Preparation method of recombinant cell, recombinant cell and application of recombinant cell

Abstract

The invention provides a preparation method of a recombinant cell, the recombinant cell and application of the recombinant cell, and relates to the technical field of biology. A target protein expressed by the recombinant cell contains one, two, three or four polypeptide chains; the preparation method comprises the following steps: integrating exogenous polynucleotides for coding a target protein into a genetic material of a starting cell in a site-specific manner, wherein each exogenous polynucleotide for site-specific integration contains coding genes of all polypeptide chains of the target protein; each exogenous polynucleotide contains at least four expression cassettes, and each polypeptide chain is encoded by at least one expression cassette. The preparation method is simple to operate, coding genes of all polypeptide chains can be integrated into genetic materials of starting cells at a time, and the expression quantity of foreign proteins of the recombinant cells obtained through the preparation method is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing a recombinant cell, the recombinant cell and applications thereof. Background Art

[0002] In the current biotherapeutic market, which is dominated by antibody molecules, bispecific antibodies are a key component of next-generation antibody therapies. It is hoped that two specific targets can produce a 1+1>2 effect, achieving exceptional efficacy in treating diseases. Bispecific antibodies were proposed three decades ago and have been extensively studied to overcome the limitation of natural monoclonal antibodies, which can only bind to a single epitope. Bispecific antibodies can simultaneously target two different antigens, such as simultaneously binding to tumor cell receptors and recruiting cytotoxic immune cells. This enhanced functionality may lead to fewer side effects and fewer injections. Unlike traditional monoclonal antibodies, the significant production challenges of bispecific and multispecific antibodies in terms of expression quantity, quality, and stability have hindered their wider clinical application and acceptance.

[0003] Conventional antibody production involves constructing single or multiple plasmid transfections during the cell line generation phase of the upstream process. Non-targeted transgenic integration methods are used to generate stably transfected cell pools. Multiple clones are then obtained through monoclonal cloning, and these clones undergo tedious screening to identify clones with suitable production characteristics. These screened cell lines are typically multi-copy, and the stability of different sites in the cell lines often varies significantly. Bispecific antibodies, in particular, often contain multiple expression chains. The uncertainty of random integration can easily lead to high mismatch rates, affecting target gene expression yields and increasing costs. Long culture times, low product yields, and phenotypic heterogeneity in transgenic cell clones are bottlenecks in the production of antibodies containing multiple polypeptide chains.

[0004] CN114258403A (application number CN202080057659.4, Chinese patent application entitled "Method for producing multivalent and multispecific antibody-expressing cells by targeted integration of multiple expression cassettes in a defined tissue form") discloses a method for preparing CHO host cells using two plasmids (one plasmid contains two expression cassettes) integrated into the same site. The site-directed integration cell line prepared by this scheme has the problems of high difficulty in plasmid integration, complex operation, and target protein expression level below 3.3 g / L, and the target protein expression level needs to be further improved.

[0005] CN116615462A (application number CN202180077498.X, Chinese patent application titled "Expression Technology of Antibody Constructs") discloses a method for preparing CHO host cells in which one mRNA (one mRNA contains one reading frame, and one reading frame is connected to four target gene fragments by 2A peptides) is integrated into the same site. The cell line prepared by this method has the problem of low expression of the target protein.

[0006] Therefore, how to increase the expression level of proteins, especially proteins containing multiple polypeptide chains, in host cells is an urgent problem to be solved.

[0007] In view of this, this application is hereby filed. Summary of the Invention

[0008] The object of the present invention is to provide a method for preparing recombinant cells to increase the expression level of proteins containing one or more polypeptide chains in host cells.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] In a first aspect, a method for preparing a recombinant cell is provided, wherein the recombinant cell expresses a target protein comprising one, two, three or four polypeptide chains;

[0011] The preparation method includes site-specific integration of an exogenous polynucleotide encoding a target protein into the genetic material of a starting cell, wherein each exogenous polynucleotide used for the site-specific integration contains genes encoding all polypeptide chains of the target protein; and each exogenous polynucleotide contains at least four expression cassettes, and each polypeptide chain is encoded by at least one expression cassette.

[0012] In a second aspect, a recombinant cell prepared by the preparation method of the first aspect is also provided.

[0013] In a third aspect, the invention also provides the use of the preparation method of the first aspect or the recombinant cell provided by the second aspect in preparing a protein.

[0014] In a fourth aspect, a method for preparing a protein is also provided, comprising culturing the recombinant cell of the second aspect.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention discloses a method for preparing a recombinant cell. The recombinant cell expresses a target protein composed of one to four polypeptide chains. The recombinant cell integrates an exogenous polynucleotide containing at least four expression cassettes, with each polypeptide chain encoded by at least one expression cassette. This allows the exogenous coding gene to be integrated into the host cell through a single introduction, thereby reducing the integration difficulty, simplifying the operation, and increasing the expression of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is an operational flow chart of Example 1;

[0019] Figure 2 This is the plasmid map of the RMCE recombinant plasmid containing the green fluorescent protein gene in Example 1;

[0020] Figure 3 is the plasmid map of the pDonor0.0-H plasmid in Examples 2 to 4;

[0021] Figure 4 is the plasmid map of the pDonor0.0-L plasmid in Examples 2 to 4;

[0022] Figure 5 is the plasmid map of the pLanding0.0 plasmid in Examples 2 to 4;

[0023] Figure 6 Plasmid map of pLanding0.0-A-LFH constructed in Example 2;

[0024] Figure 7 Plasmid map of pLanding0.0-A-FLH constructed in Example 2;

[0025] Figure 8 Plasmid map of pLanding0.0-A-LFHL constructed in Example 2;

[0026] Figure 9 Plasmid map of pLanding0.0-A-LFHF constructed in Example 2;

[0027] Figure 10 Plasmid map of pLanding0.0-A-LHFF constructed in Example 2;

[0028] Figure 11 Plasmid map of pLanding0.0-A-FLHL constructed in Example 2;

[0029] Figure 12 Plasmid map of pLanding0.0-A-FHLL constructed in Example 2;

[0030] Figure 13 This is the PGBB BXB1 integrase plasmid map used in Examples 1 to 4;

[0031] Figure 14 This is the non-reduced CE-SDS detection image of the C-LHFF-1 cell pool B10 monoclonal in Example 2;

[0032] Figure 15 This is the non-reduced CE-SDS detection image of the C-FLHL-2 cell pool B16 monoclonal in Example 2;

[0033] Figure 16 This is the CE-SDS detection image of the C-LHFF-1 cell pool B10 monoclonal reduction in Example 2;

[0034] Figure 17 This is the CE-SDS detection image of the B16 monoclonal reduction of the C-FLHL-2 cell pool in Example 2;

[0035] Figure 18 Schematic diagram of the three-chain antibody expressed in Example 2;

[0036] Figure 19 Schematic diagram of plasmid pLanding0.0-AL-VL-AH-VH in Example 3;

[0037] Figure 20 Schematic diagram of plasmid pLanding0.0-AL-VL-VH-AH in Example 3;

[0038] Figure 21 Schematic diagram of plasmid pLanding0.0-VL-AL-VH-AH in Example 3;

[0039] Figure 22 Schematic diagram of plasmid pLanding0.0-VL-AL-AH-VH in Example 3;

[0040] Figure 23 This is the SDS-PAGE electrophoresis diagram of the expression supernatants of the four molecules in Example 3 after purification using a Protein A chromatography column;

[0041] Figure 24Schematic diagram of the plasmid pDonor0.0-CTLA4-LC of Example 4;

[0042] Figure 25 Schematic diagram of the plasmid pDonor0.0-CTLA4-HC of Example 4;

[0043] Figure 26 This is a schematic diagram of the plasmid pLanding0.0-CTLA4-LH of Example 4;

[0044] Figure 27 Schematic diagram of the plasmid pLanding0.0-CTLA4-LHLH of Example 4;

[0045] Figure 28 This is the expression curve of the target protein cloned from pLanding0.0-CTLA4-LHLH-3G6 in Example 4;

[0046] Figure 29 This is the growth curve of the pLanding0.0-CTLA4-LHLH-3G6 clone in Example 4;

[0047] Figure 30 This is the cell viability curve of the pLanding0.0-CTLA4-LHLH-3G6 clone in Example 4;

[0048] Figure 31 This is the lactate concentration curve of the pLanding0.0-CTLA4-LHLH-3G6 clone in Example 4. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] Typically, a natural, intact antibody consists of two heavy (H) chains and two light (L) chains. Antibodies are classified into five major classes, or isotypes, based on the presence of α, δ, ε, γ, and μ heavy chains: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Each heavy chain in an intact antibody molecule consists of a heavy chain variable region (VH) and first, second, third, and optionally fourth constant regions (CH1, CH2, CH3, and CH4, respectively). Mammalian light chains are classified as either λ or κ, and each light chain in an intact antibody molecule consists of a light chain variable region (VL) and a constant region (CL). The variable regions of the light and heavy chains determine antigen binding.

[0051] The term "heavy chain" as used herein comprises a heavy chain variable region (VH) domain and part or all of a heavy chain constant region, such as at least one of CH1, CH2, CH3, and CH4. Part or all of the heavy chain constant region of the heavy chain is optionally derived from one or a combination of IgA, IgD, IgE, IgG, and IgM.

[0052] As used herein, the term "light chain" comprises a light chain variable region (VL) domain and part or all of a light chain constant region (CL). The constant region (CL) may be partially or completely derived from a lambda or kappa light chain.

[0053] Herein, "half antibodies" are formed by combining the above-mentioned "heavy chains" and "light chains" through disulfide bonds. Thus, the second half antibody comprises a VL domain, a VH domain, a hinge domain, a portion or all of the heavy chain constant region (at least one of CH1, CH2, CH3, and CH4), and optionally a portion or all of the light chain constant region (CL). In an optional embodiment, the half antibody is composed of one heavy chain and one light chain in a complete antibody molecule, for example, one heavy chain and one light chain in a complete IgG antibody molecule.

[0054] The term "recombinant heavy chain" used herein includes the heavy chain Fc region and the single-chain antibody scFv. The single-chain antibody refers to an antibody fragment composed of the light chain variable region and the heavy chain variable region directly connected to each other or connected through a peptide linker sequence.

[0055] The term "three-chain antibody" as used herein refers to an antibody composed of a half antibody and a recombinant heavy chain, wherein the half antibody and the recombinant heavy chain are bound by disulfide bonds to form a three-chain antibody, as shown in the structural diagram. Figure 18 shown.

[0056] The term "symmetrical antibody" as used herein refers to an antibody composed of two half antibodies, which may target the same or different antigens and have the same structure, for example, all or part of the same constant region.

[0057] The term "expression cassette" as used herein refers to a polynucleotide containing a gene encoding a protein of interest (e.g., a light chain, a heavy chain, or a recombinant heavy chain), the encoding gene being operably linked to a regulatory sequence (e.g., a promoter, an enhancer, and a signal peptide, etc.) that directs the expression of the encoding gene in a target cell, and may contain other regulatory, labeling, screening, and recombination sequences thereof. The regulatory sequence allows the gene of interest to be recombined, transcribed, translated, and / or expressed in the target cell.

[0058] As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" refer to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding may optionally be for either the sense strand or the antisense strand. Nucleic acid molecules may be naturally occurring, synthetic, recombinant, or any combination thereof. "Nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0059] In alternative embodiments, the nucleic acid molecule is RNA or DNA. The nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid molecule is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. When it is incorporated into a vector, DNA is preferably used.

[0060] In the present invention, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector or a cloning vector. In some embodiments, the vectors provided by the present invention (e.g., expression vectors) contain a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0061] In the present invention, the term "starting cell" refers to a cell used to introduce the polynucleotide encoding the target protein (a three-chain antibody in this application) and / or a vector carrying the polynucleotide encoding the target protein. The starting cell can be a natural cell or a modified cell.

[0062] In the present invention, the term "recombinant cell" refers to a cell into which a polynucleotide encoding a protein of interest (a triabody in this application) and / or a vector carrying the polynucleotide encoding the protein of interest has been introduced. The exogenous polynucleotide may or may not be integrated into the genome of the "recombinant cell."

[0063] In the present invention, "first", "second", "third" and "fourth" are only used to distinguish different entities or behaviors from another entity or behavior, and do not mean or imply the order or importance between the entities or behaviors.

[0064] In a first aspect, a method for preparing a recombinant cell is provided, wherein the target protein expressed by the recombinant cell contains one, two, three, or four polypeptide chains. The number of polypeptide chains in the target protein can be one or more. For example, it can be, but is not limited to, when the target protein contains one polypeptide chain, the target protein can be a multimer composed of one, two, three, or four such polypeptide chains; or, for example, it can be, but is not limited to, when the target protein contains two polypeptide chains, the target protein contains one, two, three, or four of one polypeptide chain, and / or further contains one, two, three, or four of another polypeptide chain. For example, it can be, but is not limited to, when the target protein contains three or four polypeptide chains, the target protein contains one, two, three, or four of any one polypeptide chain, and the remaining polypeptide chains all contain at least one. The genetic material includes endogenous or exogenous genetic material of the cell, endogenous genetic material such as the cell genome; exogenous genetic material such as an expression vector stably expressed in the cell.

