Polynucleotide for coding three-chain antibody, vector, recombinant cell as well as preparation method and application of polynucleotide

Abstract

The invention provides polynucleotide for coding a three-chain antibody, a vector, a recombinant cell as well as a preparation method and application of the polynucleotide, and relates to the technical field of biology. The three-chain antibody comprises a heavy chain, a light chain and a recombinant heavy chain; the heavy chain and the light chain form a half antibody, and the recombinant heavy chain contains a heavy chain Fc region and a single-chain antibody scFv; the polynucleotide for coding the bispecific antibody contains a coding gene of a light chain, a coding gene of a heavy chain, a coding gene of a recombinant heavy chain and a coding gene of the recombinant heavy chain which are arranged in sequence, so that the problems of low expression quantity and high mismatch rate of a host cell for expressing a three-chain antibody are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polynucleotide encoding a three-chain antibody, a vector, a recombinant cell, and a preparation method and application 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. The hope is that two specific targets can produce a 1+1>2 effect, achieving exceptional efficacy in disease treatment. 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 result in fewer side effects and fewer injections.

[0003] The two most common bispecific antibodies on the market are single-chain variable fragment (scFv)-based antibodies (without Fc fragment) and full-length IgG-like three-chain antibodies. Unlike traditional structured antibodies, the huge production challenges of three-chain antibodies in terms of expression quantity, quality and stability have hindered their wider clinical application and acceptance.

[0004] 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 are then tediously screened 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. This is especially true for three-chain antibodies, which often have multiple expression chains. The uncertainty of random integration can easily lead to a high mismatch rate, affecting the target gene expression yield and increasing costs. Long culture times, low product yields, and phenotypic heterogeneity of transgenic cell clones are bottlenecks in the production of three-chain antibodies. Therefore, improving the low expression levels and high mismatch rates in the production of three-chain antibodies is an urgent issue that needs to be addressed.

[0005] In view of this, this application is hereby filed. Summary of the Invention

[0006] The object of the present invention is to provide a polynucleotide encoding a three-chain antibody to increase the expression level of the three-chain antibody in host cells and reduce the mismatch rate.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, a polynucleotide encoding a three-chain antibody is provided, wherein the three-chain antibody comprises a heavy chain, a light chain, and a recombinant heavy chain; the heavy chain and the light chain constitute a half antibody; the recombinant heavy chain comprises a heavy chain Fc region and a single-chain antibody scFv;

[0009] The polynucleotide encoding the bispecific antibody contains the gene encoding the light chain, the gene encoding the heavy chain, the gene encoding the recombinant heavy chain, and the gene encoding the recombinant heavy chain, which are arranged in sequence.

[0010] In a second aspect, a vector is also provided, which carries the polynucleotide encoding the three-chain antibody of the first aspect.

[0011] In a third aspect, a recombinant cell is provided, wherein the polynucleotide encoding the three-chain antibody of the first aspect is integrated into the genetic material of the recombinant cell, and the recombinant cell expresses the three-chain antibody.

[0012] In a fourth aspect, a method for preparing the recombinant cell of the third aspect is also provided, the preparation method comprising introducing the polynucleotide encoding the three-chain antibody of the first aspect or the vector of the second aspect into a starting cell of the recombinant cell.

[0013] In a fifth aspect, the invention also provides the use of the polynucleotide encoding the three-chain antibody of the first aspect, or the vector of the second aspect, or the recombinant cell of the third aspect, or the preparation method of the fourth aspect in preparing a three-chain antibody.

[0014] In a sixth aspect, a method for preparing a three-chain antibody is also provided, the method comprising using cells to express the polynucleotide encoding the three-chain antibody of the first aspect.

