Co-expression of DNase in host cells

Abstract

The present disclosure relates to the introduction of expression constructs encoding different gene products, such as proteins or nucleic acids, into a host cell line containing a plurality of docking sites for insertion of nucleic acid constructs in defined ratios, and the production of proteins requiring the expression of at least two gene products.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 580,228, filed September 1, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Statement on Federally Funded Research This invention was completed with government funding granted by the National Institutes of Health (NIH) under license number CA197574. The government holds specific rights to this invention. Technical Field

[0003] This disclosure provides host cells that allow co-expression of endonucleases with target products (e.g., 3E10 antibody or its antigen-binding fragment) to improve the expression and / or purification of target products (e.g., 3E10 antibody or its antigen-binding fragment) in host cell cultures. Background Technology

[0004] Therapeutic recombinant products are widely used for human medical purposes. Recombinant proteins can be expressed in various types of living organisms. CHO and HEK cells are some of the most commonly used host cells for manufacturing biopharmaceuticals. Although using host cells to produce therapeutic agents has many advantages, contamination of biopharmaceuticals with host cell DNA is a major concern for manufacturers. Genomic DNA contamination in biopharmaceuticals has been considered a potential risk factor for patients receiving recombinant protein drugs.

[0005] Therefore, biopharmaceutical manufacturers need to monitor for DNA contamination and keep impurities in their biologics below safety limits recommended by regulatory agencies (such as the FDA), which typically must be less than 100 pg DNA per milligram of protein. Removing unwanted DNA can be expensive, time-consuming, and reduce the efficiency of biologic protein recovery.

[0006] Therefore, what is needed in the art is a method for limiting the contamination of biopharmaceutical protein formulations by host cell DNA. Summary of the Invention

[0007] This disclosure provides a host cell that allows co-expression of a DNase with a target product (e.g., a 3E10 antibody or its antigen-binding fragment) to improve the expression and / or purification of the target protein in the host cell culture.

[0008] In some embodiments, this disclosure provides a eukaryotic host cell comprising (i) an exogenous nucleic acid sequence encoding a nuclease operably linked to a first promoter sequence and (ii) an exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a second promoter sequence.

[0009] In some implementation schemes, the host cells are selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, juvenile hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

[0010] In some implementation schemes, the host cells are selected from a group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, and CAP cells.

[0011] In some implementations, the host cell line is a glutamine synthase (GS) knockout cell line.

[0012] In some implementations, the host cell line is a dihydrofolate reductase (DHFR) knockout cell line.

[0013] In some implementations, the endonuclease is DNase I endonuclease.

[0014] In some implementations, the endonuclease is human DNAase I endonuclease.

[0015] In some implementations, the sequence encoding the endonuclease can also be operatively linked to the secretion signal sequence.

[0016] In some implementations, the first promoter sequence is a weak promoter sequence.

[0017] In some implementations, the first promoter sequence is the cytomegalovirus immediate early (CMV-IE) promoter sequence.

[0018] In some implementations, the CMV-IE promoter sequence is the simian CMV-IE promoter (sCMV) sequence.

[0019] In some implementations, the first promoter sequence is not a retroviral LTR promoter.

[0020] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises (a) a heavy chain polypeptide or a fragment thereof and (b) a light chain polypeptide or a fragment thereof, and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment comprises (1) a first exogenous nucleic acid sequence encoding the heavy chain polypeptide or a fragment thereof and (2) a second exogenous nucleic acid sequence encoding the heavy chain polypeptide or a fragment thereof.

[0021] In some implementations, the ratio of (i) the number of first exogenous nucleic acid sequences integrated into the host cell genome to (ii) the number of second exogenous nucleic acid sequences integrated into the host cell genome is 1:2 to 2:1.

[0022] In some implementations, a first exogenous nucleic acid sequence is operatively linked to a second promoter sequence, and the second exogenous nucleic acid sequence is operatively linked to a third promoter sequence.

[0023] In some implementations, the third promoter sequence has the same nucleotide sequence as the first promoter.

[0024] In some implementations, the third promoter sequence has the same nucleotide sequence as the second promoter.

[0025] In some implementations, the third promoter sequence has a different nucleotide sequence from the first promoter.

[0026] In some implementations, the third promoter sequence has a different nucleotide sequence than the second promoter.

[0027] In some implementations, the third promoter sequence is the cytomegalovirus immediate early (CMV-IE) promoter sequence.

[0028] In some implementations, the CMV-IE promoter sequence is the simian CMV-IE promoter (sCMV) sequence.

[0029] In some implementations, the third promoter sequence is not a retroviral LTR promoter.

[0030] In some implementations, the 3E10 antibody or its antigen-binding fragment includes a monovalent, bivalent, or multivalent single-chain variable fragment (scFv).

[0031] In some embodiments, the 3E10 antibody or its antigen-binding fragment includes scFv-Fc peptide, CrossMab peptide, dual variable domain immunoglobulin (DVD-Ig), tandem dual scFv, (scFv)2, single-chain tandem variable (scTaFv) peptide, single-chain variable (scFv) peptide, dual antibody, tandem dual antibody (TandAb), Fabsc peptide, modular IgG-scFv, or F(ab')2.

[0032] In some implementations, the 3E10 antibody or its antigen-binding fragment is humanized.

[0033] In some implementations, the 3E10 antibody or its antigen-binding fragment is a bivalent antibody or a fragment thereof.

[0034] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: In some embodiments, the heavy chain variable region (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 58, the VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and the VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60; and In some embodiments, the light chain variable region (VL) CDR1 contains the amino acid sequence of SEQ ID NO: 61, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 62, and the VLCDR3 contains the amino acid sequence of SEQ ID NO: 63.

[0035] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 64, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and In some embodiments, VL CDR1 contains the amino acid sequence of SEQ ID NO: 9, VL CDR2 contains the amino acid sequence of SEQ ID NO: 10, and VL CDR3 contains the amino acid sequence of SEQ ID NO: 11.

[0036] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 64, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and In some embodiments, VL CDR1 contains the amino acid sequence of SEQ ID NO: 9, VL CDR2 contains the amino acid sequence of SEQ ID NO: 10, and VL CDR3 contains the amino acid sequence of SEQ ID NO: 11.

[0037] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) having at least 90% of the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 14 and a light chain variable region (VL) having at least 90% of the same amino acid sequence as SEQ ID NO: 7.

[0038] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) having at least 90% of the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 14 and a light chain variable region (VL) having at least 95% of the same amino acid sequence as SEQ ID NO: 7.

[0039] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 104-113 and a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 114-122.

[0040] In some implementations, the second promoter sequence has the same nucleotide sequence as the first promoter.

[0041] In some implementations, the second promoter sequence has a different nucleotide sequence from the first promoter.

[0042] In some implementations, the second promoter sequence is the cytomegalovirus immediate early (CMV-IE) promoter sequence.

[0043] In some implementations, the CMV-IE promoter sequence is the simian CMV-IE promoter (sCMV) sequence.

[0044] In some implementations, the third promoter sequence is not a retroviral LTR promoter.

[0045] In some implementations, 2 to 500 copies of each of the exogenous nucleic acid sequence encoding a nuclease and the exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment are stably integrated into the genome of the host cell.

[0046] In some implementations, 5 to 500 copies of each of the first exogenous nucleic acid sequence and at least the second exogenous nucleic acid sequence are stably integrated into the genome of the host cell.

[0047] In some implementations, the ratio of (i) the number of exogenous nucleic acid sequences encoding endonucleases to (ii) the number of exogenous nucleic acid sequences encoding 3E10 antibodies or their antigen-binding fragments is 1:1 to 1:100.

[0048] In some implementations, the ratio of (i) the number of exogenous nucleic acid sequences encoding endonucleases to (ii) the number of exogenous nucleic acid sequences encoding 3E10 antibodies or their antigen-binding fragments is 1:2 to 1:100.

[0049] In some implementations, the exogenous nucleic acid sequence encoding a nuclease and the exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment are stably integrated into the host cell genome at the docking site.

[0050] In some embodiments, before integrating the exogenous nucleic acid sequence encoding a nuclease and the exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof into the docking site, the docking site comprises at least one docking site insert element, and the exogenous nucleic acid sequence encoding the nuclease and the exogenous nucleic acid sequence encoding the 3E10 antibody or an antigen-binding fragment thereof are present in one or more transfer vectors, the transfer vectors comprising at least one insert element compatible with at least one docking site insert element.

[0051] In some embodiments, a foreign nucleic acid sequence encoding a nuclease is operatively linked to a first polyadenylated sequence, and a foreign nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is operatively linked to a second polyadenylated sequence.

[0052] In some implementations, the host cell genome contains 5 to 500 docking sites, and each docking site contains at least one docking site insertion element.

[0053] In some implementations, the integrated docking site is independently located throughout the host cell genome.

[0054] In some embodiments, this disclosure provides a cell culture comprising host cells as described herein.

[0055] In some embodiments, this disclosure provides a method for expressing a 3E10 antibody or an antigen-binding fragment thereof, the method comprising culturing multiple host cells as described herein in a culture medium under conditions that achieve (i) expression of a nuclease by an exogenous nucleic acid sequence encoding a nuclease and (ii) expression of a 3E10 antibody or an antigen-binding fragment thereof.

[0056] In some implementation schemes, cultivation is carried out under feed-in batch conditions.

[0057] In some embodiments, this disclosure provides a cell culture supernatant containing a 3E10 antibody or an antigen-binding fragment thereof prepared according to the methods described herein.

[0058] In some embodiments, this disclosure provides a cell culture supernatant containing an antibody titer comprising at least 0.001 μg / mL, at least 0.01 μg / mL, at least 0.1 μg / mL, at least 0.125 μg / mL, at least 0.250 μg / mL, at least 0.5 μg / mL, at least 0.75 μg / mL, at least 1 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL of 3E10 antibody or its antigen-binding fragment.

[0059] In some implementations, less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or 0% of the 3E10 antibody or its antigen-binding fragment binds to the nucleic acid.

[0060] In some embodiments, this disclosure provides a composition comprising a 3E10 antibody or an antigen-binding fragment thereof generated according to the methods described herein. In some embodiments, this disclosure provides a composition comprising a 3E10 antibody or an antigen-binding fragment thereof purified from the cell culture supernatant described herein.

[0061] In some embodiments, this disclosure provides a host cell culture comprising eukaryotic host cells containing an exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operatively linked to a promoter sequence and a purified exogenously added endonuclease.

[0062] In some embodiments, this disclosure provides a method for preparing a host cell culture comprising: a eukaryotic host cell containing a foreign nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a promoter sequence; and a purified, foreign-added endonuclease added to the host cell culture. Attached Figure Description

[0063] Figure 1A and Figure 1B The final reverse transcription vector expression construct contains the human DNase 1 nucleic acid sequence and the flanking DNA cloning junction region (SEQ ID NO: 139). Figure 1A ) and the amino acid sequence of the DNase (SEQ ID NO: 140, Figure 1B ).

[0064] Figure 2Map of the 6187bp initiation reverse transcription vector pCS-novel MCS-WPRE (novel ori).

[0065] Figure 3 GPEx ® Map of the human DNase 1 pathway gene in the vector pCS-CFSD1-WPRE (new ori) 6977bp.

[0066] Figure 4A and Figure 4B Sequence data of LC CDS in 207attB-GS-h3E10LC-WPRE. Figure 4A The nucleic acid sequence (SEQ ID NO: 141) was provided, and Figure 4B The amino acid sequence (SEQ ID NO: 142) is provided.

[0067] Figure 5 The spectrum of plasmid 207attB-GS-h3E10LC-WPRE.

[0068] Figure 6. HC CDS sequence data in 207attB-GS-h3E10HC-WPRE. Figure 6A The nucleic acid sequence (SEQ ID NO: 143) was provided, and Figure 6B The amino acid sequence (SEQ ID NO: 144) is provided.

[0069] Figure 7 Map of plasmid 207attB-GS-h3E10HC-WPRE Figure 8. Sequence data of the pathway CDS in 215-puc19attB287-GS-pathway-WPRE-TKpa. Figure 8A The nucleic acid sequence (SEQ ID NO: 145) was provided, and Figure 8B The amino acid sequence (SEQ ID NO: 146) is provided.

[0070] Figure 9 Map of plasmid 215-puc19attB287-GS-pathway-WPRE-TKpa Figure 10 SDS-PAGE results of merged cells.

[0071] Figure 11 A graph showing the density of live cells.

[0072] Figure 12 A graph showing the percentage of cell viability.

[0073] Figure 13A graph showing antibody titers.

[0074] Figure 14 A graph showing protein concentration (mg / ml) relative to clone number.

[0075] Figure 15 A graph showing the Ambr15™ IVCD relative to the clone number.

[0076] Figure 16 A graph showing Ambr15™ rQp relative to the clone number.

[0077] Figure 17 The amino acid sequence of the parental 3E10 monoclonal antibody is shown.

[0078] Figure 18A , Figure 18B and Figure 18C The D31N variant of the 3E10 monoclonal antibody is shown in some embodiments according to this disclosure. Figure 18A Other CDR variants () Figure 18B ) and other CDR variants covered ( Figure 18C The amino acid sequence of ).

[0079] Figure 19 Exemplary charge-conserving CDR variants of the 3E10 monoclonal antibody are shown according to various embodiments of the present disclosure.

[0080] Figure 20 Exemplary CDR variants containing combinations of amino acid substitutions, charged conserved amino acid substitutions, and rationally designed amino acid substitutions, according to various embodiments of the present disclosure, are shown.

[0081] Figure 21 The amino acid sequence of the humanized 3E10 variable heavy chain (3E10-VH) domain is shown according to various embodiments of the present disclosure.

[0082] Figure 22 The amino acid sequence of a mature humanized 3E10 heavy chain (3E10-HC) lacking a signal peptide is shown according to various embodiments of the present disclosure.

[0083] Figure 23 The amino acid sequences of the humanized 3E10 heavy chain (3E10-HC) according to various embodiments of the present disclosure are shown.

[0084] Figure 24 The amino acid sequence of the humanized 3E10 variable light chain (3E10-VL) domain is shown according to various embodiments of the present disclosure.

[0085] Figure 25The amino acid sequence of a mature humanized 3E10 light chain (3E10-LC) lacking a signal peptide is shown according to various embodiments of the present disclosure.

[0086] Figure 26 The amino acid sequence of the humanized 3E10 light chain (3E10-LC) according to various embodiments of the present disclosure is shown.

[0087] Figure 27 Sequence alignments of instances of humanized 3E10 heavy chain variable regions are shown, with CDRs underlined as indicated.

[0088] Figure 28 Sequence alignment of an instance of the variable region of the humanized 3E10 heavy chain is shown, with the CDR and the putative nuclear localization signal (NLS) underlined as indicated.

[0089] Figure 29A , Figure 29B , Figure 29C , Figure 29D and Figure 29E Sequence alignments of instances of humanized scFv constructs of the 3E10 monoclonal antibody are shown together.

[0090] Figure 30A and Figure 30B An electrostatic surface potential rendering of the molecular model of the 3E10-scFv construct is shown, revealing the putative nucleic acid binding pocket (NAB1). Figure 30A Additionally, the predicted structural and electrostatic potential changes induced by the amino acid substitution at residue 31 of HC CDR1 are shown. Figure 30B This is an illustration of the molecular modeling of 3E10-scFv (Pymol), in which the NAB1 amino acid residues are highlighted by dots.

[0091] Figure 31A The expression (μg / ml) of IgG1 in the supernatant of the 3E10 variant (D31N or V66) is shown.

[0092] Figure 31B The expression of SEAP (fold relative to untransfected cells) is shown in the presence of exogenous DNase treatment.

[0093] definition To facilitate understanding of this disclosure, a number of terms are defined below.

[0094] As used herein, unless the context explicitly indicates otherwise, the singular forms “a” and “the” are intended to also include the plural forms. It should also be understood that the term “and / or” as used herein refers to and covers any and all possible combinations of one or more of the relevant listed items. Unless the context requires otherwise, it will also be understood that, when used in this specification, the terms “comprising,” “including,” or any variation thereof specify the presence of the defined features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or groups thereof. Furthermore, within the scope of the use of the terms “comprising,” “having,” “having,” or variations thereof in the Detailed Description and / or Claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” Additionally, where the terms “comprising,” “including,” “having,” “having,” or variations thereof are used in the Detailed Description and / or Claims, the alternative expressions “consisting of” or “substantially consisting of” are intended to cover such disclosure.

[0095] Unless otherwise stated herein, references to value ranges herein are intended only to serve as a shorthand for each individual value falling within that range, and each individual value is incorporated into the specification as if it were referenced separately herein.

[0096] The term “about” is used to describe values ​​that are approximately + / - 10% above or below the specified value.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" is used in the broadest sense and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigenic determinant of an antibody, and antibody fragments (such as Fab, Fab', F(ab')2, Fv fragments, scFv molecules), as well as any other modified immunoglobulin molecule containing an antigen recognition site, provided they exhibit one or more of the desired biological activities. In embodiments, the "desired biological activity" of an antibody refers to the ability of the antibody to bind to its target antigen (e.g., a nucleic acid, such as DNA). In embodiments, the "desired biological activity" may also include the antibody binding to its target antigen and producing a measurable biological response, which can be measured in vitro or in vivo. This activity can be antagonistic or agonistic. In the implementation scheme, the “desired biological activity” of an antibody refers to the antibody’s ability to bind to a target (e.g., a nucleic acid molecule). In the implementation scheme, the “desired biological activity” of an antibody refers to the antibody’s ability to bind to a cell receptor (e.g., ENT2). In the implementation scheme, the “desired biological activity” of an antibody refers to the antibody’s ability to be internalized by target cells. As used herein, “target antigen” refers to a molecule that is specifically bound to an antigen-binding domain comprising a variable region of a given antibody. The term “specific binding” means that an antibody binds to its homologous antigen (e.g., a nucleic acid, such as DNA) without significantly binding to other antigens.

[0098] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their constant domains in the heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. As used herein, "isotype" refers to any of the subclasses of an immunoglobulin defined by the chemical and antigenic properties of its constant domain. The constant domains of the heavy chain corresponding to the different classes of immunoglobulins are referred to as α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known and generally described, for example, by Abbas et al. Cellular and Mol. Immunology, In the 4th edition (WBSaunders, Co., 2000). It should be understood that the antibodies disclosed herein may also include hybrids of isotypes and / or subtypes.

[0099] The antibodies disclosed herein are generally isolated or recombinant. When “isolated” is used to describe the various polypeptides disclosed herein, it refers to polypeptides that have been identified and isolated and / or recovered from cells or cell cultures expressing them. Typically, isolated polypeptides are prepared by at least one purification step. “Isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigen specificities. As used herein, “recombinant antibody” refers to an antibody generated in exogenous host cells using recombinant nucleic acid technology, and recombinant antibodies may also be isolated.

[0100] "Natural antibodies" are typically heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds varies between different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable domains of the light chain are aligned with the variable domains of the heavy chain. Specific amino acid residues are believed to form the interface between the variable domains of the light and heavy chains.

[0101] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain containing the antigen-binding site. The constant domain contains heavy chain CH1, CH2, and CH3 domains (collectively referred to as CH) and a light chain CHL (or CL) domain.

[0102] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain may be referred to as the “VH”, and the variable domain of the light chain as the “VL”. These domains are typically the most variable parts of an antibody and contain antigen-binding sites. The term “variable” refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domains of an antibody. It is concentrated in three segments within both the light and heavy chain variable domains, known as the hypervariable region (HVR) or complementarity-determining region (CDR), which confer antigen specificity. The “variable heavy chain domain” pairs with the “variable light chain domain” to form the antigen-binding domain (ABD) that specifically binds to the target antigen. The more highly conserved portions of the variable domain are called the frame region (FR). The variable domains of the natural heavy and light chains each contain four FR regions, which are mostly in a β-sheet configuration and linked by three CDR / HVRs. These CDR / HVRs form loops that connect the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDR / HVRs in each chain are tightly held together by the FR regions and, together with CDR / HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al.). Sequences of Proteins of Immunological Interest , 5th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits various effector functions, such as antibody participation in antibody-dependent cytotoxicity.

[0103] In this document, the terms “hypervariant region,” “HVR,” “HV,” “complementarity-determining region,” and “CDR” are used interchangeably to refer to regions within the variable domains of an antibody that are highly variable in sequence and / or form structurally defined loops. Generally, an antibody contains six HVRs or CDRs; three in the VH (H1, H2, H3; or VH CDR1, VH CDR2, VH CDR3), and three in the VL (L1, L2, L3; or VL CDR1, VL CDR2, VL CDR3).

[0104] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be classified into one of two distinct types based on the amino acid sequence of their constant domains: kappa (“κ”) and lambda (“λ”).

[0105] The CDRs of the VH and VL domains together form the Fv region. In an embodiment, the VH and VL domains contain six CDRs of the ABD. In the “Fab” form, the variable heavy chain domain (VH; containing VH CDR1, VH CDR2, and VH CDR3) and the variable light chain domain (VL or VL; containing VL CDR1, VL CDR2, and VL CDR3) contain a group of six CDRs, wherein the C-terminus of the VH domain is attached to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the VL domain is attached to the N-terminus of the constant light chain domain (and thus forms the light chain). In the “scFv” form, the VH and VL domains are typically covalently attached to a single polypeptide sequence using a linker (e.g., a “scFv linker”), the N-terminus to C-terminus arrangement of which can be VH-linker-VL or VL-linker-VH. Generally, the C-terminus of the scFv structural domain is attached to the N-terminus of the hinge in the second monomer.

[0106] As used herein, "Fab" or "Fab region" refers to a polypeptide that typically contains VH, CH1, VL, and CL immunoglobulin domains on two separate polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on the other). Fab can refer to this region in its isolated state or in the context of the antibody disclosed herein. In embodiments, Fab includes an Fv region in addition to the CH1 CL domain.

[0107] The other part of the heavy chain is the hinge region. As used herein, “hinge,” “hinge region,” “antibody hinge region,” or “hinge domain” refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain terminates at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Therefore, for IgG, the antibody hinge is defined herein as encompassing positions 216 (E216 in IgG1) through 230 (p230 in IgG1), where the numbering is based on the EU index in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either or both of the N-terminus and C-terminus of the hinge domain.

[0108] As used herein, a “heavy chain constant region” refers to the CH1-hinge-CH2-CH3 portion of an antibody or a fragment thereof, excluding the variable heavy chain domain. In embodiments, the heavy chain constant region comprises amino acids 118-447 of human IgG1 as designated by the EU. As used herein, a “heavy chain constant region fragment” refers to a heavy chain constant region containing fewer amino acids from either or both of the N-terminus and C-terminus but still retaining the ability to form a dimer with another heavy chain constant region.

[0109] As used herein, “Fv,” “Fv fragment,” or “Fv region” refers to a polypeptide containing the VL and VH domains of an antibody-binding domain. The Fv region can take the form of either Fab or scFv, where the VL and VH domains combine (e.g., by means of a linker, as discussed herein) to form scFv.

[0110] As used herein, “Fc,” “Fc region,” or “Fc domain” refers to a polypeptide containing the CH2-CH3 domain of an IgG molecule and, in some cases, a hinge. In the EU designation of human IgG1, the CH2-CH3 domain contains amino acids 231 to 447, and the hinge is 216 to 230. Therefore, the definition of an “Fc domain” includes both or fragments of amino acids 231-447 (CH2-CH3) and 216-447 (hinge-CH2-CH3) of IgG1. In this case, the “Fc fragment” may contain fewer amino acids from either or both of the N-terminus and C-terminus, but retains the ability to form a dimer with another Fc domain or Fc fragment, as detectable using standard size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). In embodiments, the disclosed antibody contains a human Fc domain. In embodiments, the disclosed antibody contains an Fc domain from human IgG1, IgG2, or IgG4.

[0111] Compared to the parental Fc domain, the “variant Fc domain” contains amino acid modifications. Therefore, compared to the human IgG1 Fc domain, the “variant human IgG1 Fc domain” is a domain containing amino acid modifications (typically amino acid substitutions, but including amino acid deletions in the case of ablation variants). In embodiments, the variant Fc domain has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with the corresponding parental IgG Fc domain. In embodiments, the identity percentage is calculated using an identity algorithm discussed below. In embodiments, the identity percentage is calculated using the BLAST algorithm known in the art, with default parameters. In embodiments, the variant Fc domain has 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications compared to the parental Fc domain. In the implementation, the variant Fc domain retains the ability to form a dimer with the Ir Fc domain, as measured using known techniques such as non-denaturing gel electrophoresis as described herein.

[0112] For all antibody-related positions discussed in this disclosure, unless otherwise indicated, amino acid position numbering is based on the EU index. The EU index in Kabat or the EU numbering scheme refers to the number of the EU antibody. Kabat et al. collected a large number of primary sequences of variable regions of the heavy and light chains. Based on the degree of sequence conservation, they classified the individual primary sequences into CDRs and frames and provided a list of them. See [link to relevant documentation]. SEQUENCES OF IMMUNOLOGICAL INTEREST , 5th edition, NIH Publication No. 91-3242, EA Kabat et al.; Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the contents of which are incorporated herein by reference. In embodiments of this disclosure, amino acid position numbering is based on the IMGT system.

[0113] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to an antibody in essentially its complete form, rather than an antibody fragment as defined below. Specifically, this term refers to antibodies whose heavy chains contain the Fc region.

[0114] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, biantibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0115] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, for example, individual antibodies constituting that population are identical except for possible mutations (e.g., naturally occurring mutations) that may be present in small amounts. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such a monoclonal antibody typically comprises an antibody containing a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process including selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a library of clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence may be further modified, for example, to increase affinity for the target, humanize the target-binding sequence, increase its yield in cell cultures, reduce its immunogenicity in vivo, construct multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of this disclosure. Compared to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated by other immunoglobulins.

[0116] The antibodies described herein specifically include “chimeric” antibodies, wherein a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit one or more desired biological activities (see, for example, U.S. Patent No. 4,816,567; and Morrison et al.). Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one mammalian species (e.g., mice, rats, rabbits, etc.) that possess the desired specificity, affinity, and / or capability, while the constant regions are sequence homologous to antibodies derived from another mammalian species (e.g., humans) to avoid triggering an immune response in that species. Chimeric antibodies include PRIMATTZED® antibodies, wherein the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque with a target antigen.

[0117] A “humanized” form of a nonhuman (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In embodiments, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the receptor’s CDR / HVR are replaced by residues of the nonhuman species (donor antibody) (such as mouse, rat, rabbit, or nonhuman primate) having the desired specificity, affinity, and / or capability. In some cases, FR residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, the humanized antibody may contain residues not found in the receptor or donor antibody. These modifications can be made to further optimize antibody performance. Generally, the humanized antibody will contain at least one and typically substantially all of two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the nonhuman immunoglobulin, and all or substantially all of the FRs correspond to those of the human immunoglobulin sequence. Optionally, the humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc) typically of the human immunoglobulin. See, for example, Jones et al. Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409. Examples of methods for generating humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641, which are incorporated herein by reference in their entirety.

[0118] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of a human-derived antibody and / or being produced using any technique known in the art. This definition of a human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy Alan R. Liss, p. 77 (1985); Boerner et al. J. Immunol., The method described in 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as immunized xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 relating to XENOMOUSE™ technology). Also see, for example, Li et al., regarding human antibodies generated using human B-cell hybridoma technology. Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).

[0119] "Species-dependent antibodies" are antibodies that have a stronger binding affinity for antigens from a first mammalian species than for homologs of that antigen from a second mammalian species. Typically, species-dependent antibodies exhibit "specific binding" to human antigens (e.g., a binding affinity (Kd) value not exceeding approximately 1 × 10⁻⁶). -7 M, preferably not exceeding approximately 1×10 -8 M and preferably not exceeding about 1×10 -9M), but the binding affinity for antigenic homologs from a second non-human mammal species is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity for human antigens. Species-dependent antibodies can be any of the various types of antibodies as defined above, but are preferably humanized antibodies or human antibodies.

[0120] The term "linear antibody" refers to the work of Zapata et al. (1995). Protein Eng, The antibodies described in 8(10):1057-1062) are described in short. In short, these antibodies contain a pair of tandem Fd regions (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0121] As used in this article, “modification” refers to amino acid substitutions, insertions, deletions, and / or any other mutations in a polypeptide sequence.

[0122] As used herein, “variant protein” or “protein variant” or “variant” means a protein that differs from a parent protein due to at least one amino acid modification. A protein variant has at least one amino acid modification compared to the parent protein, but not so many that the variant protein cannot be aligned with the parent protein using the alignment procedure described below. Generally, using any alignment procedure known in the art (such as BLAST), the variant proteins described herein (such as variant Fc domains, etc.) are typically at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identical to the parent polypeptide.