[0065] The preparation method includes site-specific integration of an exogenous polynucleotide encoding a target protein into the genetic material of a starting cell, wherein each exogenous polynucleotide used for site-specific integration contains genes encoding all polypeptide chains of the target protein; and each exogenous polynucleotide contains at least four expression cassettes, with each polypeptide chain encoded by at least one expression cassette. Each expression cassette contains a gene encoding at least one polypeptide chain, preferably a gene encoding one polypeptide chain.

[0066] When the exogenous polynucleotide contains multiple genes encoding the same polypeptide chain, the genes encoding the same polypeptide chain may be the same or different. The genes encoding the same polypeptide chain may be adjacent or non-adjacent in the exogenous polynucleotide.

[0067] For example, it may be, but is not limited to, that the target protein consists of one polypeptide chain, and the exogenous polynucleotide contains four genes encoding this polypeptide chain.

[0068] For example, but not limited to, the target protein may be composed of two polypeptide chains, and the exogenous polynucleotide may contain two coding genes for each polypeptide chain; or three coding genes for one polypeptide chain and only one coding gene for the other polypeptide chain.

[0069] For example, but not limited to, the target protein may be composed of three polypeptide chains, the exogenous polynucleotide may contain two or more coding genes for one of the polypeptide chains, and the remaining polypeptide chains may contain one coding gene, or two or more coding genes.

[0070] For example, but not limited to, the target protein may be composed of four polypeptide chains, and the exogenous polynucleotide may contain a coding gene for each polypeptide chain; or, some polypeptide chains in the exogenous polynucleotide may correspond to multiple coding genes, and the remaining polypeptide chains may contain only one coding gene.

[0071] The method for preparing recombinant cells provided by the present invention uses an exogenous polynucleotide for site-directed integration containing the coding genes for all polypeptide chains of the target protein. Through a single transduction, the coding genes for all polypeptide chains of the target protein can be site-directedly integrated into the genetic material of the starting cell, simplifying the integration operation. At the same time, the present invention adopts a site-directed integration method to provide a more stable cloning method, thereby reducing the cell line development timeline. A clear integration site can reduce the impact of the site effect of the integration of the exogenous target gene, improve the expression level of the target gene, and enable the host cell to express the target gene with high activity and stable expression.

[0072] In an optional embodiment, the exogenous polynucleotide further contains a polynucleotide fragment encoding a regulatory element.

[0073] In an optional embodiment, the regulatory element includes one or more of a ribosome binding site, a Kozak sequence, a promoter, an enhancer, a signal peptide and a polynucleotide.

[0074] In an optional embodiment, the signal peptide amino acid sequence is shown as SEQ ID No.9, SEQ ID No.10 or SEQ ID No.11.

[0075] In an optional embodiment, the polynucleotide gene includes polyA.

[0076] In an optional embodiment, each expression cassette independently contains one or more of a ribosome binding site, a Kozak sequence, a promoter, an enhancer, a signal peptide and a polynucleotide.

[0077] In an optional embodiment, each expression cassette independently contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and a polynucleotide gene in sequence.

[0078] In an optional embodiment, the exogenous polynucleotide is introduced into the genetic material of the starting cell mediated by a vector;

[0079] In an alternative embodiment, the vector comprises a plasmid.

[0080] In an optional embodiment, the vector further contains a recombinase recognition site or a homology arm of the genetic material of the originating cell.

[0081] In an optional embodiment, the recombinant cell comprises a prokaryotic cell or a eukaryotic cell.

[0082] In an alternative embodiment, the eukaryotic cell comprises a mammalian cell.

[0083] In an optional embodiment, the recombinant cell comprises a CHO cell or a HEK293 cell.

[0084] In an optional embodiment, the CHO cell is a CHO-K1 cell, a CHO-S cell or a CHO-GS cell.

[0085] Exemplary gene editing systems include, but are not limited to, zinc-finger nuclease systems (ZFNs), transcription activator-like effector nuclease systems (TALENs), and clustered regularly interspaced short palindromic repeat sequences (CRISPR).

[0086] Exemplary integrase systems include, but are not limited to, Cre, Dre, Vika, Bxb1, RDF, FLP, TP901-1、A118、 MR11, TG1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebcuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Hinder, ICleared, Sheen, Mundrea, BxZ2 or or mutants of any of them.

[0087] Exemplary integration sites in the integrase system include, but are not limited to, one or more of a LoxP site, a LoxPL3 site, a LoxP 2L site, a LoxFas site, a Lox511 site, a Lox2272 site, a Lox2372 site, a Lox5171 site, a Loxm2 site, a Lox71 site, a Lox66 site, a FRT site, a Bxb1 attP site, and a Bxb1 attB site.

[0088] In an optional embodiment, the starting cell is a CHO-K1 cell, and the site-directed integration comprises integrating the exogenous polynucleotide into a highly expressed fragment of the starting cell, wherein the highly expressed fragment comprises the nucleotide sequence shown in SEQ ID No. 1;

[0089] In an optional embodiment, the fixed site of the integration site is any site within the 11th to 430th base interval of the highly expressed fragment; or, any site within the 21st to 414th base interval of the highly expressed fragment; or, any site within the 38th to 402nd base interval of the highly expressed fragment; or, any site within the 53rd to 389th base interval of the highly expressed fragment; or, any site within the 73rd to 373rd base interval of the highly expressed fragment; or, any site within the 91st to 360th base interval of the highly expressed fragment; or, any site within the 108th to 342nd base interval of the highly expressed fragment; or, any site within the 53rd to 389th base interval of the highly expressed fragment. any site within the 126th to 326th base interval of the highly expressed fragment; or, any site within the 143rd to 310th base interval of the highly expressed fragment; or, any site within the 160th to 295th base interval of the highly expressed fragment; or, any site within the 178th to 274th base interval of the highly expressed fragment; or, any site within the 194th to 263rd base interval of the highly expressed fragment; or, any site within the 209th to 253rd base interval of the highly expressed fragment; or, any site within the 221st to 242nd base interval of the highly expressed fragment; or, any site within the 231st to 240th base interval of the highly expressed fragment.

[0090] In an optional embodiment, the fixed site of the integration site is the position shown in the annotation information NW_003616785.1:83044 on CHO-K1 cells.

[0091] In an optional embodiment, the starting cell is a CHO-K1 cell, the genome of the CHO-K1 cell contains a marker gene, the marker gene is integrated into a highly expressed fragment of the starting cell, and the highly expressed fragment contains the nucleotide sequence shown in SEQ ID No. 1.

[0092] In an optional embodiment, the fixed site of the integration site of the marker gene is any site within the 11th to 430th base interval of the highly expressed fragment; or, any site within the 21st to 414th base interval of the highly expressed fragment; or, any site within the 38th to 402nd base interval of the highly expressed fragment; or, any site within the 53rd to 389th base interval of the highly expressed fragment; or, any site within the 73rd to 373rd base interval of the highly expressed fragment; or, any site within the 91st to 360th base interval of the highly expressed fragment; or, any site within the 108th to 342nd base interval of the highly expressed fragment; or, any site within the 53rd to 389th base interval of the highly expressed fragment. Any site within the 126th to 326th base interval of the expression fragment; or, any site within the 143rd to 310th base interval of the highly expressed fragment; or, any site within the 160th to 295th base interval of the highly expressed fragment; or, any site within the 178th to 274th base interval of the highly expressed fragment; or, any site within the 194th to 263rd base interval of the highly expressed fragment; or, any site within the 209th to 253rd base interval of the highly expressed fragment; or, any site within the 221st to 242nd base interval of the highly expressed fragment; or, any site within the 231st to 240th base interval of the highly expressed fragment.

[0093] In an optional embodiment, the fixed site of the integration site of the marker gene is the position shown in the annotation information NW_003616785.1:83044 on CHO-K1 cells.

[0094] In an optional embodiment, the marker gene includes a fluorescent marker gene.

[0095] In some embodiments, the protein of interest expressed by the recombinant cell comprises a triabody comprising three polypeptides, including a first heavy chain, a first light chain, and a recombinant heavy chain; the first heavy chain and the first light chain constitute a first half antibody, and the recombinant heavy chain comprises a heavy chain Fc region and a single-chain antibody (scFv). The first half antibody and the recombinant heavy chain may target the same or different antigens.

[0096] In an optional embodiment, the three-chain antibody is a bispecific antibody, and the first half antibody and the recombinant heavy chain target different antigens.

[0097] In an alternative embodiment, the first half-antibody targets EpCAM, CD38 or HER2.

[0098] In an optional embodiment, the first half antibody targets EpCAM, the gene encoding the first light chain is shown as SEQ ID No. 2, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 3.

[0099] In an optional embodiment, the first half antibody targets CD38, the gene encoding the first light chain is shown as SEQ ID No. 4, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 5.

[0100] In an optional embodiment, the first half antibody targets HER2, the gene encoding the first light chain is shown as SEQ ID No. 6, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 7.

[0101] In an alternative embodiment, the recombinant heavy chain targets CD3.

[0102] In an optional embodiment, the gene encoding the recombinant heavy chain is shown as SEQ ID No.8.

[0103] In an optional embodiment, it is also found that the arrangement order of the polypeptide chain encoding genes in the exogenous polynucleotide helps to further increase the expression level of the target protein and helps to reduce the mismatch rate of the three-chain antibody.

[0104] In an optional embodiment, the exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence;

[0105] The first expression cassette contains the recombinant heavy chain encoding gene. Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the recombinant heavy chain encoding gene and a polynucleotide gene in sequence.

[0106] The second expression cassette contains the gene encoding the first light chain. Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the first light chain and a polynucleotide gene in sequence.

[0107] The third expression cassette contains the gene encoding the first heavy chain. Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the first heavy chain and a polynucleotide gene in sequence.

[0108] The fourth expression cassette contains the gene encoding the first light chain. Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the first light chain and a polynucleotide gene in sequence.

[0109] In an optional embodiment, the first expression cassette of the exogenous polynucleotide contains the gene encoding the recombinant heavy chain, the second expression cassette contains the gene encoding the first light chain, the third expression cassette contains the gene encoding the first heavy chain, and the fourth expression cassette contains the gene encoding the first light chain, each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence, and the exogenous polynucleotide further satisfies any one of ( ) to ( ):

[0110] ( ) The first half antibody targets EpCAM; the gene encoding the first light chain is shown in SEQ ID No. 2, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 3, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0111] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11;

[0112] ( ) The first half antibody targets CD38; the gene encoding the first light chain is shown in SEQ ID No. 4, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 5, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0113] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11;

[0114] ( ) The first half-antibody targets HER2; the gene encoding the first light chain is shown in SEQ ID No. 6, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 7, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0115] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown as SEQ ID No.8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID No.11.

[0116] In other optional embodiments, the exogenous polynucleotide for preparing and expressing the above-mentioned three-chain antibody contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence;

[0117] The first expression cassette contains the gene encoding the first light chain. Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the first light chain, and a polynucleotide gene in sequence;

[0118] The second expression cassette contains the gene encoding the first heavy chain. Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the first heavy chain and a polynucleotide gene in sequence;

[0119] The third expression cassette contains the recombinant heavy chain encoding gene. Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the recombinant heavy chain encoding gene and a polynucleotide gene in sequence;

[0120] The fourth expression cassette contains the recombinant heavy chain encoding gene. Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the recombinant heavy chain encoding gene and a polynucleotide gene in sequence.

[0121] Arranging the coding genes in the above order can also obtain recombinant cells with higher expression levels and reduce the mismatch rate of three-chain antibodies.

[0122] In an optional embodiment, the first expression cassette of the exogenous polynucleotide contains the gene encoding the first light chain, the second expression cassette contains the gene encoding the first heavy chain, the third expression cassette contains the gene encoding the recombinant heavy chain, and the fourth expression cassette contains the gene encoding the recombinant heavy chain, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence, and the exogenous polynucleotide is selected from any one of ( ) to ( ):

[0123] ( ) The first half antibody targets EpCAM; the gene encoding the first light chain is shown in SEQ ID No. 2, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 3, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0124] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11;

[0125] ( ) The first half antibody targets CD38; the gene encoding the first light chain is shown in SEQ ID No. 4, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 5, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0126] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11;

[0127] ( ) The first half-antibody targets HER2; the gene encoding the first light chain is shown in SEQ ID No. 6, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 7, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10;

[0128] The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown as SEQ ID No.8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID No.11.

[0129] In other embodiments, the target protein expressed by the recombinant cell contains four polypeptide chains, the target protein includes a second light chain and a second heavy chain, the second light chain and the second heavy chain constitute a second half antibody; the target protein also includes a third light chain and a third heavy chain, the third light chain and the third heavy chain constitute a third half antibody.

[0130] In an alternative embodiment, the four-chain antibody is a symmetrical antibody.

[0131] In an alternative embodiment, the second half-antibody and the third half-antibody target different antigens.

[0132] In an optional embodiment, the partial fragment of the second light chain and the partial fragment of the second heavy chain are replaced after expression, and this replacement increases the difficulty of expressing the second half antibody. Exemplary replacement methods include, but are not limited to, replacing the constant region CL of the second light chain with the constant region CH1 of the second heavy chain after expression.

[0133] In an alternative embodiment, the second substituted antibody half targets ANG-2.

[0134] In an optional embodiment, the second half-antibody targets ANG-2, the gene encoding the second light chain is shown as SEQ ID No. 12, and / or the gene encoding the second heavy chain is shown as SEQ ID No. 13.

[0135] In an alternative embodiment, the third half-antibody targets VEGF.

