoter located at 3' of the third RRS operatively linked to an alternative marker gene. In other embodiments, the first nucleic acid in the first vector may further include the 5' portion of a selective marker gene located at the 5' of the third RRS, and the second nucleic acid in the second vector may further include the remaining 3' portion of the selective marker gene located at the 3' of the third RRS; wherein optionally the third RRS in the first vector is located within the 5' portion of the intron of the selective marker gene; and the third RRS in the second vector is located within the 3' portion of the intron of the selective marker gene.

[0041] In some implementations, a nucleotide sequence encoding LCF is operatively linked to a first promoter, a nucleotide sequence encoding a first HCF is operatively linked to a second promoter, and a nucleotide sequence encoding a second HCF is operatively linked to a third promoter, wherein the first, second, and third promoters are identical and different from the promoter operatively linked to by the selective marker gene (if present in a vector).

[0042] 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, wherein the first CH3 domain and the second CH3 domain may be the same or different. In some embodiments, one of the two CH3 domains is the native CH3 domain of human IgG, and the other CH3 domain is a modified CH3 domain of human IgG. In a particular embodiment, the nucleotide sequence encoding the modified CH3 domain is optionally encoded downstream of the nucleotide sequence of the first LCF in a first vector (i.e., the vector encoding the first LCF). In other particular embodiments, the nucleotide sequence encoding the modified CH3 domain is encoded upstream of the nucleotide sequence encoding the unmodified CH3 domain in a second vector.

[0043] In another aspect, this disclosure provides a method for preparing bispecific antigen-binding proteins.

[0044] In some embodiments, the method includes providing the system described herein, the system comprising cells having RRS and a vector containing multiple exogenous nucleic acids that collectively encode a bispecific antigen-binding protein and an RRS that matches the RRS in the cells; introducing the vector into the cells by transfection; selecting transfected cells, wherein the exogenous nucleic acids in the vector are integrated into an enhanced expression locus in the cells via RRS-mediated recombination; expressing the polypeptide encoded by the nucleic acids in the transformed cells; and obtaining the bispecific antigen-binding protein from the transfected cells.

[0045] In some embodiments, the method may include a cell containing a foreign nucleic acid sequence encoding a bispecific antigen-binding protein integrated into an expression-enhancing locus; expressing the bispecific antigen-binding protein from the foreign nucleic acid sequence; and obtaining the bispecific antigen-binding protein from the cell. Attached Figure Description

[0046] Figure 1 An exemplary bispecific cloning strategy for integration into expression-enhancing loci. Light chain (“LC”) vectors (e.g., common light chain) and double-strand (“HC”) vectors (where “*” indicates that the two HCs are different, e.g., HC* contains a CH3 domain modification and / or a codon modification) are prepared by cloning the variable region of the antibody of interest into the appropriate vector. The 3′ RRS site of the LC vector and the 5′ RRS site of the double-HC vector are identical and included in the broken intron of the hygromycin resistance gene, engineered to combine and excise the intron to allow expression of the protein encoded by the hygromycin resistance gene, thereby facilitating efficient selection of recombinants. Arrows represent promoters.

[0047] Figure 2An exemplary bispecific cloning strategy for integration into expression-enhancing loci. Utilizing a universal light chain with 5′ RRS (RRS1) (see, for example, humanized universal light chain (ULC) as described in WO 2013022782). (In mice) This allows for the efficient construction of novel bispecific antibodies by inserting a heavy chain (HC*) side-attached to the third RRS (RRS3) into a pre-existing plasmid containing a universal light chain expression cassette. The second heavy chain (HC) is cloned into a second plasmid via RRS2 and RRS3 sites.

[0048] Figure 3 An exemplary bispecific cloning strategy for integration into an expression-enhancing locus. First, three distinct antibody chains (AbC1, AbC2, and AbC3) of a bispecific antibody are cloned into a single vector. The AbC1 and AbC3 vectors each have an RRS site flanking the antibody expression cassette. The AbC2 expression cassette is excised from the AbC2 plasmid and then subcloned into the AbC3 expression plasmid, resulting in a plasmid containing the RRS3 site, the AbC2 expression cassette, the AbC3 expression cassette, and the RRS2 site from 5′ to 3′. This plasmid, along with the AbC1 plasmid and recombinase, is introduced into host cells containing the RRS1 and RRS2 loci at the expression-enhancing locus. Cell lines expressing the bispecific antibody are isolated following recombinase-mediated cassette exchange.

[0049] Figure 4 An exemplary bispecific cloning strategy for integration into an expression-enhancing locus. First, three distinct antibody chains (AbC1, AbC2, and AbC3) of a bispecific antibody are cloned into a single vector. The AbC1 and AbC3 vectors each have an RRS site flanking the antibody expression cassette. The AbC2 expression cassette is excised from the AbC2 plasmid and then subcloned into the AbC1 expression plasmid, resulting in a plasmid containing the RRS1 site, the AbC1 expression cassette, the AbC2 expression cassette, and the RRS3 site from 5′ to 3′. This plasmid, along with the AbC3 plasmid and recombinase, is introduced into host cells containing the RRS1 and RRS2 expression loci. Cell lines expressing the bispecific antibody are isolated following recombinase-mediated cassette exchange.

[0050] Figure 5 From a genomic site The integrated expression cassette expresses bispecific antibodies. This is achieved through... Recombinase-mediated cassette exchange at the locus produces CHO cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1. The left side depicts... The expression cassette arrangement of the three different antibody chains (AbC1, AbC2, and AbC3) of the bispecific Ab at the locus. The titer of each bispecific antibody in the waste culture medium after 4 days of shake-flask culture was determined by HPLC and is shown in the bar chart on the right. Detailed Implementation

[0051] definition

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains (two heavy chains (H) and two light chains (L)) linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH herein) 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 as LCVR or VL herein) 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 hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2 and LCDR3).

[0053] The phrase "antigen-binding protein" includes proteins that have at least one CDR and are capable of selectively recognizing antigens, i.e., capable of binding proteins with a K+ concentration at least in the micromolar range. D Proteins that bind to antigens. Therapeutic antigen-binding proteins (e.g., therapeutic antibodies) often require K+ in the nanomolar or picomolar range. D Typically, antigen-binding proteins comprise two or more CDRs, such as 2, 3, 4, 5, or 6 CDRs. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies such as polypeptides containing variable regions of the antibody's heavy and light chains (e.g., Fab fragments, F(ab')2 fragments), and proteins containing variable regions of the antibody's heavy and light chains and additional amino acids of constant regions of the heavy and / or light chains (such as one or more constant domains, i.e., one or more of the CL, CH1, CH2, and CH3 domains).

[0054] The phrase "bispecific antigen-binding protein" includes antigen-binding proteins capable of selectively binding to two or more epitopes or having different specificities to said two or more epitopes—on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). The antigen-binding portion or fragment antigen-binding (Fab) portion of such proteins is specific to a particular antigen and is typically composed of the heavy chain variable region and the light chain variable region of an immunoglobulin. In some cases, the heavy chain variable region and the light chain variable region may not be a homologous pair; in other words, they may have different binding specificities.

[0055] An example of a bispecific antigen-binding protein is a "bispecific antibody," which comprises an antibody capable of selectively binding to two or more epitopes. A bispecific antibody typically comprises two distinct heavy chains, each specifically binding to a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). If a bispecific antigen-binding protein is capable of selectively binding 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 will generally be at least one to two, three, or four orders of magnitude lower than the affinity of the variable region of the first heavy chain for the second epitope, and vice versa. Bispecific antigen-binding proteins, such as bispecific antibodies, may include variable regions of heavy chains that recognize different epitopes of the same antigen. A typical bispecific antibody has two heavy chains and an immunoglobulin light chain. Each of the two heavy chains has three heavy chain CDRs, followed by a (N-terminal to C-terminal) CH1 domain, a hinge, a CH2 domain, and a CH3 domain. The immunoglobulin light chain does not confer antigen-binding specificity but can bind to each heavy chain, or can bind to each heavy chain and bind one or more epitopes bound by the heavy chain antigen-binding region, or can bind to each heavy chain and enable one or both heavy chains to bind to one or two epitopes. In one embodiment, the Fc domain includes at least CH2 and CH3. The Fc domain may include a hinge, a CH2 domain, and a CH3 domain.

[0056] One specific bispecific form includes a first heavy chain (HC), a second heavy chain with modified CH3 (HC*), and a common light chain (LC) (two copies of the same light chain). Another embodiment includes a first heavy chain (HC), a common LC, and an HC-ScFv fusion polypeptide (wherein the second HC is fused to the N-terminus of ScFv). Another embodiment includes a first HC, homologous LC, and an HC-ScFv fusion polypeptide (wherein the second HC is fused to the N-terminus of ScFv). Another embodiment includes a first heavy chain (HC), LC, and an Fc domain. Another embodiment includes a first HC, LC, and a ScFv-Fc fusion polypeptide (wherein the Fc is fused to the C-terminus of ScFv). Another embodiment includes a first HC, a common LC, and an Fc-ScFv fusion polypeptide (wherein the Fc is fused to the N-terminus of ScFv). Another embodiment includes a first HC, LC, and ScFv-HC (wherein the second HC is fused to the C-terminus of ScFv).

[0057] In some embodiments, one heavy chain (HC) may be a natural or "wild-type" sequence and the second heavy chain may be modified in the Fc domain. In other embodiments, one heavy chain (HC) may be a natural or "wild-type" sequence and the codon of the second heavy chain may be codon-modified.

[0058] The term "cell" includes any cell suitable for expressing recombinant nucleic acid sequences and having loci that allow for stable integration and enhanced expression of exogenous nucleic acids. Cells include mammalian cells, such as non-human animal cells, human cells, or cell fusions, such as hybridomas or tetramorphic hybridomas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are mammalian cells selected from the following: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, 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, MMT060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells contain one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6). TM cell).

[0059] "Cell density" refers to the number of cells per unit volume of sample, such as total (live and dead) cells per milliliter. Cell counts can be performed manually or automatically (e.g., using flow cytometry). Automated cell counters have been adapted to count the number of live cells, dead cells, or both live and dead cells using, for example, standard trypan blue uptake techniques. The phrase "live cell density" or "live cell concentration" refers to the number of live cells per sample volume (also known as "live cell count"). Any number of well-known manuals or automated techniques can be used to determine cell density. The online biomass of a culture can be measured, where capacitance or optical density is related to the number of cells per volume. The final cell density in cell cultures, such as production cultures, varies depending on the starting cell line, for example, from approximately 1.0 to 10 × 10⁻⁶. 6 The range is cells / mL. In some embodiments, the final cell density reaches 1.0 to 10 × 10⁻⁶ cells / mL before harvesting the target protein from the production cell culture. 6 Cells / mL. In other embodiments, the final cell density reaches at least 5.0 × 10⁶ cells / mL. 6 cells / mL, at least 6 × 10⁻⁶ 6 10 cells / mL, at least 7 × 10 6 cells / mL, at least 8 × 10⁻⁶ 6 10 cells / mL, at least 9 × 10 6 10 × 10⁶ cells / mL or at least 10 × 10⁶ cells / mL 6 Cells / mL.

[0060] The term "codon-modified" means that the nucleotide sequence encoding a protein has been modified in one or more nucleotides (i.e., one or more codons) without changing the amino acid encoded by the codon, thereby producing a codon-modified form of the nucleotide sequence. Codon modification of nucleotide sequences can provide a convenient basis for distinguishing nucleotide sequences from their codon-modified forms in nucleic acid-based assays (e.g., especially hybridization-based assays, PCR). In some cases, codon modification of nucleotide sequences using codon optimization techniques well known in the art can provide improved or optimized expression of the encoded protein in the host cell (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 that use these techniques are also well known in the field, 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 (Puigbò P et al. 2007, Nucleic Acids Research, 35:W126-W131), etc.