[0123] Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured using algorithms such as those in the following literature: Smith, TF and Waterman, MS (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [Local homology algorithm]; Needleman, SB and Wunsch, CD. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [Homology alignment algorithm]; Pearson, WR and Lipman, DJ (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (USA) 85:2444 [Similarity search method]; or Altschul, SF et al., (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10, “BLAST” algorithm, see the webpage at URL blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the above algorithms, use the default parameters (window length, space penalty, etc.). Unless otherwise explicitly stated, use the BLAST algorithm with the default parameters to determine sequence identity.

[0124] In the embodiments, the parental polypeptide (e.g., the Fc parental polypeptide) is a human wild-type sequence, such as a heavy chain constant domain or Fc region from IgG1, IgG2, IgG3, or IgG4, but human sequences with variants may also be used as "parental polypeptides". In the embodiments, the antibody sequence described herein has at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the parental polypeptide sequence. Therefore, as used herein, "antibody variant" or "variant antibody" means an antibody that is different from the parent antibody due to at least one amino acid modification; as used herein, "IgG variant" or "variant IgG" means an IgG that is different from the parent IgG (e.g., the human IgG sequence) due to at least one amino acid modification; as used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that is different from the parent immunoglobulin sequence due to at least one amino acid modification; and as used herein, "Fc variant" or "variant Fc" means an Fc that is different from the parent Fc (e.g., the Fc domain of human IgG1, IgG2, IgG3, or IgG4) due to at least one amino acid modification.

[0125] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of an IgG isotype into the corresponding amino acid in a different alignment IgG isotype. For example, because IgG1 contains tyrosine at EU position 296 and IgG2 contains phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0126] As used herein, “non-naturally occurring modification” means a non-isotype amino acid modification. For example, since no human IgG contains serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a non-naturally occurring modification.

[0127] As used herein, the interchangeable terms “oligonucleotide” and “polynucleotide” refer to a linear polymer or analogue of natural or modified nucleoside monomers linked by phosphodiester bonds. The term “oligonucleotide” generally refers to a shorter polymer, such as containing about 3 to about 100 monomers, and the term “polynucleotide” generally refers to a longer polymer, such as containing about 100 monomers to thousands of monomers, such as 10,000 monomers or more. Oligonucleotides and polynucleotides can be natural or synthetic. Oligonucleotides and polynucleotides may include deoxyribonucleosides, ribonucleosides, and / or their non-natural analogues. In embodiments, oligonucleotides or polynucleotides are capable of binding specifically to a target genome via regular patterns of inter-monomer interactions, such as Watson-Crick base pairing, base stacking, Hogstein-type or anti-Hogstein base pairing, etc. As used herein, “functional nucleic acid” refers to a nucleic acid that has a biological function in vivo or in cells, such as enzyme function, catalytic function, or biological inhibitory or enhancing function (e.g., inhibiting or enhancing transcription or translation). In the implementation plan, examples of functional nucleic acids include, but are not limited to, siRNA, ASO, shRNA, miRNA (including pri-miRNA and pre-miRNA), nucleic acid aptamers (including RNA aptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitch, U1 adaptor, molecular beacon, and transcription factor binding regions.

[0128] As used herein, “3E10 antibody” refers to an antibody having a set of heavy chain CDRs (VH CDR1, VH CDR2, and VH CDR3) identified according to the Kabat system, containing amino acid sequences that differ from SEQ ID NO: 58, 59, and 60 by no more than two amino acids, respectively; a set of light chain CDRs (VL CDR1, VL CDR2, and VL CRD3) containing amino acid sequences that differ from SEQ ID NO: 61, 62, and 63 by no more than two amino acids, respectively; capable of binding nucleic acids and possessing cell penetration at least upon binding to nucleic acids; and its antigen-binding fragment. As described herein, the 3E10 antigen is a polynucleotide.

[0129] As used herein, the term "cell-penetrating" refers to the ability of an antibody or its antigen-binding fragment to penetrate cells (e.g., mammalian cells) without the aid of an exogenous transport medium (such as liposomes or conjugated cell-penetrating peptides). Regarding the 3E10 antibody and its antigen-binding fragment, a cell-penetrating antibody or its antigen-binding fragment can penetrate cells expressing the ENT2 receptor on their cell surface in the presence of nucleic acids (e.g., nucleic acids non-covalently bound and / or conjugated to the 3E10 antibody or its antigen-binding fragment), thereby leading to the internalization of the 3E10 antibody and its antigen-binding fragment. In some embodiments, the cell-penetrating 3E10 antibody or its antigen-binding fragment is conjugated to a functional molecule (e.g., a chemical agent, polynucleotide, or polypeptide). While cell-penetrating molecules are generally referred to herein as "cell-penetrating antibodies," it will be understood that fragments for use in the compositions, conjugates, and methods disclosed herein are also explicitly provided, including antigen-binding fragments, variants, binding proteins, and fusion proteins, such as scFv, discFv, triscFv, and other single-chain variable fragments and other cell-penetrating molecules disclosed herein. Autoantibodies against double-stranded deoxyribonucleic acid (dsDNA) are frequently identified in the serum of patients with systemic lupus erythematosus (SLE) and are often associated with disease pathogenesis. Therefore, in embodiments, cell-penetrating antibodies (e.g., cell-penetrating anti-DNA antibodies) may be derived from or isolated from SLE patients or animal models of SLE.

[0130] As used herein, the term “host cell” refers to any eukaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

[0131] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., those with an immortalized phenotype), primary cell cultures, limited cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.

[0132] As used herein, the term "vector" refers to any genetic element, such as a plasmid, bacteriophage, transposon, kinase, chromosome, virus, virion, etc., which, when combined with appropriate control elements, is capable of replicating and transferring gene sequences between cells. Therefore, the term includes cloning and expression vectors as well as viral vectors.

[0133] As used in this article, the term “genome” refers to the genetic material of an organism (e.g., chromosomes).

[0134] The term "target nucleotide sequence" refers to any nucleotide sequence (e.g., RNA or DNA) that, for any reason (e.g., treating a disease, conferring improved qualities, expressing a target protein in a host cell, expressing a ribozyme, etc.), would be desirable to manipulate by someone skilled in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.) and non-coding regulatory sequences that do not encode mRNA or protein products (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.).

[0135] As used herein, the term "target product" refers to a protein or nucleic acid product encoded by a target nucleic acid, such as a viral backbone or genome. In some implementations, multiple target products are expressed in host cells.

[0136] As used in this article, the term "target protein" refers to a protein encoded by a target nucleic acid.

[0137] As used herein, the terms “nucleic acid molecule encoding…”, “DNA sequence encoding…”, “DNA encoding…”, “RNA sequence encoding…”, and “RNA encoding…” refer to the sequence of deoxyribonucleotides or ribonucleotides along the chain of deoxyribonucleic acid or ribonucleic acid. The sequence of these deoxyribonucleotides or ribonucleotides determines the sequence of amino acids along the polypeptide (protein) chain. Therefore, a DNA or RNA sequence encodes an amino acid sequence.

[0138] As used herein, the terms “promoter,” “promoter element,” or “promoter sequence” refer to a DNA sequence that, when linked to a target nucleotide sequence, controls the transcription of that target nucleotide sequence into mRNA. A promoter is typically (but not necessarily) located at the 5' (i.e., upstream) of the target nucleotide sequence it controls into mRNA and provides a site for the specific binding of RNA polymerase and other transcription factors to initiate transcription.

[0139] Transcriptional control signals in eukaryotes include “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with the cellular proteins involved in transcription (Maniatis et al., Science 236:1237

[1987] ). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (similar control elements, i.e., promoters, are also present in prokaryotes). The choice of a particular promoter and enhancer depends on the cell type used to express the target protein. Some eukaryotic promoters and enhancers have a broad host range, while others function in limited cell type subpopulations (see reviews by Voss et al., Trends Biochem. Sci., 11:287

[1986] ; and Maniatis et al., ibid.). For example, the SV40 early gene enhancer is highly active in a variety of cell types from many mammalian species and has been widely used for protein expression in mammalian cells (Dijkema et al., EMBO J.4:761

[1985] ). Two other examples of promoter / enhancer elements active in a wide range of mammalian cell types are those from the human elongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791

[1989] ; Kim et al., Gene 91:217

[1990] ; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322

[1990] ), and long terminal repeats of Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777

[1982] ) and long terminal repeats of human cytomegalovirus (Boshart et al., Cell 41:521

[1985] ).

[0140] As used herein, the term "promoter / enhancer" refers to a DNA segment containing a sequence capable of providing both promoter and enhancer functions (i.e., functions provided by promoter elements and enhancer elements, see the discussion of these functions above). For example, the long terminal repeat sequence of a retrovirus contains both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterogeneous." An "endogenous" enhancer / promoter is an enhancer / promoter naturally linked to a given gene in the genome. An "exogenous" or "heterogeneous" enhancer / promoter is an enhancer / promoter juxtaposed with a gene through genetic manipulation (i.e., molecular biology techniques such as cloning and recombination), such that the transcription of that gene is directed by the linked enhancer / promoter.

[0141] As used herein, the term "LTR" for "long terminal repeat" refers to a transcriptional control element located in or isolated from the 5' and 3' regions of the U3 region of a retroviral genome. As is known in the art, long terminal repeat sequences can be used as control elements in retroviral vectors, or isolated from retroviral genomes and used to control expression from other types of vectors.

[0142] As used herein, the terms “complementarity” or “complementarity” are used to refer to polynucleotides (i.e., nucleotide sequences) that are related by base pairing rules. For example, the sequence “5'-AGT-3'” is complementary to the sequence “3'-TCA-5'”. Complementarity can be “partial,” where only some bases of the nucleic acids match according to the base pairing rules. Alternatively, there may be “complete” or “full” complementarity between nucleic acid chains. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and in detection methods that rely on the binding between nucleic acids.

[0143] When referring to nucleic acids, the terms "homology" and "percentage of identity" refer to the degree of complementarity. Partial homology (i.e., partial identity) or complete homology (i.e., complete identity) can exist. A partially complementary sequence is a sequence that at least partially inhibits hybridization between a completely complementary sequence and a target nucleic acid sequence, and is referred to using the functional term "substantially homologous." Inhibition of hybridization between a completely complementary sequence and a target sequence can be checked using hybridization assays (DNA or RNA blotting, solution hybridization, etc.) under low-tightness conditions. A substantially homologous sequence or probe (i.e., an oligonucleotide capable of hybridizing with another target oligonucleotide) will compete with and inhibit the binding (i.e., hybridization) of a completely homologous sequence to the target sequence under low-tightness conditions. This does not mean that low-tightness conditions allow non-specific binding; low-tightness conditions require that the binding of the two sequences to each other is a specific (i.e., selective) interaction. The presence or absence of non-specific binding can be tested by using a second target that even lacks a partial degree of complementarity (e.g., identity less than about 30%); in the absence of non-specific binding, the probe will not hybridize with the second non-complementary target.

[0144] As used herein, the terms "in an operable combination," "in an operable order," and "operably linked" refer to the linking of nucleic acid sequences in a manner that produces a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linking of amino acid sequences in a manner that produces a functional protein.

[0145] As used herein, the term “selective marker” refers to a gene that encodes enzyme activity or other proteins that confers the ability to grow in a culture medium lacking nutrients that would otherwise be essential; additionally, selective markers can confer resistance to antibiotics or drugs to cells that express selective markers.

[0146] As used herein, the term "retrovirus" refers to a retroviral particle capable of entering a cell (i.e., containing membrane-binding proteins, such as envelope proteins or viral G glycoproteins, which bind to the surface of the host cell and facilitate the entry of the viral particle into the cytoplasm of the host cell) and integrating the retroviral genome (as a double-stranded provirus) into the host cell's genome. The term "retrovirus" encompasses the subfamilies of tumor viruses (e.g., Moloney mouse leukemia virus (MoMLV), Moloney mouse sarcoma virus (MoMSV), and mouse mammary tumor virus (MMTV)), foam viruses, and lentiviruses (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and goat arthritis-encephalitis virus; see, for example, U.S. Patent Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

[0147] As used herein, the term "retroviral vector" refers to a retrovirus that has been modified to express a target gene. Retroviral vectors can be used to efficiently transfer genes into host cells by utilizing the viral infection process. Foreign or heterologous genes cloned (i.e., inserted using molecular biology techniques) into the retroviral genome can be efficiently delivered to host cells susceptible to retroviral infection. The replication capacity of the retroviral genome can be disrupted through well-known genetic manipulations. The resulting replication-deficient vectors can be used to introduce new genetic material into cells, but they cannot replicate. Helper viruses or packaging cell lines can be used to allow vector particles to assemble and be expelled from the cell. Such retroviral vectors contain a replication-deficient retroviral genome containing a nucleic acid sequence encoding at least one target gene (i.e., a polycistronic nucleic acid sequence encoding more than one target gene), a 5' retroviral long terminal repeat (5' LTR), and a 3' retroviral long terminal repeat (3' LTR).

[0148] As used herein, the term "lentiviral vector" refers to a retroviral vector derived from the Lentiviralidae family (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus) that is capable of integrating into non-dividing cells (see, for example, U.S. Patent Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

[0149] As used herein, the term "transposon" refers to a transposable element (e.g., Tn5, Tn7, and Tn10) that can move or transpose from one location in the genome to another. Generally, transposition is controlled by transposases. As used herein, the term "transposon vector" refers to a vector encoding a target nucleic acid with a transposon end. Examples of transposon vectors include, but are not limited to, those described in U.S. Patents 6,027,722, 5,958,775, 5,968,785, 5,965,443, and 5,719,055, all of which are incorporated herein by reference.

[0150] As used herein, the term "adeno-associated virus (AAV) vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, and AAVX7. AAV vectors may contain one or more of the fully or partially deleted wild-type AAV genes, preferably the rep and / or cap genes, but retain functional flanking ITR sequences.

[0151] AAV vectors can be constructed using recombinant techniques known in the art to include one or more heterologous nucleotide sequences flanked at both ends (5' and 3'). In the practice of this disclosure, an AAV vector may contain at least one AAVITR and a suitable promoter sequence upstream of the heterologous nucleotide sequence and at least one AAVITR downstream of the heterologous sequence. "Recombinant AAV vector plasmid" refers to a class of recombinant AAV vectors containing a plasmid. As with general AAV vectors, the 5' and 3' ITRs are flanked by selected heterologous nucleotide sequences.

[0152] As used in this article, the term "adenovirus vector" refers to a non-enveloped double-stranded DNA vector containing an adenovirus backbone.

[0153] As used herein, the term "purified" refers to molecules (nucleic acid or amino acid sequences) that have been removed, isolated, or separated from their normal environment. Thus, an "isolated nucleic acid sequence" is a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free of, preferably at least 75% free of, and more preferably at least 90% free of other components they are typically associated with.

[0154] abbreviation AmpR = bacterial ampicillin resistance gene attB = bacterial attachment site attP = phage attachment site attR = Recombination upstream attachment site Skeleton = Plasmid skeleton CDS = Encoded Sequence EPR = MMLV Extended Packaging Area GCI = Gene Copy Index GS = Glutamine synthase H or HC = heavy chain hCMV = Human cytomegalovirus immediate early promoter I = Intron L or LC = light chain MoMuSV 5'LTR = 5' long terminal repeat sequence of Moloni mouse sarcoma virus Neo = neomycin resistance gene PA or PolyA = polyadenylation signal ProV SIN-LTR = Proviral self-inactivation long terminal repeat sequence sCMV = Immediate Early Promoter of Simian Cytomegalovirus SDS-PAGE = Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SIN-3'LTR = Self-inactivated 3' long terminal repeat sequence SV40 = Simian Virus 40 TK = thymidine kinase UTR = Non-translation area W or WPRE = Posttranscriptional regulatory element in marmots Detailed Implementation

[0155] This disclosure provides host cells that allow co-expression of a nuclease (e.g., DNase) with a target product (e.g., a 3E10 antibody or its antigen-binding fragment) to improve the expression and / or purification of the target product in host cell cultures. In some embodiments, an expression construct encoding a nuclease (e.g., DNase) and an expression construct encoding one or more target genes are introduced into the host cell at a defined ratio. In some embodiments, the host cell line contains multiple docking sites for inserting the nuclease construct. Cell lines containing multiple docking sites and expression constructs for use with these cells are described in WO2021247671 and WO2021247672, both of which are incorporated herein by reference in their entirety.

[0156] This disclosure addresses several problems by providing host cells that allow co-expression of a nuclease (e.g., DNase) with one or more target proteins (e.g., a 3E10 antibody or its antigen-binding fragment). In one aspect, co-expression of a nuclease (e.g., DNase) with one or more target products allows for more efficient removal of host cell DNA from pharmaceutical formulations. In another aspect, co-expression of a nuclease (e.g., DNase) with one or more target products allows for more efficient production of the target product (e.g., a 3E10 antibody or its antigen-binding fragment), particularly where the target product can interact with host cell DNA. In both aspects, the inventors have unexpectedly discovered that nucleases (e.g., DNases) can be stably co-expressed in host cell lines.

[0157] Therefore, in some embodiments, this disclosure provides a eukaryotic host cell comprising a first exogenous nucleic acid sequence operatively linked to a promoter sequence encoding a nuclease (e.g., a DNase, such as DNase I or DNase II) and at least a second exogenous nucleic acid sequence operatively linked to a promoter sequence encoding a first target product (e.g., a 3E10 antibody or an antigen-binding fragment thereof), wherein the first sequence encoding the nuclease (e.g., a DNase, such as DNase I or DNase II) and the at least second exogenous nucleic acid sequence encoding the first target product (e.g., a 3E10 antibody or an antigen-binding fragment thereof) are co-expressed in the host cell. In some embodiments, this disclosure provides a host cell culture comprising the host cell.

[0158] In some implementations, the host cell contains multiple exogenous nucleic acids encoding multiple target products, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 target products.

[0159] This disclosure is not limited to the use of any particular endonuclease. In some embodiments, the endonuclease sequence incorporated into the construct encodes DNase 1 (i.e., a protein with DNase activity) having at least 80% sequence identity with SEQ ID NO: 140. In some embodiments, the DNase sequence incorporated into the construct encodes a DNase having at least 90% sequence identity with SEQ ID NO: 140. In some embodiments, the DNase sequence incorporated into the construct encodes a DNase having at least 95% sequence identity with SEQ ID NO: 140. In some embodiments, the DNase sequence incorporated into the construct encodes a DNase having at least 98% sequence identity with SEQ ID NO: 140. In some embodiments, the DNase sequence incorporated into the construct encodes the DNase having SEQ ID NO: 140. In other embodiments, the DNase sequence incorporated into the construct has at least 80% sequence identity with the DNase encoding portion of SEQ ID NO: 139. In some embodiments, the DNase sequence incorporated into the construct has at least 90% sequence identity with the DNase coding portion of SEQ ID NO: 139. In some embodiments, the DNase sequence incorporated into the construct has at least 95% sequence identity with the DNase coding portion of SEQ ID NO: 139. In some embodiments, the DNase sequence incorporated into the construct has at least 98% sequence identity with the DNase 1 coding portion of SEQ ID NO: 139. In some embodiments, the DNase 1 sequence incorporated into the construct is SEQ ID NO: 139.

[0160] 3E10 antibody and its antigen-binding fragment Generally, endonucleases (e.g., DNase 1 or DNase II) can be co-expressed with the target protein, which can be any pharmaceutical or industrial protein that is expected to be expressed and produced through a host culture. In some embodiments, the target protein is a biopharmaceutical protein. In some embodiments, the target protein is a cell-penetrating immunoglobulin heavy chain and / or light chain. In some embodiments, the target protein is a cell-penetrating immunoglobulin fragment or a single-chain antibody. In some embodiments, when immunoglobulin expression is required, the host cell also contains a third exogenous nucleic acid sequence encoding a second target protein operatively linked to a promoter sequence and a secretion signal sequence, wherein the first target protein is an immunoglobulin light chain sequence and the second target protein is an immunoglobulin heavy chain sequence.

[0161] In some aspects, this disclosure relates to the co-expression of a nuclease (e.g., DNase 1 or DNase II) with a 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the compositions and methods described herein improve the productivity and / or specific productivity of cell cultures expressing a 3E10 antibody or an antigen-binding fragment thereof. For example, as reported in Example 2, co-expression of a humanized 3E10 antibody and the nuclease DNase 1 yielded a specific productivity of up to 5.1 picograms of humanized 3E10 antibody / day / cell. In contrast, expressing a humanized 3E10 antibody in the same cell type without co-expressing the nuclease yielded a specific productivity of less than 5.0 picograms of humanized 3E10 antibody / day / cell. In some embodiments, expressing a humanized 3E10 antibody in the same cell type without co-expressing the nuclease yielded a specific productivity of less than 4.0 picograms of humanized 3E10 antibody / day / cell. In some embodiments, expressing the humanized 3E10 antibody in the same cell type without co-expressing the endonuclease yields a specific productivity of less than 3.0 picograms of humanized 3E10 antibody / day / cell. In some embodiments, expressing the humanized 3E10 antibody in the same cell type without co-expressing the endonuclease yields a specific productivity of less than 2.0 picograms of humanized 3E10 antibody / day / cell. In some embodiments, expressing the humanized 3E10 antibody in the same cell type without co-expressing the endonuclease yields a specific productivity of less than 1.0 picograms of humanized 3E10 antibody / day / cell. Therefore, in some embodiments, the endonuclease is co-expressed with the heavy chain or a fragment thereof of the 3E10 antibody and a fragment thereof of the light chain.

[0162] 3E10 antibodies or their antigen-binding fragments that can be used in the compositions described herein include whole immunoglobulins typically based on IgG classes, such as intact antibodies, which have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4, their binding fragments, and synthetic proteins containing at least a nucleic acid-binding variable domain. The sequence of the variable domain varies among 3E10 antibodies and determines the binding kinetics and specificity of each particular 3E10 antibody. The variability is generally not uniformly distributed throughout the variable domains of 3E10 antibodies. It is typically concentrated in three segments called complementarity-determining regions (CDRs) in both the heavy and light chain variable domains. The more conserved portions of the variable domains are called frames (FRs). The variable domains of the native heavy and light chains each contain four FR regions, employing a β-sheet configuration linked by three CDRs that form linking loops and, in some cases, part of a β-sheet structure. The CDRs in each chain are tightly held together by the FR regions and contribute to the formation of the nucleic acid binding site of the antibody with CDRs from the other chain. Therefore, 3E10 antibodies or their antigen-binding fragments typically contain at least the CDR necessary to maintain DNA binding.

[0163] 1. 3E10 antibody While generally referred to herein as "3E10" or "3E10 antibody," it is to be understood that this phrase encompasses fragments, variants, and binding proteins, including antigen-binding fragments and fusion proteins such as scFv, discFv, triscFv, and other single-chain variable fragments and other cell-penetrating nucleic acid transport molecules disclosed herein, which are also explicitly provided for use in the compositions, conjugates, and methods disclosed herein. Therefore, when describing the characteristics of the 3E10 antibody disclosed herein, the same characteristics relating to the 3E10 antigen-binding fragment are also implicitly disclosed. In embodiments, antibodies and other binding proteins are also referred to herein as having cell-penetrating properties.

[0164] In one embodiment, the 3E10 antibody comprises the VL CDR of SEQ ID NO: 61, 62, and 63 and the VH CDR of SEQ ID NO: 58, 59, and 60. In another embodiment, the 3E10 antibody comprises the VL CDR of SEQ ID NO: 9, 10, and 11 and the VH CDR of SEQ ID NO: 3, 4, and 5. In yet another embodiment, the 3E10 antibody comprises the VL CDR of SEQ ID NO: 22, 23, and 24 and the VH CDR of SEQ ID NO: 15, 17, and 18. In yet another embodiment, the 3E10 antibody comprises the VL CDR of SEQ ID NO: 9, 10, and 11 and the VH CDR of SEQ ID NO: 16, 4, and 5. Other examples of 3E10 VL and VH CDR sequences are shown in... Figures 17-20 middle.

[0165] In the implementation plan, the 3E10 antibody has nucleic acid binding affinity.

[0166] In the implementation plan, when bound to nucleic acids, the 3E10 antibody is capable of ENT2-mediated cell internalization.

[0167] In some aspects of this disclosure, the antibody or its antigen-binding fragment is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody or its antigen-binding fragment.

[0168] In some respects, the 3E10 construct disclosed herein penetrates into the cell and nucleus in an ENT2-dependent manner.

[0169] In some embodiments, the polynucleotide binds nonvalently to the 3E10 construct of this disclosure to help promote the internalization of the 3E10 construct within cells. That is, in some embodiments, the antibody-conjugated polynucleotide (loador polynucleotide) does not interact with the complementary site of the antibody's nucleic acid binding site, and the second polynucleotide (e.g., the carrier nucleic acid) nonvalently complexes with the complementary site to help promote internalization. In some embodiments, the polynucleotide is pre-complexed with the 3E10 construct before administration to a subject. In some embodiments, the polynucleotide is an extracellular polynucleotide bound to the 3E10 construct at a target site in vivo, such as at a site of tumor ischemia and / or necrosis. In some embodiments, the second polynucleotide is DNA. In some embodiments, the polynucleotide is RNA.

[0170] In the embodiments, the 3E10 construct disclosed herein comprises the VH and VL domains of the 3E10 antibody. In the embodiments, the 3E10 construct comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VLCDR3 of the 3E10 antibody.

[0171] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain variable region (VL) containing at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the same amino acid sequence as SEQ ID NO: 21. In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) containing at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the same amino acid sequence as SEQ ID NO: 14. In yet another embodiment, the antibody or its antigen-binding fragment comprises a full-length light chain (LC) containing at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the same amino acid sequence as SEQ ID NO: 20. In the implementation scheme, the antibody or its antigen-binding fragment comprises a full-length heavy chain (HC) containing an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13.

[0172] In some respects, 3E10 antibodies or antigen-binding fragments thereof can be transported to the cytoplasm and / or nucleus of cells without the aid of a carrier or conjugate. For example, monoclonal 3E10 antibodies and their active fragments that can be transported to the nucleus of mammalian cells in vivo without cytotoxic effects are disclosed in Richard Weisbart’s U.S. Patents Nos. 4,812,397 and 7,189,396, the disclosures of which are incorporated herein by reference in their entirety.

[0173] The amino acid sequences of the 3E10 monoclonal antibody and its antigen-binding fragment are known in the art. Exemplary sequences of the 3E10 heavy and light chains are provided below.

[0174] The mouse form of the 3E10 antibody is described in Zack et al., Immunology and Cell Biology, 72:513-520 (1994), the contents of which are incorporated herein by reference in their entirety.

[0175] Amino acid variants of the 3E10 antibody are also known in the art, for example, as Zack et al. J. Immunol As described in, 157(5):2082-8 (1996). For example, amino acid position 31 in CDR1 of the heavy chain variable region of 3E10 affects the ability of nucleic acids to bind and antibodies to penetrate the cell nucleus. The substitution of aspartic acid with asparagine in “wild-type” (e.g., relative to the original mouse antibody) (“D31N” mutation) enhances nucleic acid binding and antibody penetration into the cell nucleus relative to “wild-type” mouse antibodies. See, for example, Zack et al., Immunology and Cell Biology , 72:513-520 (1994); Weisbart et al., J. Autoimmun ., 11, 539-546 (1998); and Weisbart, Int. J. Oncol ., 25, 1867-1873 (2004) (These references are incorporated herein by reference in their entirety).

[0176] This document discloses sequences of 3E10 antibodies with a D31N substitution and their antigen-binding fragments or variants. In some aspects, the 3E10 antibodies and their antigen-binding fragments disclosed herein contain a D31N substitution. In some aspects, other amino acids are substituted at position 31 in the 3E10 antibodies and their antigen-binding fragments disclosed herein. For example, in some aspects of this disclosure, a D31R, D31K, or D31R substitution is incorporated.

[0177] Other 3E10 light chain sequences are known in the art. See, for example, Zack et al. J.Immunol., 15;154(4):1987-94 (1995); GenBank: L16981.1-L chain gene of mouse Ig rearrangement, partial cds; GenBank:AAA65681.1-Immunoglobulin light chain, partial [mice ( Mus musculus )]).

[0178] Traditional antibody structural units typically comprise tetramers. Each tetramer typically consists of two pairs of identical polypeptide chains, each pair having a "light chain" (typically with a molecular weight of about 25 kDa) and a "heavy chain" (typically with a molecular weight of about 50-70 kDa). Human light chains are divided into κ light chains and λ light chains. In embodiments, the antibodies disclosed herein are IgA, IgD, IgE, IgG, or IgM antibodies, including any of their subtypes or isotypes. In embodiments, the antibodies disclosed herein are based on the IgG class. In embodiments, the antibodies disclosed herein are based on one of the subclasses of IgG, including but not limited to IgG1, IgG2, IgG3, and IgG4. Generally, IgG1, IgG2, and IgG4 are used more frequently than IgG3. It should be noted that IgG1 has different allotypes, exhibiting polymorphism at positions 356 (D or E) and 358 (L or M), and in embodiments, the antibodies disclosed herein are based on IgG1 having a D or E at position 356 and / or an L or M at position 358.