[0136] In an optional embodiment, the third half antibody targets VEGF, the gene encoding the third light chain is shown as SEQ ID No.14, and / or the gene encoding the third heavy chain is shown as SEQ ID No.15.

[0137] In an optional embodiment, it is also found that the arrangement order of the polypeptide chain encoding genes in the above-mentioned exogenous polynucleotide of the target protein containing four polypeptide chains helps to further increase the expression level of the recombinant cell target protein and helps to reduce the mismatch rate of the antibody.

[0138] In an optional embodiment, the exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence;

[0139] The first expression cassette contains the gene encoding the second light chain. Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the second light chain, and a polynucleotide gene in sequence;

[0140] The second expression cassette contains the gene encoding the third light chain. Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the third light chain, and a polynucleotide gene in sequence;

[0141] The third expression cassette contains the gene encoding the second heavy chain. Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the second heavy chain and a polynucleotide gene in sequence;

[0142] The fourth expression cassette contains the gene encoding the third heavy chain. Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the third heavy chain and a polynucleotide gene in sequence.

[0143] In an optional embodiment, the first expression cassette contains the gene encoding the second light chain, the second expression cassette contains the gene encoding the third light chain, the third expression cassette contains the gene encoding the second heavy chain, and the fourth expression cassette contains the gene encoding the third heavy chain, and each expression cassette contains, in sequence, a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene, and Poly A; and the second half-antibody targets ANG-2, the gene encoding the second light chain is set forth in SEQ ID No. 12, the amino acid sequence of the signal peptide of the second light chain is set forth in SEQ ID No. 9, the gene encoding the second heavy chain is set forth in SEQ ID No. 13, and the amino acid sequence of the signal peptide of the second heavy chain is set forth in SEQ ID No. 10; and the third half-antibody targets ANG-2, the gene encoding the third light chain is set forth in SEQ ID No. 14, the amino acid sequence of the signal peptide of the third light chain is set forth in SEQ ID No. 9, the gene encoding the third heavy chain is set forth in SEQ ID No. 15, and the amino acid sequence of the signal peptide of the third heavy chain is set forth in SEQ ID No. 10.

[0144] In other embodiments, the target protein contains two polypeptide chains, including a fourth light chain and a fourth heavy chain, the fourth light chain and the fourth heavy chain constitute a fourth half antibody, and the target protein contains two fourth half antibodies.

[0145] In an optional embodiment, the fourth half antibody targets CTLA-4;

[0146] In an optional embodiment, the fourth half antibody targets CTLA-4, the gene encoding the fourth light chain is shown as SEQ ID No.19, and the gene encoding the fourth heavy chain is shown as SEQ ID No.18.

[0147] In an optional embodiment, increasing the copy number of the polypeptide chain in the exogenous polynucleotide helps to increase the expression level of the exogenous protein in the recombinant cell.

[0148] In an optional embodiment, the exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence;

[0149] The first expression cassette contains the gene encoding the fourth light chain. Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the fourth light chain, and a polynucleotide gene in sequence;

[0150] The second expression cassette contains the gene encoding the fourth heavy chain. Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the gene encoding the fourth heavy chain, and a polynucleotide gene in sequence;

[0151] The third expression cassette contains the encoding gene of the fourth light chain. Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the encoding gene of the fourth light chain and a polynucleotide gene in sequence;

[0152] The fourth expression cassette contains the coding gene of the fourth heavy chain. Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide coding gene, the coding gene of the fourth heavy chain and a polynucleotide gene in sequence.

[0153] In an optional embodiment, the first expression cassette contains the coding gene for the fourth light chain, the second expression cassette contains the coding gene for the fourth heavy chain, the third expression cassette contains the coding gene for the fourth light chain, and the fourth expression cassette contains the coding gene for the fourth heavy chain, and each expression cassette contains, in sequence, a promoter, a Kozak sequence, a signal peptide coding gene, a polypeptide chain coding gene, and Poly A; and the fourth half antibody targets CTLA-4, each coding gene for the fourth light chain is as shown in SEQ ID No. 19, the signal peptide amino acid sequence of each fourth light chain is as shown in SEQ ID No. 9, each coding gene for the fourth heavy chain is as shown in SEQ ID No. 18, and the signal peptide amino acid sequence of each fourth heavy chain is as shown in SEQ ID No. 10.

[0154] In a second aspect, a recombinant cell prepared by the preparation method of the first aspect is also provided.

[0155] In a third aspect, the invention also provides the use of the preparation method of the first aspect or the recombinant cell provided by the second aspect in preparing a protein.

[0156] In a fourth aspect, a method for preparing a protein is provided, comprising culturing the recombinant cell of the second aspect. The method may further optionally include other conventional and known steps in the art, including but not limited to one or more steps of expanded culture, enrichment, purification, freezing, and drying, etc., which are not limited by the present invention.

[0157] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0158] Example 1 Preparation of primary cells

[0159] Specific consumables include: Neon Resuspension Buffer R (ThermoFisher), a resuspension buffer specifically for cell electroporation; E1 Buffer (ThermoFisher), a shock solution; recovery medium consisting of 80% (v / v) EX-CELL Chocloning Medium (Sigma-Aldrich) and 20% (v / v) EX-CELL Advanced CHO Fed-batch Medium (Sigma-Aldrich) supplemented with 1% GlutaMAX (ThermoFisher); expansion medium and subculture medium were both EX-CELL Advanced CHO Fed-batch CHO medium was supplemented with 1% GlutaMAX (ThermoFisher); pressurized medium 1 was passage medium supplemented with G418 at a final concentration of 800 μg / ml; pressurized medium 2 was passage medium supplemented with hygromycin at a final concentration of 250 μg / ml; the conditioned medium was the supernatant obtained by sterile filtration after inoculating the passage medium with CHO-K1 for 1 day; the cloning medium was 75% (v / v) EX-CELL CHO Cloning Medium, 20% (v / v) conditioned medium, and 5% (v / v) ClonaCell-CHO ACF Supplement supplemented with 1% GlutaMAX; the basal medium in the fed-batch medium was EX-CELL Advanced CHO Fed-batch Medium supplemented with 1% GlutaMAX (ThermoFisher), and the feed medium was Cell Boost 7a / 7b (HyClone).

[0160] The operation process of this embodiment is as follows Figure 1 As shown, it mainly includes the following steps:

[0161] (1) The green fluorescent protein gene (EGFP) was used as a screening marker gene, and the attP sequence was used as a homology arm to construct a recombinant plasmid containing RMCE. Figure 2 After linearizing the constructed plasmid, purify and recover the linear DNA. Centrifuge CHO K1 cells and discard the supernatant. Resuspend the cells in 100 μL of R Buffer, a resuspension buffer specifically for cell electroporation. Electroporate three times.

[0162] (2) Pressurization: the pressurization reagent is G418, the pressurization concentration is 800 μg / ml, and the cells are divided into minipools (i.e., 96-well plates) for culture.

[0163] (3) Check the plate after ten days. When a large number of fluorescent cell clusters are observed, they can be enriched. The one-well-to-one-well principle should be followed during enrichment.

[0164] (4) Observe the growth of enriched cells at any time and expand them when the coverage rate reaches more than 50%.

[0165] (5) After expansion to shake flasks, the cells were passaged three times to form a stable cell pool. The formation of monoclonal cells and screening of stable fluorescence were achieved by artificial intelligence. The steps are shown in Figure 1 The specific steps are shown in a~h.

[0166] a) The cells in the stable cell pool are diluted to a certain concentration and then inoculated into a culture dish containing a semi-solid culture medium. During the inoculation process, the cells are ensured to be approximately evenly distributed in all positions of the culture dish. The cells are allowed to stand for about half an hour to allow the cells to settle to the bottom of the culture dish.

[0167] b) The culture dish is transferred to the electron microscope stage, and the electron microscope performs high-throughput scanning on the cells in the culture dish;

[0168] c) The electron microscope scanned image is uploaded to the server for artificial intelligence image analysis. The analysis process includes monoclonal cell line detection, protein expression level prediction of monoclonal cell lines, protein expression level ranking, and coding and localization of screened high-protein expressing cell lines. The protein expression level prediction of the monoclonal cell line can be based on fluorescence or not. The following step c is protein expression level prediction not based on fluorescence.

[0169] Step c is based on the detection of monoclonal cell line targets using image processing technology. In this embodiment, the target detection algorithm of YOLOv8 is adopted, and the actual detection effect reaches mAP 94.2%. The target detection model for monoclonal cell lines in step c is consistent with the commonly used deep learning target detection model, so it is not described in detail. Specifically, the cell image needs to be annotated first. In order to improve the prediction accuracy of the algorithm, the bounding boxes of all monoclonal cell lines and adhesion cell lines will be marked and used as the real target bounding box (ground truth) for the loss calculation of the model output. After algorithm training, the model learns the ability to extract the border information of monoclonal cell lines. In the actual application scenario step c, the trained model can predict the border of the monoclonal cell line in the image.

[0170] Step c predicts the fluorescent protein expression level based on monoclonal cell images. In this example, the SqueezeNet deep learning network and the MSE loss function are used. Since the predicted protein expression levels in this project are ranked, the ranking result is the ultimate goal of the algorithm, so the NDCG evaluation standard of the ranking algorithm is used here. The specific calculation formula is as follows:

[0171]

[0172]

[0173] Where IDCG = best-ranked DCG. Specifically in this example, cell imaging predicted expression levels, sorted by predicted expression levels, and compared with the actual fluorescence value sorting results, the NDCG = 0.89. The expression level prediction model in this invention is described in detail in patent CN112037862B.

[0174] d) The coding and location information of the screened protein high-expressing cell lines are returned to the robot control software;

[0175] The cell codes and positions returned in step d, in this embodiment, return the top 100 cells in terms of predicted expression levels, with the codes ranging from 1 to 100. The position information includes the coordinates in the plane coordinate system relative to the center of the microscope (the depth of the culture medium is not considered for the time being, because all the cells to be selected are deposited to the bottom of the culture dish. In addition, during the photography process, cells that have not settled to the bottom of the culture dish will be out of focus, resulting in blurred cells, and cells with blurred images will also be excluded).

[0176] e) The robot control software automatically operates (or manually assists) the robotic arm to aspirate the selected monoclonal cell lines and transfer them to the designated well plate. This process is repeated until all high-expressing cell lines are transferred.

[0177] Step e: The process of the robot arm aspirating the target monoclonal cell line and transferring it to the well plate is described in detail in patents CN113821287B, CN113403431B, N113771030A, and CN113733087B.

[0178] f) After the cell clones in the well plate have grown to a certain number, they are transferred to a larger well plate for culture, and finally expanded to a shake flask for culture. The cell expression level is then tested to confirm that the cell line selected by the artificial intelligence is a high-yield cell line;

[0179] In this embodiment, step f is to transfer the cells to a 96-well plate for amplification and then transfer them to a shake flask for culture.

[0180] g) The selected high-yield cell lines are subcultured. Before each generation of cell lines is subcultured, a portion of the sample is diluted and placed in a culture dish, and images are taken using an electron microscope. Images of the cell lines are taken continuously for several generations. Currently, the expression of the selected cells can be predicted by the model learned from the cell morphology using photography, and the expression of the selected cells can also be predicted by fluorescent labeling.

[0181] Step g: Photographing Monoclonal Cells In this example, a Thermo Fisher Scientific M7000 electron microscope was used to photograph the cells.

[0182] h) The collected images of several generations of cell lines are input into an artificial intelligence algorithm to predict the cell line's passage stability based on fluorescence intensity and / or protein expression. Cell lines with high protein expression characteristics that can be stably passaged are selected as the final candidate cell lines. The prediction of the cell line's passage stability can be based on fluorescence or cell morphology instead of fluorescence. The following h steps are for predicting protein expression based on cell morphology instead of fluorescence.

[0183] Step h predicts the cell line stability, including but not limited to using traditional image recognition algorithms to extract and predict image features using histograms, automatically extracting and predicting image features using deep learning neural networks, and other image-based prediction techniques. In this embodiment, a deep learning-based method was used to automatically extract image features and predict stability for stable and unstable cell lines, achieving an accuracy rate of 84% for predictions across different cell lines. This is described in detail in patent CN114417582A.

[0184] Wherein, step d and step e can be completed manually. In this embodiment, they are completed automatically by a robotic arm.

[0185] (6) CHO-K1 cells were used as control and the seeding density was 5×10 5 cells / ml, inoculate 30 ml of the system, count the cells on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13, and culture day 14, and screen out monoclones with a growth rate lower than that of CHO-K1.

[0186] (7) Recover the remaining monoclonal clones and perform stable subculture after recovery. Subculture should be performed every 3 or 4 days. The cell density of the 4-day subculture is 3×10 5 cells / ml, and the cell density after 3 days was 5×10 5 cells / ml, and the fluorescence of the cell line was regularly monitored during subculture. The stability of the high-fluorescence cell line was studied for approximately 90 days using the subculture medium. GBB003 cells, which exhibited minimal fluorescence fluctuations and stable cell line growth, were confirmed to be stable high-fluorescence cells. NGS sequencing of the integration site of this cell line revealed the highly expressed fragment sequence located at the integration site as SEQ ID No. 1. Specifically, the annotation information for the integration site on the CHO gene is: NW_003616785.1:83044.