[0061] The phrase "complementarity-determining region" or the term "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of an immunoglobulin gene of an organism, which typically (i.e., in wild-type animals) appears 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). A CDR can be encoded by, for example, germline sequences or rearranged or unrearranged sequences, and by, for example, native or mature B cells or T cells. In some cases (e.g., for CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not adjacent (e.g., in unrearranged nucleic acid sequences) but are adjacent in a B-cell nucleic acid sequence, for example, due to splicing or joining sequences (e.g., VDJ recombination to form heavy chain CDR3).

[0062] The term "expression-enhancing locus" refers to a locus in a cell genome containing one or more sequences that, when a suitable gene or construct is exogenously added (i.e. integrated) into or near the one or more sequences, or "operably linked" to the one or more sequences, exhibits a higher level of expression compared to other regions or sequences in the genome.

[0063] The term "enhancement," when used to describe enhanced expression, includes, for example, an enhancement exceeding, typically observed by at least about 1.5-fold to at least about 3-fold, compared to a pool of random integrators of a single copy of the same expression construct, either by random integration of a foreign sequence into the genome or by integration into a different locus. The doubling of expression enhancement observed using the sequences of the present 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 present invention, for example, compared to another locus integrated into the genome of the same species. Enhanced recombination efficiency includes an enhancement of the locus's recombination capacity (e.g., using a recombinase recognition site ("RRS")). Enhancement refers to a recombination efficiency exceeding that of random recombination (e.g., without the use of a recombinase recognition site, etc.), which is typically 0.1%. Preferably, the enhanced recombination efficiency exceeds random by about 10-fold, or about 1%. Unless otherwise specified, the claimed invention is not limited to a specific recombination efficiency. Enhanced expression loci are typically supported by high productivity of the target protein by the host cell. Therefore, enhanced expression involves a high yield of the target protein per cell (an increased titer in grams of protein), rather than simply achieving a high titer through high copy numbers in the culture. Specific productivity Qp (picograms per cell per day, or pcd) is considered a measure of sustainable productivity. Recombinant host cells exhibiting Qp greater than 5 pcd, or 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 target gene insertion at enhancing loci, or "hotspots," exhibit high specific productivity.

[0064] When the phrase "exogenously added gene" or "exogenously added nucleic acid" or simply "exogenous nucleic acid" is used to refer to a target locus, the phrase refers to any DNA sequence or gene that is not present in the locus, even if it is a target locus found in nature. For example, "exogenous nucleic acid" in a CHO locus (e.g., a locus containing the sequence SEQ ID NO:1 or SEQ ID NO:2) could be a hamster gene not found in a specific 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., a human / mouse gene), or any other gene not found in nature present in the target CHO locus.

[0065] The phrase “heavy chain” or “immunoglobulin heavy chain” includes the constant region sequence of an immunoglobulin heavy chain from any organism and, unless otherwise stated, includes the variable domain of the heavy chain. Unless otherwise stated, the variable domain of the heavy chain includes three heavy chain CDRs and four FR regions. A typical heavy chain has a CH1 domain, a hinge, a CH2 domain, and a CH3 domain (from the N-terminus to the C-terminus) following the variable domain. The term “fragment of heavy chain” 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 the heavy chain and may include one or more CDRs, one or more CDRs combined with one or more FRs, one or more of CH1, hinge, CH2, or CH3, a variable region, a constant region, a fragment of a constant region (e.g., CH1, CH2, CH3), or a combination thereof. Examples of HCF include VH and all or part of the Fc region. The phrase "nucleotide sequence encoding HCF" includes nucleotide sequences encoding polypeptides composed of HCF and nucleotide sequences encoding polypeptides containing HCF, such as polypeptides containing additional amino acids besides the specified HCF. For example, nucleotide sequences encoding HCF include nucleotide sequences encoding polypeptides composed of VH, VH linked to CH3, or intact heavy chains.

[0066] In the case of nucleic acid sequences, a "homologous sequence" refers to a sequence that is substantially homologous to a reference nucleic acid sequence. In some embodiments, two sequences are considered 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 the corresponding nucleotides are identical in the relevant residue sequence segment. In some embodiments, the relevant sequence segment is a complete sequence.

[0067] The phrase "light chain" includes the constant region sequence of an immunoglobulin light chain from any organism, and, unless otherwise stated, includes human κ and λ light chains. Unless otherwise stated, the light chain variable (VL) domain typically comprises three light chain CDRs and four framework (FR) regions. Generally, a full-length light chain from the amino terminus to the carboxyl terminus includes a VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a light chain constant domain. Light chains that can be used in this invention include, for example, those that do not selectively bind to a first or second epitope selectively bound by a bispecific antibody. Suitable light chains also include one or both epitopes that can bind or facilitate binding to an antigen-binding region of an antibody. The term "fragment of light chain" or "light chain fragment" (or "LCF") includes a peptide 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, one or more CDRs bound to one or more FRs, variable regions, constant regions, fragments of constant regions, or combinations thereof. Examples of LCFs include all or part of the VL and the light chain constant region ("CL"). The phrase "nucleotide sequence encoding LCF" includes nucleotide sequences encoding polypeptides composed of LCFs and nucleotide sequences encoding polypeptides containing LCFs, such as polypeptides containing additional amino acids besides the specified LCF. For example, nucleotide sequences encoding LCFs include nucleotide sequences encoding polypeptides, particularly composed of VLs, or composed of an intact light chain.

[0068] The phrase "operably linked" refers to the connection 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, if a promoter is capable of participating in the transcription of a coding sequence, then the promoter is operably linked to that sequence; if a ribosome binding site is positioned to allow translation, then the ribosome binding site is operably linked to the coding sequence. Generally, operably linked can include, but does not require, contiguity. For sequences such as secretory leader sequences, contiguity and proper placement within the reading frame are typical characteristics. In cases where an expression-enhancing sequence at a target locus is functionally related to the target gene (GOI), for example, if its presence enhances the expression of the GOI, it is operably linked to the GOI.

[0069] When describing a target locus, such as SEQ ID NO:1 or SEQ ID NO:2 or a fragment thereof, "identity percentage" means including homologous sequences that exhibit the listed identity along adjacent homologous regions, but the presence of gaps, deletions or insertions that do not have homology in the compared sequences is not included in the calculation of the identity percentage.

[0070] As used herein, the determination of “percentage of identity” between, for example, SEQ ID NO:1 or a fragment thereof and a species homolog will not include sequence comparisons of species homologs in which there is no homologous sequence comparison (i.e., SEQ ID NO:1 or a fragment thereof has an insertion at that point, or the species homolog has a gap or deletion, as the case may be). Therefore, “percentage of identity” does not include penalties for gaps, deletions, and insertions.

[0071] A "recognition site" or "recognition sequence" is a specific DNA sequence that is recognized by nucleases or other enzymes to bind to and guide site-specific cleavage of the DNA backbone. Nucleases cleave DNA within the DNA molecule. Recognition sites are also referred to in this field as target sites.

[0072] A "recombinase recognition site" (or "RRS") is a specific DNA sequence recognized by a recombinase, such as Cre recombinase (Cre) or flipping enzyme (flp). Site-specific recombinases can perform DNA rearrangements, including deletions, inversions, and translocations, when one or more of their target recognition sequences are strategically placed in an organism's genome. In one example, Cre specifically mediates recombination events at its DNA target recognition site loxP, which consists of two 13-bp inverted repeat sequences separated by an 8-bp spacer. More than one recombinase recognition site can be used, for example, to facilitate recombination-mediated DNA exchange. Variants or mutants of recombinase recognition sites (e.g., lox sites) can also be used (Araki, N. et al., 2002, Nucleic Acids Research, 30:19, e103).

[0073] "Recombinase-mediated cassette exchange" or "RMCE" refers to a method for precisely replacing a genomic target cassette with a donor cassette. Typically, the molecular composition used for this method comprises: 1) a genomic target cassette with 5' and 3' side-attached recognition sites specific to a particular recombinase; 2) a donor cassette with a matching recognition target site; and 3) a site-specific recombinase. Recombinase proteins are well known in the art (Turan, S. and Bode J., 2011, FASEB J., 25, pp. 4088-4107) and are capable of precisely cleaving DNA (the DNA sequence) within a specific recognition target site without adding or losing nucleotides. Common recombinase / site combinations include (but are not limited to) Cre / lox and Flp / frt. Commercially available kits also provide vectors containing the R4-attP site and vectors encoding the phiC31 integrase for use in RMCE. (See also, for example, U.S. Publication No. US20130004946.)

[0074] "Site-specific integration" or "targeted insertion" refers to gene-targeting methods used to guide the insertion or integration of a gene or nucleic acid sequence into a specific location in the genome; that is, guiding DNA to a specific site between two nucleotides in a linked polynucleotide chain. Site-specific integration or targeted insertion can also target nucleotides comprising multiple expression units or cassettes, such as multiple genes, each with its own regulatory elements (e.g., promoters, enhancers, and / or transcription termination sequences). "Insertion" and "integration" are used interchangeably. It should be understood that the insertion of a gene or nucleic acid sequence (e.g., a nucleic acid sequence containing an expression cassette) may result in (or may be engineered to) the substitution or deletion of one or more nucleic acids, depending on the gene-editing technology employed.

[0075] "Stable integration" means that the foreign nucleic acid integrated into the host cell genome maintains its integration in cell culture for a sustained period of time, 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 should be understood that large-scale production and purification of bispecific antigen-binding proteins is a challenging task. Stability and clonality are crucial for the reproducibility of any biomolecule, especially those used therapeutically. The stable clones expressing bispecific antibodies prepared by the methods of this disclosure provide the same and reproducible way to produce therapeutic biomolecules.

[0076] General Instructions

[0077] This disclosure provides compositions and methods for improving the expression of multiple peptides in host cells (particularly Chinese hamster (Cricetulus griseus) cell lines) by employing expression-enhancing loci in host cells. More specifically, this disclosure provides compositions and methods designed to integrate multiple exogenous nucleic acids co-encoding a bispecific antigen-binding protein into specific sites within expression-enhancing loci in host cells (such as CHO cells). Specifically, this disclosure provides cells containing multiple exogenous nucleic acids integrated into specific sites within expression-enhancing loci, wherein the multiple exogenous nucleic acids co-encode a bispecific antigen-binding protein. This disclosure also provides nucleic acid vectors designed to specifically integrate multiple exogenous nucleic acid sites into expression-enhancing loci. This disclosure further provides systems comprising host cells containing two or more recombinase recognition sites (RRS) and a set of vectors containing matching RRS and multiple exogenous nucleic acids for specifically integrating multiple exogenous nucleic acid sites from the vectors into expression-enhancing loci. Additionally, this disclosure provides methods for preparing bispecific antigen-binding proteins using the cells, vectors, and systems disclosed herein.

[0078] Cells with multiple exogenous nucleic acids integrated into specific sites within the expression enhancement locus

[0079] In one aspect, this disclosure provides cells containing exogenous nucleic acid sequences at specific sites integrated into enhanced expression loci, wherein the exogenous nucleic acid sequences encode bispecific antigen-binding proteins.

[0080] The cells provided herein are capable of producing bispecific antigen-binding proteins (e.g., bispecific antibodies) with high titers and / or high specific productivity (picograms / cells / day). In some embodiments, the cells produce bispecific antigen-binding proteins 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 greater. In some embodiments, the cells produce bispecific antigen-binding proteins at a ratio of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or higher of bispecific antigen-binding protein titer to total antigen-binding protein titer. In some embodiments, cells that produce bispecific antigen-binding proteins have a specific productivity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 picograms / cell / day (pcd) or higher, said specific productivity being determined based on the total antigen-binding protein (in picograms) produced per cell per day.

[0081] Host cells containing exogenous nucleic acid sequences encoding bispecific antigen-binding proteins that integrate into specific loci within enhanced expression loci exhibited high cell densities in production cultures, e.g., 1 to 10 × 10⁶ cells / year. 6 Cells / mL. In other embodiments, the host cells encoding the bispecific antigen-binding protein have a density of at least 5 × 10⁻⁶ cells / mL. 6 cells / mL, 6×10 6 10 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 Cells / mL or 10 × 10 6 Final cell density of cells / mL.