[0179] Light chains typically contain two domains: a variable light chain domain (containing the light chain CDR and forming the Fv region together with the variable heavy chain domain) and a constant light chain region (often referred to as CL or Cκ). Heavy chains contain a variable heavy chain domain and a constant domain, the latter comprising the CH1-optional hinge-Fc domain containing CH2-CH3.

[0180] The hypervariable region of an antibody typically encompasses approximately amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) from the light chain variable region, and approximately amino acid residues 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) from the heavy chain variable region (see Kabat et al.). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991); and / or residues forming hypervariable rings (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region, see Chothia and Lesk (1987). J. Mol. Biol .196:901-917. Specific CDRs that can be used with the compositions, conjugates, and methods described herein are described below.

[0181] Those skilled in the art will understand that the exact numbering and location of CDRs may differ across different numbering systems. However, it should be understood that the disclosure of variable heavy chain and / or variable light chain sequences includes the disclosure of the associated (inherent) CDRs. Therefore, the disclosure of each variable heavy chain region is a disclosure of the VH CDR (e.g., VH CDR1, VH CDR2, and VH CDR3), and the disclosure of each variable light chain region is a disclosure of the VL CDR (e.g., VL CDR1, VL CDR2, and VL CDR3).

[0182] Throughout this disclosure, when referring to residues in the variable domains (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), the Kabat numbering system is generally used, and for the Fc region, the EU numbering system is used (e.g., Kabat et al., ibid. (1991)). In some aspects, this specification uses the IMGT system to define the complementarity-determining regions (CDRs) provided herein.

[0183] This disclosure provides a number of different CDR sets. In this context, a “complete CDR set” comprises three variable light chain CDRs, such as VL CDR1, VL CDR2, and VL CDR3, and three variable heavy chain CDRs, such as VH CDR1, VH CDR2, and VHCDR3. These can be part of a larger variable light chain or variable heavy chain domain, respectively. Additionally, as outlined more fully herein, when using heavy and light chains (e.g., when using Fab), the variable heavy and light chain domains can be on separate polypeptide chains, or on a single polypeptide chain in the case of an scFv sequence.

[0184] As noted above, this disclosure relates to different antibody domains of the 3E10 antibody or its antigen-binding fragment. These domains include, but are not limited to, the Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), variable heavy chain (VH) domain, variable light chain (VL) domain, light chain constant domain, Fab domain, and scFv domain.

[0185] 2. Humanized antibodies In some respects, the antibodies of this disclosure comprise heavy chain variable regions derived from specific germline heavy chain immunoglobulin genes and / or light chain variable regions derived from specific germline light chain immunoglobulin genes. For example, such antibodies may comprise or consist of mouse antibodies, chimeric antibodies, humanized antibodies, or human antibodies or their antigen-binding fragments, wherein the aforementioned antibodies or their antigen-binding fragments comprise heavy or light chain variable regions that are “products” of or “derived from” specific germline sequences (e.g., the germline sequence of the 3E10 antibody). A human antibody can be confirmed as a “product” of or “derived from” a human germline immunoglobulin sequence by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is sequence-closest to the human antibody sequence (i.e., with the greatest identity%) (using the methods outlined herein). Human antibodies that are "products" of or "derived from" a specific human germline immunoglobulin sequence may contain amino acid differences compared to that germline sequence, for example, due to naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, humanized antibodies are typically at least 90% identical in amino acid sequence to the sequence encoded by the human germline immunoglobulin gene and, when compared to germline immunoglobulin sequences from other species (e.g., mouse germline sequences), contain amino acid residues that identify the antibody as being derived from a human sequence. In some cases, humanized antibodies may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical in amino acid sequence to the sequence encoded by the germline immunoglobulin gene. Typically, humanized antibodies derived from a specific human germline sequence will exhibit an amino acid sequence differing from the sequence encoded by the human germline immunoglobulin gene by no more than 10-20 amino acids. In some cases, humanized antibodies may exhibit an amino acid sequence differing from the sequence encoded by the germline immunoglobulin gene by no more than 5 amino acids, or even no more than 4, 3, 2, or 1 amino acid.

[0186] In one respect, as is known in the art, the parent antibody is already affinity-matured. Structure-based methods can be used for humanization and affinity maturation, for example, as described in U.S. Patent Publication No. 2006 / 0008883, which is incorporated herein by reference. Selection-based methods can be used for the humanization and / or affinity maturation of the antibody variable region, including but not limited to those described in the following literature: Wu et al., 1999, J. Mol. Biol 294:151-162; Baca et al., 1997, J. Biol. Chem 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of the above references are incorporated herein by reference. Other humanization methods may involve only transplanting a portion of the CDR, including but not limited to the methods described in the following documents: U.S. Patent Publication No. 2001 / 0035606; Tan et al., 2002, J. Immunol 169:1119-1125; De Pascalis et al., 2002, J. Immunol All of the above references are incorporated herein by reference. 169:3076-3084.

[0187] 3. Fc variant In an implementation, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0188] In the implementation scheme, the Fc region variant possesses some, but not all, effector functions, making it an ideal candidate for applications where in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays known in the art can be performed to ensure that the antibody lacks FcγR binding (and therefore potentially lacks ADCC activity), but retains FcRn binding capacity. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0189] In embodiments, the antibodies provided herein may have reduced effector function and may therefore include substitutions for one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted with alanine (U.S. Patent No. 7,332,581).

[0190] In implementations, the Fc region variants provided herein may have enhanced or weakened binding to FcR. See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001), the disclosures of which are incorporated herein by reference in their entirety.

[0191] In the implementation, the Fc region variants provided herein comprise an Fc region with one or more amino acid substitutions that enhance ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbers of the residues).

[0192] In implementations, the Fc region variants provided herein contain alterations that result in changed (i.e., enhanced or weakened) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0193] In the implementations, the Fc region variants provided herein contain alterations that result in a prolonged half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976), and Kim et al., J. Immunol. 24:249 (1994)), for example, as described in US2005 / 0014934A1 (Hinton et al.). Those antibodies contain an Fc region having one or more substitutions that enhance the binding of the Fc region to the FcRn. Such Fc variants include variants with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0194] In the implementation, the Fc region variants provided herein include “mortar” or “skew” variants, which refer to amino acid engineering that produces spatial effects that favor heterodimer formation and discourage homodimer formation, as described in the following references: USSN 61 / 596,846, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, all of which are incorporated herein by reference in their entirety.

[0195] In the implementation, the Fc region variants provided herein include the changes described in Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260, 5,624,821, and WO 94 / 29351.

[0196] 4. antibody fragments In the embodiments, the antibody comprises an antigen-binding fragment of a 3E10 antibody. In the embodiments, the antigen-binding fragment retains the desired biological activity of the 3E10 antibody. In the embodiments, the antigen-binding fragment retains at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% of the desired biological activity of the 3E10 antibody. In the embodiments, the antigen-binding fragment retains the ability of the antibody to bind to its target antigen (e.g., nucleic acid, such as DNA). In the embodiments, the antigen-binding fragment retains the ability of the antibody to bind to a cell receptor (e.g., ENT2). In the embodiments, the antigen-binding fragment retains the ability of the antibody to be internalized by target cells.

[0197] In the implementation scheme, antibodies include single-chain variable fragments (scFv), tandem double scFv, (scFv)2, small antibodies, VHH, scFv-Fc, CrossMab, dual variable domain immunoglobulin (DVD-Ig), single-chain tandem variable fragments (scTaFv), double antibodies, tandem double antibodies (TandAb), Fabsc, modular IgG-scFv, Fab or F(ab')2.

[0198] In some implementations, the endonuclease is co-expressed with a single-stranded variable fragment (scFv). scFVs are small, genetically engineered antibody molecules composed of the heavy chain variable region (VH) and light chain variable region (VL) of a conventional antibody linked by a peptide linker. It retains the antigen-binding specificity of a full-length antibody but has a smaller size, making it easier to generate and modify for a variety of applications. scFvs can be used for therapeutic and diagnostic applications as well as for basic research investigating protein-protein interactions. In some implementations, scFvs contain 3E10 VH and VL CDRs.

[0199] In some embodiments, the endonuclease is co-expressed with a single-chain 3E10 fragment derived from a parental 3E10 antibody. In some embodiments, the single-chain 3E10 fragment is an scFv-Fc polypeptide. The scFv-Fc polypeptide is a fusion protein composed of a single-chain variable fragment (scFv) and a crystallizable fragment (Fc) region. The scFv portion of the protein is derived from the antibody and binds to a specific antigen, while the Fc region provides stability and biological effector functions. This fusion protein can be used for therapeutic applications targeting specific diseases or as a research tool for studying protein-protein interactions. In some embodiments, scFv-Fc contains 3E10 VH and VLCDR.

[0200] In some embodiments, the endonuclease is co-expressed with the bispecific 3E10 antibody or its antigen-binding fragment. In some embodiments, one arm of the bispecific 3E10 antibody comprises 3E10 VH and VL CRD. In some embodiments, the other arm of the bispecific antibody is specific for cell surface antigens (e.g., cell surface tumor antigens) to modulate the targeting of the bispecific antibody in vivo. For more information on bispecific antibodies, see, for example, Ahamadi-Fesharaki R. et al. Molecular Therapy Oncolytics , 14:38-56 (2019), the contents of which are cited in their entirety and incorporated into this paper.

[0201] In some implementations, the antibody is a CrossMab antibody 3E10 or its antigen-binding fragment. In the CrossMab form, complementary mutations are introduced into the heavy chain constant region of each arm to generate so-called “mortar and pestle,” resulting in preferred binding between different arms, forming a heterodimer rather than a homodimer of both in the same arm. The exact residues mutated in the heavy chain constant region of the CrossMab bispecific antibody to form the “mortar and pestle” can vary depending on the specific design and optimization goals of the antibody. For more information on CrossMab antibodies, see, for example, Huang, J. et al., Journal of Biological Chemistry, 294(50):19001–10 (2019), the contents of which are incorporated herein by reference in their entirety.

[0202] In some embodiments, the antibody is a bivalent dual-variable-domain immunoglobulin (DVD-Ig) of the 3E10 antibody or its antigen-binding fragment. In the DVD-Ig form, each arm of the antibody contains two VH / VL pairs. In some embodiments, one of the VH / VL pairs contains a 3E10 VH and a VL CDR. For more information on DVD-Ig antibodies, see, for example, Polson AG et al., Journal of Immunotherapy. 29(3):241-50 (2006) and U.S. Patent No. 7,612,181, the disclosure of which is incorporated herein by reference in its entirety.

[0203] In some embodiments, the antibody includes a single-chain variable fragment (scFv). As used herein, "single-chain Fv" or "scFv" refers to a VH domain covalently attached to a VL domain via a linker (e.g., a scFv linker) as discussed herein to form a continuous protein chain. The scFv domain can be arranged in any configuration from the N-terminus to the C-terminus (i.e., VH-linker-VL or VL-linker-VH). In the sequences shown in the sequence listing and figures herein, the order of the VH and VL domains is indicated in the names; for example, H.X_L.Y indicates an N-terminal to C-terminal arrangement of VH-linker-VL, and L.Y_H.X indicates an N-terminal to C-terminal arrangement of VL-linker-VH.

[0204] In the implementation scheme, the antigen-binding fragment of the 3E10 antibody or its antigen-binding fragment contains tandem double scFv.

[0205] In some embodiments, the antibody is a tandem dual scFv (single-chain variable fragment) antibody. A tandem dual scFv has two scFv domains connected in a linear manner. In some embodiments, each scFv domain is derived from a different antibody and provides independent antigen-binding specificity. In some embodiments, one of the scFv domains contains 3E10 VH and VL CDR. For more information on tandem dual scFvs, see, for example, Bossen C et al., MAbs, 4(2):200-08 (2012), the disclosure of which is incorporated herein by reference in its entirety.

[0206] In some respects, the antibody is a dimerized scFv antibody (scFv)2. The dimerized scFv antibody has two scFv domains linked in a dimerized arrangement. In some embodiments, each scFv domain is derived from a different antibody and provides independent antigen-binding specificity. In some embodiments, one of the scFv domains comprises 3E10 VH and VL CDR. For more information on dimerized scFv antibodies, see, for example, Llewellyn C et al., Journal of Immunological Methods, 273(1-2):33-44 (2002), the disclosure of which is incorporated herein by reference in its entirety.

[0207] In some implementations, the antibody includes scFv-Fc. As referred to herein, “scFv-Fc” is a polypeptide consisting of the heavy and light chain variable regions of the antibody linked by a linker, followed by the Fc polypeptide chain of the antibody, optionally the Fc region of a human IgG antibody (such as IgG1, IgG2, IgG3, or IgG4 antibody).

[0208] In implementations, the antibody includes a single-chain tandem variable fragment (scTaFv) antibody. A single-chain tandem variable fragment (scTaFv) antibody is a bispecific antibody consisting of two variable fragment (VH and VL) domains linked in a tandem arrangement. In some implementations, one of the variable fragment domains contains 3E10 VH and VL CDRs. For more information on scTaFv antibodies, see, for example, Schramm C et al., MAbs 5(3):442-49 (2013), the disclosure of which is incorporated herein by reference in its entirety.

[0209] In embodiments, the antibody includes a VHH, also known as a nanobody. As used herein, the term "VHH" refers to a variable domain of the heavy chain of a heavy chain antibody. A VHH is a molecule that recognizes an antigen through a single domain and is the smallest unit of antibody molecules discovered to date. In embodiments, a VHH may include one or more variable domains derived from the heavy chain of a heavy chain antibody, and the number of variable domains of the heavy chain contained in a VHH is not limited.

[0210] In implementations, the antibody includes a biantibody. As used herein, a “biantibody” refers to a bivalent antibody comprising two polypeptide chains, each of which is too short to form a pair between two domains on the same chain, such that each domain pairs with a complementary domain on the other polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-48 and Poljak et al., 1994, Structure 2: 1121-23). ​​If the two polypeptide chains of a biantibody are identical, then the biantibody will have two identical antigen-binding sites due to their pairing. In implementations, one of the antigen-binding domains of the biantibody comprises 3E10 VH and VL CDR. For more information on biantibodies, see, for example, Hoogenboom HR et al., Trends Biotechnol., 21(12):553-57 (2003), the disclosure of which is incorporated herein by reference in its entirety. Different polypeptide chains can be used to prepare biantibodies with two different antigen-binding sites. Similarly, as used herein, "tri-antibody" and "tetra-antibody" refer to antibodies containing three and four polypeptide chains, respectively, forming three and four antigen-binding sites (which may be the same or different).

[0211] The term "small antibody" is used to refer to the scFv-CH3 fusion protein, which self-assembles into an 80 kDa divalent dimer (ScFv-CH3)2.

[0212] In some embodiments, the antibody includes a tandem biantibody (TandAb). A tandem biantibody has two antigen-binding domains (VH and VL) linked in a tandem arrangement by a flexible peptide linker. In some embodiments, one of the antigen-binding domains comprises a 3E10 VH and a VL CDR. For more information on biantibodies, see, for example, Sidelmann JG et al., Mol Immunol., 45(9):2597-607 (2008), the disclosure of which is incorporated herein by reference in its entirety.

[0213] In the implementation scheme, the antibody includes Fabsc. As used herein, an antibody molecule in the form of "Fabsc" generally refers to a bispecific antibody molecule having a Fab fragment, which typically includes a hinge region located at the C-terminus of the Fab fragment connected to the N-terminus of the CH2 domain, which in turn is connected to the N-terminus of the scFv fragment.

[0214] In implementations, the antibody includes scFab. scFab, also known as a single-chain antigen-binding fragment (Fab), is a class of antibody fragments that combine variable heavy chain (VH) and variable light chain (VL) domains into a single polypeptide chain linked by a peptide linker. The domain structure of Fabsc includes variable domains (VH and VL) of both the heavy and light chains, and a peptide linker connecting these two domains. In addition to the variable domains, Fabsc also includes a constant domain (CL) of the light chain and a hinge region of the heavy chain. In some implementations, one of the antigen-binding domains includes a 3E10 VH and VL CDR. For more information on Fabsc, see, for example, Kettner, C. et al., Frontiers in Immunology, 8(8):453 (2017), the disclosure of which is incorporated herein by reference in its entirety.

[0215] In some embodiments, the antibody comprises IgG-scFv. IgG-scFv is an antibody in which scFv is fused to the light or heavy chain of IgG. In some embodiments, the scFv comprises 3E10 VH and VL CDR. In some embodiments, the IgG comprises 3E10 VH and V LCDR. In some embodiments, the antibody is F(ab')2.

[0216] 5. 3E10 sequence The mouse form of the 3E10 antibody was described by Zack et al. Immunology and Cell Biology The reference 72:513-520 (1994) is incorporated herein by reference in its entirety. Amino acid variants of the 3E10 antibody are also known in the art, such as those described by Zack et al. J. ImmunolAs described in ., 157(5):2082-8 (1996). For example, amino acid position 31 in CDR1 of the heavy chain variable region of 3E10 affects nucleic acid binding. Aspartic acid replaced by asparagine in “wild-type” (e.g., relative to the original mouse antibody) (“D31N” mutation) enhances nucleic acid binding relative to “wild-type” mouse antibodies. See, Zack et al., Immunology and Cell Biology , 72:513-520 (1994); Weisbart et al., J. Autoimmun ., 11, 539-546 (1998); and Weisbart, Int. J. Oncol These references, ., 25, 1867-1873 (2004), are incorporated into this paper in their entirety by way of citation.

[0217] Other 3E10 immunoglobulin light chain sequences are known in the art. See Zack et al. J. Immunol .,15;154(4):1987-94 (1995); GenBank: L16981.1-mouse Ig rearranged L chain gene, partial cds; GenBank: AAA65681.1-immunoglobulin light chain, partial [ House mouse ]).

[0218] As noted above, the 3E10 antibody or its antigen-binding fragment disclosed herein may refer to different antibody domains of the 3E10 antibody or its antigen-binding fragment. These domains include, but are not limited to, the Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), variable heavy chain domain, variable light chain domain, light chain constant domain, Fab domain, and scFv domain.

[0219] In some respects, the 3E10 antibody or its antigen-binding fragment of the present disclosure comprises a heavy chain variable region derived from a specific germline heavy chain immunoglobulin gene and / or a light chain variable region derived from a specific germline light chain immunoglobulin gene. For example, such target proteins may comprise or consist of mouse antibodies, chimeric antibodies, humanized antibodies, or human antibodies or their antigen-binding fragments, which contain heavy or light chain variable regions as a “product” or “derived from” a specific germline sequence (e.g., the germline sequence of the 3E10 antibody). A human antibody can be confirmed as a “product” or “derived from” a human germline immunoglobulin sequence by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is sequence-closest to the human antibody sequence (i.e., with the greatest identity%) (using the methods outlined herein). Human antibodies that are "products" of or "derived from" a specific human germline immunoglobulin sequence may contain amino acid differences compared to that germline sequence, for example, due to naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, humanized antibodies are typically at least 90% identical in amino acid sequence to the sequence encoded by the human germline immunoglobulin gene and, when compared to germline immunoglobulin sequences from other species (e.g., mouse germline sequences), contain amino acid residues that identify the antibody as being derived from a human sequence. In some cases, humanized antibodies may be at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the sequence encoded by the germline immunoglobulin gene, or even at least 96%, 97%, 98%, or 99%. Typically, humanized antibodies derived from a specific human germline sequence will exhibit an amino acid sequence differing from that encoded by the human germline immunoglobulin gene by no more than 10-20 amino acids. In some cases, humanized antibodies may exhibit amino acid sequences that differ from those encoded by germline immunoglobulin genes by no more than 5, or even no more than 4, 3, 2, or 1 amino acid.

[0220] In some implementations, the parent antibody has been affinity-matured using methods well-known in the art. Structure-based methods can be used for humanization and affinity maturation, as described in U.S. Publication US / 2006 / 0008883, which is incorporated herein by reference. Selection-based methods can be used for humanization and / or affinity maturation of the antibody variable region, including but not limited to those described in the following literature: Wu et al., 1999, J. Mol. Biol 294:151-162; Baca et al., 1997, J. Biol. Chem 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem.271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of the above references are incorporated herein by reference. Other humanization methods may involve only porting a portion of the CDR, including but not limited to the methods described in the following references: US / 2001 / 0035606; Tan et al., 2002, J. Immunol 169:1119-1125; De Pascalis et al., 2002, J. Immunol All of the above references are incorporated herein by reference. 169:3076-3084.

[0221] In some embodiments, the 3E10 antibody or antigen-binding fragment thereof described herein comprises a CDR sequence corresponding to the parental 3E10 antibody. Therefore, in some embodiments, the 3E10 antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) complementarity-determining region (CDR) 1 comprising the amino acid sequence 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence 3E10-VH-CDR1 (SEQ ID NO: 3), a VH CDR2 comprising the amino acid sequence 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence 3E10-VH-CDR3 (SEQ ID NO: 5).

[0222] In some implementations, the target protein is the 3E10 antibody or its antigen-binding fragment. In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein comprises (i) the following CDR sequences: a light chain variable region (VL) complementarity-determining region (CDR) 1 comprising the amino acid sequence 3E10-VL-CDR1m (SEQ ID NO: 61), a VL CDR2 comprising the amino acid sequence 3E10-VL-CDR2m (SEQ ID NO: 62), a VL CDR3 comprising the amino acid sequence 3E10-VL-CDR3m (SEQ ID NO: 63), and a heavy chain variable region (VH) CDR1 comprising the amino acid sequence 3E10-VH-CDR1m (SEQ ID NO: 58), a VH CDR2 comprising the amino acid sequence 3E10-VH-CDR2m (SEQ ID NO: 59), and a VH CDR3 comprising the amino acid sequence 3E10-VH-CDR3m (SEQ ID NO: 60); and (ii) a nucleic acid binding site.

[0223] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises an immunoglobulin heavy chain CDR1 with a D31N amino acid substitution. It is known in the art that a mutation of aspartic acid at residue 31 of CDR1 to asparagine increases the cationic charge of that residue and enhances nucleic acid binding (3E10-D31N). Other exemplary CDR1 variants include a mutation of aspartic acid at residue 31 to arginine (3E10-D31R), where molecular modeling indicates cationic charge expansion; and a mutation of lysine at residue 31 to lysine (3E10-D31K), where molecular modeling indicates a change in charge orientation. Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a 3E10 antigen-binding protein containing a D31R or D31K substitution. Other exemplary CDR variants include a mutation at residue 96 of arginine to asparagine (3E10-R96N), and / or a mutation at residue 30 of serine to aspartic acid (3E10-S30D), either alone or in combination with D31N, D31R, or D31K. All sequences disclosed herein having residues corresponding to 3E10-D31 or 3E10-N31 are explicitly disclosed to have D31R, D31K, N31R, or N31K substitutions. In some embodiments, the 3E10 antibody or its nucleic acid binding fragment comprises a heavy chain variable region (VH) CDR1 containing the amino acid sequence NYGMH (SEQ ID NO: 15).

[0224] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: (a) a light chain variable region (VL) complementarity-determining region (CDR) 1 containing the amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 22), (b) a VL CDR2 containing the amino acid sequence YASYLES (SEQ ID NO: 23), (c) a VL CDR3 containing the amino acid sequence QHSREFPWT (SEQ ID NO: 24), (d) a heavy chain variable region (VH) CDR1 containing the amino acid sequence NYGMH (SEQ ID NO: 15), (e) a VH CDR2 containing the amino acid sequence YISSGSSTIYYADTVKG (SEQ ID NO: 17), and (f) a VH CDR3 containing the amino acid sequence RGLLLDY (SEQ ID NO: 18).

[0225] In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a light chain variable region (VL) containing the same amino acid sequence as SEQ ID NO: 21. In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) containing the same amino acid sequence as SEQ ID NO: 14. In some embodiments, the 3E10 antibody or its nucleic acid-binding fragment comprises a full-length light chain (LC) containing the same amino acid sequence as SEQ ID NO: 20. In some embodiments, the 3E10 antibody or its nucleic acid-binding fragment comprises a full-length heavy chain (HC) containing the same amino acid sequence as SEQ ID NO: 13.

[0226] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein contains a CDR sequence from a variant 3E10 antibody that includes a D31N amino acid substitution in VH CDR1. Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: a light chain variable region (VL) complementarity-determining region (CDR) 1 containing the amino acid sequence 3E10-VL-CDR1_D31N (SEQ ID NO: 22), a VL CDR2 containing the amino acid sequence 3E10-VL-CDR2_D31N (SEQ ID NO: 23), a VLCDR3 containing the amino acid sequence 3E10-VL-CDR3_D31N (SEQ ID NO: 24), a heavy chain variable region (VH) CDR1 containing the amino acid sequence 3E10-VH-CDR1_D31N (SEQ ID NO: 15), a VH CDR2 containing the amino acid sequence 3E10-VH-CDR2_D31N (SEQ ID NO: 17), and a VH CDR2 containing the amino acid sequence 3E10-VH-CDR3_D31N (SEQ ID NO: 18). CDR3.

[0227] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein refers to the CDR sequence corresponding to the parental 3E10 antibody, optionally containing a D31N amino acid substitution in VH CDR1. Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: a light chain variable region (VL) complementarity-determining region (CDR) 1 containing the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 containing the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 containing the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 containing the amino acid sequence of 3E10-VH-CDR1a (SEQ ID NO: 16), a VH CDR2 containing the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 17), and a VH CDR3 containing the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 18).

[0228] In some embodiments, the 3E10 antibody or antigen-binding fragment thereof described herein comprises a CDR sequence corresponding to the parental 3E10 antibody, having known amino acid substitutions in one or more CDRs. Thus, in some embodiments, relative to the CDR sequence of the parental 3E10 or the 3E10-D31N variant, the 3E10 antibody or antigen-binding fragment thereof described herein comprises one or more amino acid substitutions selected from the following: G at position 5 of VH CDR2 replaced with S, T at position 14 of VH CDR2 replaced with S, S at position 5 of VL CDR1 replaced with T, M at position 14 of VL CDR1 replaced with L, H at position 15 of VL CDR1 replaced with A, and E at position 6 of VL CDR2 replaced with Q, as shown below. Figure 18B As shown in the image.

[0229] Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment contains Figure 18B One or more CDR sequences are shown. For example, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO: 26) or 3E10-VH-CDR2.2 (SEQ ID NO: 27). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3, which have one or more amino acid substitutions relative to the CDR of the parental 3E10 antibody or relative to the 3E10-D31N variant.

[0230] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises VL CDR1, which contains the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO: 28) or 3E10-VL-CDR1.2 (SEQ ID NO: 29). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDR of the parental 3E10 antibody or relative to the 3E10-D31N variant.

[0231] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR2 containing the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO: 30). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody or relative to the 3E10-D31N variant.

[0232] While some of the aforementioned amino acid substitutions are fairly conserved, such as the S substitution for T at position 5 of VL CDR1, others exhibit distinctly different properties, such as the M substitution for L at position 14 of VL CDR1, the H substitution for A at position 15 of VL CDR1, and the E substitution for Q at position 6 of VL CDR2. Unbound by theory, this suggests that at least these positions within the 3E10CDR framework tolerate other amino acid substitutions.

[0233] Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment contains conserved amino acid variations, such as... Figure 18CAs shown in the diagram. In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR2 comprising the amino acid sequence 3E10-VH-CDR2.3 (SEQ ID NO: 31). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody or relative to the 3E10-D31N variant, for example, as described herein.

[0234] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR1 containing the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO: 32). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody or relative to the 3E10-D31N variant, for example, as described herein.

[0235] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR2 comprising the amino acid sequence 3E10-VL-CDR2.2 (SEQ ID NO: 33). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody or relative to the 3E10-D31N variant, for example, as described herein.