[0187] Example 2 Expression of three-chain antibodies

[0188] This example is used to express three-chain bispecific bivalent antibody A, three-chain bispecific bivalent antibody B, and three-chain bispecific bivalent antibody C. Specific antibody A comprises a heavy chain (encoded by the gene sequence shown in SEQ ID No. 3) and a light chain (encoded by the gene sequence shown in SEQ ID No. 2) that bind to the EpCAM target, and a recombinant heavy chain scFv-Fc (encoded by the gene sequence shown in SEQ ID No. 8) that binds to the CD3 target; specific antibody B comprises a heavy chain (encoded by the gene sequence shown in SEQ ID No. 5) and a light chain (encoded by the gene sequence shown in SEQ ID No. 4) that bind to the CD38 target, and a recombinant heavy chain scFv-Fc (encoded by the gene sequence shown in SEQ ID No. 8) that binds to the CD3 target; specific antibody C comprises a heavy chain (encoded by the gene sequence shown in SEQ ID No. 7) and a light chain (encoded by the gene sequence shown in SEQ ID No. 9) that bind to the HER2 target. No. 6) and a recombinant heavy chain scFv-Fc that binds to the CD3 target (encoded by the gene sequence shown in SEQ ID No. 8). The expression cassette for the target protein corresponding to each target gene in each arrangement comprises a promoter nucleic acid sequence, a signal peptide encoding nucleic acid sequence, a target gene encoding the target protein, and a polynucleotide gene, arranged in sequence.

[0189] Among them, the light chain gene sequences of specific antibody A, specific antibody B, and specific antibody C are all preceded by a signal peptide gene sequence required for antibody secretion, and the signal peptide sequences are all:

[0190] SEQ ID No.9: MDMRVPAQLLGLLLLWLRGARC.

[0191] Among them, the heavy chain gene sequences of specific antibody A, specific antibody B, and specific antibody C are all preceded by a signal peptide gene sequence required for antibody secretion, and the signal peptide sequences are all:

[0192] SEQ ID No. 10: MGWSCIILFLVATATGVHS.

[0193] The recombinant heavy chain (scFv-Fc) gene sequences of specific antibody A, specific antibody B, and specific antibody C are preceded by a signal peptide gene sequence required for antibody secretion. The signal peptide sequences are:

[0194] SEQ ID No. 11: MRAWIFFLLCLAGRALA.

[0195] In the following examples, A represents a bispecific bivalent antibody targeting EpCAM and CD3, B represents a bispecific bivalent antibody targeting CD38 and CD3, and C represents a bispecific bivalent antibody targeting HER2 and CD3.

[0196] F represents the recombinant heavy chain expression cassette [scFv-Fc], H or HC represents the heavy chain expression cassette [HC], and L or LC represents the light chain expression cassette [LC]. The order of F, H, and L in the name represents the arrangement of the expression cassettes in the expression plasmid; or the arrangement of the expression cassettes in the expression plasmid in the host cell, as shown in Table 1.

[0197] Table 1

[0198] name Arrangement order of expression cassettes in expression vector LFH [LC]-[scFv-Fc]-[HC] FLH [scFv-Fc]-[LC]-[HC] LFHL [LC]-[scFv-Fc]-[HC]-[LC] LFHF [LC]-[scFv-Fc]-[HC]-[scFv-Fc] LHFF [LC]-[HC]-[scFv-Fc]-[scFv-Fc] FLHL [scFv-Fc]-[LC]-[HC]-[LC] FHLL [scFv-Fc]-[HC]-[LC]-[LC]

[0199] (1) Exogenous nucleic acid construction

[0200] Figure 3 As shown, the pDonor0.0-H plasmid is used to construct the expression cassette vectors of heavy chain HC and recombinant heavy chain scFv-Fc, respectively. The target gene sequence can be inserted in the middle of the Xma I and / or Hind III restriction sites, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication initiation site, which is used for plasmid replication and amplification in prokaryotic cells; the promoter segment is used to initiate the expression of the target gene; the poly (A) signal is used to terminate the transcription of the target gene; the Not I, Age I and other restriction sites are used to direct the expression of the target gene to the target gene. Figure 5 The pLangding0.0 plasmid shown was digested and ligated to insert the complete expression cassette.

[0201] like Figure 4 As shown, pDonor0.0-L is used to construct a vector for light chain expression cassette, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication initiation site, which is used for transcriptional amplification of the plasmid in prokaryotic cells; the promoter segment is used to initiate the expression of the target gene; the poly (A) signal is used to terminate the transcription of the target gene; the Xma I and / or Hind III restriction sites are used to insert the target gene sequence; other restriction sites such as Not I are used to insert the target gene sequence; Figure 5 The pLangding0.0 plasmid shown was digested and ligated to insert the complete expression cassette.

[0202] pLanding0.0 vector Figure 5As shown, the Ori segment is the plasmid replication origin, which is used for transcriptional amplification of the plasmid in prokaryotic cells; AmpR provides an ampicillin resistance gene for screening in plasmid construction, Bxb1-attB GA and attB are used for recombination with the GBB003 mother cell genome, promoter is used to drive the product screening of eukaryotic resistance genes in the mother cell genome, and Not I, BamH I and other restriction sites are used for restriction enzyme replacement in the pDonor0.0 vector.

[0203] (1) Construction of three-chain bispecific antibody molecule - specific antibody A:

[0204] Specific antibody A comprises a light chain (LC, gene sequence as shown in SEQ ID No. 2), a heavy chain (HC, gene sequence as shown in SEQ ID No. 3), and a recombinant heavy chain (scFv-Fc structure, gene sequence as shown in SEQ ID No. 8). The light chain and heavy chain combine to form a half antibody that binds to a first specific target, and the recombinant heavy chain (scFv-Fc structure) binds to a second specific target. In this embodiment, the first specific target of specific antibody A is EpCAM and the second specific target is CD3.

[0205] Light chain expression cassette [LC] construction:

[0206] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 9) and the light chain gene sequence of specific antibody A (shown by SEQ ID No. 2) were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-L plasmid by gene synthesis to form the pDonor0.0-A-LC plasmid.

[0207] Heavy chain expression cassette [HC] construction:

[0208] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 10) and the heavy chain gene sequence of specific antibody A (shown by SEQ ID No. 3) were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid by gene synthesis to form the pDonor0.0-A-HC plasmid.

[0209] Construction of recombinant heavy chain expression cassette [scFv-Fc]:

[0210] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 11) and recombinant heavy chain gene sequence (scFv-Fc sequence, as shown in SEQ ID No. 8) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-F plasmid.

[0211] Seven plasmids were constructed, and the target gene arrays within the plasmid constructs were named LFH, FLH, LFHL, LFHF, LHFF, FLHL, and FHLL. The master plasmids pDonor0.0-H and pDonor0.0-L were constructed into the pLanding0.0 plasmid by enzyme digestion and ligation.

[0212] In the names of each plasmid, F represents the recombinant heavy chain expression cassette [scFv-Fc], H or HC represents the heavy chain expression cassette [HC], and L or LC represents the light chain expression cassette [LC].

[0213] (1.1) LFH: The structure is [LC]-[scFv-Fc]-[HC]

[0214] ① After double digestion of the pDonor0.0-A-LC and pLanding0.0 plasmids with Not I / Age I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0 fragment were recovered. These fragments were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0215] ② After double digestion of the pDonor0.0-F and pLanding0.0-AL plasmids with BsiW I / Mlu I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-AL fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LF.

[0216] ③ After double digestion of pDonor0.0-A-HC and pLanding0.0-A-LF plasmids with Nhe I / Xho I, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-A-LF fragment were recovered, and then transformed into Stbl3 chemical competent cells using T4 DNA ligase. After the colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following: Figure 6 pLanding0.0-A-LFH is shown.

[0217] (1.2) FLH: The structure is [scFv-Fc]-[LC]-[HC]

[0218] ① After double digestion of the pDonor0.0-F and pLanding0.0 plasmids with Not I / Age I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0 fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-F.

[0219] ② After double digestion of the pDonor0.0-A-LC and pLanding0.0-F plasmids with BsiW I / Mlu I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0-F fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-FL.

[0220] ③ After double digestion of pDonor0.0-A-HC and pLanding0.0-A-FL plasmids with NheI / XhoI, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-A-FL fragment were recovered, and then transformed into Stbl3 chemical competent cells using T4 DNA ligase. After the colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following Figure 7 pLanding0.0-A-FLH shown.

[0221] (1.3) LFHL: The structure is [LC]-[scFv-Fc]-[HC]-[LC]

[0222] ① After double digestion of the pDonor0.0-A-LC and pLanding0.0 plasmids with Not I / Age I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0 fragment were recovered. These fragments were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0223] ② After double digestion of the pDonor0.0-F and pLanding0.0-AL plasmids with Bsiw I / Mlu I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-AL fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LF.

[0224] ③ After double digestion of the pDonor0.0-A-HC and pLanding0.0-A-LF plasmids with Nhe I / Xho I, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-A-LF fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LFH.

[0225] ④ After double digestion of pDonor0.0-A-LC and pLanding0.0-A-LFH plasmids with EcoR I / Kpn I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0-A-LFH fragment were recovered, and then transformed into Stbl3 chemically competent cells using T4 DNA ligase. After the colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following Figure 8 pLanding0.0-A-LFHL is shown.

[0226] (1.4) LFHF: The structure is [LC]-[scFv-Fc]-[HC]-[scFv-Fc]

[0227] ① After double digestion of the pDonor0.0-A-LC and pLanding0.0 plasmids with Not I / Age I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0 fragment were recovered. These fragments were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0228] ② After double digestion of the pDonor0.0-F and pLanding0.0-AL plasmids with Bsiw I / Mlu I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-AL fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LF.

[0229] ③ After double digestion of the pDonor0.0-A-HC and pLanding0.0-A-LF plasmids with Nhe I / Xho I, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-A-LF fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LFH.

[0230] ④ After double digestion of pDonor0.0-F and pLanding0.0-A-LFH plasmids with EcoR I / Kpn I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-A-LFH fragment were recovered, and then ligated with T4 DNA ligase to transform Stbl3 chemically competent cells. After the colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following Figure 9 pLanding0.0-A-LFHF is shown.

[0231] (1.5) LHFF: The structure is [LC]-[HC]-[scFv-Fc]-[scFv-Fc]

[0232] ① After double digestion of the pDonor0.0-A-LC and pLanding0.0 plasmids with Not I / AgeI, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0 fragment were recovered. These fragments were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0233] ② After double digestion of the pDonor0.0-A-HC and pLanding0.0-AL plasmids with Bsiw I / Mlu I, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-AL fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LH.

[0234] ③ After double digestion of the pDonor0.0-F and pLanding0.0-A-LH plasmids with Nhe I / Xho I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-A-LH fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-LHF.

[0235] ④ After double digestion of pDonor0.0-F and pLanding0.0-A-LHF plasmids with EcoR I / Kpn I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0-A-LHF fragment were recovered, and then transformed into Stbl3 chemical competent cells using T4 DNA ligase. After the colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following Figure 10 pLanding0.0-A-LHFF shown.

[0236] (1.6) FLHL: The structure is [scFv-Fc]-[LC]-[HC]-[LC]

[0237] pDonor0.0-A-LC and pLanding0.0-A-FLH plasmids prepared in (1.2) were double-digested with EcoR I / Kpn I, respectively, and the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0-A-FLH fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After the colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following: Figure 11 pLanding0.0-A-FLHL is shown.

[0238] (1.7) FHLL: The structure is [scFv-Fc]-[HC]-[LC]-[LC]

[0239] ① After double digestion of the pDonor0.0-F and pLanding0.0 plasmids with Not I / Age I, the F expression cassette fragment of pDonor0.0-F and the linearized pLanding0.0 fragment were recovered. They were then ligated and transformed into DH5α chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-F.

[0240] ② After double digestion of the pDonor0.0-A-HC and pLanding0.0-F plasmids with Bsiw I / Mlu I, the A-HC expression cassette fragment of pDonor0.0-A-HC and the linearized pLanding0.0-F fragment were recovered. They were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-FH.

[0241] ③ After double digestion of the pDonor0.0-A-LC and pLanding0.0-A-FH plasmids with Nhe I / Xho I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0-A-FH fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-A-FHL.

[0242] ④ After the pDonor0.0-A-LC and pLanding0.0-A-FHL plasmids were double-digested with EcoR I / Kpn I, the A-LC expression cassette fragment of pDonor0.0-A-LC and the linearized pLanding0.0-A-FHL fragment were recovered, and then ligated with T4 DNA ligase to transform Stbl3 chemical competent cells. After the colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following Figure 12 pLanding0.0-A-FHLL shown.

[0243] (2) Construction of three-chain bispecific antibody molecule - specific antibody B:

[0244] Specific antibody B includes a light chain (LC, gene sequence as shown in SEQ ID No. 4), a heavy chain (HC, gene sequence as shown in SEQ ID No. 5), and a recombinant heavy chain (scFv-Fc structure, gene sequence as shown in SEQ ID No. 8). The light chain and the heavy chain combine to form a half antibody that binds to the first specific target, and the recombinant heavy chain (scFv-Fc structure) binds to the second specific target. In this embodiment, the first specific target of specific antibody B is CD38 and the second specific target is CD3.

[0245] Light chain expression cassette [LC] construction:

[0246] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 9) and the light chain gene sequence of specific antibody B (shown by SEQ ID No. 4) were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-L plasmid by gene synthesis to form the pDonor0.0-B-LC plasmid.