[0082] In some embodiments, the bispecific antigen-binding protein contains two HC fragments (“HCF”) with different antigen specificities and two LCFs. Where two VL regions are used, they can be the same or different. In certain embodiments, the two VL regions are identical, such as a common light chain.

[0083] In some embodiments, each of the two HCFs includes an amino acid from the heavy chain constant region (such as CH1, CH2, or CH3). In a particular embodiment, each of the two HCFs includes a CH3 domain. In a specific embodiment, each of the two HCFs includes a constant region, i.e., a completely constant region.

[0084] In some implementations, each of the two HCFs includes a VH, and the two VHs can be the same or different.

[0085] In some implementations, the bispecific antigen-binding protein comprises two heavy chains (i.e., two complete heavy chains).

[0086] In some embodiments, each of the two LCFs includes a VL. In a particular embodiment, each LCF consists of a VL region operatively linked to an amino acid sequence comprising amino acids from the constant region of the light chain. In a particular embodiment, each VL region is operatively linked to a CL region, i.e., the bispecific antigen-binding protein comprises a light chain (i.e., a completely light chain).

[0087] In some implementations, the exogenous nucleic acid sequence integrated into the enhanced expression locus includes 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.

[0088] In some embodiments, the nucleotide sequence encoding the first LCF may encode a light chain variable (VL) region sequence. In a particular embodiment, the nucleotide sequence encoding the first VL region encodes the first light chain.

[0089] In some embodiments, the nucleotide sequence encoding the first HCF encodes an amino acid in the first heavy chain constant region (e.g., one or more of CH1, hinge, CH2, or CH3), and the nucleotide sequence encoding the second HCF encodes an amino acid in the second heavy chain constant region. The amino acids in the first heavy chain constant region may be the same as or different from those in the second heavy chain constant region. For example, 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, wherein the first CH3 domain and the second CH3 domain may be the same or different at one or more amino acid positions, as described below for bispecific antigen-binding proteins.

[0090] In some implementations, the nucleotide sequence encoding the first HCF encodes the first VH, and the nucleotide sequence encoding the second HCF encodes the second VH.

[0091] In some implementations, the nucleotide sequence encoding the first HCF encodes the first heavy chain, and the nucleotide sequence encoding the second HCF encodes the second heavy chain. The first and second heavy chains may have the same constant region or differ in one or more amino acids. Several examples of bispecific antigen-binding proteins with different heavy chain constant domains (such as different CH3 domains) are further described below. Regardless of the encoded amino acid sequence, the difference between the nucleotide sequences encoding amino acids from the two heavy chain constant regions lies in the fact that one of the two encoding nucleotide sequences may have a codon modification, which provides a convenient basis for distinguishing the two nucleotide sequences in nucleic acid-based detection assays.

[0092] In some embodiments, each nucleotide sequence encoding HCF or LCF is independently and operatively linked to a promoter-containing transcriptional regulatory sequence. "Independently" means that each coding sequence is operatively linked to a separate transcriptional regulatory sequence, such as a promoter, so that the transcription of the coding sequence is under separate regulation and control. In some embodiments, the promoters guiding the transcription of the two HCF-containing polypeptides are identical. In some embodiments, the promoters guiding the transcription of the two HCF-containing polypeptides, as well as the promoters guiding the transcription of the LCF-containing polypeptide, are identical, for example, the CMV promoter. In some embodiments, each nucleotide sequence encoding HCF or LCF is independently and operatively linked to an inducible or repressive promoter. Inducible or repressive promoters allow production, for example, to occur only during the production phase (batch culture) and not during the growth phase (seed culture). Fine control over the production (expression) of each gene product can be achieved through different promoters.

[0093] In one such example, the cell is first engineered to express tetracycline repressor protein (TetR), and each nucleotide sequence encoding HCF and LCF is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operons (TetO) are placed immediately downstream of the CMV promoter. In some embodiments, each nucleotide sequence encoding HCF and / or LCF is independently and operatively linked to a promoter upstream of at least one TetR operon (TetO) or Arc operon (ArcO). In other embodiments, each nucleotide sequence encoding HCF and / or LCF is independently and operatively linked to a CMV / TetO or CMV / ArcO hybrid promoter. Additional suitable promoters are described below.

[0094] The relative portions of multiple exogenous nucleic acids within a locus can vary. Without being bound by any theory, it is believed that achieving balanced (i.e., comparable) expression levels of two HCF-containing peptides is important. In some embodiments, the nucleic acid encoding LCF is upstream relative to two nucleic acids encoding HCF. Where the three promoters leading the expression of the LCF-containing peptide and the two HCF-containing peptides are identical, suitable arrangements from 5' to 3' may include the nucleotide sequence encoding LCF, the nucleotide sequence encoding a first HCF, a separate promoter operatively linked to a nucleotide sequence (such as a selective marker gene), and the nucleotide sequence encoding a second HCF. Other suitable arrangements from 5' to 3' include the nucleotide sequence encoding LCF, a separate promoter operatively linked to a nucleotide sequence (such as a selective marker gene), the nucleotide sequence encoding the first HCF, and the nucleotide sequence encoding the second HCF. In cases where the nucleotide sequence encoding HCF encodes a constant region sequence, the upstream nucleotide sequence may encode a modified form of the constant region sequence (e.g., modified CH3), or the upstream nucleotide sequence may encode a modified form of the constant region sequence while another nucleotide sequence encodes an unmodified form of the constant region sequence.

[0095] In some embodiments, the cell also contains one or more RRSs integrated into the locus. In some embodiments, the cell includes a first RRS and a second RRS that are distinct from each other and flanked by exogenous nucleic acid sequences, wherein the exogenous nucleic acid sequences contain nucleic acids encoding a first LCF, a first HCF, and a second HCF. In a particular embodiment, the nucleic acid encoding the LCF is upstream of the two nucleic acids encoding the HCF, and the cell includes a third RRS located at 3' relative to the nucleic acid encoding the first LCF and at 5' relative to one or two exogenous nucleic acids encoding the HCF, wherein the third RRS is distinct from the first and second RRSs. The third RRS can be engineered to be included within an intron of the gene, said intron being positioned between any two sequences encoding the HCF or the sequences encoding the LCF.

[0096] Bispecific antigen-binding protein

[0097] Bispecific antigen-binding proteins, such as bispecific antibodies, suitable for cloning and production in the cells, vectors, and systems described in this disclosure are not limited to any particular form of bispecific antigen-binding protein.

[0098] In several embodiments, a bispecific antigen-binding protein comprises two polypeptides, each containing an antigen-binding moiety (e.g., a VH region) and a CH3 domain, wherein the antigen-binding moieties of the two polypeptides have different antigen specificities, and wherein the two CH3 domains are heterodimers relative to each other because one of the CH3 domains is modified at at least one amino acid position to produce a protein A binding signature that is different between the two polypeptides. See, for example, a bispecific antibody described in U.S. Patent 8,586,713. In this manner, a discriminative protein A separation scheme can be used to readily separate the bispecific antigen-binding protein from the heterodimer.

[0099] In some embodiments, the bispecific antigen-binding protein comprises two heavy chains having different antigen specificities and differing at least one amino acid position in the CH3 domain, to produce a distinguishing protein A binding property between the two heavy chains.

[0100] In some embodiments, the two peptides contain a CH3 domain of human IgG, wherein one of the two peptides contains a CH3 domain of human IgG selected from IgG1, IgG2, and IgG4, and the other of the two peptides contains a modified CH3 domain of human IgG selected from IgG1, IgG2, and IgG4, wherein the modification reduces or eliminates the binding of the modified CH3 region to protein A. In a particular embodiment, one of the two peptides contains a CH3 domain of human IgG1, and the other of the two peptides contains a modified CH3 domain of human IgG1, wherein the modification is selected from (i) 95R and (ii) 95R and 96F in the IMGT exon numbering system. In other particular embodiments, the modified CH3 domain includes one to five additional modifications selected from 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system.

[0101] In various other embodiments, the two polypeptides contain a CH3 domain of mouse IgG, wherein one of the two polypeptides contains a CH3 domain of unmodified mouse IgG, and the other of the two polypeptides contains a modified CH3 domain of mouse IgG, wherein the modification reduces or eliminates the binding of the modified CH3 region to protein A. In several embodiments, the CH3 region of mouse IgG is modified to contain specific amino acids (EU numbers) at specific locations selected from: 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 a particular implementation, a specific set of modifications is made, selected from: 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.

[0102] In several 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 consists of heterodimers of two antibodies, each containing a heavy / light chain pair bound via its Fc moiety. The desired heterodimer can be readily purified from a mixture of homodimers of the two parental antibodies and the bispecific heterodimer because the binding properties of the bispecific antibody to protein A differ from those of the parental antibody to protein A: rat IgG2b does not bind to protein A, while mouse IgG2a does. Therefore, the mouse-rat heterodimer binds to protein A but is eluted at a higher pH than the mouse IgG2a homodimer, and this allows for the selective purification of the bispecific heterodimer.

[0103] In other various embodiments, the bispecific antigen-binding protein has a form known in the art as a "button-hole" (see, for example, U.S. Patent No. 7,183,076). In these embodiments, the Fc portions of two antibodies are engineered to create a protruding "button" and a complementary "hole". When produced in the same cell, by linking the engineered "button" to the engineered "hole", the heavy chain is said to preferentially form a heterodimer rather than a homodimer.

[0104] In another embodiment, the first and second heavy chains contain one or more amino acid modifications within the CH3 domain to allow interaction between the two heavy chains. The amino acid residues at the CH3-CH3 interface can be replaced with charged amino acids to provide a homodimer formation that is detrimental to electrostatics. (See, for example, PCT Publication No. WO2009089004; and European Patent Publication No. EP1870459.)

[0105] In other embodiments, the first heavy chain comprises a CH3 domain of homotype IgA, and the second heavy chain comprises a CH3 domain of IgG (or vice versa) to promote preferential formation of heterodimers. (See, for example, PCT Publication No. WO2007110205.)

[0106] In other embodiments, various forms can be integrated into the immunoglobulin chain through engineered methods to promote the formation of heterodimers, such as Fab-arm exchanges (PCT Publication No. WO2008119353; PCT Publication No. WO2011131746), coil-coil domain interactions (PCT Publication No. WO2011034605), or leucine zipper peptides (Kostelny et al. J. Immunol. 1992, 148(5): 1547-1553).

[0107] The immunoglobulin heavy chain variable regions that can be used to generate bispecific antigen-binding proteins can be generated using any method known in the art. For example, a first heavy chain contains a variable region encoded by a nucleic acid derived from the genome of a mature B cell from a first animal immunized with a first antigen, the first heavy chain specifically recognizing the first antigen; and a second heavy chain contains a variable region encoded by a nucleic acid derived from the genome of a mature B cell from a second animal immunized with a second antigen, the second heavy chain specifically recognizing the second antigen. The immunoglobulin heavy chain variable region sequences can also be obtained by any other method known in the art, such as by phage display. In other instances, the nucleic acids encoding the heavy chain variable regions include the nucleic acids of antibodies that have been described in the art or are otherwise available. In some embodiments, one of the two heavy chain coding sequences is codon-modified to provide a convenient basis for distinguishing the two coding sequences in a nucleic acid-based assay.

[0108] Bispecific antibodies comprising two heavy chains recognizing two different epitopes (or two different antigens) are more easily separated, where they can pair with the same light chain (i.e., a light chain having the same 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 with, or substantially without interfering with, the selectivity and / or affinity of the heavy chain's variable domains for their target antigens, as described, for example, in U.S. Patent 8,586,713 and the techniques disclosed therein.

[0109] The bispecific antigen-binding protein may have a variety of dual antigen specificities and related useful applications.