[0236] Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1, which comprises the amino acid sequence of 3E10-VH-CDR1.c1 (SEQ ID NO: 34), 3E10-VH-CDR1.c2 (SEQ ID NO: 35), 3E10-VH-CDR1.c3 (SEQ ID NO: 36), 3E10-VH-CDR1.c4 (SEQ ID NO: 37), or 3E10-VH-CDR1.c5 (SEQ ID NO: 38). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 2 and 3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 2 and 3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0237] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises VH CDR2, which comprises the amino acid sequence of 3E10-VH-CDR2.c1 (SEQ ID NO: 39), 3E10-VH-CDR2.c2 (SEQ ID NO: 40), or 3E10-VH-CDR2.c3 (SEQ ID NO: 41). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0238] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises VH CDR3, which comprises the amino acid sequence of 3E10-VH-CDR3.c1 (SEQ ID NO: 42), 3E10-VH-CDR3.c2 (SEQ ID NO: 43), or 3E10-VH-CDR3.c3 (SEQ ID NO: 44). In some embodiments, the 3E10 antibody or its antigen-binding fragment comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 2, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0239] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR1, which comprises the amino acid sequence of 3E10-VL-CDR1.c1 (SEQ ID NO: 45), 3E10-VL-CDR1.c2 (SEQ ID NO: 46), 3E10-VL-CDR1.c3 (SEQ ID NO: 47), 3E10-VL-CDR1.c4 (SEQ ID NO: 48), 3E10-VL-CDR1.c5 (SEQ ID NO: 49), or 3E10-VL-CDR1.c6 (SEQ ID NO: 50). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VHCDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0240] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.c1 (SEQ ID NO: 51). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0241] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR3, which comprises the amino acid sequence of 3E10-VL-CDR3.c1 (SEQ ID NO: 52), 3E10-VL-CDR3.c2 (SEQ ID NO: 53), 3E10-VL-CDR3.c3 (SEQ ID NO: 54), 3E10-VL-CDR3.c4 (SEQ ID NO: 55), 3E10-VL-CDR3.c5 (SEQ ID NO: 56), or 3E10-VL-CDR3.c6 (SEQ ID NO: 57). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VHCDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0242] It is also expected that the 3E10 antibody or its antigen-binding fragment as described herein may contain any combination of the above-described 3E10 CDR amino acid substitutions.

[0243] Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 containing the amino acid sequence 3E10-VH-CDR1m (SEQ ID NO: 58). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 2 and 3 according to the parental 3E10 antibody, such as Figure 19 As described herein. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 2 and 3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein and Figure 19 As described in [the text].

[0244] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO: 59). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0245] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises VH CDR3, which contains the amino acid sequence 3E10-VH-CDR3m (SEQ ID NO: 60). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1-3 and VH CDRs 1 and 2, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0246] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR1 containing the amino acid sequence of 3E10-VL-CDR1m (SEQ ID NO: 61). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 2 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0247] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO: 62). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 3 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0248] Similarly, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: VL CDR3 containing the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO: 63). In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VH CDRs 1-3 according to the parental 3E10 antibody. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VH CDRs 1-3 according to the 3E10-D31N variant. In some embodiments, the 3E10 antibody or its antigen-binding fragment further comprises VL CDRs 1 and 2 and VH CDRs 1-3, which have one or more amino acid substitutions relative to the CDRs of the parental 3E10 antibody, for example, as described herein.

[0249] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein comprises: VL CDR1 containing the amino acid sequence 3E10-VL-CDR1m (SEQ ID NO: 61), VL CDR2 containing the amino acid sequence 3E10-VL-CDR2m (SEQ ID NO: 62), VLCDR3 containing the amino acid sequence 3E10-VL-CDR3m (SEQ ID NO: 63), heavy chain variable region (VH) CDR1 containing the amino acid sequence 3E10-VH-CDR1m (SEQ ID NO: 58), VH CDR2 containing the amino acid sequence 3E10-VH-CDR2m (SEQ ID NO: 59), and VH CDR3 containing the amino acid sequence 3E10-VH-CDR3m (SEQ ID NO: 60).

[0250] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein refers to a CDR sequence having no more than one amino acid substitution relative to the parental 3E10 antibody, which optionally includes a D31N amino acid substitution in VH CDR1. Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: a VL CDR1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR1a (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR3 (SEQ ID NO: 5).

[0251] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein refers to a CDR sequence having no more than two amino acid substitutions relative to the parental 3E10 antibody, which optionally includes a D31N amino acid substitution in VH CDR1. Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises: a VL CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR1a (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR3 (SEQ ID NO: 5).

[0252] 3E10 antibodies or variants thereof, or other variants of their antigen-binding fragments, are also known in the art, such as, for example, Zack et al. J. Immunol As disclosed in ., 157(5):2082-8 (1996). For example, the amino acid position 31 of the heavy chain variable region of 3E10 has been determined to have an impact on the ability of antibodies and their fragments to penetrate the cell nucleus and bind to DNA. The D31N mutation in CDR1 penetrates the cell nucleus and binds to DNA with much higher efficiency than the original antibody (Zack et al., Immunology and Cell Biology , 72:513-520 (1994); Weisbart et al., J. Autoimmun ., 11, 539-546(1998); Weisbart, Int. J. Oncol ., 25, 1867-1873 (2004)). In some embodiments, the 3E10 antibody or a variant thereof or an antigen-binding fragment thereof described herein has a D31N substitution.

[0253] In some implementations, the 3E10 antibody or its antigen-binding fragment is humanized. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “input” residues, and they are typically derived from an “input” variable domain. Antibody humanization techniques typically involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of the antibody molecule.

[0254] In some embodiments, the 3E10 antibody or its antigen-binding fragment is a humanized antibody or fragment thereof known in the art. Examples of humanization can be found, for example, US 10,221,250, US / 2016 / 033324, US / 2020 / 0216567 and US / 2020 / 0216568 (each of which is expressly and entirely incorporated herein by reference).

[0255] In some embodiments, this disclosure provides the humanized VL and VH sequences disclosed herein, and any combination of VL and VH sequences that have sequence identity with them (e.g., have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VH or VL sequences described herein).

[0256] In some embodiments, the humanized 3E10 antibody or its antigen-binding fragment comprises a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the light chain variable domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence selected from the group consisting of: 3E10-VL-h1 (SEQ ID NO: 86), 3E10-VL-h2 (SEQ ID NO: 87), 3E10-VL-h3 (SEQ ID NO: 88), 3E10-VL-h4 (SEQ ID NO: 89), 3E10-VL-h5 (SEQ ID NO: 90), and 3E10-VL-h6 (SEQ ID NO: 91), and the heavy chain variable domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of: 3E10-VH-h1 (SEQ ID NO: 65), 3E10-VH-h2 (SEQ ID NO: 90), and 3E10-VL-h6 (SEQ ID NO: 91). 66), 3E10-VH-h3 (SEQ ID NO: 67), 3E10-VH-h4 (SEQ ID NO: 68), 3E10-VH-h5 (SEQ ID NO: 69), 3E10-VH-h6 (SEQ ID NO: 70) and 3E10-VH-h7 (SEQ ID NO: 71).

[0257] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h1 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h1 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h1 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h1 (SEQ ID NO: 86).

[0258] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h2 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h2 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h2 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h2 (SEQ ID NO: 87).

[0259] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h3 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h3 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h3 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h3 (SEQ ID NO: 88).

[0260] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h4 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h4 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h4 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h4 (SEQ ID NO: 89).

[0261] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h5 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h5 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h5 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h5 (SEQ ID NO: 90).

[0262] In some embodiments, the sequence of 3E10-VL is at least 97% identical to that of 3E10-VL-h6 (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-VL is at least 98% identical to that of 3E10-VL-h6 (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-VL is at least 99% identical to that of 3E10-VL-h6 (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h6 (SEQ ID NO: 91).

[0263] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h1 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h1 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h1 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h1 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h1 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h1 (SEQ ID NO: 65).

[0264] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h2 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h2 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h2 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h2 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h2 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h2 (SEQ ID NO: 66).

[0265] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h3 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h3 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h3 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h3 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h3 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h3 (SEQ ID NO: 67).

[0266] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h4 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h4 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h4 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h4 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h4 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h4 (SEQ ID NO: 68).

[0267] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h5 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h5 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h5 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h5 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h5 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h5 (SEQ ID NO: 69).

[0268] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h6 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h6 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h6 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h6 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h6 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h6 (SEQ ID NO: 70).

[0269] In some embodiments, the sequence of 3E10-VH is at least 95% identical to that of 3E10-VH-h7 (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-VH is at least 96% identical to that of 3E10-VH-h7 (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-VH is at least 97% identical to that of 3E10-VH-h7 (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-VH is at least 98% identical to that of 3E10-VH-h7 (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-VH is at least 99% identical to that of 3E10-VH-h7 (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h7 (SEQ ID NO: 71).

[0270] In some embodiments, the 3E10 antibody or its antigen-binding fragment described herein comprises a light chain (3E10-LC) and a heavy chain (3E10-HC), the light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of: 3E10-LC-h1m (SEQ ID NO: 92), 3E10-LC-h2m (SEQ ID NO: 93), 3E10-LC-h3m (SEQ ID NO: 94), 3E10-LC-h4m (SEQ ID NO: 95), 3E10-LC-h5m (SEQ ID NO: 96) and 3E10-LC-h6m (SEQ ID NO: 97), the heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of: 3E10-HC-h1m (SEQ ID NO: 72), 3E10-HC-h2m (SEQ ID NO: 73), 3E10-HC-h3m (SEQ ID NO: 97), and 3E10-LC-h6m (SEQ ID NO: 97). NO: 74), 3E10-HC-h4m (SEQ ID NO: 75), 3E10-HC-h5m (SEQ ID NO: 76), 3E10-HC-h6m (SEQ ID NO: 77) and 3E10-HC-h7m (SEQ ID NO: 78).

[0271] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h1m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h1m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h1m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h1m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h1m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h1m (SEQ ID NO: 92).

[0272] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h2m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h2m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h2m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h2m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h2m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h2m (SEQ ID NO: 93).

[0273] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h3m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h3m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h3m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h3m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h3m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h3m (SEQ ID NO: 94).

[0274] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h4m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h4m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h4m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h4m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h4m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h4m (SEQ ID NO: 95).

[0275] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h5m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h5m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h5m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h5m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h5m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h5m (SEQ ID NO: 96).

[0276] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h6m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h6m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h6m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h6m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h6m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h6m (SEQ ID NO: 97).

[0277] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h1m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h1m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h1m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h1m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h1m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h1m (SEQ ID NO: 72).

[0278] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h2m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h2m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h2m (SEQ ID NO: X73). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h2m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h2m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h2m (SEQ ID NO: 73).

[0279] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h3m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h3m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h3m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h3m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h3m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h3m (SEQ ID NO: 74).

[0280] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h4m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h4m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h4m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h4m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h4m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h4m (SEQ ID NO: 75).

[0281] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h5m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h5m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h5m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h5m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h5m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h5m (SEQ ID NO: 76).

[0282] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h6m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h6m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h6m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h6m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h6m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h6m (SEQ ID NO: 77).

[0283] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h7m (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h7m (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h7m (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h7m (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h7m (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h7m (SEQ ID NO: 78).

[0284] In some embodiments, the eukaryotic host cell provided herein comprises: a 3E10 antibody or an antigen-binding fragment thereof, comprising a light chain (3E10-LC) and a heavy chain (3E10-HC), wherein the light chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of: 3E10-LC-h1 (SEQ ID NO: 98), 3E10-LC-h2 (SEQ ID NO: 99), 3E10-LC-h3 (SEQ ID NO: 100), 3E10-LC-h4 (SEQ ID NO: 101), 3E10-LC-h5 (SEQ ID NO: 102), and 3E10-LC-h6 (SEQ ID NO: 103), and the heavy chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of: 3E10-HC-h1 (SEQ ID NO: 79), 3E10-HC-h2 (SEQ ID NO: 80), 3E10-HC-h3 (SEQ ID NO: 79), 3E10-HC-h2 (SEQ ID NO: 80), 3E10-HC-h3 (SEQ ID NO: 99), 3E10-LC ... ID NO: 81), 3E10-HC-h4 (SEQ ID NO: 82), 3E10-HC-h5 (SEQ ID NO: 83), 3E10-HC-h6 (SEQ ID NO: 84) and 3E10-HC-h7 (SEQ ID NO: 85).

[0285] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h1 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h1 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h1 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h1 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h1 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h1 (SEQ ID NO: 98).

[0286] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h2 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h2 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h2 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h2 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h2 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h2 (SEQ ID NO: 99).

[0287] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h3 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h3 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h3 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h3 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h3 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h3 (SEQ ID NO: 100).

[0288] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h4 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h4 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h4 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h4 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h4 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h4 (SEQ ID NO: 101).

[0289] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h5 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h5 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h5 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h5 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h5 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h5 (SEQ ID NO: 102).

[0290] In some embodiments, the sequence of 3E10-LC is at least 95% identical to that of 3E10-LC-h6 (SEQ ID NO: 103). In some embodiments, the sequence of 3E10-LC is at least 96% identical to that of 3E10-LC-h6 (SEQ ID NO: 103). In some embodiments, the sequence of 3E10-LC is at least 97% identical to that of 3E10-LC-h6 (SEQ ID NO: 103). In some embodiments, the sequence of 3E10-LC is at least 98% identical to that of 3E10-LC-h6 (SEQ ID NO: 103). In some embodiments, the sequence of 3E10-LC is at least 99% identical to that of 3E10-LC-h6 (SEQ ID NO: 103). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h6 (SEQ ID NO: 103).

[0291] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h1 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h1 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h1 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h1 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h1 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h1 (SEQ ID NO: 79).

[0292] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h2 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h2 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h2 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h2 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h2 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h2 (SEQ ID NO: 80).

[0293] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h3 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h3 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h3 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h3 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h3 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h3 (SEQ ID NO: 81).

[0294] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h4 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h4 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h4 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h4 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h4 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h4 (SEQ ID NO: 82).

[0295] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h5 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h5 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h5 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h5 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h5 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h5 (SEQ ID NO: 83).

[0296] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h6 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h6 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h6 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h6 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h6 (SEQ ID NO: 84). In some aspects, the sequence of 3E10-HC is 3E10-HC-h6 (SEQ ID NO: 84).

[0297] In some embodiments, the sequence of 3E10-HC is at least 95% identical to that of 3E10-HC-h7 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-HC is at least 96% identical to that of 3E10-HC-h7 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-HC is at least 97% identical to that of 3E10-HC-h7 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-HC is at least 98% identical to that of 3E10-HC-h7 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-HC is at least 99% identical to that of 3E10-HC-h7 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h7 (SEQ ID NO: 85).

[0298] In some embodiments, the eukaryotic host cell comprising the 3E10 antibody or its antigen-binding fragment described herein includes a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of: 3E10-VL-h1 (SEQ ID NO: 86), 3E10-VL-h2 (SEQ ID NO: 87), 3E10-VL-h3 (SEQ ID NO: 88), 3E10-VL-h4 (SEQ ID NO: 89), 3E10-VL-h5 (SEQ ID NO: 90), and 3E10-VL-h6 (SEQ ID NO: 89). 91); wherein the light chain variable domain (3E10-VL) further comprises one or more amino acid residues selected from the following: proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76, alanine (Ala) at position 80, Asn at position 81, Thr at position 83, Asn at position 85, and valine (Val) at position 104, according to Kabat numbering; and a set of 3E10-VL CDRs, which have a total of no more than 6 amino acid substitutions relative to the CDR group having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11); and wherein the antibody comprises a set of 3E10-VL The CDR group has a total of no more than 6 amino acid substitutions relative to the CDR group having the amino acid sequences 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NO:10), and 3E10-VL-CDR3 (SEQ ID NO:11).

[0299] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs containing no more than 5 amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0300] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs containing no more than four amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0301] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs, which contain no more than three amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0302] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs, which contain no more than two amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0303] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs, which contain no more than one amino acid substitution relative to a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0304] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VL CDRs, which contain a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0305] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a lysine (Lys) residue at position 49 of 3E10-VL according to the Kabat number.

[0306] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a glutamic acid (Glu) residue at position 81 of 3E10-VL according to the Kabat number.

[0307] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a proline (Pro) residue at position 15 of 3E10-VL according to the Kabat number.

[0308] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a valine (Val) residue at position 104 of the 3E10-VL according to the Kabat number.

[0309] In some embodiments, the eukaryotic host cell comprising the 3E10 antibody or its antigen-binding fragment described herein includes a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: 3E10-VH-h1 (SEQ ID NO: 65), 3E10-VH-h2 (SEQ ID NO: 66), 3E10-VH-h3 (SEQ ID NO: 67), 3E10-VH-h4 (SEQ ID NO: 68), 3E10-VH-h5 (SEQ ID NO: 69), 3E10-VH-h6 (SEQ ID NO: 70), and 3E10-VH-h7 (SEQ ID NO: 69). 71), wherein the heavy chain variable domain (3E10-VH) further comprises one or more amino acid residues selected from the following, according to Kabat numbering: glutamine (Gln) at position 13, leucine (Leu) at position 18, arginine (Arg) at position 19, glycine (Gly) at position 42, serine (Ser) at position 49, Ser at position 77, tyrosine (Tyr) at position 79, Asn at position 82, Ala at position 84, Val at position 89, leucine (Leu) at position 108, Val at position 109, and Ser at position 113, and wherein the antibody comprises a set of 3E10-VH CDRs, which are relative to 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 4). 5) The CDR group of the amino acid sequence has a total of no more than 6 amino acid substitutions.

[0310] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs containing no more than 5 amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0311] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs containing no more than four amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0312] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs containing no more than three amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0313] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs containing no more than two amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0314] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs that contain no more than one amino acid substitution relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0315] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment comprises: a set of 3E10-VH CDRs that contain no more than 5, 4, 3, 2 or 1 amino acid substitutions relative to a set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4) and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0316] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having an arginine (Arg) residue at position 18 of 3E10-VH according to the Kabat number.

[0317] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a (Lys) residue at position 19 of 3E10-VH according to the Kabat number.

[0318] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having an alanine (Ala) residue at position 49 of 3E10-VH according to the Kabat number.

[0319] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a glutamine (Gln) residue at position 13 of 3E10-VH according to the Kabat number.

[0320] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a leucine (Leu) residue at position 108 of the 3E10-VH according to the Kabat number.

[0321] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a valine (Val) residue at position 109 of 3E10-VH according to the Kabat number.

[0322] In one aspect, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a serine (Ser) residue at position 113 of 3E10-VH according to the Kabat number.

[0323] In some embodiments, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having a crystallizable fragment (Fc) region.

[0324] In some embodiments, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof having an Fc region selected from human IgG1Fc, human IgG2aFc, human IgG2bFc, human IgG3Fc and human IgG4Fc.

[0325] In some embodiments, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof containing a heavy chain constant domain (CH).

[0326] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment contains an Fc region selected from human γ1 CH1, human γ2 CH1, human γ3 CH1 and human γ4 CH1.

[0327] In some embodiments, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof containing a light chain constant domain (CL).

[0328] In some embodiments, this disclosure provides a eukaryotic host cell comprising a 3E10 antibody or an antigen-binding fragment thereof comprising an Fc region selected from the group consisting of human λCL and human κCL.

[0329] In some embodiments, the 3E10 antibody or its antigen-binding fragment contained in the eukaryotic host cell comprises a combination of a light chain variable domain (VL) and a heavy chain variable domain (VH) selected from the following: 3E10-VL-h1 (SEQ ID NO: 86) and 3E10-VH-h1 (SEQ ID NO: 65), 3E10-VL-h1 (SEQ ID NO: 86) and 3E10-VH-h2 (SEQ ID NO: 66), 3E10-VL-h1 (SEQ ID NO: 86) and 3E10-VH-h3 (SEQ ID NO: 67), 3E10-VL-h1 (SEQ ID NO: 86) and 3E10-VH-h4 (SEQ ID NO: 68), 3E10-VL-h2 (SEQ ID NO: 87) and 3E10-VH-h1 (SEQ ID NO: 68). 65), 3E10-VL-h2 (SEQ ID NO: 87) and 3E10-VH-h2 (SEQ ID NO: 66), 3E10-VL-h3 (SEQ ID NO: 88) and 3E10-VH-h1 (SEQ ID NO: 65), 3E10-VL-h5 (SEQ ID NO: 90) and 3E10-VH-h5 (SEQ ID NO: 69), 3E10-VL-h5 (SEQ ID NO: 90) and 3E10-VH-h6 (SEQ ID NO: 70), 3E10-VL-h6 (SEQ ID NO: 91) and 3E10-VH-h5 (SEQ ID NO: 69), and 3E10-VL-h6 (SEQ ID NO: 91) and 3E10-VH-h6 (SEQ ID NO: 70).

[0330] In some embodiments, the eukaryotic host cell containing the 3E10 antibody or its antigen-binding fragment contains a combination of the light chain variable domain (VL) of 3E10-VL-h6 (SEQ ID NO: 103) and the heavy chain variable domain (VH) of 3E10-VH-h6 (SEQ ID NO: 70).

[0331] 3E10 antibodies or their antigen-binding fragments or humanized forms may include scFv-Fc peptides, CrossMab peptides, dual variable domain immunoglobulins (DVD-Ig), tandem dual scFv, (scFv)2, single-chain tandem variable (scTaFv) peptides, single-chain variable (scFv) peptides, biantibodies, tandem dual antibodies (TandAb), Fabsc peptides, modular IgG-scFv, or F(ab')2.

[0332] In accordance with the terms of the Budapest Treaty, the deposit of the hybridoma cell line producing monoclonal antibody 3E10 was received on September 6, 2000, by the American Center for Type Culture Collection (ATCC) (10801 University Blvd., Manassas, VA 20110-2209, USA) and granted patent accession number PTA-2439. Therefore, the antibody may have the same or different epitope specificity as monoclonal antibody 3E10 produced by hybridoma with ATCC accession number PTA 2439.

[0333] In some embodiments, the 3E10 antibody or its antigen-binding fragment is a humanized antibody. Methods for humanizing nonhuman antibodies, as discussed herein, are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source (e.g., a chimera). These nonhuman amino acid residues are often referred to as “input” residues, and they are typically derived from an “input” variable domain. Antibody humanization techniques generally involve using recombinant DNA techniques to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.

[0334] Examples of humanized 3E10 sequences are discussed in WO 2015 / 106290, WO 2016 / 033324, WO 2019 / 018426, and WO / 2019 / 018428, the disclosures of which are incorporated herein by reference in their entirety for all purposes, and particularly with respect to their humanized 3E10 sequences and methods of their generation. Other examples of humanized 3E10 heavy chain variable regions (SEQ ID NO: 104-113) and humanized 3E10 light chain variable regions (SEQ ID NO: 114-122) are shown in [references to be inserted here]. Figure 27 and Figure 28 middle.

[0335] Molecular modeling of 3E10 (Pymol) revealed the putative nucleic acid binding pocket (NAB1), such as Figure 30A and Figure 30B As shown. Furthermore, Figure 30B Molecular modeling (Pymol) of 3E10-scFv is shown, with NAB1 amino acid residues highlighted in dots. In some embodiments, the disclosed 3E10 antibody or its antigen-binding fragment contains some or all of the underlined NAB1 sequence. In some embodiments, the 3E10 antibody or its antigen-binding fragment contains variant sequences with altered ability to bind nucleic acids. In some embodiments, mutations in NAB1 (e.g., substitutions, insertions, and / or deletions) enhance antibody binding to nucleic acids (such as RNA). In some embodiments, the mutation is a conserved substitution. In some embodiments, the mutation increases the cationic charge in the NAB1 pocket.

[0336] As illustrated in this paper, the aspartic acid mutation at residue 31 of CDR1 to asparagine increases the cationic charge of that residue and enhances nucleic acid binding and in vivo delivery (3E10-D31N). The aspartic acid mutation at residue 31 of CDR1 to asparagine (3E10-D31R) also expands the cationic charge, while the mutation to lysine (3E10-D31K) alters the charge orientation. Figure 30A ).

[0337] Therefore, in some embodiments, the 3E10 antibody or its antigen-binding fragment comprises a substitution of aspartic acid at residue 31 of CDR1 with arginine (3E10-D31R), modeled as a cationic charge expansion, or a substitution with lysine (3E10-D31K), modeled as a charge orientation change. Thus, in some embodiments, the 3E10 binding protein comprises either a D31R or D31K substitution. Therefore, it is contemplated that all sequences disclosed herein containing residues corresponding to 3E10 D31 or N31 may include either a D31R or D31K substitution.

[0338] The host cell may preferably contain multiple integration sequences. In some embodiments, at least 2 to at least 500 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell. In some embodiments, no more than 1000 to no more than 2 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell. In some embodiments, the number of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is in the range of 2 to 1000 copies in the genome of the host cell. In some embodiments, 2 to 500 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell. In some embodiments, 5 to 500 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell. In some embodiments, 10 to 500 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell. In some embodiments, 10 to 200 copies of each of a first exogenous nucleic acid sequence encoding a nuclease and at least a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof are stably integrated into the genome of the host cell.

[0339] The ratio of the integrated sequences in the host cell can vary. In some embodiments, the ratio of a first exogenous nucleic acid sequence (e.g., encoding a nuclease, such as DNase 1 or DNase II) to a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is 1:1 to 1:10,000. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease to a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is 1:1 to 1:1000. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease to a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is 1:1 to 1:100. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease (i.e., DNase 1 or DNase II) to a second exogenous nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof is 1:2 to 1:10,000. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence is 1:2 to 1:1000. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:2 to 1:100. In some embodiments, the ratio of (i) the number of exogenous nucleic acid sequences encoding the endonuclease to the number of exogenous nucleic acid sequences encoding the 3E10 antibody or its antigen-binding fragment is at least 1:2 to at least 1:1000. In some embodiments, the ratio of (i) the number of exogenous nucleic acid sequences encoding the endonuclease to the number of exogenous nucleic acid sequences encoding the 3E10 antibody or its antigen-binding fragment is no more than 1:250 to no more than 1:2. In some embodiments, the ratio of (i) the number of exogenous nucleic acid sequences encoding the endonuclease to the number of exogenous nucleic acid sequences encoding the 3E10 antibody or its antigen-binding fragment is in the range of 1:2 to 1:250. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease (i.e., DNase 1 or DNase II) to a second exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment is 1:5 to 1:10,000. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease to a second exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment is 1:5 to 1:1000. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease to a second exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment is 1:5 to 1:100. In some embodiments, the ratio of a first exogenous nucleic acid sequence encoding a nuclease to a second exogenous nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment is 1:1 to 1:50.In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:2 to 1:50. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:5 to 1:50. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:1 to 1:20. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:2 to 1:20. In some embodiments, the ratio of the first exogenous nucleic acid sequence encoding the endonuclease to the second exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment is 1:5 to 1:20.

[0340] As mentioned above, in some embodiments, a second exogenous sequence encoding the antibody heavy chain and a third exogenous sequence encoding the antibody light chain are co-expressed with a nuclease (i.e., the first exogenous nucleic acid sequence). In these embodiments, the ratio of these three integrated sequences in the host cell may also vary. In some embodiments, the ratio of the first exogenous sequence:second exogenous sequence:third exogenous sequence is 1:1:1 to 1:100:100. In some embodiments, the ratio is 1:2:2 to 1:100:100. In some embodiments, the ratio is 1:5:5 to 1:100:100. In some embodiments, the ratio is 1:10:10 to 1:100:100. In some embodiments, the ratio is 1:1:1 to 1:50:50. In some embodiments, the ratio of the first exogenous sequence:second exogenous sequence:third exogenous sequence is 1:2:2 to 1:50:50. In some embodiments, the ratio is 1:5:5 to 1:50:500. In some embodiments, the ratio is 1:10:10 to 1:50:50. In some embodiments, the ratio is 1:1:1 to 1:20:20. In some embodiments, the ratio is 1:2:2 to 1:20:20. In some embodiments, the ratio is 1:5:5 to 1:20:20. In some embodiments, the ratio of the first exogenous sequence:second exogenous sequence:third exogenous sequence is 1:10:10 to 1:20:20. In some embodiments, the ratio of the first exogenous sequence:second exogenous sequence:third exogenous sequence is 1:2:2 to 1:15:15. In some embodiments, the ratio of the first exogenous sequence:second exogenous sequence:third exogenous sequence is 1:5:5 to 1:15:15.

[0341] When expressing 4, 5, 6, 7, 8, 9 or 10 target products, similar ratios can be used.

[0342] Host cells and cell culture methods In some embodiments, this disclosure provides methods for producing one or more target products. In these embodiments, host cells, as described above, are cultured in a culture medium under conditions that produce a first target product (e.g., a 3E10 antibody or its antigen-binding fragment) and / or two or more target products and / or their assembled complexes. The target product, such as the 3E10 antibody or its antigen-binding fragment, is then isolated or purified from the culture medium. In some embodiments, the target product (or multiple products) is operatively associated with a signal sequence such that the product (or multiple products) is secreted into and then isolated from the culture medium.