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

[0016] The polynucleotide encoding a three-chain antibody provided by the present invention specifically arranges the light chain encoding gene, the heavy chain encoding gene, the recombinant heavy chain encoding gene, and the recombinant heavy chain encoding gene so that it is integrated into the genetic material of the host cell, thereby increasing the expression level of the three-chain antibody by the host cell and reducing the mismatch rate of the host cell-expressed protein to form a three-chain antibody. Recombinant cells based on the polynucleotide encoding the three-chain antibody and their use in the preparation of three-chain antibodies are conducive to the development and preparation of three-chain antibodies, such as the development and preparation of bispecific antibody products. 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 Schematic diagram of three-chain antibody;

[0019] Figure 2 is the plasmid map of the pDonor0.0-H plasmid in Examples 1 to 27;

[0020] Figure 3 is the plasmid map of the pDonor0.0-L plasmid in Examples 1 to 27;

[0021] Figure 4 is the plasmid map of the pLanding0.0 plasmid in Examples 1 to 27;

[0022] Figure 5 Plasmid map of pLanding0.0-A-LHFF constructed in Example 1;

[0023] Figure 6 Plasmid map of pLanding0.0-A-LFH constructed for Comparative Example 1;

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

[0025] Figure 8 Plasmid map of pLanding0.0-A-LFHL constructed for Comparative Example 3;

[0026] Figure 9 Plasmid map of pLanding0.0-A-LFHF constructed for Comparative Example 4;

[0027] Figure 10 Plasmid map of pLanding0.0-A-FLHL constructed for Comparative Example 5;

[0028] Figure 11 Plasmid map of pLanding0.0-A-FHLL constructed for Comparative Example 6;

[0029] Figure 12 This is the PGBB BXB1 integrase plasmid map used in the effect examples;

[0030] Figure 13This is the non-reduced CE-SDS detection image of the C-LHFF-1 cell pool B10 monoclonal in Example 3;

[0031] Figure 14 This is the non-reduced CE-SDS detection image of the C-FLHL-2 cell pool B16 monoclonal in Example 3;

[0032] Figure 15 This is the CE-SDS detection image of the C-LHFF-1 cell pool B10 monoclonal reduction in Example 3;

[0033] Figure 16 This is the CE-SDS detection image of the B16 monoclonal reduction of the C-FLHL-2 cell pool in Example 3;

[0034] Figure 17 Prepare an operational flow chart for GBB003 cells;

[0035] Figure 18 This is the plasmid map of the RMCE recombinant plasmid containing the green fluorescent protein gene. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] As used herein, the term "triabody" refers to an antibody consisting of a half antibody and a recombinant heavy chain, wherein the half antibody consists of a heavy chain and a light chain.

[0039] 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. The term "light chain" as used herein 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 derived in part or in whole from a λ or κ type light chain. As used herein, a "half antibody" is formed by disulfide bonds between the above-mentioned "heavy chain" and "light chain," so that the second half antibody comprises a VL domain, a VH domain, a hinge domain, part or all of a heavy chain constant region (at least one of CH1, CH2, CH3, and CH4), and optionally part or all of a light chain constant region (CL). In an optional embodiment, a half antibody is composed of a heavy chain and a light chain in a complete IgG type antibody molecule.

[0040] 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 linked to each other or connected through a peptide linker sequence.

[0041] The half antibody and the recombinant heavy chain are combined through disulfide bonds to form a three-chain antibody. The structural diagram is shown in Figure 1 shown.

[0042] 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.

[0043] In the present invention, the term "nucleic acid molecule" refers 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 encode a sense strand or an antisense strand. Nucleic acid molecules may be naturally occurring, synthetic, recombinant, or any combination thereof. "Nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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."

[0048] In a first aspect, a polynucleotide encoding a three-chain antibody is provided, wherein the polynucleotide encoding a bispecific antibody comprises a gene encoding the light chain, a gene encoding the heavy chain, a gene encoding the recombinant heavy chain, and a gene encoding the recombinant heavy chain, arranged in sequence. The two recombinant heavy chain encoding genes encode polypeptides having the same amino acid sequence, and the polynucleotide sequences of the two recombinant heavy chain encoding genes may be the same or different.

[0049] In an optional embodiment, the polynucleotide further contains a polynucleotide fragment encoding a regulatory element.

[0050] 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.

[0051] In an optional embodiment, one or several coding genes are regulated by the same regulatory element, for example, a promoter regulates one or more coding genes downstream thereof, and each coding gene is optionally connected by a cleavable polypeptide coding sequence.

[0052] In an alternative embodiment, the expression of each coding gene is independently regulated.

[0053] In an optional embodiment, the amino acid sequence of the signal peptide is selected from the sequence shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11.

[0054] In an optional embodiment, the signal peptide amino acid sequence of the light chain is shown as SEQ ID NO.9; the signal peptide amino acid sequence of the heavy chain is shown as SEQ ID NO.10; and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID NO.11.