[0110] In some instances, a bispecific antigen-binding protein can be prepared that contains binding specificity against both tumor antigens and T-cell antigens, targeting both cellular antigens, such as CD20, and T-cell antigens, such as T-cell receptors, such as CD3. In this manner, the bispecific antigen-binding protein targets both the patient's target cells (e.g., B cells of a lymphoma patient, via CD20 binding) and the patient's T cells. In several embodiments, the bispecific antigen-binding protein is designed to activate T cells upon binding to T-cell receptors (such as CD3), thereby activating the T cells to bind to specific selected tumor cells.

[0111] In the case of bispecific antigen-binding proteins, one part binds to CD3, and the other part binds to a target antigen, which can be a tumor-associated antigen. Non-restricted 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, CTLA4, and cyclin-B1. CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1, GAGE-2), GD2, GD3, GloboH, phosphatidylinositol proteoglycan-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-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, PAX3, 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 urothelial differentiation-specific glycoprotein-3.

[0112] In some embodiments, the bispecific antigen-binding protein is selected from anti-CD3×anti-CD20 bispecific antibodies (as described in U.S. Patent Application Publications Nos. US2014 / 0088295A1 and US20150266966A1, which are incorporated herein by reference), anti-CD3×antimucin 16 bispecific antibodies (e.g., anti-CD3×anti-Muc16 bispecific antibodies), and anti-CD3×antiprostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3×anti-PSMA bispecific antibodies). In other embodiments, the bispecific antigen-binding protein comprises a portion that binds to CD3. Illustrated anti-CD3 antibody portions are described in U.S. Patent Application Publications Nos. US2014 / 0088295A1 and US20150266966A1, and in International Patent Publication No. WO 2017 / 053856, published March 30, 2017, all of which are incorporated herein by reference. In other embodiments, the bispecific antigen-binding protein comprises a portion that binds CD3 and a portion that binds BCMA, CD19, CD20, CD28, CLEC-12, Her2, HLA protein, MAGE protein, Muc16, PSMA, or Steap-2.

[0113] In the case of bispecific antigen-binding proteins, one portion binds to a T-cell receptor, such as CD3, and the other portion binds to a target antigen, which may be an infectious disease-related antigen. Non-limiting examples of infectious disease-related antigens include, for example, antigens expressed on the surface of viral particles or preferentially expressed on virus-infected cells, wherein the virus is selected from: HIV, hepatitis (A, B, or C), herpesviruses (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, EBV), adenoviruses, influenza viruses, yellow fever viruses, echoviruses, rhinoviruses, Coxsackieviruses, coronaviruses, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, human papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and aromatic viral encephalitis virus. Alternatively, the target antigen may be an antigen expressed on the surface of bacteria or preferentially expressed on cells infected with bacteria, wherein the bacteria are selected from: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Choleraesuis, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Leptospira, and Lyme. In some embodiments, the target antigen is an antigen expressed on the surface of a fungus or preferentially expressed on cells infected with a fungus, wherein the fungus is selected from: Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Rhizopus, etc.), Sporothrix schenkii, Blastomyces dermatitidis, Cryptosporium brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.In some embodiments, the target antigen is an antigen expressed on the surface of the parasite or preferentially expressed on cells infected with the parasite, wherein the parasite is selected from: Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, and Taenia fatensis. Brugia malayi (a type of filariasis). Non-restricted examples of pathogen-associated antigens include, for example, HIV gp120, HIV CD4, hepatitis B glycoprotein L, hepatitis B glycoprotein M, hepatitis B glycoprotein S, hepatitis C E1, hepatitis C E2, hepatocyte-specific proteins, herpes simplex virus gB, cytomegalovirus gB, and HTLV envelope proteins.

[0114] Bispecific binding proteins containing two binding moieties, each pointing to a binding chaperone on the same cell surface (i.e., each pointing to a different target), can also be prepared. This design is particularly suitable for targeting specific cells or cell types that express two targets on the same cell surface. Although the target may be present alone on other cells, the binding moieties of these binding proteins are selected such that each binding moiety binds to its target with a relatively low affinity (e.g., low micromolar or high nanomolar – e.g., over one hundred nanomolar KD, such as 500, 600, 700, 800 nanomolar). In this way, long-term target binding is advantageous when the two targets are adjacent on the same cell.

[0115] Bispecific binding proteins can be prepared, comprising two binding moieties that bind to the same target, each at a different epitope of the same target. This design is particularly suitable for maximizing the probability of the binding protein successfully blocking the target. Multiple extracellular circulations, such as transmembrane channels or cell surface receptors, can be targeted by the same bispecific binding molecule.

[0116] Bispecific binding proteins containing two binding moieties can be prepared, which cluster and activate negative regulators of immune signaling to produce immunosuppression. Cis-blockade can be achieved when the target is on the same cell; inverse blockade can be achieved when the target is on different cells. Cis-blockade can be achieved using bispecific binding proteins with both anti-IgGRIIb and anti-FelD1 binding moieties, causing IgGRIIb to cluster only in the presence of FelD1 to downregulate the immune response to FelD1. Inverse blockade, for example, can be achieved using bispecific binding proteins with both anti-BTLA binding moieties and binding moieties that specifically bind to target tissue-specific antigens, causing the clustering of inhibitory BTLA molecules to occur only in selected target tissues, potentially addressing autoimmune diseases.

[0117] Bispecific binding proteins that activate multicomponent receptors can be prepared. In this design, two binding moieties targeting the two components of the receptor bind to, cross-link, and activate the receptor's signal transduction. This can be achieved, for example, using a bispecific binding protein having a binding moiety that binds to IFNAR1 and a binding moiety that binds to IFNAR2, wherein the binding cross-links the receptor. Such bispecific binding proteins can provide an alternative to interferon therapy.

[0118] Bispecific binding proteins capable of crossing semi-permeable barriers, such as the blood-brain barrier, can be prepared. In this design, one binding moiety binds to a target capable of delivering a specific selective barrier; the other binding moiety targets a therapeutically active molecule that cannot normally cross the barrier. Such bispecific binding proteins can be used to deliver therapeutic agents to tissues that the therapeutic agent cannot reach. Some examples include targeting the pIGR receptor to deliver therapeutic agents to the intestine or lungs, or targeting the transferrin receptor to deliver therapeutic agents across the blood-brain barrier.

[0119] Bispecific binding proteins can be prepared to deliver the binding moiety to specific cells or cell types. In this design, one binding moiety targets cell surface proteins (e.g., receptors) that are readily internalized into the cell. The other binding moiety targets intracellular proteins, wherein binding to the intracellular proteins produces a therapeutic effect.

[0120] Bispecific binding proteins that bind to both surface receptors on phagocytic immune cells and surface molecules of infectious pathogens (such as yeast or bacteria) bring the pathogens to the vicinity of phagocytic immune cells, thereby promoting phagocytosis. An example of this design is a bispecific antibody targeting both CD64 or CD89 molecules and the pathogen.

[0121] Bispecific binding proteins have an antibody variable region as one binding part and a non-Ig region as a second binding part. The antibody variable region enables targeting, while the non-Ig region is an effector or toxin linked to the Fc. In this way, ligands (e.g., effectors or toxins) are delivered to the target bound by the antibody variable region.

[0122] Bispecific binding proteins have two parts, each binding to an Ig region (e.g., an Ig sequence containing CH2 and CH3 regions), such that any two protein parts can be adjacent to each other relative to the Fc region. Examples of this design include traps, such as homo- or heterodimeric trap molecules.

[0123] Enhanced expression locus

[0124] Expression-enhancing loci suitable for this invention include, for example, loci comprising a nucleotide sequence substantially homologous to SEQ ID NO:1 as described in U.S. Patent 8,389,239 (also referred to herein as "..."). The locus described in U.S. Patent Application Serial No. 14 / 919,300 that contains a nucleotide sequence substantially homologous to SEQ ID NO:2 or SEQ ID NO:3 (also referred to herein as the “YARS locus”), as well as other expression-enhancing loci and sequences described in the art (e.g., US 20150167020A1 and U.S. Patent 6,800,457).

[0125] In some embodiments, the expression-enhancing loci used in this invention are selected from loci containing nucleotide sequences substantially homologous to SEQ ID NO:1, or loci containing nucleotide sequences substantially homologous to SEQ ID NO:2 or SEQ ID NO:3. These loci contain sequences that not only provide enhanced expression of genes operatively linked to the sequence (i.e., within or adjacent to the sequence) but also exhibit higher recombination efficiency and improved integration stability compared to other sequences in the genome.

[0126] SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 have been identified from CHO cells. Limited homology to the identified expression-enhancing regions was found in other mammalian species (such as humans or mice); however, homologous sequences can be found in cell lines derived from other tissue types of grey hamsters or other homologous species and can be isolated using techniques well-known in the art. For example, other homologous sequences can be identified by cross-species hybridization or PCR-based techniques. Furthermore, alterations can be made to the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 using site-directed or random mutagenesis techniques well-known in the art. The expression-enhancing activity of the resulting sequence variants can then be tested. DNA with expression-enhancing activity and at least approximately 90% nucleotide identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 can be isolated using routine experiments and is expected to exhibit expression-enhancing activity.

[0127] Integration sites, or the sites or nucleotide positions where one or more exogenous nucleic acids are inserted, can be located within or adjacent to any expression-enhancing sequence (such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3). Whether a specific chromosomal location in or adjacent to the target locus supports stable integration and efficient transcription of the exogenous gene can be determined according to standard procedures well known in the art, such as those described in U.S. Patent 8,389,239 and U.S. Application Serial No. 14,919,300.

[0128] The integration sites considered herein are located within or immediately adjacent to the expression-enhancing sequence, for example, less than about 1 kb, 500 base pairs (bp), 250 bp, 100 bp, 50 bp, 25 bp, 10 bbp, or less than about 5 bp, upstream (5') or downstream (3') of the expression-enhancing sequence on the chromosomal DNA. In some other embodiments, the integration sites employed are located about 1000, 2500, 5000, or more base pairs upstream (5') or downstream (3') of the expression-enhancing sequence on the chromosomal DNA.

[0129] It should be understood in this art that large genomic regions, such as scaffold / matrix attachment regions, are employed for efficient replication and transcription of chromosomal DNA. Scaffold / matrix attachment regions (S / MARs), also known as scaffold attachment regions (SARs) or matrix-associated or matrix-attached regions (MARs), are eukaryotic genomic DNA regions attached to the nuclear matrix. Without being bound by any particular theory, S / MARs are typically located in non-coding regions, separating a given transcribed region (e.g., chromatin domains) from its neighbors, and providing a platform for the machining and / or binding of factors that enable transcription (such as recognition sites for DNases or polymerases). Some S / MARs have been characterized as approximately 14–20 kb long (Klar et al., 2005, Gene 364:79–89). Therefore, gene integration at expression-enhancing loci (e.g., within or near SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) is expected to confer enhanced expression. In some embodiments, host cells containing exogenous nucleic acid sequences exhibit high specific productivity, said exogenous nucleic acid sequences encoding bispecific antigen-binding proteins integrated at specific sites in enhanced-expression loci. In other embodiments, host cells encoding bispecific antigen-binding proteins have specific productivity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 picograms / cell / day (pcd).

[0130] In some embodiments, the integration site is located within a locus containing the nucleotide sequence of SEQ ID NO:1. In a particular embodiment, the integration site is located within or immediately adjacent to the nucleotide sequence of SEQ ID NO:1. In a specific implementation, the integration site is located within SEQ ID NO:1 and is selected from nucleotides spanning the following positions: 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 located in a sequence selected from: nucleotides 5,000-7,400, 5,000-6,500, and 6,400-7,400 of SEQ ID NO:1; and nucleotides 6,400-6,500 of SEQ ID NO:1. In a particular embodiment, the integration site is located before, after, or within the "act" triplet of nucleotides 6471 to 6473 of SEQ ID NO:1.