[0343] In some embodiments, the host cell (and its culture) is engineered to include multiple integrated docking sites. For example, in some embodiments, the genome of the host cell of this disclosure preferably includes 1 to 1000 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes 1 to 500 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes at least 2 to at least 500 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes no more than 1000 to no more than 2 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes between 2 and 1000 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes 5 to 500 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome includes 5 to 250 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome contains 5 to 250 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome contains 5 to 100 integrated docking sites, each containing at least one docking site insertion element. In other embodiments, the host cell genome contains 5 to 50 integrated docking sites, each containing at least one docking site insertion element. In some embodiments, the integrated docking sites are independent integrated docking sites, separate from each other and located at independent sites within the genome. For example, the integrated docking sites may preferably be scattered across numerous chromosomes in the genome. In other embodiments, the integrated docking sites may exist as multimers comprising multiple copies of the same DNA sequence linked in tandem.

[0344] The integrated docking site preferably includes one or more insertion elements (which may be referred to as "docking site insertion elements"). The docking site insertion elements are preferably nucleic acid sequences that facilitate the insertion of a nucleic acid sequence encoding a target protein at the docking site. Nucleic acid constructs that can be inserted into docking sites in host cells according to this disclosure are described in detail below.

[0345] This disclosure is not limited to the use of any particular insert element. In fact, a variety of insert elements are contemplated for use. In some embodiments, the insert element is a recombinase docking site insert element. A recombinase docking site insert element is a nucleic acid sequence that is recognized and utilized by a recombinase.

[0346] For example, in some embodiments, the recombinase docking site insert element includes an attachment site (att). In some embodiments, the attachment site is attP. These attachment sites are utilized by PhiC31 integrase, which is a recombinase and is provided to the host cell via a vector in some embodiments. These docking sites serve as acceptors for integrating nucleic acid constructs containing the attB attachment site. In other embodiments, attR and attL attachment sites are utilized. In other embodiments, the recombinase docking site insert element includes an Flp recombination target (FRT) site. These sites are utilized by a recombinase, which is a recombinase and is provided to a host cell via a vector in each embodiment. These docking sites serve as acceptors for integration into nucleic acid constructs containing FRT sites.

[0347] In other embodiments, the recombinase docking site insert element includes a LoxP site. In these embodiments, these sites are utilized by the Cre recombinase, which can be delivered to a host cell via a vector. These docking sites serve as acceptors for integration into nucleic acid constructs containing LoxP sites.

[0348] In other embodiments, the insert element is an HDR (Homologous Directed Repair) docking site insert element. An HDR docking site insert element is a nucleic acid sequence that provides a homologous region (“homologous arm”) that pairs with the corresponding homologous arm base on a nucleic acid construct inserted at that site. These systems are preferably used in conjunction with a nuclease that introduces a double-strand break at one or more target sites (preferably flanked by a homologous arm). In some embodiments, the HDR docking site insert element is the AAVS1 safe harbor locus. In these embodiments, the docking site is utilized by a Rep 78 nuclease (nickase), which can be introduced into a host cell via a vector. The Rep 78 nuclease facilitates site-specific integration of a nucleic acid sequence carrying a homologous arm corresponding to the AAVS1 safe harbor locus.

[0349] In other embodiments, the HDR docking site insert element comprises one or more homologous arms, which are exogenous sequences of 30 to 1000 base pairs in length. These docking sites are preferably used in conjunction with a CRISPR gene editing system. In some embodiments, the docking site also comprises one or more sequences homologous to the guide RNA sequence. In these embodiments, the nucleic acid construct inserted at the docking site preferably comprises homologous arms that are homologous to and base-paired with the homologous arms in the docking site. To facilitate use with the CRISPR gene editing system, a CRISPR-compatible nuclease is introduced into the host cell. The CRISPR-compatible nuclease can be a wild-type endonuclease that produces double-strand breaks at the location defined by the guide RNA (and within the docking site), or a mutant nuclease (i.e., a nicking enzyme) that produces single-strand breaks at an alternating location within the docking sites defined by two guide RNAs. Suitable nucleases are described in detail in the discussion of nucleic acid expression constructs below.

[0350] In some implementations, the docking site may preferably contain a suitable promoter, enabling the use of a promoter capture protocol when a suitable nucleic acid construct is introduced at the docking site. Suitable promoters include, but are not limited to, SIN-LTR, SV40, EF1α, and *E. coli*. E. coli ) lac、 E. coli The promoter sequences include trp, phage λ PL, phage λ PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, α-lactalbumin, and mouse metallothionein-I. In some embodiments, the promoter sequences are oriented at the docking site such that the promoter drives the expression of the inserted nucleic acid construct. In some embodiments, the promoter is oriented relative to the 5' of the docking site. In some embodiments, the promoter is SIN LTR. In these embodiments, SIN-LTR and EPR are located at the 5' of the docking site, while SIN LTR is located at the 3' of the docking site.

[0351] The docking site can be introduced into any suitable host cell line. Suitable host cell lines include, but are not limited to: Chinese hamster ovary cells (CHO-K1, ATCC CCl-61); bovine mammary epithelial cells (ATCC CRL 10274); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subclones of 293 cells for suspension culture growth; see, for example, Graham et al., J. Gen Virol., 36:59

[1977] ); young hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251

[1980] ); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 10000). 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68

[1982] ); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP (CEVEC amniotic fluid cell production line) cells; and human hepatocellular carcinoma cell line (Hep G2).

[0352] In some embodiments, the host cells are modified to lack or naturally lack enzyme activity essential for cell growth or survival in the presence of a selector and provided by a selective marker. For example, Chinese hamster ovary (CHO) cells have been modified to lack GS. In some embodiments where the vector includes a GS selective marker, the host cell line lacks GS. In some embodiments, the GS-deficient host cell line is CHOZN® GS. - / - The cell line can be obtained from Merck KGaA. In other embodiments, where the selective marker is, for example, DHFR, the cell line may preferably be a DHFR-deficient type (i.e., DHFR-deficient). - Suitable DHFR-cell lines include, but are not limited to, CHO-DG44 and its derivatives.

[0353] The docking site sequence can be introduced into host cells using any suitable genome modification system. In some embodiments, the docking site is incorporated into host cells using an integrative vector. The use of integrative vectors to introduce high copy number target sequences (such as docking sites) is described in detail in U.S. Patents 6,852,510 and 7,332,333, and U.S. Publications 2003 / 0092882, 2003 / 0224415, 2004 / 0235173, and 2005 / 0100952, all of which are incorporated herein by reference in their entirety.

[0354] According to this disclosure, host cells such as those described above can be transduced or transfected using an integration vector containing a docking site, provided that multiple copies of the docking site are integrated into the genome of the host cell. Examples of integration vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adeno-associated virus vectors, and transposon vectors.

[0355] In some embodiments, a nucleic acid construct for expressing a target product (e.g., a 3E10 antibody and its antigen-binding fragment) is introduced into a host cell line containing multiple docking sites. As described above, in embodiments, the nucleic acid construct preferably contains a nucleic acid sequence (which may be referred to as an "expression construct insert element") compatible with the docking site insert element as described above.

[0356] Therefore, in some embodiments, this disclosure provides nucleic acid expression constructs for expressing target proteins in host cells, particularly for expressing two or more target proteins, wherein the nucleic acid expression construct encoding the two or more target proteins is integrated into the genome of the host cell at a desired ratio as described in detail above.

[0357] In some implementations, when the docking site does not contain a promoter, the nucleic acid expression construct contains, for example, the following elements that are operatively associated, preferably in a 5' to 3' order: First promoter sequence - selective marker sequence - second promoter sequence - nucleic acid sequence encoding first target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0358] First promoter sequence - selective marker sequence - second promoter sequence - nucleic acid sequence encoding second target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0359] First promoter sequence - selective marker sequence - second promoter sequence - nucleic acid sequence encoding third target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0360] First promoter sequence - selective marker sequence - second promoter sequence - nucleic acid sequence encoding fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0361] In some implementations, when the docking site contains a foreign promoter, the nucleic acid expression construct contains, for example, the following elements that are operatively associated, preferably in a 5' to 3' order: Selective marker sequence - second promoter sequence - nucleic acid sequence encoding first target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0362] Selective labeling sequence - second promoter sequence - nucleic acid sequence encoding second target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0363] The selectively labeled sequence is a second promoter sequence that encodes a third target product (e.g., a 3E10 antibody or its antigen-binding fragment) and a poly A signal sequence.

[0364] The selectively labeled sequence is a second promoter sequence that encodes a fourth target product (e.g., a 3E10 antibody or its antigen-binding fragment) and a poly A signal sequence.

[0365] In some embodiments, the constructs of this disclosure do not include a poly A signaling sequence between the selective marker sequence and the second promoter sequence. This disclosure is not limited to any particular mechanism of action. In fact, an understanding of the mechanism of action is not necessary to practice this disclosure. Nevertheless, it has been found that constructs lacking the poly A signaling sequence after the selective marker can provide better selection and production of the target protein in host cell cultures. In other embodiments, the selective marker is adjacent to the second promoter. In other embodiments, the second promoter is adjacent to the nucleic acid sequence encoding the first target protein. In this case, the term "adjacent" means that there are no intermediate functional elements or introns between the listed components.

[0366] In some embodiments, the nucleic acid expression construct further comprises at least one expression construct insertion element located at one or more positions selected from the group consisting of: the 5' of a first promoter, the 3' of a poly A signal sequence, between the first promoter and the poly A signal sequence, between a selective marker and a second promoter sequence, and both the 5' of the first promoter and the 3' of the poly A signal sequence. Suitable constructs are shown in the following non-limiting examples: Expression construct insertion elements: - First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) - Selective marker sequence - Second (i.e., internal) promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - Poly A signal sequence First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) – alternative marker sequence – second promoter sequence – nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) – poly A signal sequence – expression construct insertion element Expression construct insert element – ​​First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) – Selective marker sequence – Second promoter sequence – Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) – Poly A signal sequence – Expression construct insert element.

[0367] First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - selective marker sequence - expression construct insertion element - second promoter sequence - nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0368] Expression construct insertion elements: - First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) - Selective marker sequence - Second (i.e., internal) promoter sequence - Nucleic acid sequence encoding a second target product (e.g., 3E10 antibody or its antigen-binding fragment) - Poly A signal sequence First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) – alternative marker sequence – second promoter sequence – nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) – poly A signal sequence – expression construct insertion element Expression construct insert element – ​​First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) – Selective marker sequence – Second promoter sequence – Nucleic acid sequence encoding a second target product (e.g., 3E10 antibody or its antigen-binding fragment) – Poly A signal sequence – Expression construct insert element.

[0369] First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - selective marker sequence - expression construct insertion element - second promoter sequence - nucleic acid sequence encoding a second target product (e.g., 3E10 antibody or its antigen-binding fragment) - poly A signal sequence.

[0370] In some embodiments, the construct may contain nucleic acid sequences encoding multiple target products, such as 2, 3, 4, or 5 (or more) target products. Suitable constructs for expressing two target products are shown in the following non-limiting examples. These expression constructs may be used in different ratios with expression constructs encoding an additional third target product (e.g., a 3E10 antibody or its antigen-binding fragment) or third and fourth target products as illustrated below.

[0371] Expression construct insertion element - First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) - Selective marker sequence - Second (i.e., internal) promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence or IRES - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Intron (optional) - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insertion element Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Intron (optional) - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insert element.

[0372] First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Expression construct insertion element - Second promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE-poly A signal sequence - Third promoter sequence or IRES - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE-poly A signal sequence Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insert element.

[0373] Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding the first target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Intron - Nucleic acid sequence encoding the second target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insert element.

[0374] Expression construct insertion element - First promoter sequence (optional, depending on whether the docking site already contains a foreign promoter sequence) - Selective marker sequence - Second (i.e., internal) promoter sequence - Nucleic acid sequence encoding a third target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence or IRES - Nucleic acid sequence encoding a fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) – Selective marker sequence – Second promoter sequence – Nucleic acid sequence encoding the third target product (e.g., 3E10 antibody or its antigen-binding fragment) – WPRE (optional) – Poly A signal sequence – Third promoter sequence – Intron (optional) – Nucleic acid sequence encoding the fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) – WPRE (optional) – Poly A signal sequence – Expression construct insertion element Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding a third target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Intron (optional) - Nucleic acid sequence encoding a fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insert element.

[0375] First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Expression construct insertion element - Second promoter sequence - Nucleic acid sequence encoding the third target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE-poly A signal sequence - Third promoter sequence or IRES - Nucleic acid sequence encoding the fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE-poly A signal sequence Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding a third target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Third promoter sequence - Nucleic acid sequence encoding a fourth target product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - Poly A signal sequence - Expression construct insert element.

[0376] Expression construct insert element - First promoter sequence (optional, depending on whether the docking site already contains an exogenous promoter sequence) - Selective marker sequence - Second promoter sequence - Nucleic acid sequence encoding the target third product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - poly A signal sequence - Third promoter sequence - Intron - Nucleic acid sequence encoding the target fourth product (e.g., 3E10 antibody or its antigen-binding fragment) - WPRE (optional) - poly A signal sequence - Expression construct insert element.

[0377] In some embodiments, a mixture of different constructs is utilized. In some embodiments, the mixture of different constructs may include constructs as described above and constructs that begin with an internal or second promoter (i.e., those that begin after a selective marker and do not include a selective marker). Higher insertion rates are expected to be achieved by using mixtures of constructs (some of which do not include selective markers).

[0378] In some implementations, the expression construct insert element is an element used in conjunction with or recognized by a transposon, integrase, recombinase, or CRISPR system. Suitable insert elements include, but are not limited to, inverted terminal repeat sequences, integrase attachment sites (att), and homologous recombination arms, which, in the case of the construct described herein, can be described as homologous recombination insert elements.

[0379] Nucleic acid constructs can be used with a variety of different vectors and vector systems. These vectors and vector systems are preferably used to introduce nucleic acid expression constructs into the aforementioned host cells. Suitable vectors and vector systems include, but are not limited to, viral gene insertion technologies, such as retroviruses, lentiviruses, and AAV systems, and non-viral gene insertion technologies, such as transposases, recombinases, integrases, or CRISPR gene insertions. Specific examples of technologies / enzymes that can be used with the nucleic acid constructs of this disclosure include transposase systems, Sleeping Beauty transposase systems, Mos1 transposase systems, Tol2 transposase systems, Leapin transposase systems, λ recombinase systems, FLP / FRT systems, Cre / Lox systems, MMLV integrase systems, Rep 78 integrase systems, and CRISPR systems (which may include nucleases or nickases and guide sequences). In some embodiments, the system is a nucleic acid integration system, provided that the system is not a retroviral or lentiviral system utilizing a retrovirus or lentiviral LTR.

[0380] As discussed above, in some embodiments, the expression construct insert element includes an attachment site (att). In some specific embodiments, the attachment site is attB. These attachment sites are utilized by PhiC31 integrase, a recombinase, and in some embodiments, can be provided to the host cell via a vector. These sites facilitate the integration of the nucleic acid construct into a docking site containing the attP attachment site. In other embodiments, attR and attL attachment sites may be utilized.

[0381] In other embodiments, the expression construct insert element includes Flp recombination target (FRT) sites. These sites are utilized by a recombinase, which is a recombinase and is provided to the host cell via a vector in the embodiments. These sites facilitate the integration of the nucleic acid construct into a docking site containing the corresponding FRT site.

[0382] In other embodiments, the expression construct insert element includes a LoxP site. In these embodiments, these sites are utilized by a Cre recombinase, which can be delivered to the host cell via a vector. These sites facilitate the integration of the nucleic acid construct into a docking site containing the corresponding LoxP site.

[0383] In other embodiments, the expression construct insert element is an HDR (homology-directed repair) expression construct insert element. An HDR expression construct insert element is a nucleic acid sequence providing a homologous region (“homologous arm”) that pairs with the corresponding homologous arm base in the docking site. These systems are preferably used in conjunction with a restriction endonuclease that introduces a double-strand break at one or more target sites (preferably flanked by a homologous arm). In some embodiments, the HDR expression construct insert element comprises a homologous arm of the AAVS1 safe harbor locus. In these embodiments, the expression construct is specifically integrated into the docking site containing the AAVS1 safe harbor locus. Integration is facilitated by a Rep 78 protein restriction endonuclease (nickase), which can be introduced into the host cell via a vector. The Rep 78 protein nickase promotes site-specific integration of a nucleic acid sequence carrying the homologous arm corresponding to the AAVS1 safe harbor locus.

[0384] In other embodiments, the HDR expression construct insert element comprises one or more homologous arms, which are exogenous sequences of 30 to 1000 base pairs in length. These expression constructs are preferably used in conjunction with a CRISPR gene editing system. In these embodiments, the nucleic acid construct is inserted at a docking site containing homologous arms that are homologous to and base-paired with the homologous arms in the nucleic acid construct. To facilitate use with the CRISPR gene editing system, a CRISPR-compatible nuclease is introduced into the host cell. The CRISPR-compatible nuclease can be a wild-type endonuclease that produces double-strand breaks at a location defined by the guide RNA (and within the docking site), or a mutant nuclease (i.e., a nicking enzyme) that produces single-strand breaks at an alternating location within docking sites defined by two guide RNAs. Suitable nucleases are described in detail in the discussion of nucleic acid expression constructs below.

[0385] As discussed above, integration at the docking site typically requires the expression of a foreign enzyme in the host cell. Suitable enzymes include, but are not limited to, recombinases (including integrases), endonucleases, and nickases. Therefore, in some embodiments, the host cell of this disclosure contains a foreign nucleic acid sequence (or expression construct) for the expression of recombinases (including integrases), endonucleases, and nickases. In some embodiments, the construct for expressing the foreign enzyme can be stably integrated into the genome of the host cell. In other embodiments, a vector for expressing the foreign enzyme is transiently introduced into the host cell, for example using an extrachromosomal vector (such as a plasmid).

[0386] In some embodiments, a vector containing a foreign enzyme and a vector containing a nucleic acid construct for expressing the target protein are transiently introduced into a host cell, for example, by transfection. In some embodiments, the ratio of the vector encoding the foreign enzyme to the target gene vector is 1:1000 to 1:10. In some more embodiments, this ratio is 1:100 to 1:750. In some still more embodiments, this ratio is 1:400 to 1:600. This is surprising because literature on other integrase systems generally indicates the need for higher levels of vectors encoding the foreign enzyme to the target gene construct.

[0387] In some implementations, the integrase is phiC31 integrase (BioCat GmbH, Heidelberg, DE, or System Biosciences, Palo Alto, CA). phiC31 integrase is a sequence-specific recombinase encoded within the genome of the bacteriophage phiC31. phiC31 integrase mediates recombination between two 34-base-pair sequences called attachment sites (att), one present in the bacteriophage and the other in the host. This serine integrase has been shown to function efficiently in a variety of cell types, including mammalian cells. In the presence of phiC31 integrase, donor plasmids containing attB can be unidirectionally integrated into the target genome via recombination at sites with sequence similarity to the native attP site (called pseudo-attP sites). phiC31 integrase can integrate plasmids of any size in single-copy form and does not require cofactors. The integrated transgene is stably expressed and heritable.

[0388] Other suitable recombinase-based systems include the CRISPR gene editing system, CRE-Lox, FLP-FRT, and λ recombinase system.

[0389] Cre-Lox recombination is a site-specific recombinase technology used to perform deletions, insertions, translocations, and inversions at specific sites in a cell's DNA. It allows DNA modifications to be targeted at specific cell types or triggered by specific external stimuli. It can be performed in both eukaryotic and prokaryotic systems. The Cre-Lox recombination system is particularly useful for neuroscientists studying the brain, where complex cell types and neural circuits come together to produce cognition and behavior. The system consists of a single enzyme, Cre recombinase, which recombines a pair of short target sequences called Lox sequences. The system can be performed without inserting any additional supporting proteins or sequences. The Cre enzyme and the original Lox site, called the LoxP sequence, are derived from bacteriophage P1. See, for example, Targeted integration of DNA using mutant lox sites in embryonic stem cells. Araki et al., Nucleic Acids Res, February 1997, Vol. 25, No. 4, pp. 868-872; High-Resolution Labeling and Functional Manipulation of Specific Neuron Types in Mouse Brain by Cre-Activated Viral Gene Expression. Kuhlman et al., PLoS One, April 2008, Vol. 3, e2005; When reverse genetics meets physiology: the use of site-specific recombinases in mice. Tronche et al., FEBS Letters, August 2002, Vol. 529, No. 1, pp. 116-121.

[0390] The FLP-FRT recombination system is another site-directed recombination technique conceptually very similar to Cre-lox, where the flippant enzyme (Flp) and the short flippant enzyme target recognition (FRT) sites are similar to Cre and loxP, respectively. See, for example, Candice et al., Cre / loxP, Flp / FRT Systems and Pluripotent Stem Cell Lines (2012) Topics in Current Genetics, Vol. 23. FLP-FRT technology can be an efficient alternative to Cre-lox and has also been used in combination with it, allowing for the parallel control of two separate recombination events.

[0391] The nucleic acid constructs disclosed herein can be used in conjunction with the CRISPR homologous recombination (HDR) system. HDR is triggered by the presence of double-strand breaks (DSBs) in DNA. The CRISPR / Cas9 system is preferably used to generate targeted double-strand breaks via guide RNA sequences, enabling insertion into the nucleic acid constructs disclosed herein. See, for example, Zhang et al., Efficient precise knockin with a double-cut HDR donor after CRISPR / Cas9-mediated double-stranded DNA cleavage (2017) Genome Biol. 18:35; Mali et al., Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957–963 (2013); Mali et al., RNA-Guided Human Genome Engineering via Cas9. Science 339(6121), 823-826 (2013); Ran et al., Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 155(2), 479-480 (2013). Suitable guide RNA sequences (gRNAs) can be designed, as known in the art. In some implementations, the CRISPR system for HDR utilizes one or two guide sequences. In some embodiments, when using a single guide RNA sequence, a nuclease, such as Cas9, is used, which is guided by the guide RNA sequence to produce a single double-strand break. In some embodiments, when using two guide sequences, a nicking enzyme, which may be a mutant Cas9 nuclease, is used, which produces a single-strand break only in the target DNA sequence guided by each of the guide RNA sequences. The single-strand breaks are preferably located at intersections on different strands of the target DNA sequence (i.e., the sense and antisense strands). This arrangement generally improves HDR efficiency.

[0392] Generally, the term "CRISPR system" refers to transcripts and other elements involved in the expression of CRISPR-related ("Cas") genes or guiding their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr partner sequences (in the case of endogenous CRISPR systems, encompassing "direct repeat sequences" and partially direct repeat sequences processed by tracrRNA), guide sequences (also referred to as "spacers" in the case of endogenous CRISPR systems), or other sequences and transcripts derived from CRISPR loci. In some embodiments, one or more elements of the CRISPR system are derived from a type I, II, or III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism containing an endogenous CRISPR system, such as *Streptococcus pyogenes* (…). Streptococcus pyogenes Generally, a CRISPR system is characterized by elements (also called protospacers in the case of endogenous CRISPR systems) that promote the formation of the CRISPR complex at a target sequence site. In the case of CRISPR complex formation, a "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Perfect complementarity is not required, as long as there is sufficient complementarity to induce hybridization and promote CRISPR complex formation. The target sequence can contain any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the cell nucleus or cytoplasm. In some embodiments, the target sequence can be within organelles (e.g., mitochondria or chloroplasts) of eukaryotic cells. A sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "edit template," "edit polynucleotide," or "edit sequence." In aspects of this disclosure, exogenous template polynucleotides may be referred to as edit templates. In one aspect of this disclosure, the recombination is homologous recombination.

[0393] Typically, in the case of an endogenous CRISPR system, the formation of a CRISPR complex (containing a guide sequence that hybridizes with the target sequence and complexes with one or more Cas proteins) results in the cleavage of one or both strands of the target sequence in or near it (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). Not wishing to be bound by theory, the tracr sequence may contain all or part of, or be composed of, the wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence), and may also form part of the CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence with all or part of a tracr-pair sequence operatively linked to the guide sequence. In some embodiments, the tracr sequence and the tracr-pair sequence are sufficiently complementary to hybridize and participate in the formation of the CRISPR complex. As with the target sequence, complete complementarity is believed not to be required, only sufficient for functionality. In some embodiments, when optimally aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr partner sequence. In some embodiments, one or more vectors driving the expression of one or more elements of a CRISPR system are introduced into a host cell, such that the expression of the CRISPR system elements is directed to form a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr partner sequence, and the tracr sequence may each be operatively linked to a separate regulatory element on a separate vector. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, wherein one or more additional vectors provide any components of the CRISPR system not included in the first vector. The CRISPR system elements combined in a single vector may be arranged in any suitable orientation, such as one element being located at 5′ (“upstream”) or 3′ (“downstream”) relative to the second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of the second element and oriented in the same or opposite directions. In some embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme and one or more of the following: a guide sequence, a tracr partner sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each located in a different intron, two or more located in at least one intron, or all located in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr partner sequence, and tracr sequence are operably linked to the same promoter and expressed from there.

[0394] Non-limiting examples of Cas proteins that can be used in this disclosure include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs or modified forms thereof. These enzymes are known; for example, Streptococcus pyogenes The amino acid sequence of the Cas9 protein can be found in the SwissProt database with accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme is Cas9, and may be derived from... Streptococcus pyogenes or Streptococcus pneumoniae ( S. pneumoniae The CRISPR enzyme directs the cleavage of one or both strands at a target sequence location (such as within the target sequence and / or within the complementary sequence of the target sequence). In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartic-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes transforms Cas9 from a two-strand cleaving nuclease into a nicking enzyme (cleaving a single strand). Other examples of mutations that transform Cas9 into a nicking enzyme include, but are not limited to, H840A, N854A, and N863A. In this disclosure, nickases can be used for genome editing via homologous recombination.

[0395] In some implementations, the HDR insert element comprises a homologous arm of the AAVS1 safe harbor locus and is used in conjunction with the Rep 78 endonuclease (nickase). The adeno-associated virus serotype 2 (AAV2) Rep 78 protein is a chain-specific endonuclease (nickase) that facilitates site-specific integration of transgenic sequences carrying homologous arms corresponding to the AAVS1 safe harbor locus. See, for example, Ramachandra et al., Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors (2011) Nucleic Acids Research, 39(16):e107;WO1998027207).

[0396] As noted above, in some embodiments, the nucleic acid constructs of this disclosure include optional first and second promoter sequences. The first and second promoter sequences may be the same or different. Suitable first and second promoter sequences include, but are not limited to, MMLV LTR promoters, MoMuSV LTR promoters, RSVLTR promoters, SIN LTR promoters, SV40 promoters, cytomegalovirus (CMV) immediate early promoters, herpes simplex virus (HSV) thymidine kinase promoters, α-lactalbumin promoters, mouse metallothionein-I promoters, dihydrofolate reductase promoters, β-actin promoters, glycerol phosphokinase (PGK) promoters, and EF1α promoter sequences, and combinations thereof. In some embodiments, the first promoter sequence is not a retroviral LTR promoter; that is, the first promoter is a promoter sequence other than a retroviral LTR promoter sequence. However, when the promoter is a retroviral promoter sequence, it can be a SIN (self-inactivating) LTR promoter sequence. See, for example, co-pending application PCT / US2019 / 064423, which is incorporated herein by reference in its entirety. Suitable Sin LTR promoters are known in the art and are prepared by removing all or part of the U3 region of the LTR.

[0397] As described in PCT / US2019 / 064423, in some embodiments, the first promoter driving the selective marker is a weak promoter. In some embodiments, the weak promoter is a promoter, preferably a constitutive promoter, whose activity, when operably linked to the selective marker sequence, is equal to or less than that of the SIN LTR promoter in the target host (e.g., CHO cells). In other embodiments, the weak promoter is a promoter, preferably a constitutive promoter, whose activity, when operably linked to the selective marker sequence, is equal to or less than that of the human ubiquitin C (UBC) promoter in the target host (e.g., CHO cells). Suitable methods for assessing promoter strength are known in the art. See, for example, Dandindorj et al. (2014) A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors, PLoS One 9(8): e106472; Zhang and Baum (2005) Evaluation of Viral and Mammalian Promoters for Use in Gene Delivery to Salvary Glands Mol. Ther. 12(3):528-536; Qin et al. (2010) Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter PLoS 5(5):e10611; Jeyaseelan et al. (2001) Real-time detection of gene promoter activity: quantitation of toxin gene transcription, Nucleic Acids Research. 29(12):58e–58. In some implementations, weak promoters have been modified to reduce promoter activity. Therefore, in some embodiments, this disclosure provides a vector for expressing a target protein, the vector comprising: a nucleic acid sequence encoding a selective marker, operatively associated with a first weak promoter sequence or a promoter sequence modified to reduce promoter activity compared to an unaltered or wild-type version of the first promoter sequence; and a nucleic acid sequence encoding the target protein, operatively linked to a second promoter sequence. The SIN LTR promoter sequence is one such example.Other promoter sequences mentioned above can also be modified to reduce activity and provide a weak promoter, or the weak promoter can be a naturally occurring weak promoter, such as the UBC promoter.