[0055] In an alternative embodiment, the polynucleotide comprises polyA.

[0056] The half antibody and the recombinant heavy chain in the triabody can target the same or different antigens.

[0057] In an optional embodiment, the half-antibody targets EpCAM, CD38 or HER2;

[0058] In an optional embodiment, the half-antibody targets EpCAM, and the gene encoding the light chain of the half-antibody targeting EpCAM is exemplarily shown in SEQ ID NO.2; the gene encoding the heavy chain of the half-antibody targeting EpCAM is exemplarily shown in SEQ ID NO.3.

[0059] In an optional embodiment, the half antibody targets CD38, and the gene encoding the light chain of the half antibody targeting CD38 is exemplarily shown in SEQ ID NO.4; the gene encoding the heavy chain of the half antibody targeting CD38 is exemplarily shown in SEQ ID NO.5.

[0060] In an optional embodiment, the half antibody targets HER2, and the gene encoding the light chain of the half antibody targeting HER2 is exemplified as SEQ ID NO.6; the gene encoding the heavy chain of the half antibody targeting HER2 is exemplified as SEQ ID NO.7.

[0061] In an optional embodiment, the recombinant heavy chain targets CD3, and its exemplary encoding gene is shown in SEQ ID NO.8.

[0062] In an optional embodiment, the three-chain antibody is a bispecific antibody, and the half antibody and the recombinant heavy chain target different antigens.

[0063] In an optional embodiment, the half antibody targets EpCAM and the recombinant heavy chain targets CD3; or the half antibody targets CD38 and the recombinant heavy chain targets CD3; or the half antibody targets HER2 and the recombinant heavy chain targets CD3.

[0064] In an optional embodiment, the polynucleotide contains a first expression cassette, a second expression cassette, a third expression cassette and a fourth expression cassette arranged in sequence.

[0065] The first expression cassette contains the light chain encoding gene. Optionally, the first expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the light chain encoding gene and a polynucleotide gene in sequence.

[0066] The second expression cassette contains the heavy chain encoding gene; optionally, the second expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, the heavy chain encoding gene and a polynucleotide gene in sequence.

[0067] 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.

[0068] The fourth 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.

[0069] In an optional embodiment, the polynucleotide encoding the three-chain antibody is selected from any one of (I) to (III):

[0070] (I) The half antibody of the three-chain antibody targets EpCAM, and the recombinant heavy chain targets CD3. The polynucleotide comprises a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette arranged in sequence, and each expression cassette comprises a promoter, a signal peptide encoding gene, a coding gene, and a polynucleotide gene in sequence; wherein the light chain encoding gene is shown in SEQ ID NO.2, and the light chain signal peptide amino acid sequence is shown in SEQ ID NO.9; the heavy chain encoding gene is shown in SEQ ID NO.3, and the heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.10; the recombinant heavy chain encoding gene is shown in SEQ ID NO.8, and the recombinant heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.11; the first expression cassette expresses the light chain encoding gene shown in SEQ ID NO.2, the second expression cassette expresses the heavy chain encoding gene shown in SEQ ID NO.3, the third expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8, and the fourth expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8.

[0071] (II) The half antibody of the three-chain antibody targets CD38, and the recombinant heavy chain targets CD3. The polynucleotide comprises a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette arranged in sequence, and each expression cassette comprises a promoter, a signal peptide encoding gene, a coding gene, and a polynucleotide gene in sequence; wherein the light chain encoding gene is shown in SEQ ID NO.4, and the light chain signal peptide amino acid sequence is shown in SEQ ID NO.9; the heavy chain encoding gene is shown in SEQ ID NO.5, and the heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.10; the recombinant heavy chain encoding gene is shown in SEQ ID NO.8, and the recombinant heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.11; the first expression cassette expresses the light chain encoding gene shown in SEQ ID NO.4, the second expression cassette expresses the heavy chain encoding gene shown in SEQ ID NO.5, the third expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8, and the fourth expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8.