[0131] In some embodiments, the integration site is located within a locus containing the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3. In a particular embodiment, the integration site is located within or immediately adjacent to the nucleotide sequence of SEQ ID NO:2. In a specific embodiment, the integration site is located within or immediately adjacent to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the integration site is located within the nucleotide sequence of SEQ ID NO:3. (The remaining text appears to be a list of dates and dates, possibly related to SEQ ID NO:3, but without further context, it's impossible to translate accurately.) 007-2008, 2008-2009, 2009-2010, 2010-2011, 2011-2012, 2012-2013, 2013-2014, 2014-2015, 2015-2016, 2016-2017, 2017-2018, 2018-2019, 2019-2020, 2020-2021, or 2021-2022. In a particular embodiment, 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 2021-2022 of SEQ ID NO:3, and nucleotides 2002-2021 of SEQ ID NO:3 are deleted due to the insertion.

[0132] Site-specific integration into expression-enhancing loci

[0133] Multiple exogenous nucleic acids can be integrated into an expression-enhancing locus in a site-specific manner, i.e., into a specific site within the expression-enhancing locus disclosed herein. This can be achieved in a variety of ways, including, for example, through homologous recombination and recombinase-mediated cassette exchange as described in the art (see, for example, U.S. Patent 8,389,239 and the field disclosed herein).

[0134] In some embodiments, cells containing at least two (i.e., two or more) distinct recombinase recognition sequences (RRS) within an expression-enhancing locus are provided to facilitate the integration of nucleic acid sequences containing multiple exogenous nucleic acids or genes of interest. Such cells can be obtained by introducing exogenous nucleic acid sequences containing two or more RRSs into the desired locus through various methods, including homologous recombination, as described below and in the art, such as U.S. Patent 8,389,239 and the techniques disclosed therein.

[0135] In a particular embodiment, cells containing more than two different recombinase recognition sequences (RRS) at an expression-enhancing locus are provided, the recombinase recognition sequences facilitating the integration of multiple exogenous nucleic acids. In another embodiment, cells containing three different recombinase recognition sequences (RRS) within an expression-enhancing locus are provided, the recombinase recognition sequences mediating the integration of two separate exogenous nucleic acids, for example, wherein the 5' RRS and the middle RRS in the genome receive the 5' RRS and 3' RRS of a first exogenous nucleic acid to be integrated, and the middle RRS and the 3' RRS in the genome receive the 5' RRS and 3' RRS of a second exogenous nucleic acid to be integrated.

[0136] Suitable recombinase recombinases (RRSs) can be selected from: LoxP, Lox511, Lox5171, Lox2272, Lox2372, Loxm2, Lox-FAS, Lox71, Lox66, and their mutants, wherein the site-specific recombinase is Cre recombinase or a derivative thereof to achieve recombinase-mediated cassette exchange (RMCE). In other examples, suitable RRSs can be selected from: FRT, F3, F5, FRT mutant-10, FRT mutant+10, and their mutants, in which case the site-specific recombinase Flp recombinase or its derivative is used to achieve RMCE. In yet another example, RRSs can be selected from: attB, attP, and their mutants, in which case the site-specific recombinase phiC31 integrase or its derivative is used to achieve RMCE.

[0137] In other implementations, natural cells are modified using homologous recombination technology to integrate a nucleic acid sequence containing multiple exogenous nucleic acids into a specific site within an expression-enhancing locus.

[0138] In homologous recombination, homologous polynucleotide molecules (i.e., homologous arms) align and exchange a segment of their sequence. If the transgene is side-joined with a homologous genomic sequence, then the transgene can be introduced during this exchange. In one instance, a recombinase recognition site can be introduced into the integration site of the host cell genome via homologous recombination. In other instances, a nucleic acid sequence containing multiple exogenous nucleic acids, such as multiple nucleic acids co-encoding a bispecific antigen-binding protein, is inserted into the host genome, wherein said nucleic acid sequence is side-joined with a sequence homologous to the target locus (“homologous arm”).

[0139] Homologous recombination in eukaryotic cells can be promoted by introducing breaks at integration sites in chromosomal DNA. This can be achieved by targeting certain nucleases to specific integration sites. DNA-binding proteins that recognize DNA sequences at target loci are known in the art. Gene-targeting vectors are also used to promote homologous recombination.

[0140] The construction of gene-targeting vectors and the selection of nucleases for achieving homologous recombination are within the skill of those skilled in the art to which this invention pertains. In some instances, zinc finger nucleases (ZFNs) with modular structures and containing individual zinc finger domains recognize specific 3-nucleotide sequences (e.g., targeting integration sites) in target sequences. Some embodiments may utilize ZFNs with combinations of individual zinc finger domains targeting multiple target sequences. Transcription activator-like (TAL) effector nucleases (TALENs) can also be used for site-specific genome editing. TAL effector protein DNA-binding domains are typically used in combination with non-specific cleavage domains of restriction nucleases such as FokI. In some embodiments, fusion proteins containing TAL effector protein DNA-binding domains and restriction nuclease cleavage domains are used to recognize and cleave DNA at target sequences within the loci of this invention (Boch J et al., 2009 Science 326:1509-1512). RNA-directed endonucleases (RGENs) are programmable genome engineering tools developed from bacterial adaptive immune mechanisms. In this system (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-Associated (Cas) Immunoreaction), the protein Cas9 forms a sequence-specific endonuclease when it complexes with two RNAs (one of which guides target selection). RGEN consists of components (Cas9 and tracrRNA) and target-specific CRISPR RNA (crRNA). The efficiency of DNA target cleavage and the location of the cleavage site vary based on the position of the pre-intercalation sequence neighbor motif (PAM), which is an additional requirement for target recognition (Chen, H. et al., J. Biol. Chem. 2014, 3 / 14, published online as manuscript M113.539726). Unique sequences of specific target loci (such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) can be identified by aligning many of these sequences with the CHO genome; this alignment can reveal potential off-target sites with 16-17 base pairings.

[0141] In some embodiments, a targeting vector carrying the nucleic acid of interest (e.g., a nucleic acid containing one or more RRSs optionally side-gated with one or more selective marker genes, or a nucleic acid containing multiple exogenous nucleic acids co-encoding a bispecific antigen-binding protein) (the nucleic acid being side-gated with 5' and 3' homologous arms) is introduced into a cell having one or more additional vectors or mRNAs. In one embodiment, the one or more additional vectors or mRNAs contain a nucleotide sequence encoding a site-specific nuclease, including (but not limited to) zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, and RNA-directed DNA endonucleases. In some embodiments, the one or more vectors or mRNAs contain a first vector comprising a guide RNA, tracrRNA, and a nucleotide sequence encoding a Cas enzyme, and a second vector comprising a donor (exogenous) nucleotide sequence. Such donor sequences contain a nucleotide sequence encoding a target gene, or a recognition sequence, or a gene cassette designed for targeting any of these exogenous elements. When using mRNA, the mRNA can be transfected into cells using common transfection methods known to those skilled in the art and can encode enzymes such as transposases or endonucleases. While the mRNA introduced into the cells may be transient and not integrated into the genome, it may carry exogenous nucleic acids necessary or beneficial for integration. In some cases, if only short-term expression is required to achieve the desired integration of nucleic acids, mRNA is chosen to eliminate any risk of persistent side effects from helper polynucleotides.

[0142] Vectors for site-specific integration

[0143] This article provides nucleic acid vectors for introducing exogenous nucleic acids into expression enhancement loci via site-specific integration. Suitable vectors include: vectors designed to contain exogenous nucleic acid sequences, said exogenous nucleic acid sequences being side-joined with RRS for integration via RMCE; and vectors designed to contain target exogenous nucleic acid sequences, said target exogenous nucleic acid sequences being side-joined with homologous arms for integration via homologous recombination.

[0144] In several embodiments, vectors are provided for site-specific integration via RMCE. In some embodiments, the vectors are designed to enable the simultaneous integration of multiple nucleic acids into the target locus. Compared to sequential integration, simultaneous integration allows for efficient and rapid isolation of the desired clones that produce bispecific antigen-binding proteins.

[0145] In some embodiments, a set of vectors is provided, comprising two or more vectors, each containing at least two RRSs side-attached to one or more nucleic acids, wherein the nucleic acids in the vectors of the set co-encode a bispecific antigen-binding protein.

[0146] In one embodiment, a set of vectors includes a first vector comprising, from 5' to 3': a first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF, and a third RRS; a second vector comprising, from 5' to 3': a third RRS, a second nucleic acid comprising a nucleotide sequence encoding a first HCF, and a second RRS; wherein the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF; and wherein the first HCF, the second HCF, and the first LCF encode a region (e.g., a variable region) of a bispecific antigen-binding protein. In some embodiments, the nucleotide sequence encoding the second HCF is included 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 included in the second nucleic acid on the second vector (the first HCF and the second HCF on one vector).

[0147] The nucleotide sequence encoding HCF can encode an amino acid, such as an amino acid in the constant region or one or more domains, or encode the entire constant region. In a particular embodiment, the nucleotide sequence encoding HCF or LCF can encode one or more constant domains, such as CL, CH1, hinge, CH2, CH3, or combinations thereof. In some embodiments, the nucleotide sequence encoding HCF can encode a CH3 domain. For example, the nucleotide sequence encoding a first HCF can encode a first CH3 domain, and the nucleotide sequence encoding a second HCF can encode a second CH3 domain. The first CH3 domain and the second CH3 domain can be identical or differ in at least one amino acid. The difference in the CH3 domain or constant region can take any form of bispecific antigen-binding protein as described herein, such as producing different protein A binding characteristics, electrostatic guidance, or differences in the form of a “buttonhole.” Regardless of any amino acid sequence difference, the difference between the two nucleotide sequences encoding HCF can also lie in the fact that one of the two nucleotide sequences is codon-modified.

[0148] In some embodiments, each HCF-encoding nucleotide sequence is independently and operatively linked to a transcriptional regulatory sequence, including, for example, a promoter. In some embodiments, the promoters leading the transcription of the two HCF-containing polypeptides are identical. In some embodiments, the promoters leading the transcription of the two HCF-containing polypeptides, as well as the promoters leading the transcription of the LCF-containing polypeptide, are identical (e.g., the CMV promoter). In some embodiments, each HCF-encoding or LCF-encoding nucleotide sequence is independently and operatively linked to an inducible or repressive promoter. Inducible or repressive promoters allow production, for example, to occur only during the production phase (batch culture) and not during the growth phase (seed culture). Inducible or repressive promoters also allow for differential expression of one or more target genes. In some embodiments, each HCF-encoding and / or LCF-encoding nucleotide sequence is independently and operatively linked to a promoter upstream of at least one TetR operon (TetO) or Arc operon (ArcO). In other embodiments, each nucleotide sequence encoding HCF and / or LCF is independently and operatively linked to a CMV / TetO or CMV / ArcO heterozygous promoter. Examples of heterozygous promoters (also known as regulators of fusion proteins) can be found in International Patent Publication No. WO03101189A1, published on December 11, 2003 (incorporated herein by reference).

[0149] In some embodiments, the first nucleic acid in the first vector further comprises the 5' portion of a selective marker gene located at the 5' of the third RRS in the first vector; and the second nucleic acid further comprises the remaining 3' portion of the selective marker gene located at the 3' of the third RRS in the second vector. In these embodiments, the first, second, and third RRS mediate site-specific integration of the first and second nucleic acids, allowing the 5' and 3' portions of the selective marker gene to be ligated in a suitable and simultaneously integrated clone that is readily selected. In some embodiments, the third RRS in the first vector is designed within the 5' portion of an intron of the selective marker gene; and the third RRS in the second vector is designed within the 3' portion of an intron of the selective marker gene. In other embodiments, the third RRS in the first vector is designed to be located between the promoter and the selective marker gene to which it is operatively linked (but is segregated on another vector); the third RRS in the first vector is designed at the 3' of the promoter; and the third RRS in the second vector is designed at the 5' of the selective marker gene.