[0398] In some embodiments, the nucleic acid construct contains a selective marker. Suitable selective markers include, but are not limited to, glutamine synthase (GS), dihydrofolate reductase (DHFR), etc. These genes are described in U.S. Patents 5,770,359, 5,827,739, 4,399,216, 4,634,665, 5,149,636, and 6,455,275, all of which are incorporated herein by reference. In some embodiments, the selective marker utilized is compatible with host cell lines that are defective in the production of enzymes encoded by the selective marker nucleic acid sequence. Suitable host cell lines are described in more detail below. In other embodiments, the selective marker is an antibiotic resistance marker, i.e., a gene that produces a protein that provides antibiotic resistance to cells expressing that protein. Suitable antibiotic resistance markers include genes that provide resistance to: neomycin (neomycin resistance gene), hygromycin (hygromycin B phosphotransferase gene), puromycin (puromycin N-acetyltransferase), etc.

[0399] In other embodiments of this disclosure, where secretion of a target protein is desired, the nucleic acid construct includes a signal peptide sequence operatively associated with the target protein. Sequences of several suitable signal peptides are known to those skilled in the art, including but not limited to those derived from tissue plasminogen activator, human growth hormone, lactoferrin, α-casein, and α-lactalbumin.

[0400] In other embodiments of this disclosure, the nucleic acid construct includes an RNA output element at the 3' or 5' of the nucleic acid sequence encoding the target protein (see, for example, U.S. Patents 5,914,267, 6,136,597, and 5,686,120; and WO99 / 14310, all of which are incorporated herein by reference). The use of the RNA output element is intended to allow for high-level expression of the target protein without incorporating splicing signals or introns into the nucleic acid sequence encoding the target protein.

[0401] In other embodiments, the nucleic acid construct includes at least one internal ribosome entry site (IRES) sequence. Several suitable IRES sequences are available, including but not limited to those derived from foot-and-mouth disease virus (FDV), encephalomyocarditis virus, and poliovirus. The IRES sequence may be located between two transcription units (e.g., nucleic acids encoding subunits of different target proteins or multi-subunit proteins such as antibodies) to form a polycistronic sequence, such that both transcription units are transcribed from the same promoter.

[0402] In some embodiments, nucleic acid constructs are incorporated into nucleic acid expression vectors. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One class of vectors is the “plasmid,” which refers to a circular double-stranded DNA loop in which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another class of vectors is the viral vector, in which a virally derived DNA or RNA sequence is present in a vector intended for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by the virus to achieve transfection into host cells. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) integrate into the host cell’s genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operatively linked. This article refers to such vectors as "expression vectors". Common expression vectors useful in recombinant DNA technology are usually in the form of plasmids. Other suitable vectors include, but are not limited to, granules and yeast artificial chromosomes.

[0403] Therefore, suitable nucleic acid expression vectors include, but are not limited to, transposon vectors as described above, as well as plasmid vectors, retroviral vectors, lentiviral vectors, AAV vectors, phage vectors, etc. Any vector is intended to be used, as long as it can replicate and survive in the host. In the embodiment, the vector is a mammalian expression vector that includes, among the other elements described herein, an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and a 5' flanked non-transcriptional sequence.

[0404] Suitable plasmid vectors that can be incorporated into the nucleic acid constructs of this disclosure include specific plasmid systems for transposon vectors, FLP-FLT systems, Cre-lox systems, CRISPR-Cas9 systems, recombinase systems and integrase systems, as well as plasmid vectors derived from pCIneo, pVAX1, pACT, Gateway plasmid, pAdvantage, pBIND, pG5luc, pTNT, pTarget, pCat3, pSI, pCMV, pSV, etc.

[0405] In some embodiments, this disclosure provides host cells and host cell cultures, wherein the host cells express the target protein from the aforementioned nucleic acid constructs. In some embodiments, the host cells are mammalian host cells. Many mammalian host cell lines are known in the art. Generally, these host cells are capable of growth and survival when placed in a culture medium containing appropriate nutrients and growth factors for monolayer or suspension culture, as described in more detail below. Typically, the cells are capable of expressing and secreting large amounts of a specific target protein into the culture medium. Examples of suitable mammalian host cells include, but are not limited to: Chinese hamster ovary cells (CHO-K1, ATCC CCl-61); bovine mammary epithelial cells (ATCCCRL 10274); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subclones of 293 cells for suspension culture growth; see, for example, Graham et al., J. Gen Virol., 36:59

[1977] ); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251

[1980] ); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 10). 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68

[1982] ); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP (CEVEC amniotic fluid cell production line) cells; and human hepatocellular carcinoma cell line (HepG2).

[0406] In some embodiments, the host cells are modified to lack or naturally lack enzyme activity essential for cell growth or survival in the presence of a selector and provided by a selective marker. For example, Chinese hamster ovary (CHO) cells have been modified to lack GS. In some embodiments where the vector includes a GS selective marker, the host cell line lacks GS. In some embodiments, the GS-deficient host cell line is CHOZN® GS. - / -The cell line can be obtained from Merck KGaA. In other embodiments, where the selective marker is, for example, DHFR, the cell line may preferably be a DHFR-deficient type (i.e., DHFR-deficient). - ). Suitable DHFR - Cell lines include, but are not limited to, CHO-DG44 and its derivatives.

[0407] The nucleic acid constructs and vectors disclosed herein can be introduced into host cells by any suitable method, such as by transfection, transformation, or transduction. In some embodiments, after transfection or transduction, cells are proliferated, then trypsinized and replate. Individual colonies are then selected to provide clonal-selected cell lines. In a further embodiment, clonal-selected cell lines are screened by RNA blotting or PCR assay to verify that the desired number of integration events have occurred. Clonal selection is also expected to allow the identification of superior protein-producing cell lines. In other embodiments, clonal selection is not performed on cells after transfection.

[0408] In some implementations, nucleic acid constructs encoding different target proteins are introduced into host cells, for example, by transfection or electroporation. Nucleic acid constructs encoding different target proteins can be introduced into host cells simultaneously or sequentially (e.g., introducing a nucleic acid construct encoding a first target protein, waiting for a period of time, and then introducing a nucleic acid construct encoding a second target protein).

[0409] In some embodiments of this disclosure, after transforming a suitable host cell line and allowing the host cell line to grow to an appropriate cell density in a culture medium, the target protein is secreted during the culture of the host cells. In some embodiments utilizing amplifiable markers, it is anticipated that the transduced host cells are cultured in a culture medium containing a gene inhibitor. Suitable inhibitors include, but are not limited to, methotrexate for inhibiting DHFR and methionine sulfoxide (Msx) or phosphinothricin for inhibiting GS. It is anticipated that as the concentration of these inhibitors increases in the cell culture system, cells with higher copy numbers of the amplifiable marker (and therefore one or more target genes) or containing higher yields of the insert will be selected.

[0410] Therefore, it is preferable to culture host cells containing the vectors described above according to methods known in the art. Suitable culture conditions for mammalian cells are well known in the art (see, for example, J. Immunol. Methods (1983) 56:221-234

[1983] , Animal Cell Culture: A Practical Approach 2nd ed., Rickwood, D. and Hames, BD ed., Oxford University Press, New York

[1992] ).

[0411] The host cell cultures disclosed herein are prepared in a medium suitable for the specific cells being cultured. Commercially available mediums such as ActiPro (HyClone), ExCell Advanced Fed Batch (SAFC), Ham's F10 (Sigma, St. Louis, MO), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Durbeco Modified Eagle Medium (DMEM, Sigma) are exemplary nutrient solutions. Suitable mediums are also described in U.S. Patents 4,767,704, 4,657,866, 4,927,762, 5,122,469, 4,560,655, and WO 90 / 03430 and WO 87 / 00195; the disclosures of these patents are incorporated herein by reference. Any of these culture media may be supplemented as needed with serum, hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin (Gentamicin), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), lipids (such as linoleic acid or other fatty acids) and their suitable carriers, and glucose or equivalent energy. In some embodiments utilizing selective labeling (such as GS), for example, the culture medium will be deficient in glutamine. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.

[0412] This disclosure also considers various culture systems (e.g., culture dishes, 96-well plates, roller flasks, and bioreactors) for using transfected host cells. For example, transfected host cells can be cultured in a perfusion system. Perfusion culture refers to providing a continuously flowing culture medium in a culture that maintains a high cell density. Cells are in suspension and do not require solid support for growth. Typically, a continuous supply of fresh nutrients must be provided while toxic metabolites are removed, and ideally, dead cells are selectively removed. Filtration, retention, and microencapsulation methods are all suitable for updating the culture environment at a sufficient rate.

[0413] As another example, in some implementations, fed-batch culture procedures may be employed. In fed-batch culture, host cells (e.g., mammalian host cells) and culture medium are typically initially supplied to the culture vessel, and additional culture nutrients are added to the culture in continuous or discrete increments during culture, with or without periodic cell and / or product harvesting before culture termination. Fed-batch culture may include, for example, semi-continuous fed-batch culture, in which the entire culture (including cells and culture medium) is periodically removed and replaced with fresh culture medium. Fed-batch culture differs from simple batch culture, in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process. Fed-batch culture can be further distinguished from perfusion culture because the supernatant is not removed from the culture vessel during this process (in perfusion culture, cells are retained in the culture through, for example, filtration, encapsulation, anchoring to microcarriers, etc., while culture medium is continuously or intermittently introduced into and removed from the culture vessel). In some implementations, batch culture is performed in roller flasks.

[0414] Furthermore, the cells in the culture can be propagated according to any protocol or procedure that may be suitable for a particular host cell and a specific production plan. Therefore, this disclosure contemplates single-step or multi-step culture procedures. In a single-step culture, host cells are seeded into a culture environment, and the methods of this disclosure are employed in a single production stage of cell culture. Alternatively, multi-stage cultures are contemplated. In a multi-stage culture, cells can be cultured in multiple steps or stages. For example, cells can be grown in a first-step or growth-phase culture, where cells that may be taken from storage are seeded into a medium suitable for promoting growth and high viability. By adding fresh medium to the host cell culture, the cells can be maintained in the growth phase for a suitable period of time.

[0415] Fed-batch or continuous cell culture conditions are designed to enhance the growth of mammalian cells during the growth phase of cell culture. In the growth phase, cells are grown for a period of time under conditions that maximize growth promotion. Culture conditions, such as temperature, pH, and dissolved oxygen (dO2), are those tailored to specific hosts and are readily apparent to those skilled in the art. Typically, pH is adjusted to a level between approximately 6.5 and 7.5 using an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH). Suitable temperature ranges for culturing mammalian cells (such as CHO cells) are between approximately 30°C and 38°C, and suitable dO2 is between 5% and 90% of air saturation.

[0416] Following the peptide production phase, the target peptide is recovered from the culture medium using techniques well-established in the art. The target protein is preferably recovered from the culture medium as a secreted peptide (e.g., secretion of the target protein is guided by a signal peptide sequence), but it can also be recovered from host cell lysates. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. Subsequently, the peptide is purified from contaminating soluble proteins and peptides, with the following procedures being examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica gel or cation exchange resins (such as DEAE), chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, and protein A agarose gel column to remove contaminants such as IgG. Protease inhibitors such as benzyl sulfonyl fluoride (PMSF) can also be used to inhibit proteolytic degradation during purification. Additionally, the target protein can be fused within a marker sequence frame that allows for the purification of the target protein. Non-limiting examples of tag sequences include a hexahistine tag (which can be supplied by a vector, preferably the pQE-9 vector) and a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from influenza hemagglutinin protein (see, for example, Wilson et al.). Cell , 37:767

[1984] ). Those skilled in the art will understand that the purification methods suitable for the target peptide may need to be modified to take into account changes in peptide properties after expression in recombinant cell culture.

[0417] In some embodiments, nucleic acid constructs are incorporated into the system. In some embodiments, the system comprises multiple nucleic acid constructs or vectors as described above, intended for introduction into host cells. In other embodiments, in addition to nucleic acids or vectors encoding enzymes necessary for incorporating nucleic acid constructs into the host cell genome, the system also comprises one or more nucleic acid constructs or vectors as described above, intended for introduction into host cells. Exemplary enzymes include, but are not limited to, transposons used in conjunction with transposon vector systems, integrases for systems utilizing integrated sequences (such as the PhiC31 system, MMLV system, etc.), recombinases for vector systems (such as Cre-loc, FLP-FRT, etc.), and Cas9 nucleases for CRISPR-based systems.

[0418] Example This disclosure provides a unique method for combining the expression of human DNase 1 with another product in a cellular production system to allow for the removal of DNA from the final product to be produced. The production level of the DNase may need to vary depending on the product and its potential use. Human DNase 1 can be replaced by any other DNase or many other endonuclease molecules.

[0419] Example 1: Production of CHO cells that generate human DNA enzyme 1 The full-length human DNase 1 encoding DNA sequence, including the endogenous signal peptide region, was identified from NCBI Genbank and the following publication (Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 9188-9192, December 1990). The human DNase 1 gene was engineered, with an optimized Kozak translation initiation sequence added to the flanking clones. A Hind III cloning site was added to the 5' end of the human DNase 1 gene sequence, and an Xho I cloning site was added to the 3' end to accelerate expression cloning. See also... Figure 1A The nucleic acid sequence (SEQ ID NO: 139) and Figure 1B The protein sequence (SEQ ID NO: 140) was obtained. Codon optimization was also performed on this sequence to enhance expression. The full-length CDS encoding human DNase 1 with the added flanking sequences was synthesized and cloned into the pUC57 vector.

[0420] The synthesized plasmid was digested with Hind III and Xho I restriction enzymes to release a DNA fragment encoding human DNase 1. This fragment was then purified by gel electrophoresis and ligated into the pCS-novel MCS-WPRE plasmid digested with these two enzymes. See the plasmid map below. Figure 2 and Figure 3The final clone was sequenced using human DNA enzyme 1 CDS to verify its consistency with the predicted DNA sequence and named pCS-CFSD1-WPRE (new ori).

[0421] Table 1. Characteristics of the initiating reverse transcription vector pCS-novel MCS-WPRE (novel ori)

[0422] Table 2. Characteristics of pCS-CFSD1-WPRE (new ori)

[0423] 1.1 Overview of the development of DNAse1 expression clones.

[0424] A Chinese hamster ovary (CHO-S) production cell line was prepared by three rounds of transduction of the CHO-S parental cell line using a reverse transcription vector prepared from a human DNase 1 expression gene construct. The merged population was expanded after each transduction for cryopreservation.

[0425] The (3X) pooled population was diluted in 96-well cell culture plates to establish single-cell clonal cell lines. Protein titers of the clonal cell lines were screened using Pico Green DNA assays. The top thirteen clones were amplified and subjected to overgrowth productivity tests in triplicate T175 culture flasks, and then cryopreserved. QC analysis of the clonal cell lines was performed after cryopreservation. QC tests included viability, gene copy index, reverse transcription vector composition, bioburden, and mycoplasma.

[0426] 1.2 Materials.

[0427] The plasmid (pHCMV-G) used to express the envelope glycoprotein of vesicular stomatitis virus (Indiana) was initially developed by Pangenix (San Diego, CA) and prepared by Bayou Biolabs (Harahan, LA). The reverse transcription vector plasmid was prepared in-house using the manufacturer's protocol via endotoxin-free maxi-prep (Qiagen, Valencia, CA). Quality control analyses included determination of DNA concentration and sequencing of protein-coding and clonal linker regions. DNA sequencing was performed by ACGT, Inc. (Wheeling, IL) using primers manufactured by Invitrogen (Carlsbad, CA).

[0428] The reverse transcription vector-packaged cell line 293GP was developed by Pangenix (San Diego, CA) and has been characterized for CPS-M and a master cell bank established at BioReliance (Rockville, MD). The suspension-adapted Chinese hamster ovary cell line (CHO-S) was received from GIBCO Life Technologies Inc. (Rockville, MD, catalog number 11619012) and has been characterized for CPS-M and a master cell bank established at BioReliance (Rockville, MD). DF medium was Durbeco modified Eagle medium (HyClone, catalog number SH30243) with 10% fetal bovine serum (FBS) added to HyClone (catalog number SH30070). DFP medium was DF medium with 10 µg / mL cymoxanil. PF CHO liquid soybean medium (PF CHO LS) was purchased from HyClone (catalog number SH30359).

[0429] 1.3 Methods.

[0430] 1.3.1 Reverse transcription vector production.

[0431] 293GP cells were cultured in DFP medium and then passaged into 16 T150 culture flasks using trypsin (HyClone catalog number SH30042). Two hours before transfection, the medium in the culture flasks was replaced with 25 mL of DF medium. Following SOP STM-CEL-0325, transfection was performed using 864 µg of the reverse transcription vector construct DNA plasmid pCS-CFSD1-WPRE (neori) and 54 µg of the expression plasmid for the vesicular stomatitis virus envelope glycoprotein. The plasmid solution was combined with 1:10 TE (total volume 17.47 mL) and 2.52 mL of 2M CaCl2, and then precipitated by adding it dropwise to 19.92 mL of 2X HBS solution. Then, 2.5 mL of the suspension was added to each of the 16 T150 culture flasks, and the cells were incubated at 37°C in a 5% CO2 atmosphere for six hours. From this moment on, growth at 37℃±1℃ and in a 5%±1% CO2 atmosphere will be referred to as standard conditions.

[0432] Six hours later, the medium was replaced with 20 mL of fresh DF medium. The culture flasks were incubated under standard conditions until the day after transfection. Medium was collected from 16 T150 culture flasks and filtered through a 0.45 μm filter, then through a 0.2 μm filter. The reverse transcription vector was concentrated from 320 mL of the harvested medium by centrifugation at 18,750 rpm (40,000 x G) for 90 minutes at 4 °C in a Beckman J-30I centrifuge equipped with a JA-30.5 rotor. The supernatant was aspirated from the centrifuge tubes, and the precipitate in each tube was resuspended in 25 µL of PF CHO LS medium. The concentrated vector was used for the CHO-S transduction step.

[0433] 1.3.2 Transduction of CHO-S cells using a reverse transcription vector Parental CHO-S cells were established in the culture and prepared for transduction according to SOP STM-CEL-0350. 4 × 10⁴ cells were prepared in 5 mL PF CHO LS medium containing 8 µg / mL polybrene. 4 A suspension of live CHO-S cells was prepared. This cell suspension was incubated under standard conditions for at least two hours before adding the reverse transcription vector. Shortly before adding the reverse transcription vector, the cell suspension was centrifuged at 1500 rpm (500 x G) for four minutes in a benchtop centrifuge (Beckman Coulter Allegra 6, equipped with a GH 3.8 rotor) to remove the supernatant, without disturbing the cell pellet. The reverse transcription vector was added to the CHO-S cell pellet, mixed, and incubated under standard conditions.

[0434] One day later, add 5 mL of PF CHO LS medium to the tube containing the cell-reverse transcription vector mixture and mix. Then, centrifuge the mixture in a benchtop centrifuge at 1500 rpm (500 x G) for four minutes. Remove the supernatant containing any residual reverse transcription vector, and wash and centrifuge again. Remove the supernatant, without disturbing the cell pellet, and resuspend the cells in 2 mL of PF CHO LS medium. Transfer the cell suspension to 12-well cell culture plates and gradually expand to increasingly larger cell culture flasks using PF CHO LS medium through continuous passages.

[0435] Using cells from previously transduced cultures, each subsequent transduction was performed using the same method as before. The merged populations from each transduction were amplified and cryopreserved. Additionally, cell samples were submitted for gene copy index testing.

[0436] 1.3.3 Establishment of Clonal Cell Lines Clonal selection was performed on aliquots of sCHO-S / sC-CFSD1-R (3X) cells from passage 6. Cells were diluted to 0.5 and 0.75 viable cells per 200 µL in PF CHO LS medium containing 2% FBS. The addition of FBS allowed cells to grow as adherent cultures and facilitated the growth of founder colonies derived from single cells. For each dilution, twenty 96-well plates were seeded with 200 µL of cell suspension per well.

[0437] 1.3.4 Selection and testing of top-level clonal cell lines.

[0438] The seeded 96-well plates were incubated under standard conditions, and colony development derived from single parental cells was observed under a microscope on two separate days (SOP STM-CEL-0330). On day 14, culture medium was collected from 218 wells where single colonies were observed. Culture medium samples were screened by activity assay to determine protein titers. The medium was replaced with FBS-free PF CHO LS, and cells were cultured in FBS-free PF CHO LS from this point onwards. The top 24 clones were selected based on human DNase 1 titer. Eleven clones failed to survive the transition from adherent to suspension and were discarded. The remaining 13 clones were amplified in an overgrowth study for productivity testing. Triplets of T175 culture flasks were seeded with 50 mL working volumes of PF CHO LS at 300,000 viable cells / mL. Viable cell density (VCD) was measured on day 3, and samples were collected on days 3 and 14 for protein analysis by activity assay. Culture was terminated on day 14.

[0439] 1.4 Results and Discussion.

[0440] Human DNase 1 expression cell lines were created by transducing the sCHO-S parental cell line multiple times using a reverse transcription vector prepared with construct DNA plasmids to achieve pCS-CFSD1-WPRE (neoori). The amplified and merged populations after each transduction were cryopreserved, and cell samples were submitted for gene copy index analysis. The results show the names and gene copy indexes used to identify the sCHO-S cell lines after each transduction round.

[0441] Table 3. Gene copy index results of human DNase 1 expression cell lines

[0442] Each transduction created cell populations with human DNase 1 genes inserted into production cell lines in varying numbers and locations. Clonal selection was performed by limiting dilution plating to identify candidate clonal cell lines for production. The pooled sCHO-S / sC-CFSD1-R (3X) cell populations were diluted to 0.5 and 0.75 viable cells per 200 µL of medium and seeded in 96-well cell culture plates to establish single-cell derived clonal cell lines. Forty 96-well plates were screened twice under a microscope, identifying 218 wells with single colonies.

[0443] In the clonal selection process, the number of wells containing single cells corresponds to the expected number based on seeding density; however, the number of wells with two or more cell colonies is typically greater than the number of wells with single cells. The selection process is very rigorous, so any well containing colonies slightly exceeding the "standard" is classified as a 2+ cell colony well (i.e., larger than normal colonies, satellite cells, irregularly shaped colonies, floating cells). Due to this selection process, many cells that might be single-cell colonies are classified as >1 cell colony per well.

[0444] Table 4. Results of Microscopic Clon Screening

[0445] Fourteen days post-inoculation, culture medium samples were collected from 96-well plates, and protein yield was screened by activity assay. The top 24 clones from the 96-well plate samples ranged from 32 to 47 µg / mL of human DNase 1 protein.

[0446] Table 5. Activity assay results from 96-well plates

[0447] Discarded due to poor growth.

[0448] The top 13 clones were inoculated into triplicate T175 culture flasks for overgrowth productivity testing.

[0449] Table 6. Productivity results of the top 12 clones in triplicate T175 culture flasks

[0450] The top five clones based on titer (PCD second) were identified. ).

[0451] 1.5 Conclusion.

[0452] Clonal cell lines were prepared by limiting dilution of the sCHO-S / sC-CFSD1-R (3X) pooled population. Twelve clonal cell lines were screened by overgrowth analysis. Protein titers were determined by ELISA. The mean highest protein level of the top 12 clones was 40 mg / L. The top five clones, #1, #20, #66, #85, and #98, were selected primarily based on titer. The highest expression levels of these top five clone lines ranged from 42 mg / L for clone #85 to 47 mg / L for clone #1. On day 3, the mean PCD of the top 12 clones was 8.43. The PCD of the top five clones ranged from 10.64 for clone #66 to 16.67 for clone #98. The clones produced high levels of human DNase 1, and CHO cell growth or behavior was not affected by product production. After further cell culture optimization, protein production levels reached up to 2 g / L in fed-batch culture.

[0453] Example 2: Production of CHO cells expressing human DNase 1 and antibody products The presence of residual DNA during cell culture can, in some cases, inhibit the production of specific recombinant proteins, or in others, bind tightly to recombinant protein products, thus preventing effective DNA removal during product purification. Co-expression of human DNase 1 or other nucleases with these types of products can improve product expression and purity. In this experiment, different levels of human DNase 1 were co-expressed with antibody products to determine the success of this method. Human DNase 1, antibody heavy chain, and antibody light chain were co-expressed in CHO cells using GPExLightning technology.

[0454] 2.1 Summary.

[0455] Based on the obtained antibody sequence information, Catalent designed the coding DNA sequences (CDS) for optimal expression of the antibody light chain (LC) and heavy chain (HC) of GMAB, as well as the human DNase 1 gene. Then, each of the three genes was cloned into GPEx. ® The LC and HC CDS in the Lightning expression vector (plasmid) were each confirmed by DNA sequencing.

[0456] Nine independent production cell lines were prepared by single-round transfection of the 1F7 CHO parental cell line using gene constructs developed for expressing GMAB and human DNase 1. The nine merged populations were selected by glutamine removal and amplified for gene copy index (GCIV) testing and cryopreservation. The CHOZn / GA04-LC / HC / DNase merged population #6 cell line showed the optimal GCI ratio among LC, HC, and DNase 1 and was further tested in fed-batch productivity studies.

[0457] Using the Beacon® platform, a single-round clonal selection was conducted on population #6, which combined CHOZn / GA04-LC / HC / DNase, to establish clonal cell lines derived from single cells expressing the target protein. The clones reported in this paper have an average calculated probability of >99% monoclonalness. The top 12 clones were grown under normal conditions in fed-batch productivity culture studies to identify the cell lines optimal for GMAB production.

[0458] Based on protein production and cell line quality, clones #31, 37, and 1328 were identified as the top three Master Cell Bank (MCB) candidates, while clone #11 was considered as a backup candidate.

[0459] 2.2 DNA construction and cloning.

[0460] 2.2.1 Materials and methods.

[0461] Based on the starting antibody sequence information, CDSs for optimal expression of antibodies LC and HC were designed. The unique CDSs for GMAB LC and HC were designed by Catalent using proprietary Triplet-Fix® codon optimization technology and publicly available sources, including the NCBI and IMGT websites. Unique restriction sites were added to the 5' and 3' ends of the CDSs to clone the complete LC and HC CDSs into the Catalent expression vector. Additionally, Kozak sequences for efficient protein expression and two tandem stop codons to prevent translation readthrough were introduced at the 5' and 3' ends, respectively. The human DNase 1 “pathway” will be co-expressed with the antibody strands to potentially enhance their expression and secretion. The human DNase 1 CDSs were subcloned into the Lightning expression vector as described.

[0462] Each designed CDS was synthesized using IDT. For each full-length CDS, cloning was performed in a similar manner. IDTgBlock ® Alternatively, the parental vector can be digested with HindIII and XhoI (HC and pathway) or NotI and BglII (LC) restriction endonucleases, and the released CDS can be ligated to GPEx. ®In the Lightning expression vector, the vector is also digested with the same two corresponding restriction endonucleases. Ligation reactions are performed using NEBuilder HiFi DNA assembly premix according to the manufacturer's procedure. The Lightning expression vector 207puc19attB287-GS-new MCS-WPRE-TKpa was previously generated in Catalent.

[0463] 2.2.2 Results.

[0464] The clones were identified as encoding full-length GMAB LC and HC CDS, as well as the DNase pathway CDS. New plasmids encoding each of the three CDS were named: 207attB-GS-h3E10LC-WPRE, 207attB-GS-h3E10HC-WPRE, and 215-puc19attB287-GS-pathway-WPRE-TKpa. All three CDS and flanking DNA cloning junctions in the final expression construct are shown. Cloning restriction sites are also displayed. The expression vector was sequenced using all three CDS and cloning junctions to exclude mutations.

[0465] Figure 4A The nucleic acid sequence of the h3E10LC insert (SEQ ID NO: 141) is provided, while Figure 4B The amino acid sequence of the h3E10LC insert is provided (SEQ ID NO: 142). Figure 5 The plasmid map of plasmid 207attB-GS-h3E10LC-WPRE is provided. Figure 6A The nucleic acid sequence of the h3E10HC insert (SEQ ID NO: 143) is provided, while Figure 6B The amino acid sequence of the h3E10HC insert is provided (SEQ ID NO: 144). Figure 7 The plasmid map of plasmid 207attB-GS-h3E10HC-WPRE is provided. Figure 8A The nucleic acid sequence (SEQ ID NO: 145) of the 1008-215-pathway-207 insert is provided, while Figure 8B The amino acid sequence of the 1008-215-pathway-207 insert is provided (SEQ ID NO: 146). Figure 9 A plasmid map of plasmid 215-puc19attB287-GS-pathway-WPRE-TKpa is provided. Tables 7, 8, and 9 provide a summary of the characteristics of these expression vectors.