[0072] (III) The half antibody of the three-chain antibody targets HER2, and the recombinant heavy chain targets CD3. The polynucleotide comprises a first expression cassette, a second expression cassette, a third expression cassette, and a fourth expression cassette arranged in sequence, and each expression cassette comprises a promoter, a signal peptide encoding gene, a coding gene, and a polynucleotide gene in sequence; wherein the light chain encoding gene is shown in SEQ ID NO.6, and the light chain signal peptide amino acid sequence is shown in SEQ ID NO.9; the heavy chain encoding gene is shown in SEQ ID NO.7, and the heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.10; the recombinant heavy chain encoding gene is shown in SEQ ID NO.8, and the recombinant heavy chain signal peptide amino acid sequence is shown in SEQ ID NO.11; the first expression cassette expresses the light chain encoding gene shown in SEQ ID NO.6, the second expression cassette expresses the heavy chain encoding gene shown in SEQ ID NO.7, the third expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8, and the fourth expression cassette expresses the recombinant heavy chain encoding gene shown in SEQ ID NO.8.

[0073] In a second aspect, a vector is also provided, which carries the polynucleotide encoding the three-chain antibody of the first aspect.

[0074] In an optional embodiment, the vector is a plasmid.

[0075] In an optional embodiment, the plasmid further contains an integration site for integrase.

[0076] In a third aspect, a recombinant cell is provided, wherein the polynucleotide encoding the three-chain antibody of the first aspect is integrated into the genetic material of the recombinant cell, and the recombinant cell expresses the three-chain antibody. The genetic material includes endogenous or exogenous genetic material of the cell, such as the cell genome; exogenous genetic material, such as an expression vector stably expressed in the cell.

[0077] In an optional embodiment, the recombinant cell is a prokaryotic cell or a eukaryotic cell.

[0078] In an alternative embodiment, the eukaryotic cell comprises a mammalian cell.

[0079] In an optional embodiment, the recombinant cell comprises a CHO cell or a HEK293 cell.

[0080] In an optional embodiment, the CHO cell is a CHO-K1 cell, a CHO-S cell or a CHO-GS cell.

[0081] In an optional embodiment, the polynucleotide encoding the triabody is site-specifically integrated into the genome of the recombinant cell. Site-specific integration provides a means to generate more stable clones, thereby reducing the timeline for cell line development. A well-defined integration site can reduce the impact of site effects caused by 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 stability.

[0082] In an optional embodiment, the recombinant cell is a CHO-K1 cell, the integration site of the polynucleotide encoding the three-chain antibody is within the high expression fragment of the starting cell, and the high expression fragment comprises the nucleotide sequence shown in SEQ ID NO.1.

[0083] 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.

[0084] 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.

[0085] In a fourth aspect, a method for preparing the recombinant cell of the third aspect is also provided, the preparation method comprising introducing the polynucleotide encoding the three-chain antibody of the first aspect or the vector of the second aspect into a starting cell of the recombinant cell.

[0086] In an optional embodiment, the polynucleotide encoding the three-chain antibody is site-specifically integrated into the genome of the starting cell.

[0087] In an optional embodiment, the starting cell contains a marker gene, and the marker gene is knocked out after the polynucleotide encoding the three-chain antibody is integrated into the genome of the cell, and the preparation method includes sorting recombinant cells that do not contain the marker gene.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] In an optional embodiment, the marker gene includes a fluorescent marker gene.

[0092] In an optional embodiment, the site-specific integration is performed using a gene editing system or an integrase system.

[0093] 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).

[0094] 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.

[0095] 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.

[0096] In a fifth aspect, the invention also provides the use of the polynucleotide encoding the three-chain antibody of the first aspect, or the vector of the second aspect, or the recombinant cell of the third aspect, or the preparation method of the fourth aspect in preparing a three-chain antibody.

[0097] In a sixth aspect, a method for preparing a triabody is provided, comprising using cells to express a polynucleotide encoding the triabody of the first 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.

[0098] 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.

[0099] The following examples and comparative examples are 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 CD3 target, and a recombinant heavy chain scFv-Fc (encoded by the gene sequence shown in SEQ ID NO.8) that binds to the CD38 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. 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.

[0100] 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:

[0101] SEQ ID NO.9: MDMRVPAQLLGLLLLWLRGARC.

[0102] 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:

[0103] SEQ ID NO. 10: MGWSCIILFLVATATGVHS.