[0150] The aforementioned group of vectors may include more than two vectors. For example, in addition to the two vectors mentioned above, the group may also include a third vector containing at least two RRSs flanked by nucleotide sequences encoding a second LCF. The group may also include vectors encoding one or more recombinases that recognize RRSs.

[0151] In other embodiments, vectors are provided for site-specific integration via homologous recombination. In some instances, the polynucleotide sequence integrated into the host genome can be a DNA sequence, such as an RRS or multiple RRSs flanked by one or more selective marker genes to generate one or more RRSs integrated into the desired locus for subsequent integration into the cell of a nucleic acid encoding a bispecific antigen-binding protein. In other instances, the polynucleotide sequence to be integrated into the host genome comprises multiple nucleic acids co-encoding a 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 co-encoding a bispecific antigen-binding protein are each independently (i.e., separately) operably linked to a regulatory sequence (such as a promoter, enhancer, transcription termination sequence, or a combination thereof), meaning that the regulatory sequence (such as a promoter) for each of the multiple nucleic acids is separate, and these nucleic acids can be the same or different (i.e., containing the same or different nucleotide sequences). In cases where one of the multiple nucleic acids comprises multiple coding sequences, each nucleotide sequence of each coding sequence or the N-terminal portion encoding a polypeptide is independently and operably linked to its own regulatory sequence (such as a promoter).

[0152] Selecting sequences homologous to the sequence within the enhancing locus and including the selected sequence as a homologous arm in the targeting vector is entirely within the skill of a person skilled in the art. In some embodiments, the vector or construct comprises a first homologous arm and a second homologous arm, wherein the merged first and second homologous arms comprise a targeting sequence that replaces the endogenous sequence within the locus. In other embodiments, the first and second homologous arms comprise a targeting sequence integrated into or inserted into the endogenous sequence within the locus. In some embodiments, the homologous arms contain nucleotide sequences homologous to the nucleotide sequences present in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In a particular embodiment, the vector comprises: a 5' homologous arm having a nucleotide sequence corresponding to nucleotides 1001-2001 of SEQ ID NO:3; and a 3' homologous arm having a nucleotide sequence corresponding to nucleotides 2022-3022 of SEQ ID NO:3. The homologous arms, such as the first homologous arm (also referred to as the 5' homologous arm) and the second homologous arm (also referred to as the 3' homologous arm), are homologous to the targeting sequence within the locus. The 5' to 3' homologous arms can amplify regions or target sequences within the locus that contain 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. In other embodiments, the total number of nucleotides selected for the target sequences used in the first and second homologous arms comprises 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. In some cases, the distance between the 5' homologous arm and the 3' homologous arm (homological to the target sequence) includes 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. When nucleotides 1001-2001 and 2022-3022 of SEQ ID NO:3 are selected as 5' and 3' homologous arms, the distance between the two homologous arms can be 20 nucleotides (corresponding to nucleotides 2002-2021 of SEQ ID NO:3); such homologous arms can mediate the integration of exogenous nucleic acid sequences into loci containing SEQ ID NO:3, for example, within nucleotides 1990-2021 or 2002-2021 of SEQ ID NO:3, while simultaneously deleting nucleotides 2002-2021 of SEQ ID NO:3.

[0153] The vectors disclosed herein for introducing exogenous nucleic acids by site-specific integration into expression-enhancing loci may include additional genes and sequences for guiding the expression of the target exogenous nucleic acid and its encoded polypeptide, and for selecting and identifying cells in which the target exogenous nucleic acid has been successfully integrated. Other such sequences are also described below, including, for example, transcriptional and translational regulatory sequences, selective marker genes, and so on.

[0154] Regulatory sequence

[0155] The vectors disclosed herein for introducing exogenous nucleic acids into expression-enhancing loci in a site-specific manner, and the cells obtained through site-specific integration, may include regulatory sequences for guiding the expression of target exogenous nucleic acids and encoded polypeptides. Regulatory elements include transcription promoters, enhancers, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling transcription and translation termination. Control sequences for transcription and translation may be provided from viral sources. For example, commonly used promoters and enhancers are derived from viruses such as polyomavirus, adenovirus 2, simian virus 40 (SV40), mouse or human cytomegalovirus (CMV), CMV immediate early (CMV-IE) or CMV major early IE (CMV-MIE) promoters, as well as RSV, SV40 late promoters, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters. Viral genomic promoters, control and / or signaling sequences may be used to drive expression, and such control sequences provided are compatible with the selected host cell. Non-viral cell promoters (e.g., β-globulin and EF-1α promoters) can also be used, depending on the cell type in which the target protein will be expressed. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early and late promoters, enhancers, splicing sites, and polyadenylation sites, can be used to provide additional gene elements useful for the expression of exogenous DNA sequences. Early and late promoters are particularly useful because both are readily active from the SV40 virus as fragments that also contain the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments can also be used. Typically, this includes a sequence of approximately 250 bp extending from the Hind III site located at the SV40 origin of replication to the BglI site. Inducible promoters (e.g., induced by compounds, cofactors, or regulatory proteins) can be used, and are particularly useful for allowing the production of antigen-binding proteins that occur only during the production phase (batch culture) rather than the growth phase (seed culture). Examples of inducible or repressive promoters include the alcohol dehydrogenase I gene promoter, the tetracycline-responsive promoter system, the glucocorticoid receptor promoter, the estrogen receptor promoter, the ecdysone receptor promoter, metallothionein-based promoters, and T7 polymerase-based promoters. Sequences suitable for expressing multiple transcripts via bicistronic vectors have previously been described (Kim SK and Wold B.J., Cell 42:129, 1985) and can be used in this invention. Examples of suitable strategies for expressing proteins in polycistronic form include the use of a 2A peptide (Szymczak et al., Expert Opin Biol Ther 5:627–638 (2005)) and the use of ribosomal entry sites ("IRES"), both well known in the art.Other types of expression vectors would also be useful, such as those described in U.S. Patent No. 4,634,665 (Axel et al.) and U.S. Patent No. 4,656,134 (Ringold et al.).

[0156] Selective Marking

[0157] The vectors disclosed herein for introducing exogenous nucleic acids into expression enhancement loci in a site-specific manner, and the cells obtained due to site-specific integration, may include one or more optional marker genes.

[0158] In some implementations, optional marker genes confer resistance, such as those described in Table 1 of Kaufman, RJ (1988) Meth. Enzymology 185:537, and include DHFR-MTX resistance, P-glycoprotein and multidrug resistance (MDR) – various lipophilic cytotoxic agents (e.g., adriamycin, colchicine, vincristine), and adenosine deaminase (ADA) – Xyl-A or adenosine and 2'-deoxycoformycin. Other dominant-selective markers include antibiotic resistance genes derived from microorganisms, such as neomycin, kanamycin, or hygromycin resistance. Several suitable selection systems exist in mammalian hosts (Sambrook, ibid., pp. 16.9–16.15). Co-transfection protocols using two dominant-selective markers have also been described (Okayama and Berg, Mol. Cell Biol 5:1136, 1985).

[0159] In other embodiments, the optional marker gene encodes a polypeptide that provides or is capable of generating a detectable signal for identifying a gene cassette that has been or has not yet been successfully inserted and / or replaced, as appropriate. Suitable examples include fluorescent markers or proteins, enzymes that catalyze chemical reactions to generate a detectable signal, and so on. Examples of fluorescent labels are well known in the art, including (but not limited to) Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), blue-green fluorescent protein (CFP), enhanced blue-green fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), and far-infrared fluorescent proteins (e.g., mKate, mKate2, mPlum, mRaspberry, or E2-crimson). See also, for example, Nagai, T. et al., 2002 Nature Biotechnology 20:87-90; Heim, R. et al., February 23, 1995 Nature 373:663-664; and Strack, RL et al., 2009 Biochemistry 48:8279-81.

[0160] System for preparing bispecific antigen-binding proteins

[0161] In another aspect, this disclosure provides a system comprising a combination of cells and one or more vectors, and which can be used to prepare cells in which exogenous nucleic acids are integrated into an expression-enhancing locus to co-encode a bispecific antigen-binding protein. For example, the system may be provided in the form of a kit.

[0162] In some implementations, the system is designed to allow efficient vector construction and simultaneous integration of multiple exogenous nucleic acids into specific sites within an enhanced expression locus via RMCE. Simultaneous integration allows for rapid isolation of desired clones, and using a single enhanced expression locus is also important for generating stable cell lines.

[0163] In some embodiments, a system is provided comprising any one of a set of vectors designed to integrate multiple exogenous nucleic acids via RMCE as described above, and a cell containing an RRS, the RRS being integrated into a specific site within an enhancement expression locus and matching an RRS in the set of vectors. For example, the system comprises a cell and a set of vectors, wherein the cell contains, from 5' to 3', an enhancement expression locus integrated into its genome containing: 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 distinct from each other; wherein the set of vectors comprises a first vector, from 5' to 3' comprising a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF (e.g., a first VL), and a second RRS; a second vector, the second vector comprising a second RRS, a second nucleic acid containing a nucleotide sequence encoding a first HCF (e.g., a first VH), and a third RRS; and wherein the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF (e.g., a second VH). When the vector is introduced into cells, the first and second nucleic acids in the vector are integrated into the enhanced expression locus via recombination mediated by the first, second, and third RRS. To facilitate screening transfectants from which nucleic acids have been appropriately integrated from the vector into the locus, the first exogenous nucleic acid in the system's cells may include a first selective marker gene, and the second exogenous nucleic acid in the cells may include a second selective marker gene, wherein the first and second selective marker genes are distinct from each other and also distinct from any selective marker genes provided by the vector; in a particular embodiment, the first and second selective marker genes encode a fluorescent protein (which provides negative selection), and the first and second nucleic acids on the vector provide additional selective marker genes in fragmented form to provide positive selection. Negative selection alone can provide rapid clonal isolation, but the efficiency of clonal isolation by expected recombination can be limited (approximately 1%). Combining negative selection with positive selection (novel fluorescence or resistance to a drug or antibiotic) can significantly improve the isolation efficiency of positive clones (up to approximately 80%).

[0164] The system may include additional components, reagents, or information, such as protocols for introducing (multiple) vectors into cells within the system via transfection. Non-restrictive transfection methods include chemical-based methods, including 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: WH Freeman and Company. pp. 96-97); dendritic polymers; or cationic polymers, such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation; acoustic perforation; and optical transfection. Particle-based transfection includes the use of gene guns, magnet-assisted transfection (Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methods may also be used for transfection. mRNA delivery includes the use of TransMessenger. TM and The method described in the article (Bire et al., BMC Biotechnology 2013, 13:75) is used to introduce heterologous DNA into cells. A common method for introducing heterologous DNA into cells is calcium phosphate precipitation, as described by 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 suitable method for introducing heterologous DNA. DNA can also be introduced directly into the cytoplasm of host cells using electroporation, as described by Potter et al. (Proc. Natl. Acad. Sci. USA 81:7161, 1988) or Shigekawa et al. (BioTechniques 6:742, 1988). Other reagents suitable for introducing heterologous DNA into mammalian cells have been described, such as Lipofectin. TM Reagents and Lipofectamine TM Reagents (Gibco BRL, Gaithersburg, Md.). Both of these commercially available reagents are used to form lipid-nucleic acid complexes (or liposomes), which, when applied to cultured cells, facilitate nucleic acid uptake into the cells.

[0165] Methods for preparing bispecific antigen-binding proteins

[0166] This disclosure also provides a method for preparing bispecific antigen-binding proteins. By utilizing the method of the present invention, bispecific antigen-binding proteins (e.g., bispecific antibodies) can be produced at high titers and / or high specific productivity (picograms / cells / 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 greater. In some embodiments, the bispecific antigen-binding proteins are produced at a ratio of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or higher of bispecific antigen-binding protein titer to total antigen-binding protein titer. In some implementations, bispecific antigen-binding proteins are produced at a specific productivity of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 picograms / cell / day or higher, said specific productivity being determined based on the total antigen-binding protein (in picograms) produced per cell per day.