[0466] Table 7. Characteristics of expression vector 207attB-GS-h3E10LC-WPRE

[0467] Table 8. Characteristics of expression vector 207attB-GS-h3E10HC-WPRE

[0468] Table 9. Characteristics of expression vector 215-puc19attB287-GS-pathway-WPRE-TKpa

[0469] 2.3 Cell line development 2.3.1 Materials and methods.

[0470] Next, parental GPEx Lightning 1F7 CHO cells were established in the culture and prepared for transfection. EX-CELL cells supplemented with 6 mM L-glutamine were used. ® Advanced CHO fed-batch medium was used as the basal medium for culture maintenance. The required volume of 1F7 parental cell suspension was centrifuged at 500 x 10⁻⁶ in a benchtop centrifuge (Sorvall Legend XT equipped with a TX-750 rotor) just before the addition of transfection reagents and DNA. g Centrifuge for five minutes. Remove as much supernatant as possible, without disturbing the cell pellet. Then, wash the cells with 5 mL of CHOGro expression medium and centrifuge at 500 x 1000 ml in a benchtop centrifuge. g Centrifuge and aspirate a second time. Then, add fresh CHOGro expression medium to the cell pellet to achieve 200 × 10⁻⁶ cells / mL CHOGro expression medium in 2 mL of medium. 5 The final suspension of viable 1F7 cells was prepared. LC / HC / DNase 207puc19attB287-GS-new MCS-WPRE-TKpa plasmid was combined at ratios of 1:1:1, 5:5:1, and 10:10:1 (LC:HC:DNase), where the total amount of DNA remained constant across the three DNA formulations. The DNA plasmid and recombinase DNA plasmid were mixed in Optipro medium at a ratio of 500:1 in tube #1. Expifectamine reagent was diluted in Optipro medium in a second tube (tube #2). Tubes #1 and #2 were combined and allowed to stand for 1–5 min before being added to 50 mL culture vessels containing 30 × 10⁶ 1F7 cells. 5 (each cell) was then shaken at 250 rpm and 37°C. Three independent transfections were performed, resulting in three separate merged populations.

[0471] After incubation for 2–4 hours, centrifuge the cell-transfection reagent mixture at 500 x 1000 rpm in a benchtop centrifuge. gCentrifuge for five minutes. Remove the supernatant and resuspend the transfected cells in 10 mL of EX-CELL electrolyte solution supplemented with 2% ACF and 6 mM L-glutamine. ® Advanced CHO fed-batch culture medium. Transfected cells were allowed to recover for three days post-transfection before the selection process began. After the cells had fully recovered from transfection, the culture was centrifuged at 500 x 10⁻⁶ in a benchtop centrifuge. g Centrifuge for five minutes; discard all culture medium, do not disturb the pellet, and resuspend the cells in 10 mL EX-CELL cells without L-glutamine or ACF. ® Advanced CHO fed-batch medium was used. Cultures were then counted at least every other day to monitor viable cell density, viability percentage, and doubling time. Typically, transfected cells exhibited a decline in viability from the start of selection until day 10 post-transfection (the lowest point). After reaching the lowest viability, the selection process was complete, and successfully transfected cells began to recover. Cultures were allowed to recover for another 10 days until viability was fully restored and doubling time stabilized.

[0472] The merged cell populations from the transfection were amplified and cryopreserved. Supernatant samples were collected for protein titer analysis by BioHT and protein quality analysis by SDS-PAGE. Cell samples were submitted for gene copy index (GCIV) analysis and residual recombinase analysis.

[0473] 2.3.2 Generation of GMAB-expressing cell lines.

[0474] Next, GMAB-expressing cell lines were created by transfecting the 1F7 parental cell lines with three different DNA plasmids in one round. The names used to identify each of the nine 1F7 cell lines after transfection, along with the GCIV results, are shown. See Table 10. SDS-PAGE results are also shown. See [link to table]. Figure 10 .

[0475] Table 10. Transfection of GMAB-expressing cell lines

[0476] 2.4 Feed batch production from merged cell populations.

[0477] 2.4.1 Materials and methods.

[0478] The cell line CHOZn / GA04-LC / HC / DNase was scaled up in population #6 for preliminary protein production. Feed-and-batch analysis was performed in 500 mL shake flasks at 6.0 × 10⁻⁶. 5 One live cell / mL was seeded in 120 mL ActiPro medium supplemented with 4% ps307.

[0479] Viable cell density (VCD) and viability were measured daily after day 2. Protein titers were determined by BioHT IgG analysis. Protein yield (picograms produced per cell per day, pcd) was determined by the following method: protein concentration (in pg / nL) relative to integral cell density (cells per day). Plot the results (days / nL) and record the slope of the linear fit curve. Terminate the culture when the viability drops below 90% but no later than day 14. See [link / reference] Figures 11-13 .

[0480] 2.4.2. Results Gel analysis showed that each of the three protein products (LC / HC / DNase 1) was expressed at the expected ratio based on plasmid transfection. DNase 1 production in cells did not appear to inhibit the production of high levels of functional antibodies. These cells behaved similarly to standard CHO cell lines that only produce antibodies. The CHOZn / GA04-LC / HC / DNase pooled population reached 254 × 10⁻⁶ on day 10. 5 The peak viable cell density was [number] cells / mL, and the cell line was harvested on day 14 with a viability of 96.4%. The cell line produced 5.1 picograms of antibody / cell / day (pg / c / d).

[0481] The antibody concentration in the supernatant is at least 0.001 μg / mL, at least 0.01 μg / mL, at least 0.1 μg / mL, at least 0.125 μg / mL, at least 0.250 μg / mL, at least 0.5 μg / mL, at least 0.75 μg / mL, at least 1 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.001 μg / mL and 100 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.01 μg / mL and 50 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.1 μg / mL and 25 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.125 μg / mL and 10 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.250 μg / mL and 5 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.5 μg / mL and 1 μg / mL. In some embodiments, the antibody concentration in the supernatant is between 0.75 μg / mL and 1 μg / mL.

[0482] 2.5 Clonal selection using Beacon instruments.

[0483] 2.5.1 Materials and methods.

[0484] A single-round clonal selection was performed using a Beacon instrument (Berkeley Lights Inc (BLI), Emeryville, CA) to identify candidate clonal cell lines. The Beacon photofluidic platform utilizes OptoSelect™ photoelectric positioning technology (optical cage) to place single cells into 1 nL microwells on the chip. The instrument can place cells into wells, culture, measure, image, sort, and then output highly expressing clonal cell lines. CHOZn / GA04-LC / HC / DNAase cells were seeded at a concentration of one to two million cells / mL in 10 mL of dynamic culture. One day later, the cells were loaded onto the Beacon instrument containing G12.1 medium supplemented with 6 mM L-glutamine, 0.25 g / L PS307, and 2.5% animal-free fraction (ACF) supplement. The viable cell density of the cell suspension after loading was 38.2 × 10⁻⁶ cells / mL. 5 Cells / ml were collected with a viability of 99.9%. The microwell filling efficiency (percentage of wells containing a single cell) was calculated to be 80.7% (2837 / 3516). Each well was manually verified by the operator to ensure the presence of a single cell. Cells were then cultured on the chip for three days in G12.1 medium supplemented with 6 mM L-glutamine and 0.25 g / L PS307 with 2.5% ACF supplement. Microwells were confirmed to be free of overgrowth (confluence not exceeding 50%). The percentage of clones expanded on the chip (OCCE; percentage of wells containing 4 or more cells by day 3) was calculated to be 96.3% (2731 / 2837).

[0485] On day 3 of culture, micro-diffusion assays were performed using a Spotlight Hu3™ (BLI) apparatus, and parameters were set and applied to select colonies for output. Clones meeting the criteria were ranked by titer scores. The top 96 colonies were output to optical 96-well plates (VWR, catalog number 82050-772), each containing 160 µL of G12.1 medium containing 6 mM L-glutamine, 0.25 g / L PS307, and 2.5% ACF supplement.

[0486] Colonies were cultured for eleven days, and growth was monitored by imaging using an Operetta imaging system (PerkinElmer) on days 3, 7, 9, and 10 post-output. Clones were expanded from 96-well plates to 24-well plates (VWR, catalog number 82050-846), and then to 6-well plates. Clones were submitted for gene copy index (GCIV) analysis. Subsequently, clones were expanded into shaking cultures in 50 mL bioreactor tubes, and then to a volume of 120 mL in 500 mL shake flasks for cryopreservation. Cell counting was performed at each passage in dynamic culture. Twenty-three vials were prepared for cryopreservation for each cell line; three vials were prepared for QC testing for each clone. QC testing included viability after thawing, MET (metabolism-weighted emulsion), and mycoplasma.

[0487] Table 11. Beacon used to select clones to be exported ® parameter

[0488] 2.5.2 Clonal Analysis The transfection process produced cell populations in the production cell lines with GMAB CDS inserted in variable amounts and locations. A total of approximately 2731 colonies were evaluated during clonal selection.

[0489] The top 96 clones, ranked by AU score, were output to 96-well plates with optical apertures. Growth of these clones was observed using an Operetta imaging system (Perkin Elmer) on days 3, 7, 9, and 10 post-output. Of the top 96 clones, a total of 74 were successfully scaled up from the 96-well plates to 24-well plates, and then to 6-well plates. The top 48 clones selected by GCI value were then evaluated in a fed-batch productivity study.

[0490] Table 12. Beacon Results – Top 71 Clones

[0491] 2.5.3 Productivity Study in 24-well Plates The top 71 clones, ranked by GCI value, were tested in dynamic batch cultures in 24-well deep plates. VCD and protein titers were assessed on days 3 and 6 to identify the top 12 clones for cryopreservation and further characterization in a microbial reactor (Ambr® 15; Sartorius AG). VCD, viability, and titers were determined for 48 clones. See Table 13.

[0492] Table 13. Productivity and Harvest Data for 24-hole Plates

[0493] 2.5.4 Testing and selecting clonal cell lines in Ambr15™.

[0494] The productivity of the top 12 selected clonal cell lines was tested in the Ambr15™ microbioreactor system. The Ambr15™ simulates the operating parameters of smaller-scale (10–15 mL) classic bioreactors (2 L and 10 L). The automated workstation consists of 12 disposable microbial reactor blocks, each with independent environmental (aeration and pH), feeding, and sampling controls. Clones were evaluated in ActiPro medium with Cell Boost™ 7a / 7b feed and ExCell Advance fed batch medium with Feed 1 / 4 FEED. For both ActiPro and fed batch cultures, the temperature was transitioned to 34°C on day 6. Culture was terminated when viability dropped below 90% and did not exceed day 14.

[0495] For scale-up, cells from the CHOZn / GA04-LC / HC / DNase cell line were used for clones #5, 11, 12, 28, 31, 37, 66, 317, 419, 1328, 1476, and 1730. Cells were maintained in Actipro medium supplemented with 0.25 g / L PS307, and then cells from each clone cell line were adapted to fed-batch medium (without supplement). The top 12 clones were passaged three times in ActiPro medium or fed-batch medium, and then seeded into Ambr15™ tubes.

[0496] Clones were seeded into Ambr15™ tubes at a cell density of 6.0 × 10⁶ cells / year. 5 10.0 × 10⁻⁶ cells / mL or 10.0 × 10⁻ 5 Cells / mL. Sample daily for VCD, viability, and metabolite levels (glucose, lactate, and ammonia). Measure titers on days 4, 8, 12, 14, or at harvest.

[0497] Table 14. Ambr15™ Experimental Design

[0498] The results show comparisons of clone number relative to cumulative cell density (CCD), clone number relative to titer i, and clone number relative to rQp. Based on analysis of Ambr15™ production data, the top 4 clone candidates based on titer yield (BioHT) are clones #31, #37, #1328, and #11. On average, fed-batch conditions produced higher titers than ActiPro conditions, but the highest overall titer was observed in ActiPro. Further optimization of these conditions and other culture conditions could lead to additional protein yields.

[0499] 2.6 Overall Conclusion The results demonstrate that co-expression of the nuclease human DNase 1 and a second recombinant protein or antibody in this embodiment is possible. Antibody production in these cell lines is at a commercially viable level. Based on activity assays of the culture medium harvested from the culture, human DNase 1 is active and capable of cleaving DNA in the environment of the cell culture medium. The level of DNase 1 produced has been shown to be modulated to achieve the desired effect for this particular product.

[0500] All publications and patents mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the methods and systems described in this disclosure will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Although this disclosure has been described in conjunction with specific embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes of implementation that will be apparent to those skilled in the art are intended to fall within the scope of the claims below.

[0501] Example 3: Comparison of stable CHO cell lines expressing 3E10 antibody with and without human DNase 1 The GPEx Lightning process was used to generate two stable cell libraries expressing the 3E10 antibody. One cell library, PP5, was programmed to express only the heavy and light chains of the 3E10 antibody. The second cell library, PP6, was programmed to express both the heavy and light chains of the 3E10 antibody, as well as human DNase 1. After generating the two stable cell libraries, their heavy and light chain gene copy index values ​​and productivity performance in fed-batch culture were tested.

[0502] Table 15. Copy index of heavy and light chain genes in PP5 and PP6

[0503] The results showed that the gene copy index values ​​of the PP5 and PP6 cell banks were similar. Fed-batch productivity studies were performed on each cell bank on day 14. The results for PP5 and PP6 are compared below: Table 16. Productivity Study of PP5 and PP6 in Feed-in Batches on Day 14

[0504] The results showed that expression of the 3E10 antibody alone produced antibody product titers far lower than expected, and antibody-producing cells exhibited poorer growth characteristics compared to cells producing conventional antibody products. Since the 3E10 antibody binds to nucleic acids to form a complex that can be internalized, it was hypothesized that DNA from dead cells in the culture might bind to the secreted antibody and enter the cells, leading to expression and cell growth problems. Further hypothesies were made that removing DNA by having cells secrete human DNase 1 in addition to the 3E10 antibody could improve antibody production and cell health. Comparisons of two cell lines, PP5 and PP6, supported these hypotheses. The gene copy index of the GPEx Lightning technology was directly correlated with product titers. For both stable cell lines, PP5 (the DNase 1-free line) was expected to produce more antibodies than PP6 (the DNase 1-containing line) because it has a slightly higher transgene number for both heavy and light chain genes. However, the opposite was observed; the PP6 cell line produced antibody levels three times higher than the PP5 cell line. Furthermore, the cell growth behavior of the PP6 cell line was more similar to that of CHO cells producing other types of antibodies than that of the PP5 cell line. These results indicate that the DNase 1 co-expression method is a more feasible way to generate 3E10 antibodies than the conventional method of expressing only the heavy and light chains in CHO cells.

[0505] Example 4: Achieving 3E10 expression in mammalian cell cultures using exogenous DNase In the experiments, purified exogenous endonucleases, such as deoxyribonuclease-I (DNase I), were added directly to the culture medium of mammalian cell cultures transiently or stably transfected to express the 3E10 antibody or fragments thereof. Specifically, one day after transfection, purified DNase I was added directly to the cell culture medium of HEK293 cells transiently transfected with plasmids encoding chimeric or humanized antibody variants of 3E10. As a control, a mutant variant called R92, which lacks the potential to bind nucleic acids, was included in the experiments. To assess the effect of DNase treatment on protein secretion, a plasmid encoding the secreted alkaline phosphatase protein (SEAP) was incorporated. Antibody production in the supernatant was assessed at cell harvest. Figure 31AAs shown, significant expression (µg / ml) of IgG1 in the supernatant was observed for the 3E10 variants (D31N or V66) in the presence of exogenous DNase treatment. For these variants, reduced expression (µg / ml) of IgG1 in the supernatant was detected in untreated samples (without DNase), and the R92 control showed IgG1 expression in the supernatant regardless of treatment. It was also observed that administration of DNase to transfected cells did not significantly affect SEAP expression and secretion (Figure, further supporting the beneficial effect of DNase on the expression and retention of secreted anti-DNA-binding 3E10 antibodies). If the incubation period is longer than 3–4 days, dosing may be more frequent in transiently transfected cells. Furthermore, preferred concentrations of DNase I may be 5–10 µg / ml, 10–25 µg / ml, 25–50 µg / ml, 50–100 µg / ml, or higher than 100 µg / ml. This methodology is also applicable to production cell lines with stable expression of 3E10 and derived antibodies. DNase can be administered once at these concentrations or at regular intervals to ensure its continued presence in the medium throughout the culture period (e.g., up to 14–21 days).

Claims

1. A eukaryotic host cell comprising (i) a foreign nucleic acid sequence encoding a nuclease operably linked to a first promoter sequence and (ii) a foreign nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a second promoter sequence.

2. The host cell of claim 1, wherein the host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, juvenile hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

3. The host cell as described in claim 1, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells, and CAP cells.

4. The host cell line of claim 1 or 2, wherein the host cell line is a glutamine synthase (GS) knockout cell line.

5. The host cell line according to any one of claims 1 to 3, wherein the host cell line is a dihydrofolate reductase (DHFR) knockout cell line.

6. The host cell according to any one of claims 1 to 5, wherein the endonuclease is DNase I endonuclease.

7. The host cell of claim 6, wherein the endonuclease is human DNAase I endonuclease.

8. The host cell according to any one of claims 1 to 5, wherein the endonuclease is a DNase II endonuclease.

9. The host cell of claim 8, wherein the endonuclease is human DNAase II endonuclease.

10. The host cell of any one of claims 1 to 9, wherein the sequence encoding the endonuclease is also operatively linked to a secretion signal sequence.

11. The host cell of any one of claims 1 to 10, wherein the first promoter sequence is a weak promoter sequence.

12. The host cell of any one of claims 1 to 10, wherein the first promoter sequence is a cytomegalovirus immediate early (CMV-IE) promoter sequence.

13. The host cell of claim 12, wherein the CMV-IE promoter sequence is a simian CMV-IE promoter (sCMV) sequence.

14. The host cell of any one of claims 1 to 11, wherein the first promoter sequence is not a retroviral LTR promoter.

15. The host cell according to any one of claims 1 to 14, wherein: The 3E10 antibody or its antigen-binding fragment comprises (a) a heavy chain polypeptide or a fragment thereof and (b) a light chain polypeptide or a fragment thereof; and The exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment comprises (1) a first exogenous nucleic acid sequence encoding the heavy chain polypeptide or a fragment thereof and (2) a second exogenous nucleic acid sequence encoding the light chain polypeptide or a fragment thereof.

16. The host cell of claim 15, wherein (i) the ratio of the number of first exogenous nucleic acid sequences integrated into the host cell genome to (ii) the number of second exogenous nucleic acid sequences integrated into the host cell genome is 1:2 to 2:

1.

17. The host cell of claim 15 or 16, wherein the first exogenous nucleic acid sequence is operatively linked to the second promoter sequence, and the second exogenous nucleic acid sequence is operatively linked to the third promoter sequence.

18. The host cell of claim 17, wherein the third promoter sequence has the same nucleotide sequence as the first promoter.

19. The host cell of claim 17 or 18, wherein the third promoter sequence has the same nucleotide sequence as the second promoter.

20. The host cell of claim 17, wherein the third promoter sequence has a different nucleotide sequence from the first promoter.

21. The host cell of claim 17 or 20, wherein the third promoter sequence has a different nucleotide sequence from the second promoter.

22. The host cell of any one of claims 17 to 21, wherein the third promoter sequence is a cytomegalovirus immediate early (CMV-IE) promoter sequence.

23. The host cell of claim 22, wherein the CMV-IE promoter sequence is a simian CMV-IE promoter (sCMV) sequence.

24. The host cell of any one of claims 17 to 21, wherein the third promoter sequence is not a retroviral LTR promoter.

25. The host cell of any one of claims 1 to 24, wherein the 3E10 antibody or its antigen-binding fragment comprises a monovalent, bivalent, or multivalent single-chain variable fragment (scFv).

26. The host cell of any one of claims 1 to 24, wherein the 3E10 antibody or its antigen-binding fragment comprises scFv-Fc polypeptide, CrossMab polypeptide, dual variable domain immunoglobulin (DVD-Ig), tandem dual scFv, (scFv)2, single-chain tandem fragment variable (scTaFv) polypeptide, single-chain fragment variable (scFv) polypeptide, dual antibody, tandem dual antibody (TandAb), Fabsc polypeptide, modular IgG-scFv or F(ab')2.

27. The host cell of any one of claims 1 to 26, wherein the 3E10 antibody or its antigen-binding fragment is humanized.

28. The host cell of any one of claims 1 to 27, wherein the 3E10 antibody or its antigen-binding fragment is a bivalent antibody or a fragment thereof.

29. The host cell of any one of claims 1 to 28, wherein the 3E10 antibody or its antigen-binding fragment is a 3E10 antibody or its antigen-binding fragment.

30. The host cell of claim 29, wherein the 3E10 antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) complementarity-determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 58, the VH CDR2 containing the amino acid sequence of SEQ ID NO: 59, and the VH CDR3 containing the amino acid sequence of SEQ ID NO: 60; and The light chain variable region (VL) CDR1 containing the amino acid sequence of SEQ ID NO: 61, the VL CDR2 containing the amino acid sequence of SEQ ID NO: 62, and the VL CDR3 containing the amino acid sequence of SEQ ID NO:

63.

31. The host cell of claim 29, wherein the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 containing the amino acid sequence of SEQ ID NO: 64, VHCDR2 containing the amino acid sequence of SEQ ID NO: 4, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 5; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 9, VLCDR2 containing the amino acid sequence of SEQ ID NO: 10, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

11.

32. The host cell of claim 29, wherein the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 containing the amino acid sequence of SEQ ID NO: 64, VHCDR2 containing the amino acid sequence of SEQ ID NO: 15, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 5; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 9, VLCDR2 containing the amino acid sequence of SEQ ID NO: 10, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

11.

33. The host cell of any one of claims 29 to 32, wherein the 3E10 antibody or its cell-penetrating variant comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having at least 90% identical amino acid sequence to SEQ ID NO: 2 or SEQ ID NO: 14, and the light chain variable region (VL) having at least 90% identical amino acid sequence to SEQ ID NO:

7.

34. The host cell of any one of claims 29 to 32, wherein the 3E10 antibody or a cell-penetrating variant thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having at least 90% identical amino acid sequence to SEQ ID NO: 2 or SEQ ID NO: 14, and the light chain variable region (VL) having at least 95% identical amino acid sequence to SEQ ID NO:

7.

35. The host cell of any one of claims 29 to 32, wherein the 3E10 antibody or a cell-penetrating variant thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 104-113, and the light chain variable region (VL) having an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 114-122.

36. The host cell of any one of claims 1 to 35, wherein the second promoter sequence has the same nucleotide sequence as the first promoter.

37. The host cell of any one of claims 1 to 35, wherein the second promoter sequence has a nucleotide sequence different from that of the first promoter.

38. The host cell of any one of claims 1 to 37, wherein the second promoter sequence is a cytomegalovirus immediate early (CMV-IE) promoter sequence.

39. The host cell of claim 38, wherein the CMV-IE promoter sequence is a simian CMV-IE promoter (sCMV) sequence.

40. The host cell of any one of claims 1 to 37, wherein the third promoter sequence is not a retroviral LTR promoter.

41. The host cell of any one of claims 1 to 40, wherein 2 to 500 copies of each of the exogenous nucleic acid sequence encoding the endonuclease and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment are stably integrated into the genome of the host cell.

42. The host cell of any one of claims 15 to 40, wherein 5 to 500 copies of each of the first exogenous nucleic acid sequence and at least the second exogenous nucleic acid sequence are stably integrated into the genome of the host cell.

43. The host cell according to any one of claims 1 to 42, wherein the ratio of (i) the number of exogenous nucleic acid sequences encoding the endonuclease to (ii) the number of exogenous nucleic acid sequences encoding the 3E10 antibody or its antigen-binding fragment is 1:1 to 1:

100.

44. The host cell according to any one of claims 1 to 42, wherein the ratio of (i) the number of exogenous nucleic acid sequences encoding the endonuclease to (ii) the number of exogenous nucleic acid sequences encoding the 3E10 antibody or its antigen-binding fragment is 1:2 to 1:

100.

45. The host cell of any one of claims 1 to 44, wherein the exogenous nucleic acid sequence encoding the endonuclease and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment are stably integrated into the genome of the host cell at the docking site.

46. ​​The host cell of claim 45, wherein before integrating the exogenous nucleic acid sequence encoding the endonuclease and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment into the docking site: The docking site includes at least one docking site insertion element; and The exogenous nucleic acid sequence encoding the endonuclease and the exogenous nucleic acid sequence encoding the 3E10 antibody or its antigen-binding fragment are present in one or more transfer vectors, the transfer vectors containing at least one insert element compatible with the at least one docking site insert element.

47. The host cell of any one of claims 1 to 46, wherein the exogenous nucleic acid sequence encoding the endonuclease is operatively linked to a first polyadenylated sequence, and the exogenous nucleic acid sequence encoding the 3E10 antibody or an antigen-binding fragment thereof is operatively linked to a second polyadenylated sequence.

48. The host cell of any one of claims 45 to 47, wherein the host cell genome comprises 5 to 500 docking sites, and each docking site comprises at least one docking site insertion element.

49. The host cell of claim 48, wherein the integrated docking site is independently located throughout the host cell genome.

50. A cell culture comprising a host cell as claimed in any one of claims 1 to 49.

51. A method for expressing a 3E10 antibody or an antigen-binding fragment thereof, the method comprising: Multiple host cells according to any one of claims 1 to 49 are cultured in a culture medium under conditions that achieve (i) the expression of the endonuclease derived from the exogenous nucleic acid sequence encoding the endonuclease and (ii) the 3E10 antibody or its antigen-binding fragment.

52. The method of claim 51, wherein the cultivation is carried out under fed-batch conditions.

53. A cell culture supernatant comprising the 3E10 antibody or its antigen-binding fragment prepared according to the method of claim 51.

54. A cell culture supernatant comprising at least 0.001 μg / mL, at least 0.01 μg / mL, at least 0.1 μg / mL, at least 0.125 μg / mL, at least 0.250 μg / mL, at least 0.5 μg / mL, at least 0.75 μg / mL, at least 1 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL of the 3E10 antibody or its antigen-binding fragment.

55. The cell culture of claim 53 or 54, wherein less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or 0% of the 3E10 antibody or its antigen-binding fragment is bound to the nucleic acid.

56. A composition comprising a 3E10 antibody or an antigen-binding fragment thereof generated according to the method of claim 51 or 52.

57. A composition comprising a 3E10 antibody or an antigen-binding fragment thereof purified from a cell culture supernatant as described in any one of claims 53-55.

58. A cell culture comprising: a eukaryotic host cell containing a foreign nucleic acid sequence encoding a 3E10 antibody or an antigen-binding fragment thereof operably linked to a promoter sequence, and a purified, foreign-added endonuclease.

59. The eukaryotic host cell of claim 58, wherein the eukaryotic host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, young hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

60. The host cell of claim 59, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells and CAP cells.

61. The host cell of claim 58 or 59, wherein the host cell line is a glutamine synthase (GS) knockout cell line.

62. The host cell of any one of claims 58 to 60, wherein the host cell line is a dihydrofolate reductase (DHFR) knockout cell line.

63. The host cell of claim 58, wherein the exogenous nucleic acid sequence encoding the 3E10 antibody or antigen-binding fragment is stably integrated into the host genome.

64. The host cell of claim 58, wherein the exogenous nucleic acid sequence encoding the 3E10 antibody or antigen-binding fragment is expressed on one or more DNA plasmids transiently transfected into the cell.

65. The host cell of claim 64, wherein the DNA plasmid is transiently transfected into the cell by electroporation, PEI, lipid transfection, or calcium phosphate precipitation.

66. The host cell of any one of claims 58 to 65, wherein the purified exogenously added endonuclease is DNase I endonuclease.

67. The host cell of claim 66, wherein the purified exogenously added endonuclease is human DNAase I endonuclease.

68. The host cell of claim 66 or 67, wherein the purified exogenously added endonuclease is added to the host cell culture at least once at a concentration of 10 µg / ml.

69. The host cell of claim 66 or 67, wherein the purified exogenously added endonuclease is added to the host cell culture more than once at a concentration of 10 µg / ml.

70. The host cell of claim 66 or 67, wherein the purified exogenously added endonuclease is added at a concentration of about 5-10 µg / ml, about 10-25 µg / ml, about 25-50 µg / ml, about 50-100 µg / ml, or higher than 100 µg / ml.