[0247] Heavy chain expression cassette [HC] construction:

[0248] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 10) and the heavy chain gene sequence of specific antibody B (shown by SEQ ID No. 5) were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid by gene synthesis to form the pDonor0.0-B-HC plasmid.

[0249] Construction of recombinant heavy chain expression cassette [scFv-Fc]:

[0250] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 11) and recombinant heavy chain gene sequence (scFv-Fc sequence, as shown in SEQ ID No. 8) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-F plasmid.

[0251] Seven plasmids were constructed, and the target gene arrangement within the plasmid structure was named LFH, FLH, LFHL, LFHF, LHFF, FLHL, and FHLL. The master plasmids pDonor0.0-H and pDonor0.0-L were constructed into the pLanding0.0 plasmid by enzyme digestion and ligation. LFH was constructed according to the method described in (1.1), FLH was constructed according to the method described in (1.2), LFHL was constructed according to the method described in (1.3), LFHF was constructed according to the method described in (1.4), LHFF was constructed according to the method described in (1.5), FLHL was constructed according to the method described in (1.6), and FHLL was constructed according to the method described in (1.7). The seven plasmids expressing specific antibody B differed from the plasmid construction method expressing specific antibody A in (1) only in that the nucleotide sequences of the genes encoding the light chain and heavy chain were different.

[0252] (3) Construction of three-chain bispecific antibody molecule - specific antibody C:

[0253] Specific antibody C includes a light chain (LC, gene sequence as shown in SEQ ID No. 6), a heavy chain (HC, gene sequence as shown in SEQ ID No. 7), and a recombinant heavy chain (scFv-Fc structure, gene sequence as shown in SEQ ID No. 8). The light chain and the heavy chain combine to form a half antibody that binds to a first specific target, and the recombinant heavy chain (scFv-Fc structure) binds to a second specific target. In this embodiment, the first specific target of specific antibody C is HER2 and the second specific target is CD3.

[0254] Light chain expression cassette [LC] construction:

[0255] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 9) and the light chain gene sequence of specific antibody C (shown by SEQ ID No. 6) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-L plasmid to form the pDonor0.0-C-LC plasmid.

[0256] Heavy chain expression cassette [HC] construction:

[0257] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 10) and the heavy chain gene sequence of specific antibody C (SEQ ID No. 7) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-C-HC plasmid.

[0258] Construction of recombinant heavy chain expression cassette [scFv-Fc]:

[0259] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 11) and recombinant heavy chain gene sequence (scFv-Fc sequence, as shown in SEQ ID No. 8) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-F plasmid.

[0260] Seven plasmids were constructed, and the target gene arrangement within the plasmid structure was named LFH, FLH, LFHL, LFHF, LHFF, FLHL, and FHLL. The master plasmids pDonor0.0-H and pDonor0.0-L were constructed into the pLanding0.0 plasmid by enzyme digestion and ligation. LFH was constructed according to the method described in (1.1), FLH was constructed according to the method described in (1.2), LFHL was constructed according to the method described in (1.3), LFHF was constructed according to the method described in (1.4), LHFF was constructed according to the method described in (1.5), FLHL was constructed according to the method described in (1.6), and FHLL was constructed according to the method described in (1.7). The seven plasmids expressing specific antibody C differed from the plasmid construction method expressing specific antibody A in (1) only in that the nucleotide sequences of the genes encoding the light chain and heavy chain were different.

[0261] (2) Verification of expression effect

[0262] In the bispecific bivalent antibody formed after the expression of the seven molecules constructed in each molecule constructed in step (a), the light chain sequences of specific antibody A, specific antibody B, and specific antibody C are all combined with their corresponding heavy chain sequences through disulfide bonds to form half IgG-like antibodies, and the recombinant heavy chain (F antibody chain) is a scFv-Fc structure. Half of the IgG-like antibody and scFv-Fc are combined through disulfide bonds to form the target protein. There are many mismatch modes in the combination of light chain sequence, heavy chain sequence and scFv-Fc structure. Therefore, different construction methods of the target gene in the target protein gene expression can affect the expression ratio and results of each chain in the light chain sequence, heavy chain sequence and scFv-Fc structure, thereby affecting the production of specific antibody A, specific antibody B, and specific antibody C.

[0263] (1) Transient expression verification

[0264] The stable high fluorescence cells GBB003 prepared in Example 1 were selected for transient transfection and verification. The recombinant plasmids containing the seven bispecific bivalent antibody expression genes corresponding to the specific antibodies A, B, and C in the above step (1) were amplified and transfected into the stable high fluorescence cells GBB003 prepared in Example 1. After transfection, the cells were shaken and cultured for 7 days. On the 7th day, the expression levels of the specific antibodies A, B, and C in the supernatant were measured. The specific antibodies A, B, and C all had the highest expression levels in the cells when the target genes were arranged in the LFH manner. The transient transfection results are shown in Table 2.

[0265] Table 2. Statistical results of expression levels in transiently transfected cell pools on day 7

[0266]

[0267] (2) Select the stable high fluorescence cell GBB003 from (1) for transfection and integration verification

[0268] a) The recombinant plasmids expressing the bispecific bivalent antibody genes of the seven arrangements of the specific antibody C and the LFH arrangement of the specific antibody A and the LFH arrangement of the specific antibody B in step (1) above were amplified and linearized, and then co-transfected with the PGBB BXB1 integrase plasmid into the stable high fluorescence cell GBB003. The PGBB BXB1 integrase plasmid map is shown in FIG. Figure 13 As shown;

[0269] b) One day after transfection, the cell pool was used to construct a minipool and pressure screening was performed using pressure medium 2;

[0270] c) After 14 days, the non-fluorescent cells in the minipool were expanded and cultured in a 96-well plate-24-well plate-6-well plate. When expanding to a 6-well plate, a 6-well batch culture was performed. The 6-well batch culture was performed by seeding 5×10 5 Cells / ml, 2ml system, 37°C, 5% CO2, 120 rpm. Only C-FHLL molecule (not counted in Table 3) was not enriched in the cell pool with expression. The results of the 6-well batch culture experiment of the remaining cell pools are shown in Table 3:

[0271] Table 3. Statistical results of expression levels in six-well batch culture of cell pools

[0272]

[0273]

[0274] d) For each arrangement of specific antibody A, specific antibody B, and specific antibody C, the top two cell pools in 6-well batch culture expression (i.e., A-LFH-3 cell pool, A-LFH-7 cell pool, B-LFH-4 cell pool, B-LFH-6 cell pool, C-LFH-16 cell pool, C-LFH-6 cell pool, C-FLH-5 cell pool, C-FLH-7 cell pool, C-LFHL-6 cell pool, C-LFHL-12 cell pool, C-LFHF-2 cell pool, C-LFHF-5 cell pool, C-LHFF-1 cell pool, C-LHFF-3 cell pool, C-FLHL-1 cell pool, and C-FLHL-2 cell pool) were selected and expanded to T125 shake flasks. After two passages, shake flasks were fed for 14 days. The seeding density was 5×10 5 / ml, the inoculation system was 30ml, and the cells were counted on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13, and culture day 14. 3% Cell Boost 7a and 0.3% Cell Boost 7b were supplemented on the 3rd day, and 5% Cell Boost 7a and 0.5% Cell Boost 7b were supplemented on the 5th, 7th, 9th, 11th, and 13th culture days. During this period, the glucose concentration was controlled at 2-8g / L. After 14 days, the expression levels of specific antibody A, specific antibody B, and specific antibody C were determined by Octet, and the shake flask feeding expression results are shown in Table 4.

[0275] Table 4. Statistical results of shake flask feeding expression

[0276]

[0277]

[0278] e) After purification using a Protein A column, the grayscale value of each lane was analyzed by SDS-PAGE to determine the purity of the corresponding antibody. The statistical results are shown in Table 5. The purity of C-FLHL-2 was 90.7%, and the purity of C-LHFF-1 was 85.6%. Based on the expression level and purity, C-FLHL-2 and C-LHFF-1 were selected for monoclonal cloning.

[0279] Table 5. Purity statistics of cell pools by SDS-PAGE

[0280] Cell pool number Purity of target molecule (%) Cell pool number Purity of target molecule (%) A-LFH-3 41.3 C-LFHL-6 37.0 A-LFH-7 44.7 C-LFHL-12 37.0 B-LFH-4 51.7 C-FLHL-1 84.1 B-LFH-6 32.8 C-FLHL-2 90.7 C-LFH-6 46.2 C-LHFF-1 85.6 C-LFH-16 38.4 C-LHFF-3 90 C-FLH-5 25.5 C-LFHF-2 66.2 C-FLH-7 38.0 C-LFHF-5 62.9

[0281] f) Select FLHL and LHFF arrays for monoclonal

[0282] The C-LHFF-1 and C-FLHL-2 cell pools expressing specific antibody C were diluted to a limit, and a 96-well plate was plated at a density of 0.8 cells / well. The cell growth status was photographed with VIPS at 1 hour, 20 hours, 2 days, 3 days, 5 days, 7 days, and 14 days. The non-fluorescent monoclonal clones were counted, and the expression level was measured with Octet after 14 days. The top 12 non-fluorescent monoclonal wells were expanded and cultured in the manner of 96-well plate-24-well plate-6-well plate-shake flask. During the expansion process, clones with low growth rates were eliminated. After the 6-well expansion, a six-well batch culture was performed. The 6-well batch culture was performed by inoculating 5×10 5 / ml, 2ml system, 37°C 5% carbon dioxide 120 rpm conditions for 7 days, the statistical results of the expression of single clone wells in each cell pool in 6-well batch culture are shown in Table 6.

[0283] Table 6. Statistical results of expression levels of monoclonal 6-well batch culture

[0284]

[0285]

[0286] g) Select clones F4, E3, and B10 from the cell pool C-LHFF-1 and clones F11, E3, and B6 from the cell pool C-FLHL-2, subculture them twice, and then feed them in shake flasks for 14 days. The seeding density is 5×10 5 / ml, inoculate 30ml of the system, count on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13 and culture day 14, and supplement 3% Cell Boost 7a and 0.3% Cell Boost 7b on the 3rd day, 5% Cell Boost 7a and 0.5% Cell Boost 7b on the 5th day, culture day 7, culture day 9, culture day 11 and culture day 13, during which the glucose concentration was controlled at 2-8g / L. After 14 days, use The expression levels of the monoclonal shake flask feed-specific antibody C were measured and the expression results are shown in Table 7. Among them, the expression level of the monoclonal B10 of C-LHFF-1 was the highest, at 5592.9 mg / l, and the expression level of the monoclonal B6 of C-FLHL-2 was the highest, at 4147.1 mg / l.

[0287] Table 7. Statistical results of monoclonal shake flask feeding expression

[0288]

[0289] The supernatant of the shake flask feed was purified using a protein A chromatography column and then analyzed by SDS-PAGE for the purity of specific antibody C. The statistical results are shown in Table 8. The purity of specific antibody C expressed by clone B10 of C-LHFF was 82.7%, and the purity of specific antibody C expressed by clone B6 of C-FLHL was 72.3%.

[0290] Table 8. Monoclonal shake flask feed SDS-PAGE purity statistics

[0291]

[0292] The purified samples were analyzed by non-reduced CE-SDS method. The analysis results are shown in Table 9. Among them, the quality attributes of C-LHFF clones F4, B10, and F3 were excellent. Taking B10 as an example, the non-reduced CE-SDS detection image is shown in Figure 14 Among them, the quality attributes of C-FLHL clones B6, E3, and F11 are excellent. Taking B6 as an example, the non-reduced CE-SDS detection image is shown as follows. Figure 15 Show.

[0293] Table 9. Monoclonal non-reduced CE-SDS purity statistics

[0294]

[0295] The purified samples were analyzed by reduced CE-SDS method. The analysis results are shown in Table 10. The molecular mass of LC is 23.4 kDa, the molecular mass of HC is 49.3 kDa, and the molecular mass of F is 52.3 kDa. Therefore, the molecular mass ratio of LC:HC:F is approximately 1:2:2, which corresponds to the peak area proportion of each chain. Taking the monoclonal B10 of C-LHFF-1 as an example, the reduced CE-SDS detection image is shown in Figure 16 As shown. Taking the monoclonal B6 of C-FLHL-2 as an example, the CE-SDS detection image is as follows Figure 17 shown.

[0296] Table 10. Results of monoclonal reduced CE-SDS analysis

[0297]

[0298] Through the above experiments, we screened for a bispecific antibody-expressing cell line consisting of a single heavy chain (HC), a single light chain (LC), and a recombinant heavy chain (scFv-Fc, single-chain variable fragment (scFv)-based (Fc fragment) antibody) that met our expectations. The experimental process is controllable and the workload is significantly reduced compared to traditional methods. The antibody mismatch rate of this combination of a single heavy chain (HC), a single light chain (LC), and a recombinant heavy chain (scFv-Fc, single-chain variable fragment (scFv)-based (Fc fragment) antibody) is lower. Since heavy chain expression in conventional antibody expression depends on the proper folding of the light chain, which assists in the secretion of the heavy chain, placing the light chain expression cassette at the front of the plasmid design in the seven molecules produced three-chain antibodies, thereby further increasing the yield of the target molecule. This explains why placing the light chain expression cassette first and increasing light chain expression in the preferred molecular design results in higher target protein expression and lower mismatch rates. The cell line constructed with the target gene arrangement in LHFF had the highest expression level and the lowest mismatch rate. Specifically, it was found that when an exogenous nucleic acid fragment was integrated into the fixed site within the expression vector, and the exogenous nucleic acid fragment contained a gene encoding a light chain (LC), a gene encoding a heavy chain (HC), a gene encoding a recombinant heavy chain (scFv-Fc), and a gene encoding a recombinant heavy chain (scFv-Fc), the expression level of the three-chain antibody was high and the mismatch rate was low.