[0167] In one embodiment, the method utilizes the system disclosed herein and introduces a vector from the system into cells via transfection. Transfected cells in which the exogenous nucleic acid has been appropriately integrated into the target-enhancing expression locus of the cell via RMCE can be screened and identified. Two polypeptides containing HCF and at least one polypeptide containing LCF can be expressed from the integrated nucleic acid, and a bispecific antigen-binding protein containing all three polypeptides can be obtained from the identified transfected cells and purified using known methods.

[0168] In some embodiments, the method includes (i) providing a system comprising cells and a set of vectors, wherein the cells contain, from 5' to 3', an enhanced expression locus integrated into their 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 distinct from each other; wherein the set of vectors comprises a first vector, from 5' to 3' comprising a first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF (e.g., a first VL), and a second RRS; a second vector, the second vector comprising a second RRS, a second nucleic acid comprising a nucleotide sequence encoding a first HCF (e.g., a first VH), and a third RRS; and wherein the first or second nucleic acid further comprises a nucleotide sequence encoding a second HCF (e.g., a second VH); (ii) simultaneously introducing the vectors into the cells; and (iii) screening for transformed cells in which the first and second nucleic acids in the vectors are simultaneously integrated into the enhanced expression locus via recombination mediated by the first, second, and third RRSs.

[0169] In a specific embodiment of the method, to facilitate screening transformants in which nucleic acids are appropriately integrated from the vector into the locus, the first exogenous nucleic acid in the system's cells may include a first selective marker gene, and the second exogenous nucleic acid in the cells may include a second selective marker gene, wherein the first and second selective marker genes are distinct from each other; and the first and second nucleic acids on the vector jointly encode an additional selective marker in a fragmented form, wherein simultaneous integration provides a complete sequence encoding the additional selective marker gene. Transformants can be screened for selection targeting the first and second selective markers (negative selection) and for the additional selective marker (positive selection).

[0170] In another embodiment, the method uses only cells with exogenous nucleic acid sequences integrated into specific sites within enhanced expression loci of the cells, wherein the exogenous nucleic acid sequences encode bispecific antigen-binding proteins, and the bispecific antigen-binding proteins are expressed from the cells. The clonal expression cassettes within the specific integration site are sequential.

[0171] The present specification is further illustrated by the following examples, which should not be construed as limiting in any way. All references cited herein (including references cited throughout this application, published patents, and published patent applications) are hereby expressly incorporated by reference.

[0172] Example

[0173] Example 1: Cloning of a bispecific antibody expression plasmid:

[0174] The heavy and light chain components of bispecific antibodies can be cloned from hybridoma cells, B cells, plasma cells, or recombinant antibody gene libraries using methods known in the art. For example, antibodies can be cloned from hybridomas or B cells via five primary RACE PCRs or PCR using primers with a leader peptide, frame 1 sequence, frame 4 sequence, or constant region sequence. Alternatively, antibody genes or mRNA in antibody-expressing cells can be sequenced using next-generation sequencing technologies and subsequently identified by bioinformatics. Sequencing antibody proteins and cloning the corresponding antibody genes using synthetic DNA techniques is also feasible. Recombinant antibody libraries (such as yeast or phage libraries) are also sources of antibody genes.

[0175] CHO-expressing cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 each produced bispecific antibodies composed of three different polypeptides: AbC1, AbC2, and AbC3. Figure 5To generate plasmids for constructing RSX4189-1, the AbC1 plasmid was linearized by Mfe I digestion, with Mfe I located at the 3' of the AbC1 gene. The AbC2 expression cassette, excised from the AbC2 plasmid via Mfe I digestion, was ligated to the Mfe I site of the linearized AbC1 plasmid. The ligation product was transformed into DH10B *E. coli*. After transformation and growth in LB medium containing ampicillin, single *E. coli* clones containing the desired plasmids of both AbC1 and AbC2 genes were analyzed. The sequences of the bulk-produced DNA from 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 *E. coli* with DH10B *E. coli* using lipofectamine. RRS1 and RRS3 loci at the gene locus Host cells. Transfected cells were selected with antibiotics for 12 days, and then the recombinant cells were merged into RSX4189-1.

[0176] To generate the plasmid for constructing RSX4187-1, the AbC3 expression cassette flanked at the Mlu I and Nhe I sites was cloned into the 3' region of the AbC2 gene at the Mlu I and Spe I sites in the AbC2 plasmid. The combined AbC2-AbC3 plasmid, AbC1 plasmid, and Cre plasmid pRG858 were co-transfected into the AbC2 plasmid using lipofectamine. Host cells. Cells that have undergone RMCE are merged into RSX4187-1.

[0177] To generate the plasmid for constructing RSX4191-1, the AbC3 expression cassette was cloned into the 3' region of the AbC1 gene at the Mfe I site in the AbC1 plasmid. The combined AbC1-AbC3 plasmid, AbC2 plasmid, and Cre plasmid pRG858 were co-transfected into the RSX4191-1 plasmid using lipofectamine. Host cells. Cells that have undergone RMCE are merged into RSX4191-1.

[0178] To generate the plasmid for constructing RSX4188-1, the AbC2 expression cassette flanked at the Mlu I and Nhe I sites was cloned into the 3' region of the AbC3 gene at the Mlu I and Spe I sites in the AbC3 plasmid. The combined AbC3-AbC2 plasmid, AbC1 plasmid, and Cre plasmid pRG858 were co-transfected into the AbC3 plasmid using lipofectamine. Host cells. Cells that have undergone RMCE are merged into RSX4188-1.

[0179] Example 2: From Expression of bispecific antibodies at loci

[0180] Bispecific antibody expression cell lines RSX4189-1, RSX4187-1, RSX4191-1, and RSX4188-1 were cultured in suspension in serum-free medium. To quantify bispecific antibody expression levels, cell counts were performed using a Guava Flow flow cytometer, and cultures were initiated in fresh shake flasks containing 2 million cells / ml of medium. After 4 days, waste medium was collected after centrifugation to remove cells. Bispecific antibody titers were determined using HPLC analysis of bispecific antibody-specific protein A. The titers of 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 expressed from these cell lines (including bispecific and monospecific antibody proteins), and the ratio of bispecific antibody protein titers to total antibody protein titers, are shown in the table below.

[0181]

[0182] sequence list

[0183] SEQ ID NO:1

[0184] 13515 bases

[0185] DNA

[0186] Chinese hamster

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] SEQ ID NO:2

[0193] 14931 bases

[0194] DNA

[0195] Chinese hamster

[0196] misc_feature(2176)..(2239)

[0197] n represents a, c, g, t, or a nucleotide deletion.

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] SEQ ID NO:3

[0204] 4001 bases

[0205] DNA

[0206] Chinese hamster

[0207]

[0208]

[0209] This application includes the following implementation scheme:

[0210] 1. A cell comprising a foreign nucleic acid sequence at a specific site integrated into an enhanced expression locus, wherein the foreign nucleic acid sequence encodes a bispecific antigen-binding protein.

[0211] 2. The cell according to embodiment 1, wherein the exogenous nucleic acid sequence 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.

[0212] 3. The cell according to embodiment 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.

[0213] 4. The cell according to embodiment 3, wherein the first CH3 domain and the second CH3 domain differ at at least one amino acid position.

[0214] 5. The cell according to embodiment 3, wherein the first CH3 domain and the second CH3 domain are CH3 domains of human IgG, and one of the two CH3 domains is modified at at least one amino acid position to produce a protein A binding characteristic different from that of the unmodified CH3 domain.

[0215] 6. The cell according to embodiment 3, wherein the nucleotide sequences encoding the first CH domain and the second CH domain differ from each other in that one of the nucleotide sequences is codon-modified.

[0216] 7. The cell according to embodiment 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.

[0217] 8. The cell according to embodiment 2, wherein the nucleotide sequence encoding the first LCF encodes the first VL.

[0218] 9. The cell according to embodiment 1, wherein the exogenous nucleic acid sequence further comprises an additional exogenous nucleic acid, the additional exogenous nucleic acid comprising a nucleotide sequence encoding a second LCF.

[0219] 10. The cell according to embodiment 9, wherein the nucleotide sequence encoding the second LCF encodes the second VL.

[0220] 11. The cell according to embodiment 2, wherein the first exogenous nucleic acid further comprises a first promoter operatively linked to a nucleotide sequence encoding a first LCF, the second exogenous nucleic acid further comprises a second promoter operatively linked to a nucleotide sequence encoding a first HCF, and the third exogenous nucleic acid comprises a third promoter operatively linked to a nucleotide sequence encoding a second HCF, wherein the first promoter, the second promoter, and the third promoter are the same or different.

[0221] 12. The cell according to embodiment 2, wherein the first exogenous nucleic acid is located upstream of the second exogenous nucleic acid and the third exogenous nucleic acid.

[0222] 13. The cell according to embodiment 12 further comprises a first recombinase recognition site (RRS) located at 5' relative to the first exogenous nucleic acid, and a second recombinase recognition site (RRS) located at 3' relative to both the second and third exogenous nucleic acids, wherein the first RRS and the second RRS are different.

[0223] 14. The cell according to embodiment 12 further comprises a third RRS located at 3' relative to the first exogenous nucleic acid and at 5' relative to one or both of the second and third exogenous nucleic acids, wherein the third RRS is different from the first and second RRS.

[0224] 15. The cell according to embodiment 13 further comprises a fourth exogenous nucleic acid, said fourth exogenous nucleic acid comprising a selective marker gene.

[0225] 16. The cell according to embodiment 15, wherein the fourth exogenous nucleic acid further comprises a third RRS and is located at 3' relative to the first exogenous nucleic acid.

[0226] 17. The cell according to embodiment 2, wherein the second exogenous nucleic acid is located upstream of the first exogenous nucleic acid and the third exogenous nucleic acid.

[0227] 18. The cell according to embodiment 16, wherein the sequence of the exogenous nucleic acids at the locus from 5' to 3' is: 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 the human IgG.

[0228] 19. The cell according to embodiment 16, wherein the sequence of the exogenous nucleic acids at the locus from 5' to 3' is: 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 a natural CH3 domain of human IgG, and the third exogenous nucleic acid comprises a nucleotide sequence encoding a modified CH3 domain of the human IgG.

[0229] 20. The cell according to embodiment 18 or 19, wherein the first promoter, the second promoter and the third promoter are identical and different from the promoter to which the selective marker gene is operatively linked.

[0230] 21. The cell according to embodiment 1, wherein the bispecific antigen-binding protein specifically binds to T cell antigen and tumor cell antigen.

[0231] 22. The cell according to embodiment 1, wherein the enhanced expression locus is selected from: a locus containing a nucleotide sequence having at least 90% identity with SEQ ID NO:1, and a locus containing a nucleotide sequence having at least 90% identity with SEQ ID NO:2.

[0232] 23. The cell according to any one of the foregoing embodiments, wherein the cell is a CHO cell.

[0233] 24. A set of carriers, which contain

[0234] The first vector, from 5' to 3', comprises: a first RRS, a first nucleic acid containing a nucleotide sequence encoding a first LCF, and a third RRS;

[0235] The second vector, from 5' to 3', comprises: the third RRS, a second nucleic acid containing a nucleotide sequence encoding the first HCF, and the second RRS;

[0236] The first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF; and

[0237] The first HCF, the second HCF, and the first LCF encode a bispecific antigen-binding protein.

[0238] 25. A set of vectors according to embodiment 24, wherein the nucleotide sequence encoding the second HCF is included within the first nucleic acid.

[0239] 26. A set of vectors according to embodiment 25, wherein the nucleotide sequence encoding the second HCF is located upstream or downstream of the nucleotide sequence encoding the first LCF.