71. A method for preparing a host cell culture, the host cell culture comprising: a. A eukaryotic host cell containing a foreign nucleic acid sequence encoding a 3E10 antibody or its antigen-binding fragment operatively linked to a promoter sequence; and b. A purified exogenous nuclease added to the host cell culture.

72. The method of claim 71, wherein the eukaryotic host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, young hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

73. The host cell of claim 72, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells and CAP cells.

74. The host cell of claim 71 or 72, wherein the host cell line is a glutamine synthase (GS) knockout cell line.

75. The host cell of any one of claims 71 to 73, wherein the host cell line is a dihydrofolate reductase (DHFR) knockout cell line.

76. The host cell of claim 71, wherein the exogenous nucleic acid sequence encoding the 3E10 antibody or antigen-binding fragment is stably integrated into the host genome.

77. The host cell of claim 71, wherein the exogenous nucleic acid sequence encoding the 3E10 antibody or antigen-binding fragment is expressed on one or more DNA plasmids transiently transfected into the cell.

78. The host cell of claim 77, wherein the DNA plasmid is transiently transfected into the cell by electroporation, PEI, lipid transfection, or calcium phosphate precipitation.

79. The host cell according to any one of claims 71 to 78, wherein the purified exogenously added endonuclease is DNase I endonuclease.

80. The host cell of claim 79, wherein the purified exogenously added endonuclease is human DNAase I endonuclease.

81. The host cell of claim 79 or 80, wherein the purified exogenously added endonuclease is added to the host cell culture at least once at a concentration of 10 µg / ml.

82. The host cell of claim 79 or 80, wherein the purified exogenously added endonuclease is added to the host cell culture more than once at a concentration of 10 µg / ml.

83. The host cell of claim 79 or 80, wherein the purified exogenously added endonuclease is added at a concentration of about 5-10 µg / ml, about 10-25 µg / ml, about 25-50 µg / ml, about 50-100 µg / ml, or higher than 100 µg / ml.

84. A eukaryotic host cell comprising (i) multiple copies of a first exogenous nucleic acid sequence integrated into the genome of the host cell, the first exogenous nucleic acid sequence encoding a first polypeptide, and (ii) one or more copies of a second exogenous nucleic acid sequence integrated into the genome of the host cell, the second exogenous nucleic acid sequence encoding a second polypeptide, wherein: (a) the ratio of the copy number of the first exogenous nucleic acid sequence to (b) the copy number of the second exogenous nucleic acid sequence is at least 2:1; and The first polypeptide is the 3E10 polypeptide.

85. The eukaryotic host cell of claim 84, wherein the first polypeptide comprises a 3E10 heavy chain polypeptide or a fragment thereof, and the second polypeptide comprises a 3E10 light chain polypeptide or a fragment thereof.

86. The eukaryotic host cell of claim 84, wherein the first polypeptide comprises a 3E10 light chain polypeptide or a fragment thereof, and the second polypeptide comprises a 3E10 heavy chain polypeptide or a fragment thereof.

87. The eukaryotic host cell of claim 84, wherein the first polypeptide comprises a 3E10 heavy chain polypeptide or a fragment thereof, and the second polypeptide comprises a nuclease.

88. The eukaryotic host cell of claim 84, wherein the first polypeptide comprises a 3E10 light chain polypeptide or a fragment thereof, and the second polypeptide comprises a nuclease.

89. The host cell of claim 87 or 88, wherein the endonuclease is DNase I endonuclease.

90. The host cell of claim 89, wherein the endonuclease is human DNAase I endonuclease.

91. The host cell of any one of claims 87 or 88, wherein the endonuclease is a DNase II endonuclease.

92. The host cell of claim 91, wherein the endonuclease is human DNAase II endonuclease.

93. The host cell of any one of claims 84 to 92, wherein the host cell encodes a 3E10 antibody or an antigen-binding fragment thereof.

94. The host cell of claim 93, wherein the 3E10 antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) complementarity-determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 58, the VH CDR2 containing the amino acid sequence of SEQ ID NO: 59, and the VH CDR3 containing the amino acid sequence of SEQ ID NO: 60; and The light chain variable region (VL) CDR1 containing the amino acid sequence of SEQ ID NO: 61, the VL CDR2 containing the amino acid sequence of SEQ ID NO: 62, and the VL CDR3 containing the amino acid sequence of SEQ ID NO:

63.

95. The host cell of claim 93, wherein the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 containing the amino acid sequence of SEQ ID NO: 64, VHCDR2 containing the amino acid sequence of SEQ ID NO: 4, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 5; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 9, VLCDR2 containing the amino acid sequence of SEQ ID NO: 10, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

11.

96. The host cell of claim 93, wherein the 3E10 antibody or its antigen-binding fragment comprises: VH CDR1 containing the amino acid sequence of SEQ ID NO: 64, VHCDR2 containing the amino acid sequence of SEQ ID NO: 15, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 5; and VL CDR1 containing the amino acid sequence of SEQ ID NO: 9, VLCDR2 containing the amino acid sequence of SEQ ID NO: 10, and VL CDR3 containing the amino acid sequence of SEQ ID NO:

11.

97. The host cell of any one of claims 93 to 96, wherein the 3E10 antibody or its cell-penetrating variant comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having at least 90% identical amino acid sequence to SEQ ID NO: 2 or SEQ ID NO: 14, and the light chain variable region (VL) having at least 90% identical amino acid sequence to SEQ ID NO:

7.

98. The host cell of any one of claims 93 to 96, wherein the 3E10 antibody or its cell-penetrating variant comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having at least 90% identical amino acid sequence to SEQ ID NO: 2 or SEQ ID NO: 14, and the light chain variable region (VL) having at least 95% identical amino acid sequence to SEQ ID NO:

7.

99. The host cell of any one of claims 93 to 96, wherein the 3E10 antibody or a cell-penetrating variant thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) having at least 95% identical amino acid sequences to those selected from the group consisting of SEQ ID NO: 104-113, and the light chain variable region (VL) having at least 95% identical amino acid sequences to those selected from the group consisting of SEQ ID NO: 114-122.

100. The host cell of any one of claims 84 to 99, wherein the ratio of (a) the copy number of the first exogenous nucleic acid sequence to (b) the copy number of the second exogenous nucleic acid sequence is at least 3:1, at least 5:1, at least 10:1, at least 25:1, at least 50:1, at least 100:1, at least 250:1, at least 500:1, or at least 1000:

1.

101. The host cell of any one of claims 84 to 99, wherein the ratio of (a) the copy number of the first exogenous nucleic acid sequence to (b) the copy number of the second exogenous nucleic acid sequence is 2:1 to 1000:1, 2:1 to 500:1, 2:1 to 250:1, 2:1 to 100:1, 2:1 to 50:1, 2:1 to 25:1, 2:1 to 10:1, or 2:1 to 5:

1.

102. A cell culture comprising a host cell as claimed in any one of claims 84 to 101.

103. A method for expressing a 3E10 antibody or an antigen-binding fragment thereof, the method comprising: Multiple host cells according to any one of claims 84 to 101 are cultured in a culture medium under conditions that achieve (i) the expression of the endonuclease derived from the exogenous nucleic acid sequence encoding the endonuclease and (ii) the 3E10 antibody or its antigen-binding fragment.

104. The method of claim 103, wherein the cultivation is carried out under fed-batch conditions.

105. A cell culture supernatant comprising the 3E10 antibody or its antigen-binding fragment prepared according to the method of claim 103 or 104.

106. A cell culture supernatant comprising at least 0.001 μg / mL, at least 0.01 μg / mL, at least 0.1 μg / mL, at least 0.125 μg / mL, at least 0.250 μg / mL, at least 0.5 μg / mL, at least 0.75 μg / mL, at least 1 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL of antibody or antigen-binding fragment thereof.

107. The cell culture of claim 105 or 106, wherein less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or 0% of the 3E10 antibody or its antigen-binding fragment is bound to the nucleic acid.

108. A composition comprising a 3E10 antibody or an antigen-binding fragment thereof generated by the method according to claim 103 or 104.

109. A composition comprising a 3E10 antibody or an antigen-binding fragment thereof purified from a cell culture supernatant as described in any one of claims 105-107.

110. A method comprising: At least a first nucleic acid construct encoding a first target protein or nucleic acid and a second nucleic acid construct encoding a second target protein or nucleic acid are introduced into a host cell having a genome containing 1 to 500 integrated docking sites at a ratio of 1 to 1000:1, each docking site containing at least one docking site insertion element, and each nucleic acid construct containing at least one insertion element compatible with at least one docking site insertion element in the integrated docking sites, provided that the nucleic acid expression construct is inserted at the docking site at a ratio of at least 1:1, wherein the first target protein or the second target protein contains (i) a 3E10 heavy chain or a CDR-containing fragment thereof, or (ii) a 3E10 light chain or a CDR-containing fragment thereof.

111. The method of claim 110, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 2:1 to 1000:

1.

112. The method of claim 110, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 5:1 to 500:

1.

113. The method of claim 110, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 10:1 to 200:

1.

114. The method of claim 110, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 10:1 to 100:

1.

115. The method of any one of claims 110 to 114, wherein the first target protein is a 3E10 heavy chain or a CDR-containing fragment thereof, and the second target protein is a 3E10 light chain or a CDR-containing fragment thereof.

116. The method of any one of claims 110 to 114, wherein the first target protein comprises (i) a 3E10 heavy chain or a CDR-containing fragment thereof, or (ii) a 3E10 light chain or a CDR-containing fragment thereof, and the second target protein is a nuclease.

117. The method of any one of claims 110 to 116, wherein only the first nucleic acid construct and the second nucleic acid construct are introduced into the host cell.

118. The method of any one of claims 110 to 116, further comprising introducing a third nucleic acid construct encoding a third target protein, wherein the ratio of the first or second nucleic acid construct to the third nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

119. The method of claim 118, further comprising introducing a fourth nucleic acid construct encoding a fourth target protein, wherein the ratio of the first, second, or third nucleic acid construct to the fourth nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

120. The method of claim 119, further comprising introducing a fifth nucleic acid construct encoding a fifth target protein, wherein the ratio of the first, second, third, or fourth nucleic acid construct to the fifth nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

121. The method of any one of claims 110 to 120, wherein at least the first nucleic acid construct and the second nucleic acid construct further comprise at least the following elements operably associated in a 5' to 3' sequence: Internal startup sequence; A nucleic acid sequence encoding the first target protein or the second protein that is operably linked to the internal promoter; and poly A signal sequence.

122. The method of claim 121, wherein at least the first nucleic acid construct and the second nucleic acid construct contain a selective marker sequence.

123. The method of claim 122, wherein at least the first nucleic acid construct and the second nucleic acid construct contain different selective marker sequences.

124. The method of claim 121, wherein one of the first nucleic acid construct and the second nucleic acid construct comprises a selective marker sequence, and the other of the first nucleic acid construct and the second nucleic acid construct does not comprise a selective marker sequence.

125. The method of any one of claims 121 to 122, wherein the selective marker sequence is located at 5' of the internal promoter sequence and is operatively connected to the 5' promoter sequence.

126. The method of any one of claims 121-124, wherein the nucleic acid construct comprises an extended packaging region (EPR) between the 5' promoter and the selective marker.

127. The method of claim 126, wherein the EPR comprises a plurality of potential Kozak sequences and / or ATG translation start sites.

128. The method of any one of claims 121 to 127, wherein the promoter sequence is selected from the group consisting of: SIN-LTR, SV40, EF1α, E. coli lac、 E. coli trp, phage λ PL, phage λ PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, α-lactalbumin and mouse metallothionein-I promoter sequences.

129. The method of any one of claims 121 to 127, wherein the first promoter sequence is a weak promoter sequence.

130. The method of any one of claims 121 to 129, wherein the first promoter sequence is not a retroviral LTR promoter.

131. The method of any one of claims 121 to 130, wherein the integrated docking site further comprises an exogenous promoter.

132. The method of claim 131, wherein the exogenous promoter is selected from the group consisting of: SIN-LTR, SV40, EF1α, E. coli lac、 E. coli trp, phage λ PL, phage λ PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, α-lactalbumin and mouse metallothionein-I promoter sequences.

133. The method of claim 132, wherein the promoter is a retroviral LTR.

134. The method of claim 133, wherein the retroviral LTR is a SIN LTR.

135. The method of any one of claims 110 to 134, wherein the nucleic acid expression construct is provided in a vector.

136. The method of claim 135, wherein the vector is a plasmid vector.

137. The method of any one of claims 135 to 136, wherein the vector is transiently introduced into the host cell.

138. The method of any one of claims 110 to 137, wherein the host cell line comprises a nucleic acid construct encoding an enzyme that facilitates the insertion of the nucleic acid expression construct at the docking site.

139. The method of claim 138, wherein the nucleic acid construct encoding an enzyme that promotes the insertion of the nucleic acid expression construct at the docking site is transiently introduced into the host cell.

140. The method of claim 138 or 139, wherein the nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the docking site is provided in a vector.

141. The method of claim 140, wherein the vector is a plasmid vector.

142. The method of any one of claims 138 to 141, wherein the ratio of the nucleic acid construct encoding an enzyme that promotes the insertion of the nucleic acid expression construct at the docking site to the nucleic acid expression construct encoding the first target protein, which is transiently introduced into the host cell, is 1:1000 to 1:

10.

143. The method of any one of claims 138 to 141, wherein the enzyme is selected from the group consisting of integrase, recombinase, nuclease and nickase.

144. The method of any one of claims 138 to 143, wherein the nucleic acid construct encoding an enzyme that facilitates insertion of the nucleic acid expression construct at the docking site is provided in a vector.

145. The method of any one of claims 138 to 144, wherein the host cell genome comprises 5 to 500 integrated docking sites, each docking site comprising at least one docking site insertion element.

146. The method of any one of claims 138 to 145, wherein the host cell genome comprises 5 to 250 integrated docking sites, each docking site comprising at least one docking site insertion element.

147. The method of any one of claims 110 to 146, wherein the host cell genome comprises 5 to 100 integrated docking sites, each docking site comprising at least one docking site insertion element.

148. The method of any one of claims 110 to 147, wherein the integrated docking site is independently located throughout the host cell genome.

149. The method of any one of claims 110 to 148, wherein the docking site insertion element is targeted by an enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase.

150. The method of any one of claims 110 to 149, wherein the docking site insertion element is selected from the group consisting of recombinase docking site insertion elements and HDR docking site insertion elements.

151. The method of claim 150, wherein the docking site insertion element is a recombinase docking site insertion element.

152. The method of claim 151, wherein the recombinase docking site insertion element comprises an attachment site (att).

153. The method of claim 152, wherein the attachment site (att) is selected from the group consisting of attB, attP, attR, and attL.

154. The method of claim 151, wherein the recombinase docking site insertion element comprises a LoxP sequence.

155. The method of claim 151, wherein the recombinase docking site insertion element is an Flp recombinant target (FRT) site.

156. The method of claim 150, wherein the mating site insertion element is an HDR mating site insertion element.

157. The method of claim 156, wherein the HDR docking site insertion element comprises one or two docking site homologous arms.

158. The method of claim 157, wherein the HDR docking site insertion element further comprises one or more sequences homologous to the guide RNA sequence.

159. The method of any one of claims 157 to 158, wherein the length of the homologous arm at the docking site is about 30 to 1000 bases.

160. The method of claim 159, wherein the integrase docking site insert element comprises the AAVS1 safe harbor locus sequence.

161. The method of any one of claims 110 to 160, wherein each docking site is side-attached with an exogenous integration vector sequence.

162. The method of claim 161, wherein the exogenous integration vector sequence is selected from the group consisting of viral vector sequences and transposon vector sequences.

163. The method of any one of claims 110 to 162, wherein each docking site further comprises a sequence of encoded selectivity markers operatively linked to a promoter.

164. The method of any one of claims 110 to 163, wherein the host cell further comprises an expression construct encoding a foreign enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase.

165. The method of claim 164, wherein the expression construct encoding the exogenous enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase is provided in an add-on expression vector.

166. The method of claim 165, wherein the expression construct encoding a foreign enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase is integrated into the host cell genome.

167. The method of any one of claims 110 to 166, wherein the mating site insertion element is positioned to facilitate cassette exchange.

168. The method of any one of claims 110 to 167, wherein each docking site comprises two docking site insertion elements.

169. The method of claim 168, wherein the two mating site insertion elements are positioned to facilitate cassette exchange.

170. The method of any one of claims 168 to 169, wherein the two docking sites are inserted with a sequence encoding a selective marker, an enzyme, or a combination thereof.

171. The method of any one of claims 168 to 169, wherein the nucleic acid expression construct further comprises a signal peptide sequence operatively linked to the first target protein.

172. The method of claim 171, wherein the signal peptide sequence is selected from the group consisting of: tissue plasminogen activator, human growth hormone, lactoferrin, α-casein, and α-lactalbumin signal peptide sequences.

173. The method of any one of claims 110 to 172, wherein the nucleic acid expression construct further comprises a protein purification marker sequence.

174. The method of claim 173, wherein the protein purification tag sequence is a hexahistine tag or a hemagglutinin (HA) tag.

175. The method of any one of claims 110 to 174, wherein the host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, juvenile hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

176. The method of claim 175, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells and CAP cells.

177. The method of any one of claims 175 to 176, wherein the host cell is a GS knockout cell line.

178. The method of any one of claims 175 to 176, wherein the host cell is a DHFR knockout cell line.

179. A cell culture comprising host cells prepared by the method of any one of claims 110 to 178.

180. A method for producing a target protein, comprising culturing a host cell according to claim 179 under conditions that allow the target protein to be expressed, and purifying the target protein from the host cell culture.

181. The method of claim 180, wherein the host cells are grown in a culture medium containing a selectively labeled inhibitor.

182. The method of claim 181, wherein the selective marker is GS and the inhibitor is phosphatidylin or methionine sulfoxide (Msx).

183. The method of claim 181, wherein the selective marker is DHFR and the inhibitor is methotrexate.

184. A host cell comprising: Multiple docking sites integrated into the genome of the host cell, each docking site containing at least one docking site insertion element; At least an integrated first nucleic acid construct comprising at least one insert element compatible with the docking site insert element and encoding a target first protein or nucleic acid, and At least one integrated second nucleic acid construct, comprising at least one insertion element compatible with the said docking site and encoding a second target protein or nucleic acid, wherein: The first target protein or the second target protein comprises (i) a 3E10 heavy chain or a CDR-containing fragment thereof, or (ii) a 3E10 light chain or a CDR-containing fragment thereof, and The at least integrated first nucleic acid construct and the at least integrated second nucleic acid construct are integrated at the plurality of docking sites at a ratio of at least 1:1 for the first nucleic acid construct to the second nucleic acid construct.

185. The host cell of claim 184, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 2:1 to 1000:

1.

186. The host cell of claim 184, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 5:1 to 500:

1.

187. The host cell of claim 184, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 10:1 to 200:

1.

188. The host cell of claim 184, wherein the ratio of the first nucleic acid construct to the second nucleic acid construct is from 10:1 to 100:

1.

189. The method of any one of claims 184 to 188, wherein the first target protein is a 3E10 heavy chain or a CDR-containing fragment thereof, and the second target protein is a 3E10 light chain or a CDR-containing fragment thereof.

190. The method of any one of claims 184 to 188, wherein the first target protein comprises (i) a 3E10 heavy chain or a CDR-containing fragment thereof, or (ii) a 3E10 light chain or a CDR-containing fragment thereof, and the second target protein is a nuclease.

191. The host cell of any one of claims 184 to 190, wherein only the first nucleic acid construct and the second nucleic acid construct are introduced into the host cell.

192. The host cell of any one of claims 184 to 191, further comprising a third nucleic acid construct encoding a third target protein, wherein the ratio of the first or second nucleic acid construct to the third nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

193. The host cell of claim 192, further comprising a fourth nucleic acid construct encoding a fourth target protein, wherein the ratio of the first, second, or third nucleic acid construct to the fourth nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

194. The host cell of claim 193, further comprising a fifth nucleic acid construct encoding a fifth target protein, wherein the ratio of the first, second, third, or fourth nucleic acid construct to the fifth nucleic acid construct is selected from the group consisting of: at least 1:1, at least 2:1, 2:1 to 1000:1, 5:1 to 500:1, 10:1 to 200:1, and 10:1 to 100:

1.

195. The host cell of any one of claims 184 to 194, wherein at least the first nucleic acid construct and the second nucleic acid construct further comprise at least the following elements operably associated in a 5' to 3' sequence: Internal startup sequence; A nucleic acid sequence encoding the first target protein or the second protein that is operably linked to the internal promoter; and poly A signal sequence.

196. The host cell of claim 195, wherein at least the first nucleic acid construct and the second nucleic acid construct contain a selective marker sequence.

197. The host cell of claim 196, wherein at least the first nucleic acid construct and the second nucleic acid construct contain different selective marker sequences.

198. The host cell of claim 196, wherein one of the first nucleic acid construct and the second nucleic acid construct comprises a selective marker sequence, and the other of the first nucleic acid construct and the second nucleic acid construct does not comprise a selective marker sequence.

199. The host cell of any one of claims 195 to 198, wherein the selective marker sequence is located at the 5' of the internal promoter sequence and is operatively linked to the 5' promoter sequence.

200. The host cell of any one of claims 195 to 198, wherein the nucleic acid construct comprises an extended packaging region (EPR) between the 5' promoter and the selective marker.

201. The host cell of claim 200, wherein the EPR comprises a plurality of potential Kozak sequences and / or ATG translation initiation sites.

202. The host cell of any one of claims 195 to 201, wherein the promoter sequence is selected from the group consisting of: SIN-LTR, SV40, EF1α, E. coli lac、 E. coli trp, phage λ PL, phage λ PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, α-lactalbumin and mouse metallothionein-I promoter sequences.

203. The host cell of any one of claims 195 to 202, wherein the first promoter sequence is a weak promoter sequence.

204. The host cell of any one of claims 195-203, wherein the first promoter sequence is not a retroviral LTR promoter.

205. The host cell of any one of claims 195 to 204, wherein the integrated docking site further comprises an exogenous promoter.

206. The host cell of claim 205, wherein the exogenous promoter is selected from the group consisting of: SIN-LTR, SV40, EF1α, E. coli lac、 E. coli trp, phage λ PL, phage λ PR, T3, T7, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, α-lactalbumin and mouse metallothionein-I promoter sequences.

207. The host cell of claim 206, wherein the promoter is a retroviral LTR.

208. The host cell of claim 207, wherein the retroviral LTR is a SIN LTR.

209. The host cell of any one of claims 184 to 208, wherein the nucleic acid expression construct is provided in a vector.

210. The host cell of claim 209, wherein the vector is a plasmid vector.

211. The host cell of any one of claims 184 to 210, wherein the host cell comprises a nucleic acid construct encoding an enzyme that facilitates the insertion of the nucleic acid expression construct at the docking site.

212. The host cell of claim 211, wherein the nucleic acid construct encoding an enzyme that promotes the insertion of the nucleic acid expression construct at the docking site is provided in a vector.

213. The host cell of claim 211, wherein the vector is a plasmid vector.

214. The host cell of any one of claims 210 to 213, wherein the ratio of the nucleic acid construct encoding an enzyme that promotes the insertion of the nucleic acid expression construct at the docking site to the nucleic acid expression construct encoding the first target protein, transiently introduced into the host cell, is 1:1000 to 1:

10.

215. The host cell according to any one of claims 210-214, wherein the enzyme is selected from the group consisting of integrase, recombinase, nuclease and nickase.

216. The host cell of any one of claims 210 to 215, wherein the nucleic acid construct encoding an enzyme that facilitates the insertion of the nucleic acid expression construct at the docking site is provided in a vector.

217. The host cell of any one of claims 184 to 216, wherein the host cell genome comprises 5 to 500 integrated docking sites, each docking site comprising at least one docking site insertion element.

218. The host cell of any one of claims 184 to 217, wherein the host cell genome comprises 5 to 250 integrated docking sites, each docking site comprising at least one docking site insertion element.

219. The host cell of any one of claims 184 to 218, wherein the host cell genome comprises 5 to 100 integrated docking sites, each docking site comprising at least one docking site insertion element.

220. The host cell of any one of claims 184 to 219, wherein the integrated docking site is independently located throughout the host cell genome.

221. The host cell of any one of claims 184 to 220, wherein the docking site insertion element is targeted by an enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase.

222. The host cell of any one of claims 184 to 221, wherein the docking site insertion element is selected from the group consisting of recombinase docking site insertion elements and HDR docking site insertion elements.

223. The host cell of claim 222, wherein the docking site insertion element is a recombinase docking site insertion element.

224. The host cell of claim 223, wherein the recombinase docking site insertion element comprises an attachment site (att).

225. The host cell of claim 224, wherein the attachment site (att) is selected from the group consisting of attB, attP, attR, and attL.

226. The host cell of claim 223, wherein the recombinase docking site insert element comprises a LoxP sequence.

227. The host cell of claim 223, wherein the recombinase docking site insertion element is an Flp recombination target (FRT) site.

228. The host cell of claim 222, wherein the docking site insertion element is an HDR docking site insertion element.

229. The host cell of claim 228, wherein the HDR docking site insertion element comprises one or two docking site homologous arms.

230. The host cell of claim 229, wherein the HDR docking site insertion element further comprises one or more sequences homologous to the guide RNA sequence.

231. The host cell of any one of claims 229 to 230, wherein the length of the homologous arm at the docking site is about 30 to 1000 bases.

232. The host cell of claim 221, wherein the integrase docking site insert element comprises the AAVS1 safe harbor locus sequence.

233. The host cell of any one of claims 184 to 232, wherein each docking site is side-attached with an exogenous integration vector sequence.

234. The host cell of claim 233, wherein the exogenous integration vector sequence is selected from the group consisting of viral vector sequences and transposon vector sequences.

235. The host cell of any one of claims 184 to 234, wherein each of the docking sites further comprises a sequence encoding a selectable marker operatively linked to a promoter.

236. The host cell of any one of claims 184-235, wherein the host cell further comprises an expression construct encoding a foreign enzyme, the foreign enzyme being selected from the group consisting of integrase, recombinase, nuclease and nickase.

237. The host cell of claim 236, wherein the expression construct encoding the exogenous enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase is provided in an add-on expression vector.

238. The host cell of claim 236, wherein the expression construct encoding a foreign enzyme selected from the group consisting of integrase, recombinase, nuclease and nickase is integrated into the host cell genome.

239. The host cell of any one of claims 184 to 238, wherein the docking site insertion element is positioned to facilitate cassette exchange.

240. The host cell of any one of claims 184 to 239, wherein each docking site comprises two docking site insertion elements.

241. The host cell of claim 240, wherein the two docking site insertion elements are positioned to facilitate cassette exchange.

242. The host cell of any one of claims 240 to 241, wherein the two docking sites are inserted with a sequence encoding a selective marker, an enzyme, or a combination thereof.

243. The host cell of any one of claims 184 to 242, wherein the nucleic acid expression construct further comprises a signal peptide sequence operatively linked to the first target protein.

244. The host cell of claim 243, wherein the signal peptide sequence is selected from the group consisting of: tissue plasminogen activator, human growth hormone, lactoferrin, α-casein, and α-lactalbumin signal peptide sequences.

245. The host cell of any one of claims 184 to 244, wherein the nucleic acid expression construct further comprises a protein purification marker sequence.

246. The host cell of claim 245, wherein the protein purification marker sequence is a hexahistine tag or a hemagglutinin (HA) tag.

247. The host cell according to any one of claims 184 to 246, wherein the host cell is selected from the group consisting of: Chinese hamster ovary (CHO) cells, HEK 293 cells, CAP cells, bovine mammary epithelial cells, SV40-transformed monkey kidney CV1 line, young hamster kidney cells, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells, human cervical cancer cells, canine kidney cells, Buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, MRC 5 cells, FS4 cells, rat fibroblasts, MDBK cells, and human hepatocellular carcinoma lineage cells.

248. The host cell of claim 247, wherein the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, HEK 293 cells and CAP cells.

249. The host cell of any one of claims 247 to 248, wherein the host cell is a GS knockout cell line.

250. The host cell of any one of claims 247 to 248, wherein the host cell is a DHFR knockout cell line.

251. A cell culture comprising a host cell as described in any one of claims 184 to 250.

252. A method for producing a target protein, comprising culturing a host cell according to any one of claims 184 to 250 under conditions that allow the target protein to be expressed, and purifying the target protein from the host cell culture.

253. The method of claim 252, wherein the host cells are grown in a culture medium containing a selectively labeled inhibitor.

254. The method of claim 252, wherein the selective marker is GS and the inhibitor is phosphatidylin or methionine sulfoxide (Msx).

255. The method of claim 252, wherein the selective marker is DHFR and the inhibitor is methotrexate.

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