[0299] Example 3 Expression of Four-Chain Bispecific Antibodies

[0300] This example expresses an ANG-2 / VEGF bispecific bivalent antibody. The antibody comprises heavy and light chain sequences corresponding to the ANG-2 target, and heavy and light chain sequences corresponding to the VEGF target. All genes were synthesized by GenScript. The light chain gene sequence corresponding to the ANG-2 target is shown in SEQ ID No. 12, the heavy chain gene sequence corresponding to the ANG-2 target is shown in SEQ ID No. 13, the light chain gene sequence corresponding to the VEGF target is shown in SEQ ID No. 14, and the heavy chain gene sequence corresponding to the VEGF target is shown in SEQ ID No. 15.

[0301] In the ANG-2 / VEGF bispecific bivalent antibody formed after expression of the four molecules, the constant region CL (SEQ ID No. 17) of the ANG-2 antibody light chain sequence and the constant region CH1 (SEQ ID No. 16) of the ANG-2 antibody heavy chain sequence were replaced with each other. Therefore, the expression of the ANG-2 antibody light chain sequence was more difficult than that of the VEGF antibody light chain sequence, and the expression of the ANG-2 antibody heavy chain sequence was more difficult than that of the VEGF antibody heavy chain sequence.

[0302] The antibody light chain sequence is preceded by a signal peptide sequence required for antibody secretion, the sequence is:

[0303] SEQ ID No.9MDMRVPAQLLGLLLLWLRGARC;

[0304] The antibody heavy chain sequence is preceded by a signal peptide sequence required for antibody secretion, the sequence is:

[0305] SEQ ID No. 10MGWSCIILFLVATATGVHS.

[0306] (1) Exogenous nucleic acid construction

[0307] Figure 3 As shown, the pDonor0.0-H plasmid is used to construct a vector for the heavy chain expression cassette, and the target gene sequence can be inserted in the middle of the XmaI and / or HindIII restriction sites, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication initiation site, which is used for plasmid replication and amplification in prokaryotic cells; the CMV segment is used to initiate the expression of the target gene; the SV40 poly (A) is used to terminate the transcription of the target gene; NotI and other restriction sites are used to cut and connect the complete expression cassette into the pLangding0.0 plasmid.

[0308] like Figure 4 As shown, pDonor0.0-L is used to construct a vector for the light chain expression cassette, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication initiation site, used for transcriptional amplification of the plasmid in prokaryotic cells; the CMV segment is used to initiate the expression of the target gene; the SV40 poly (A) is used to terminate the transcription of the target gene; the XmaI and / or HindIII restriction sites are used to insert the target gene sequence; and other restriction sites such as NotI are used to cut and connect the complete expression cassette into the pLangding0.0 plasmid.

[0309] like Figure 5As shown, pLangding0.0 is used to accept the expression cassettes of pDonor0.0-H and / or pDonor0.0-L, wherein the Ori segment is the plasmid replication origin site for transcriptional amplification of the plasmid in prokaryotic cells; AmpR provides an ampicillin resistance gene for screening in plasmid construction, Bxb1-attB GA and attB are used for recombination with the GBB003 mother cell genome, promoter is used to drive the screening of eukaryotic resistance genes in the products of the mother cell genome, and restriction sites such as Not I and BamH I are used for restriction enzyme substitution in the pDonor0.0 vector.

[0310] In this example, four molecules were constructed according to the order of the exogenous gene expression cassettes in pLangding0.0, named Molecule 1, Molecule 2, Molecule 3, and Molecule 4. Each molecule contained four expression cassettes, each housing a target gene, each encoding one chain of a four-chain bispecific antibody. The order of the expression cassettes in the four molecules is shown in Table 11.

[0311] Table 11. Arrangement order of expression cassettes in four molecules

[0312] Serial number name The order of target genes in exogenous nucleic acid 1 Molecule No. 1 [ANG-2LC]-[VEGF LC]-[ANG-2HC]-[VEGF HC] 2 Molecule No. 2 [ANG-2LC]-[VEGF LC]-[VEGF HC]-[ANG-2HC] 3 Molecule No. 3 [VEGF LC]-[ANG-2LC]-[VEGF HC]-[ANG-2HC] 4 Molecule No. 4 [VEGF LC]-[ANG-2LC]-[ANG-2HC]-[VEGF HC]

[0313] Wherein, [ANG-2LC] represents the expression cassette encoding the light chain gene corresponding to the ANG-2 target, abbreviated as AL, [ANG-2HC] represents the expression cassette encoding the heavy chain gene corresponding to the ANG-2 target, abbreviated as AH, [VEGF LC] represents the expression cassette encoding the light chain gene corresponding to the VEGF target, abbreviated as VL, and [VEGF HC] represents the expression cassette encoding the heavy chain gene corresponding to the VEGF target, abbreviated as AH.

[0314] (1) Molecule No. 1:

[0315] The kozak sequence, signal peptide sequence (SEQ ID No. 9) and ANG-2 antibody light chain sequence (SEQ ID No. 12) were synthetically inserted into the Hind III and Xma I restriction sites of the pDonor0.0-L plasmid to form the pDonor0.0-ANG-2-LC plasmid.

[0316] The kozak sequence, signal peptide sequence (SEQ ID No. 9) and VEGF antibody light chain sequence (SEQ ID No. 14) were synthetically inserted into the HindIII and XmaI restriction sites of the pDonor0.0-L plasmid to form the pDonor0.0-VEGF-LC plasmid.

[0317] The kozak sequence, signal peptide sequence (SEQ ID No. 10) and ANG-2 antibody heavy chain sequence (SEQ ID No. 13) were synthetically inserted into the HindIII and XmaI restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-ANG-2-HC plasmid.

[0318] The kozak sequence, signal peptide sequence (SEQ ID No. 10) and VEGF antibody heavy chain sequence (SEQ ID No. 15) were synthetically inserted into the HindIII and XmaI restriction sites of the pDonor0.0-H plasmid to form the pDonor0.0-VEGF-HC plasmid.

[0319] After double digestion of the pDonor0.0-ANG-2-LC and pLanding0.0 plasmids with NotI / AgeI, the ANG-2-LC expression cassette fragment of pDonor0.0-ANG-2-LC and the linearized pLanding0.0 fragment were recovered. They were then ligated and transformed into DH5α chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0320] After double digestion of the pDonor0.0-VEGF-LC and pLanding0.0-AL plasmids with BsiwI / MluI, the VEGF-LC expression cassette fragment of pDonor0.0-VEGF-LC and the linearized pLanding0.0-AL fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL.

[0321] The pDonor0.0-ANG-2-HC and pLanding0.0-AL-VL plasmids were double-digested with NheI / XhoI, and the ANG-2-HC expression cassette fragment of pDonor0.0-ANG-2-HC and the linearized pLanding0.0-AL-VL fragment were recovered. These fragments were then ligated and transformed into StB13 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL-AH.

[0322] After the pDonor0.0-VEGF-HC and pLanding0.0-AL-VL-AH plasmids were double-digested with EcoRI / KpnI, the VEGF-HC expression cassette fragment of pDonor0.0-VEGF-HC and the linearized pLanding0.0-AL-VL-AH fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After the colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL-AH-VH. The schematic diagram of the plasmid pLanding0.0-AL-VL-AH-VH is shown in the figure. Figure 19 shown.

[0323] (2) Molecule No. 2:

[0324] The preparation method of pDonor0.0-ANG-2-LC plasmid, pDonor0.0-VEGF-LC plasmid, pDonor0.0-ANG-2-HC plasmid and pDonor0.0-VEGF-HC plasmid is the same as molecule No. 1.

[0325] The pDonor0.0-ANG-2-LC and pLanding0.0 plasmids were double-digested with Not I / Age I, and the ANG-2-LC expression cassette fragment of pDonor0.0-ANG-2-LC and the linearized pLanding0.0 fragment were recovered. They were then ligated and transformed into DH5α chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL.

[0326] After double digestion of the pDonor0.0-VEGF-LC and pLanding0.0-AL plasmids with BsiwI / MluI, the VEGF-LC expression cassette fragment of pDonor0.0-VEGF-LC and the linearized pLanding0.0-AL fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL.

[0327] After double digestion of the pDonor0.0-VEGF-HC and pLanding0.0-AL-VL plasmids with NheI / XhoI, the VEGF-HC expression cassette fragment of pDonor0.0-VEGF-HC and the linearized pLanding0.0-AL-VL fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL-VH.

[0328] After double digestion of the pDonor0.0-ANG-2-HC and pLanding0.0-AL-VL-VH plasmids with EcoRI / KpnI, the ANG-2-HC expression cassette fragment of pDonor0.0-ANG-2-HC and the linearized pLanding0.0-AL-VL-VH fragment were recovered. They were then ligated with T4 DNA ligase and transformed into StBl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-AL-VL-VH-AH. A schematic diagram of the plasmid pLanding0.0-AL-VL-VH-AH is shown in the figure. Figure 20 shown.

[0329] (3) Molecule No. 3:

[0330] The preparation method of pDonor0.0-ANG-2-LC plasmid, pDonor0.0-VEGF-LC plasmid, pDonor0.0-ANG-2-HC plasmid and pDonor0.0-VEGF-HC plasmid is the same as molecule No. 1.

[0331] After double digestion of pDonor0.0-VEGF-LC and pLanding0.0 plasmids with NotI / AgeI, the VEGF-LC expression cassette fragment of pDonor0.0-VEGF-LC and the linearized pLanding0.0 fragment were recovered, and then ligated with T4 DNA ligase to transform DH5α chemically competent cells. After colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL.

[0332] The pDonor0.0-ANG-2-LC and pLanding0.0-VL plasmids were double-digested with BsiwI / MluI, and the ANG-2-LC expression cassette fragment of pDonor0.0-ANG-2-LC and the linearized pLanding0.0-VL fragment were recovered. These fragments were then ligated and transformed into Stbl3 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL.

[0333] After double digestion of the pDonor0.0-VEGF-HC and pLanding0.0-VL-AL plasmids with NheI / XhoI, the VEGF-HC expression cassette fragment of pDonor0.0-VEGF-HC and the linearized pLanding0.0-VL-AL fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL-VH.

[0334] After double digestion of the pDonor0.0-ANG-2-HC and pLanding0.0-VL-AL-VH plasmids with EcoRI / KpnI, the ANG-2-HC expression cassette fragment of pDonor0.0-ANG-2-HC and the linearized pLanding0.0-VL-AL-VH fragment were recovered. They were then ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL-VH-AH. The schematic diagram of the plasmid pLanding0.0-VL-AL-VH-AH is shown in the figure. Figure 21 shown.

[0335] (4) Molecule No. 4:

[0336] The preparation method of pDonor0.0-ANG-2-LC plasmid, pDonor0.0-VEGF-LC plasmid, pDonor0.0-ANG-2-HC plasmid and pDonor0.0-VEGF-HC plasmid is the same as molecule No. 1.

[0337] After double digestion of pDonor0.0-VEGF-LC and pLanding0.0 plasmids with NotI / AgeI, the VEGF-LC expression cassette fragment of pDonor0.0-VEGF-LC and the linearized pLanding0.0 fragment were recovered, and then ligated with T4 DNA ligase to transform DH5α chemically competent cells. After colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL.

[0338] The pDonor0.0-ANG-2-LC and pLanding0.0-VL plasmids were double-digested with BsiwI / MluI, and the ANG-2-LC expression cassette fragment of pDonor0.0-ANG-2-LC and the linearized pLanding0.0-VL fragment were recovered. These fragments were then ligated and transformed into StB13 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL.

[0339] The pDonor0.0-ANG-2-HC and pLanding0.0-VL-AL plasmids were double-digested with NheI / XhoI, and the ANG-2-HC expression cassette fragment of pDonor0.0-ANG-2-HC and the linearized pLanding0.0-VL-AL fragment were recovered. These fragments were then ligated and transformed into StB13 chemically competent cells using T4 DNA ligase. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL-AH.

[0340] After the pDonor0.0-VEGF-HC and pLanding0.0-VL-AL-AH plasmids were double-digested with EcoRI / KpnI, the VEGF-HC expression cassette fragment of pDonor0.0-VEGF-HC and the linearized pLanding0.0-VL-AL-AH fragment were recovered, and then ligated with T4 DNA ligase to transform StBl3 chemically competent cells. After the colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-VL-AL-AH-VH. The schematic diagram of the plasmid pLanding0.0-VL-AL-AH-VH is shown in the figure. Figure 22 shown.

[0341] (2) Verification of expression effect

[0342] (1) The stable high-fluorescence cell GBB003 prepared in Example 1 was selected for transfection and integration verification. The recombinant plasmids expressing the ANG-2 / VEGF bispecific bivalent antibody genes in the four arrangements contained in step (1) were amplified and linearized, and then co-transfected with Bxb-1 integrase into the stable high-fluorescence cell GBB003. The Bxb-1 integrase plasmid map is shown in FIG. Figure 13 shown.