[0240] 27. A set of vectors according to embodiment 24, wherein the nucleotide sequence encoding the second HCF is included within the second nucleic acid.

[0241] 28. A set of vectors according to embodiment 24, 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.

[0242] 29. A set of carriers according to embodiment 28, wherein the first CH3 domain and the second CH3 domain differ at at least one amino acid position.

[0243] 30. A set of vectors according to embodiment 28, wherein the first CH3 domain and the second CH3 domain are CH3 domains of human IgG, and one of the two CH3 domains is modified at at least one amino acid position to produce a protein A binding characteristic different from that of the unmodified CH3 domain.

[0244] 31. A set of vectors according to embodiment 28, wherein the nucleotide sequences encoding the first CH domain and the second CH domain differ from each other in that one of the nucleotide sequences is codon-modified.

[0245] 32. A set of vectors according to embodiment 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.

[0246] 33. A set of vectors according to embodiment 24, wherein the nucleotide sequence encoding the first LCF encodes the first VL.

[0247] 34. A set of vectors according to embodiment 24, wherein the nucleotide sequence encoding a first light chain LCF is operatively linked to a first promoter, the nucleotide sequence encoding a first HCF is operatively linked to a second promoter, and the nucleotide sequence encoding a second HCF is operatively linked to a third promoter.

[0248] 35. A set of carriers according to embodiment 34, wherein the first promoter, the second promoter and the third promoter are the same or different.

[0249] 36. A set of vectors according to embodiment 24, wherein the first nucleic acid further comprises a second promoter at the 5' of the third RRS located in the first vector; and the second nucleic acid further comprises a selective marker gene at the 3' of the third RRS located in the second vector.

[0250] 37. A set of vectors according to embodiment 24, wherein the first nucleic acid further comprises a second promoter in the first vector located at the 5' of the 5' portion of the selective marker gene; and the 3' portion of the selective marker in the second vector is present at the 3' of the homologous arm of the 5' portion of the selective marker gene.

[0251] 38. A set of carriers according to implementation scheme 24, wherein

[0252] The first vector comprises, from 5' to 3', the first RRS, the first nucleic acid, and the third RRS; and

[0253] The second vector from 5' to 3' contains the third RRS and the second nucleic acid, wherein the second nucleic acid contains a nucleotide sequence or fragment thereof encoding a first heavy chain and a nucleotide sequence or fragment thereof encoding a second heavy chain.

[0254] 39. A set of carriers according to implementation scheme 24, wherein

[0255] The first vector, from 5' to 3', comprises the first RRS, the first nucleic acid, wherein the first nucleic acid comprises the nucleotide sequence encoding the first LCF and the nucleotide sequence encoding the second HCF, and the third RRS; and

[0256] The second vector from 5' to 3' contains the third RRS and the second nucleic acid, wherein the second nucleic acid contains a nucleotide sequence encoding the first HCF.

[0257] 40. A set of vectors according to embodiment 38 or 39, wherein the first nucleic acid further comprises a 5' portion of a selective marker gene located at the 5' of the third RRS in the first vector; and the second nucleic acid further comprises the remaining 3' portion of the selective marker gene located at the 3' of the third RRS in the second vector; and wherein optionally the third RRS in the first vector is located within the 5' portion of an intron of the selective marker gene, and the third RRS in the second vector is located within the 3' portion of an intron of the selective marker gene.

[0258] 41. The set of vectors according to embodiment 24 further includes a third vector, the third vector comprising one or more RRS and a nucleotide sequence encoding a second LCF.

[0259] 42. The set of vectors according to embodiment 24 further comprises a third vector encoding one or more recombinases that recognize RRS.

[0260] 43. A vector comprising a foreign nucleic acid sequence encoding a bispecific antigen-binding protein, said foreign nucleic acid sequence being side-joined by a 5' homologous arm and a 3' homologous arm for integration into a cell-expressing enhancement locus.

[0261] 44. A system comprising cells and a set of carriers,

[0262] The cell from 5' to 3' contains, within its genome, an enhanced expression locus: a first RRS, a first exogenous nucleic acid, a third RRS, a second exogenous nucleic acid, and a second RRS, wherein the three RRSs are distinct from one another;

[0263] The set of carriers includes

[0264] A first vector, comprising from 5' to 3': a first RRS, a first nucleic acid comprising a nucleotide sequence encoding a first LCF, and the third RRS;

[0265] The second vector comprises: the third RRS, a second nucleic acid containing a nucleotide sequence encoding the first HCF, and the second RRS; and

[0266] The first nucleic acid or the second nucleic acid further comprises a nucleotide sequence encoding a second HCF; and

[0267] When the vector is introduced into the cell, the first nucleic acid and the second nucleic acid in the vector are integrated into the enhanced expression locus through recombination mediated by the first RRS, the second RRS and the third RRS.

[0268] 45. The system according to embodiment 44, wherein the first exogenous nucleic acid comprises a first selective marker gene, and the second exogenous nucleic acid comprises a second selective marker gene, wherein the first selective marker gene and the second selective marker gene are different.

[0269] 46. ​​The system according to implementation scheme 44, wherein

[0270] The first vector, from 5' to 3', comprises: the first RRS, the first nucleic acid containing the first LCF, and the third RRS; and

[0271] The second vector from 5' to 3' includes the third RRS, the second nucleic acid, wherein the second nucleic acid includes both the nucleotide sequence encoding the first HCF and the nucleotide sequence encoding the second HCF, and the second RRS.

[0272] 47. The system according to embodiment 44, wherein

[0273] The first vector, from 5' to 3', comprises: a first RRS, a first nucleic acid comprising the nucleotide sequence encoding the first LCF and the nucleotide sequence encoding the second HCF, and the third RRS; and

[0274] The second vector from 5' to 3' comprises: the third RRS, the second nucleic acid comprising the nucleotide sequence encoding the first HCF, and the second RRS.

[0275] 48. The system according to embodiment 46 or 47, wherein the first nucleic acid in the first vector further comprises a 5' portion of a selective marker gene located at the 5' of the third RRS; and the second nucleic acid in the second vector further comprises the remaining 3' portion of the selective marker gene located at the 3' of the third RRS.

[0276] 49. The system according to embodiment 48, wherein the third RRS in the first vector is located within the 5' portion of the intron of the selective marker gene; and the third RRS in the second vector is located within the 3' portion of the intron of the selective marker gene.

[0277] 50. The system according to embodiment 48, wherein the nucleotide sequence encoding the LCF is operatively linked to a first promoter, the nucleotide sequence encoding the first HCF is operatively linked to a second promoter, and the nucleotide sequence encoding the second HCF is operatively linked to a third promoter, wherein the first promoter, the second promoter, and the third promoter are the same or different, and / or the promoters are the same or different from the promoters to which the selective marker gene is operatively linked.

[0278] 51. The system according to embodiment 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.

[0279] 52. The system according to embodiment 51, wherein one of the first CH3 domain and the second CH3 domain is a CH3 domain of human IgG, and the other is a modified CH3 domain of the human IgG containing a modification at at least one amino acid position.

[0280] 53. The system according to embodiment 52, wherein the nucleotide sequence encoding the modified CH3 domain is in the first vector.

[0281] 54. The system according to embodiment 52, wherein the nucleotide sequence encoding the modified CH3 domain is in the second vector and upstream of the nucleotide sequence encoding the unmodified CH3 domain.

[0282] 55. A method comprising:

[0283] (i) Provide a system according to any one of embodiments 44-54;

[0284] (ii) Simultaneously introducing the vector into the cells via transfection; and

[0285] (iii) Select transfected cells in which the first and second nucleic acids in the vector have been integrated into the enhanced expression locus of the cell via recombination mediated by the first RRS, the second RRS and the third RRS.

[0286] 56. The method according to embodiment 55 further includes:

[0287] (iv) Expressing the first LCF, the first HCF, and the second HCF in selected transfected cells; and

[0288] (v) Obtain a bispecific antigen-binding protein containing the first LCF, the first HCF and the second HCF from selected transfected cells.

[0289] 57. A method for preparing a bispecific antigen-binding protein, comprising:

[0290] (i) Provide cells according to any one of embodiments 1-23;

[0291] (ii) expressing the bispecific antigen-binding protein from an exogenous nucleic acid sequence; and

[0292] (iii) Obtain the bispecific antigen-binding protein from the cell.

Claims

1. A combination comprising: (i) A set of carriers comprising: A first vector, comprising from 5' to 3': a first recombinase recognition site (RRS), a first nucleic acid, and a third RRS; The second vector, from 5' to 3', comprises: the third RRS, the second nucleic acid, and the second RRS; 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). The first nucleic acid or one of the second nucleic acids further includes a nucleotide sequence encoding a second HCF. In the first vector, the first nucleic acid further comprises a 5' portion of a selective marker gene located at the 5' of the third RRS, wherein the 5' portion of the selective marker gene comprises the 5' portion of an intron, and wherein the third RRS in the first vector is located within the 5' portion of the intron of the selective marker gene; In the second vector, the second nucleic acid further comprises the remaining 3' portion of a selective marker gene located at the 3' of the third RRS, wherein the remaining 3' portion of the selective marker gene comprises the remaining 3' portion of an intron, and wherein the third RRS in the second vector is present within the remaining 3' portion of the intron of the selective marker gene. Wherein the first HCF, the second HCF, and the first LCF are fragments of a bispecific antibody; and (ii) A mammalian cell comprising, from 5' to 3', the following from a 5' to 3' region within an enhanced expression locus integrated into its genome: a first RRS, a first exogenous nucleic acid, a third RRS, a second exogenous nucleic acid, and a second RRS, wherein the three RRSs are distinct from one another; The enhanced expression locus consists of the nucleotide sequence of SEQ ID NO:

2.

2. The combination according to claim 1, wherein the first nucleic acid comprises a nucleotide sequence encoding a first LCF, and the second nucleic acid comprises a nucleotide sequence encoding a first HCF.

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

4. The combination according to any one of claims 1-3, wherein the selective marker gene confers antibiotic resistance.

5. The combination according to claim 4, wherein the antibiotic is hygromycin, neomycin, or kanamycin.

6. The combination of claim 1, wherein each of the first HCF, the first LCF, and the second HCF comprises a variable structural domain.

7. The combination according to claim 6, wherein the variable structural domains included in the first HCF are different from the variable structural domains of the second HCF.

8. The combination according to claim 1, wherein the first nucleic acid and the second nucleic acid together encode a full-length bispecific antibody.

9. The combination according to claim 1, wherein the mammalian cell is a CHO cell.

10. A mammalian cell comprising, from 5' to 3', the following, integrated into an enhanced expression locus: First recombinase recognition site (RRS). First nucleic acid, Includes selective marker genes containing introns with a third RRS. The second nucleic acid, and Second RRS, 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). The first nucleic acid or one of the second nucleic acids contains a nucleotide sequence encoding a second HCF. The first HCF, the second HCF, and the first LCF are fragments of a bispecific antibody. The first RRS, the second RRS, and the third RRS are different from each other, and The enhanced expression locus consists of the nucleotide sequence of SEQ ID NO:

2.

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

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

13. The cell according to any one of claims 10-12, wherein the selective marker gene confers antibiotic resistance.

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

15. The cell of claim 10, wherein each of the first HCF, the first LCF, and the second HCF comprises a variable domain.

16. The cell of claim 15, wherein the variable domains contained in the first HCF are different from the variable domains in the second HCF.

17. The cell of claim 10, wherein the first nucleic acid and the second nucleic acid together encode a full-length bispecific antibody.

18. The cell of claim 10, wherein the cell is a CHO cell.

19. A method for generating bispecific antibodies, comprising: Culture mammalian cells according to any one of claims 10-18; and The bispecific antibody is generated from the cells.

20. A method for generating bispecific antibodies, comprising: To obtain the combination of any one of claims 1-9, Introducing the aforementioned set of vectors into the mammalian cells, and The bispecific antibody is generated from the cells.

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