[0343] (2) One day after transfection, a minipool was constructed from the cell pool and pressure screening was performed using pressurized medium 2.

[0344] (3) After 14 days, the non-fluorescent cells in the minipool were expanded and cultured in the following manner: 96-well plate → 24-well plate → 6-well plate. When expanding to a 6-well plate, a 6-well batch culture was performed. The 6-well batch culture was performed by inoculating 5×10 5 / ml, 2ml system, 37°C 5% carbon dioxide 120 rpm condition culture, 6-well batch culture experimental results are shown in Table 12.

[0345] Table 12. Statistical results of expression levels in six-well batch culture of cell pools

[0346]

[0347] (4) For each molecule, select the top 1 cell pool (i.e., 1-9 cell pool, 2-5 cell pool, 3-4 cell pool, 4-6 cell pool) and expand it to T125 shake flasks. After two subcultures, shake flasks are fed for 14 days. The inoculation density is 5×10 5 / ml, the inoculation system was 30ml, and the cells were counted on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13, and culture day 14. 3% Cell Boost 7a and 0.3% Cell Boost 7b were supplemented on the 3rd day, and 5% Cell Boost 7a and 0.5% Cell Boost 7b were supplemented on the 5th, 7th, 9th, 11th, and 13th culture days. During this period, the glucose concentration was controlled at 2-8g / L. The expression level was measured by Octect after 14 days. The shake flask feeding expression results are shown in Table 13.

[0348] Table 13. Statistical results of shake flask feeding expression

[0349] Molecular number Cell pool number Shake flask expression (mg / L) Molecular number Cell pool number Shake flask expression (mg / L) Molecule No. 1 1-9 3915.6 Molecule No. 2 2-5 3496.4 Molecule No. 3 3-4 4077.4 Molecule No. 4 4-6 2321.1

[0350] (5) The supernatant of the shake flask feed was purified by Protein A chromatography column and analyzed by SDS-PAGE (the results are shown in Figure 5). Figure 23The SEC purity is shown in Table 14. The SEC purity of molecule No. 1 is 83.47%, which is significantly better than other molecular designs. The ratio of each chain was analyzed by reducing CE-SDS. As shown in Table 15, the ratio of the two light chains and the two heavy chains of molecule No. 1 is closer to 1:1, which is more in line with the expected theoretical value. Comprehensive analysis shows that the purity and molecular expression of molecule No. 1 are the best. Therefore, cell pools 1-9 of molecule No. 1 were selected for monocloning, and cell pools 2-5, 3-4, and 4-6 were used as controls for monocloning.

[0351] Table 14. SEC purity analysis results of cell pool

[0352] Molecular number Cell pool number SEC purity (%) Molecular number Cell pool number SEC purity (%) Molecule No. 1 1-9 83.47 Molecule No. 2 2-5 53.12 Molecule No. 3 3-4 70.12 Molecule No. 4 4-6 51.38

[0353] Table 15. CE-SDS purity analysis results of cell pool reduction

[0354] Molecular number Cell pool number L1(％) L2(％) H1(％) H2(％) Molecule No. 1 1-9 15.1 14 30.9 40 Molecule No. 2 2-5 0 23.9 22.8 53.3 Molecule No. 3 3-4 21.7 7.7 31.8 38.8 Molecule No. 4 4-6 15.6 12 27.2 45.1

[0355] (6) The cell pools 1-9, 2-5, 3-4, and 4-6 were subjected to limiting dilution and plated on a 96-well plate at a density of 0.8 Cell / well. The cell growth status was photographed with VIPS at the first hour, first day, second day, third day, fifth day, seventh day, and fourteenth day, and the non-fluorescent monoclonal clones were counted. The non-fluorescent monoclonal wells were expanded, and clones with low growth rates were eliminated during the expansion process in the manner of 96-well plate → 24-well plate → 6-well plate → shake flask. No monoclonal clones were formed in 4-6, and the cells did not grow and reproduce. Therefore, the cell pools 4-6 could not be screened by six-well batch culture. Among them, six-well batch culture was performed after the 6-well expansion. The 6-well batch culture was performed by inoculating 5×10 5 / ml, 2ml system, 37°C 5% carbon dioxide 120 rpm conditions for 7 days, 6-well batch culture of each cell pool, the top 5 single clones with the highest expression are shown in Table 16.

[0356] Table 16. Statistical results of expression levels of monoclonal six-well batch culture

[0357]

[0358]

[0359] (7) Select the top three cell lines with the highest expression in each cell pool and culture them in six-well batches. After two subcultures, perform shake flask feeding for 14 days. The seeding density is 5×10 5 / ml, inoculate 30ml of the system, count on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13 and culture day 14, and supplement 3% Cell Boost 7a and 0.3% Cell Boost 7b on the 3rd day, 5% Cell Boost 7a and 0.5% Cell Boost 7b on the 5th day, culture day 7, culture day 9, culture day 11 and culture day 13, during which the glucose concentration was controlled at 2-8g / L. After 14 days, use The expression level was determined, and the results of monoclonal shake flask feeding expression are shown in Table 17.

[0360] Table 17. Statistical results of monoclonal shake flask feeding expression

[0361]

[0362] The supernatant from the shake flask feed was captured using Protein A media and then analyzed by SEC. The results are shown in Table 18. The quality attributes of the monoclonal clones produced by each cell pool were relatively uniform, with the cell pool containing molecule No. 1 showing the highest SEC purity. Calculation of the target molecule yield showed that both 1-9-1 and 1-9-2 performed well, with a theoretical yield of approximately 4.7 g. The calculated results are shown in Table 19.

[0363] Table 18. Monoclonal SEC analysis results

[0364]

[0365] Table 19. Statistics of relative yields of monoclonal target products

[0366]

[0367]

[0368] The experiments described in Example 3 screened a cell line expressing a bispecific antibody that met the expectations. The experimental process is controllable, the workload is significantly reduced compared to traditional methods, and the antibody mismatch rate is lower. In the ANG-2 / VEGF bivalent antibodies formed after expression of the four molecules, the constant region CL of the ANG-2 antibody light chain sequence is replaced with the constant region CH1 of the ANG-2 antibody heavy chain sequence. Therefore, expression of the ANG-2 antibody light chain sequence is more challenging than that of the VEGF antibody light chain sequence, and expression of the ANG-2 antibody heavy chain sequence is more challenging than that of the VEGF antibody heavy chain sequence. Furthermore, since artificial sequence swapping is more likely to result in mismatches, placing the ANG-2 expression cassette at the front of the plasmid design minimizes the impact of the preceding expression cassette. Since heavy chain expression in conventional antibody expression depends on the proper folding of the light chain, and the light chain assists in heavy chain secretion, placing the light chain expression cassette at the front of the plasmid design can produce more light chain antibody fragments, thereby further increasing the yield of the target molecule, explaining the higher expression level and lower mismatch rate of the first molecule design.

[0369] Example 4 Expression of Four-chain Monoclonal Antibodies

[0370] In this example, the monoclonal antibody target constructed is CTLA-4, the heavy chain gene sequence is shown in SEQ ID No. 18, and the light chain gene sequence is shown in SEQ ID No. 19.

[0371] (1) Exogenous nucleic acid construction

[0372] Figure 3 As shown, the pDonor0.0-H plasmid is used to construct the expression cassette vector of the heavy chain HC, and the target gene sequence can be inserted in the middle of the Xma I and / or Hind III restriction sites, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication origin site, which is used for plasmid replication and amplification in prokaryotic cells; the promoter segment is used to initiate the expression of the target gene; the poly (A) signal is used to terminate the transcription of the target gene; the restriction sites such as NotI and Age I are used to transfer the target gene to the target gene. Figure 5 The pLangding0.0 plasmid shown was digested and ligated to insert the complete expression cassette.

[0373] like Figure 4As shown, pDonor0.0-L is used to construct a vector for light chain expression cassette, wherein KanR provides a kanamycin resistance gene for screening in plasmid construction; the Ori segment is the plasmid replication initiation site, which is used for transcriptional amplification of the plasmid in prokaryotic cells; the promoter segment is used to initiate the expression of the target gene; the poly (A) signal is used to terminate the transcription of the target gene; the Xma I and / or Hind III restriction sites are used to insert the target gene sequence; other restriction sites such as Not I are used to insert the target gene sequence; Figure 5 The pLangding0.0 plasmid shown was digested and ligated to insert the complete expression cassette.

[0374] like Figure 5 As shown, pLangding0.0 is used to accept the expression cassettes of pDonor0.0-H and / or pDonor0.0-L, wherein the Ori segment is the plasmid replication origin site for transcriptional amplification of the plasmid in prokaryotic cells; AmpR provides an ampicillin resistance gene for screening in plasmid construction, Bxb1-attB GA and attB are used for recombination with the GBB003 mother cell genome, promoter is used to drive the screening of eukaryotic resistance genes in the products of the mother cell genome, and restriction sites such as Not I and BamH I are used for restriction enzyme substitution in the pDonor0.0 vector.

[0375] In this example, the master plasmids pDonor0.0-H and pDonor0.0-L were constructed into the pLanding0.0 plasmid by enzyme digestion and ligation. The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID No. 9), and light chain gene sequence were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-L plasmid by gene synthesis to form the pDonor0.0-CTLA4-LC plasmid. Figure 24 The kozak sequence, signal peptide sequence (SEQ ID No. 10) and heavy chain gene sequence were inserted into the Hind III and Xma I restriction sites of the pDonor0.0-H plasmid by gene synthesis to form the pDonor0.0-CTLA4-HC plasmid. Figure 25 shown.

[0376] In this example, two molecules were constructed according to the number of exogenous gene expression cassettes in pLangding0.0:

[0377] (1) pLangding0.0 contains two expression cassettes, which are arranged in sequence as a light chain gene expression cassette and a heavy chain gene expression cassette (LH). The construction method is as follows:

[0378] After double digestion of the pDonor0.0-CTLA4-LC and pLanding0.0 plasmids with Not I / Age I, the LC expression cassette fragment of pDonor0.0-CTLA4-LC and the linearized pLanding0.0 fragment were recovered, and then ligated with T4 DNA ligase to transform DH5α chemically competent cells. After colonies grew on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-CTLA4-LC.

[0379] The pDonor0.0-CTLA4-HC and pLanding0.0-CTLA4-LC plasmids were double-digested with Bsiw I / Mlu I, and the HC expression cassette fragment of pDonor0.0-CTLA4-HC and the linearized pLanding0.0-CTLA4-LC fragment were recovered. Then, they were ligated with T4 DNA ligase and transformed into Stbl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-CTLA4-LH. The plasmid structure is shown in FIG. Figure 26 shown.

[0380] (2) pLangding0.0 contains four expression cassettes, which are arranged in sequence as light chain gene expression cassette, heavy chain gene expression cassette, light chain gene expression cassette and heavy chain gene expression cassette (LHLH). The construction method is as follows:

[0381] After double digestion of pDonor0.0-CTLA4-LC and the pLanding0.0-CTLA4-LH plasmid prepared in step (1) with Nhe I / Xho I, the CTLA4-LC expression cassette fragment of pDonor0.0-CTLA4-LC and the linearized pLanding0.0-CTLA4-LH fragment were recovered, and then ligated with T4 DNA ligase to transform Stbl3 chemically competent cells. After colonies were grown on LB ampicillin-resistant plates, the cells were washed and collected with LB medium, and the plasmid was extracted to form pLanding0.0-CTLA4-LHL.

[0382] The pDonor0.0-CTLA4-HC and pLanding0.0-CTLA4-LHL plasmids were double-digested with EcoR I / Kpn I, and the CTLA4-HC expression cassette fragment of pDonor0.0-CTLA4-HC and the linearized pLanding0.0-CTLA4-LHL fragment were recovered. Then, they were ligated with T4 DNA ligase to transform Stbl3 chemically competent cells. After the colonies grew on LB ampicillin-resistant plates, they were washed and collected with LB medium, and the plasmids were extracted to form the following: Figure 27 pLanding0.0-CTLA4-LHLH is shown.

[0383] (2) Verification of expression effect

[0384] (1) The stable high fluorescence cell GBB003 prepared in Example 1 was selected for transfection and integration verification. The recombinant plasmids of the CTLA4 antibody expression genes in the two arrangements contained in the above step (1) were amplified and linearized, and then co-transfected with Bxb-1 integrase into the stable high fluorescence cell GBB003. The Bxb-1 integrase plasmid map is shown in FIG. Figure 13 shown.

[0385] (2) One day after transfection, a minipool was constructed from the cell pool and pressure screening was performed using pressurized medium 2.

[0386] (3) After 14 days, the non-fluorescent cells in the minipool were expanded and cultured, and then monocloned. After the monoclonal clones were expanded, six-well batch culture was performed. The data of the six-well batch culture of the monoclonal clones are shown in Table 20.

[0387] Table 20. Statistical results of expression levels of monoclonal six-well batch culture

[0388]

[0389]

[0390] (4) Select the top two cell lines in the six-well batch culture for each molecular monoclonal clone, subculture them twice, and then feed them in shake flasks for 14 days. The inoculation density is 5×10 5 / ml, inoculate 30ml of the system, count on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13 and culture day 14, and supplement 3% Cell Boost 7a and 0.3% Cell Boost 7b on the 3rd day, 5% Cell Boost 7a and 0.5% CellBoost 7b on the 5th day, culture day 7, culture day 9, culture day 11, culture day 13, culture day 15 and culture day 17, during which the glucose concentration was controlled at 2-8g / L. After 18 days, use The expression level was determined, and the results of monoclonal shake flask feeding expression are shown in Table 21.

[0391] Table 21. Statistical results of monoclonal shake flask feeding expression

[0392]

[0393] Taking pLanding0.0-CTLA4-LHLH-3G6 with the highest expression level as an example, its expression curve is as follows Figure 28 As shown in Figure 2, the highest expression level reached 25g. Figure 29 As shown, the highest cell density was 1.16×10 7 Cells / ml, so the yield of the cell line is very high. The cell viability curve during the culture process is as follows Figure 30 As shown in Figure 2, the cell viability after 18 days of culture was maintained at 90%, which is suitable for the long production cycle of the scale-up process. Figure 31 As shown, lactate is maintained at a low level, which is a lactate-consuming cell and meets the needs of industrial production. The results show that the highest expression level of pLanding0.0-CTLA4-LHLH is 1.7 times the highest expression level of pLanding0.0-CTLA4-LH, and the cells are in good condition during the culture process, and the lactate concentration is controlled at a relatively low level. Since the transcription and translation levels of the cell line are not unlimited, the probability of a double copy being twice or more than a single copy is extremely low. In this embodiment, the double copy is 1.7 times the single copy, which is an expected level, and has been significantly improved compared to the single copy, and has reached a higher level compared to the industry average. Therefore, it is shown that the use of a plasmid design with four expression cassettes to express symmetrical monoclonal antibodies can significantly increase the yield of symmetrical monoclonal antibodies and is suitable for subsequent process scale-up production.

[0394] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a recombinant cell, characterized in that: The target protein expressed by the recombinant cell contains one, two, three or four polypeptide chains; The preparation method includes site-specific integration of an exogenous polynucleotide encoding a target protein into the genetic material of a starting cell, wherein each exogenous polynucleotide used for the site-specific integration contains genes encoding all polypeptide chains of the target protein; and each exogenous polynucleotide contains at least four expression cassettes, and each polypeptide chain is encoded by at least one expression cassette.

2. The preparation method according to claim 1, characterized in that The exogenous polynucleotide further contains a polynucleotide fragment encoding a regulatory element; Optionally, the regulatory element comprises one or more of a ribosome binding site, a Kozak sequence, a promoter, an enhancer, a signal peptide and a polynucleotide; Optionally, the signal peptide amino acid sequence is shown as SEQ ID No.9, SEQ ID No.10 or SEQ ID No.11; Optionally, the polynucleotide gene comprises polyA.

3. The preparation method according to claim 1, characterized in that Each expression cassette contains a gene encoding a polypeptide chain; Optionally, each expression cassette independently contains one or more of a ribosome binding site, a Kozak sequence, a promoter, an enhancer, a signal peptide and a polynucleotide; Optionally, each expression cassette independently contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and a polynucleotide gene in sequence.

4. The preparation method according to claim 1, characterized in that The exogenous polynucleotide is introduced into the genetic material of the starting cell through the mediation of the vector; Optionally, the vector comprises a plasmid.

5. The preparation method according to claim 1, characterized in that The recombinant cells include prokaryotic cells or eukaryotic cells; Optionally, the eukaryotic cells include mammalian cells; Optionally, the recombinant cells include CHO cells or HEK293 cells; Optionally, the CHO cells are CHO-K1 cells, CHO-S cells or CHO-GS cells.

6. The preparation method according to claim 1, characterized in that Including using a gene editing system or an integrase system to perform the site-specific integration; Optionally, the enzymes in the integrase system include Cre, Dre, Vika, Bxb1, RDF, FLP, TP901-1、A118、 MR11, TG1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebcuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Hinder, ICleared, Sheen, Mundrea, BxZ2 or or mutants of any of them; Optionally, the integration sites in the integrase system include one or more of a LoxP site, a LoxPL3 site, a LoxP 2L site, a LoxFas site, a Lox511 site, a Lox2272 site, a Lox2372 site, a Lox5171 site, a Loxm2 site, a Lox71 site, a Lox66 site, a FRT site, a Bxb1 attP site, and a Bxb1 attB site.

7. The preparation method according to claim 6, characterized in that The starting cell is a CHO-K1 cell, and the site-directed integration comprises integrating the exogenous polynucleotide into a highly expressed fragment of the starting cell, wherein the highly expressed fragment comprises the nucleotide sequence shown in SEQ ID No. 1; Optionally, the fixed site of the integration site is any site within the range of bases 11 to 430 of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 21st to 414th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 38th to 402nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 53rd to 389th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 73rd to 373rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 91st to 360th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 108th to 342nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 126th to 326th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 143rd to 310th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 160th to 295th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 178th to 274th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 194th to 263rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 209th to 253rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 221st to 242nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is any site within the 231st to 240th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site is the position shown in the annotation information NW_003616785.1:83044 on CHO-K1 cells.

8. The preparation method according to claim 7, characterized in that The starting cell is a CHO-K1 cell, the genome of which contains a marker gene, and the marker gene is integrated into the highly expressed fragment according to claim 7; Optionally, the marker gene includes a fluorescent marker gene.

9. The preparation method according to any one of claims 1 to 8, characterized in that The target protein includes a three-chain antibody, which contains three polypeptide chains, including a first heavy chain, a first light chain and a recombinant heavy chain; the first heavy chain and the first light chain constitute a first half antibody, and the recombinant heavy chain contains a heavy chain Fc region and a single-chain antibody scFv; Optionally, the three-chain antibody is a bispecific antibody, wherein the first half antibody and the recombinant heavy chain target different antigens.

10. The preparation method according to claim 9, characterized in that The first half-antibody targets EpCAM, CD38 or HER2; Optionally, the first half antibody targets EpCAM, the gene encoding the first light chain is shown as SEQ ID No. 2, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 3; Optionally, the first half antibody targets CD38, the gene encoding the first light chain is shown as SEQ ID No. 4, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 5; Optionally, the first half-antibody targets HER2, the gene encoding the first light chain is shown as SEQ ID No. 6, and / or the gene encoding the first heavy chain is shown as SEQ ID No. 7; Optionally, the recombinant heavy chain targets CD3; Optionally, the gene encoding the recombinant heavy chain is shown as SEQ ID No.

8.

11. The preparation method according to claim 9, characterized in that The exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence; The first expression cassette contains the gene encoding the recombinant heavy chain; The second expression cassette and the fourth expression cassette contain the gene encoding the first light chain; The third expression cassette contains the gene encoding the first heavy chain; Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the recombinant heavy chain, and a polynucleotide gene in sequence; Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the first light chain, and a polynucleotide gene in sequence; Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the first heavy chain, and a polynucleotide gene in sequence; Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the first light chain and a polynucleotide gene in sequence.

12. The preparation method according to claim 11, characterized in that Each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence, and the exogenous polynucleotide is selected from any one of ( ) to ( ): ( ) The first half-antibody targets EpCAM; the gene encoding the first light chain is shown in SEQ ID No. 2, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 3, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11; ( ) The first half antibody targets CD38; the gene encoding the first light chain is shown in SEQ ID No. 4, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 5, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11; ( ) The first half-antibody targets HER2; the gene encoding the first light chain is shown in SEQ ID No. 6, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 7, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown as SEQ ID No.8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID No.

11.

13. The preparation method according to claim 9, characterized in that The exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence; The first expression cassette contains the gene encoding the first light chain; The second expression cassette contains the gene encoding the first heavy chain; The third expression cassette and the fourth expression cassette contain the gene encoding the recombinant heavy chain. Optionally, the first expression cassette contains a promoter, a kozak sequence, a signal peptide encoding gene, a gene encoding the first light chain, and a polynucleotide gene in sequence; Optionally, the second expression cassette contains a promoter, a kozak sequence, a signal peptide encoding gene, a gene encoding the first heavy chain, and a polynucleotide gene in sequence; Optionally, the third expression cassette contains a promoter, a kozak sequence, a signal peptide encoding gene, a gene encoding the recombinant heavy chain, and a polynucleotide gene in sequence; Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the recombinant heavy chain encoding gene and a polynucleotide gene in sequence.

14. The preparation method according to claim 13, characterized in that Each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence, and the exogenous polynucleotide is selected from any one of ( ) to ( ): ( ) The first half-antibody targets EpCAM; the gene encoding the first light chain is shown in SEQ ID No. 2, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 3, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11; ( ) The first half antibody targets CD38; the gene encoding the first light chain is shown in SEQ ID No. 4, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 5, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown in SEQ ID No. 8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown in SEQ ID No. 11; ( ) The first half-antibody targets HER2; the gene encoding the first light chain is shown in SEQ ID No. 6, and the signal peptide amino acid sequence of the first light chain is shown in SEQ ID No. 9; the gene encoding the first heavy chain is shown in SEQ ID No. 7, and the signal peptide amino acid sequence of the first heavy chain is shown in SEQ ID No. 10; The recombinant heavy chain targets CD3, the encoding gene of the recombinant heavy chain is shown as SEQ ID No.8, and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID No.

11.

15. The preparation method according to any one of claims 1 to 8, characterized in that: The target protein contains four polypeptide chains, including a second light chain and a second heavy chain, wherein the second light chain and the second heavy chain constitute a second half antibody; the target protein also includes a third light chain and a third heavy chain, wherein the third light chain and the third heavy chain constitute a third half antibody; Optionally, the second half-antibody and the third half-antibody target different antigens.

16. The preparation method according to claim 15, characterized in that The partial fragment of the second light chain and the partial fragment of the second heavy chain are replaced after expression; Optionally, the constant region CL of the second light chain and the constant region CH1 of the second heavy chain are replaced after expression.

17. The preparation method according to claim 16, characterized in that The second half-antibody targets ANG-2; Optionally, the second half-antibody targets ANG-2, the gene encoding the second light chain is shown as SEQ ID No. 12, and / or the gene encoding the second heavy chain is shown as SEQ ID No.

13.

18. The preparation method according to claim 15, characterized in that The third half antibody targets VEGF; Optionally, the third half antibody targets VEGF, the gene encoding the third light chain is shown as SEQ ID No.14, and / or the gene encoding the third heavy chain is shown as SEQ ID No.

15.

19. The preparation method according to claim 15, characterized in that The exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence; The first expression cassette contains the gene encoding the second light chain; The second expression cassette contains the gene encoding the third light chain; The third expression cassette contains the gene encoding the second heavy chain; The fourth expression cassette contains the gene encoding the third heavy chain; Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a second light chain encoding gene and a polynucleotide gene in sequence; Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the third light chain, and a polynucleotide gene in sequence; Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a second heavy chain encoding gene and a polynucleotide gene in sequence; Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the third heavy chain and a polynucleotide gene in sequence.

20. The preparation method according to claim 19, characterized in that Each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence; The second half-antibody targets ANG-2, the gene encoding the second light chain is shown in SEQ ID No. 12, the amino acid sequence of the signal peptide of the second light chain is shown in SEQ ID No. 9, the gene encoding the second heavy chain is shown in SEQ ID No. 13, and the amino acid sequence of the signal peptide of the second heavy chain is shown in SEQ ID No. 10; The third half-antibody targets ANG-2, the gene encoding the third light chain is shown in SEQ ID No. 14, the signal peptide amino acid sequence of the third light chain is shown in SEQ ID No. 9, the gene encoding the third heavy chain is shown in SEQ ID No. 15, and the signal peptide amino acid sequence of the third heavy chain is shown in SEQ ID No.

10.

21. The preparation method according to any one of claims 1 to 8, characterized in that: The target protein contains two polypeptide chains, including a fourth light chain and a fourth heavy chain. The fourth light chain and the fourth heavy chain constitute a fourth half antibody. The target protein contains two fourth half antibodies.

22. The preparation method according to claim 21, characterized in that The fourth half-antibody targets CTLA-4; Optionally, the fourth half antibody targets CTLA-4, the gene encoding the fourth light chain is shown as SEQ ID No.19, and the gene encoding the fourth heavy chain is shown as SEQ ID No.

18.

23. The preparation method according to claim 22, characterized in that The exogenous polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence; The first expression cassette and the third expression cassette contain the gene encoding the fourth light chain; The second expression cassette and the fourth expression cassette contain the gene encoding the fourth heavy chain; Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the fourth light chain, and a polynucleotide gene in sequence; Optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the fourth heavy chain, and a polynucleotide gene in sequence; Optionally, the third expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the fourth light chain, and a polynucleotide gene in sequence; Optionally, the fourth expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a gene encoding the fourth heavy chain and a polynucleotide gene in sequence.

24. The preparation method according to claim 23, characterized in that Each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a polypeptide chain encoding gene and Poly A in sequence; The fourth half antibody targets CTLA-4, the encoding gene of each of the fourth light chains is shown as SEQ ID No.19, the signal peptide amino acid sequence of each of the fourth light chains is shown as SEQ ID No.9, the encoding gene of each of the fourth heavy chains is shown as SEQ ID No.18, and the signal peptide amino acid sequence of each of the fourth heavy chains is shown as SEQ ID No.

10.

25. The recombinant cell produced by the production method according to any one of claims 1 to 24.

26. The preparation method according to any one of claims 1 to 24, or use of the recombinant cell according to claim 25 in preparing a protein.

27. A method for preparing a protein, characterized in that: Comprising culturing the recombinant cell of claim 25.

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