[0104] 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:

[0105] SEQ ID NO. 11: MRAWIFFLLCLAGRALA.

[0106] The target gene vector plasmids used to construct multiple expression cassettes in Examples 1 to 27 are described as follows:

[0107] Figure 2 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.

[0108] like Figure 3 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.

[0109] pLanding0.0 vector Figure 4As 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.

[0110] 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.

[0111] 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.

[0112] Table 1

[0113]

[0114]

[0115] Example 1

[0116] In this example, an expression vector for expressing specific antibody A, named A-LHFF, was constructed to express a three-chain bispecific bivalent antibody comprising a half-antibody targeting EpCAM and a recombinant heavy chain scFv-Fc targeting CD3 (encoded by the gene sequence shown in SEQ ID NO. 8). The half-antibody is composed of 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).

[0117] The expression vector contains a light chain expression cassette [LC], a heavy chain expression cassette [HC], a recombinant heavy chain expression cassette [scFv-Fc] and a second recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0118] 1. Construction of expression cassette:

[0119] Light chain expression cassette [LC] construction:

[0120] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID NO.9) and light chain gene sequence (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.

[0121] Heavy chain expression cassette [HC] construction:

[0122] The kozak sequence, signal peptide sequence (SEQ ID NO.10) and heavy chain gene sequence (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.

[0123] Construction of recombinant heavy chain expression cassette [scFv-Fc]:

[0124] 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.

[0125] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LHFF:

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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 5 pLanding0.0-A-LHFF shown.

[0130] Example 2

[0131] In this example, an expression vector, named B-LHFF, was constructed to express a three-chain bispecific bivalent antibody comprising a CD38-targeting half-antibody and a recombinant heavy chain scFv-Fc targeting CD3 (encoded by the gene sequence set forth in SEQ ID NO. 8). The half-antibody is composed of a heavy chain (encoded by the gene sequence set forth in SEQ ID NO. 5) and a light chain (encoded by the gene sequence set forth in SEQ ID NO. 4).

[0132] The expression vector contains a light chain expression cassette [LC], a heavy chain expression cassette [HC], a recombinant heavy chain expression cassette [scFv-Fc] and a second recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0133] 1. Construction of expression cassette:

[0134] Light chain expression cassette [LC] construction:

[0135] The gene sequence of the kozak sequence, signal peptide sequence (SEQ ID NO.9) and light chain gene sequence (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.

[0136] Heavy chain expression cassette [HC] construction:

[0137] The kozak sequence, signal peptide sequence (SEQ ID NO.10) and heavy chain gene sequence (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.

[0138] Construction of recombinant heavy chain expression cassette [scFv-Fc]:

[0139] 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.

[0140] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LHFF:

[0141] The construction method is the same as that of Example 1, except that the expression cassette replaces the corresponding expression cassette in step 1 of Example 2.

[0142] Example 3

[0143] In this example, an expression vector, named C-LHFF, was constructed to express a three-chain bispecific bivalent antibody comprising a HER2-targeting half-antibody and a recombinant heavy chain scFv-Fc targeting CD3 (encoded by the gene sequence set forth in SEQ ID NO. 8). The half-antibody is composed of a heavy chain (encoded by the gene sequence set forth in SEQ ID NO. 7) and a light chain (encoded by the gene sequence set forth in SEQ ID NO. 6).

[0144] The expression vector contains a light chain expression cassette [LC], a heavy chain expression cassette [HC], a recombinant heavy chain expression cassette [scFv-Fc] and a second recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0145] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LHFF:

[0146] The construction method is the same as that of Example 1, except that the expression cassette replaces the corresponding expression cassette in step 1 of Example 3.

[0147] Comparative Example 1

[0148] In this comparative example, an expression vector was constructed, named A-LFH, for expressing the same three-chain bispecific bivalent antibody as in Example 1.

[0149] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0150] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0151] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LFH:

[0152] 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.

[0153] 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.

[0154] 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.

[0155] Comparative Example 2

[0156] In this comparative example, an expression vector for expressing specific antibody A was constructed, named A-FLH, and was used to express the same three-chain bispecific bivalent antibody as in Example 1.

[0157] The expression vector contains a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0158] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0159] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to FLH:

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Comparative Example 3

[0164] In this comparative example, an expression vector for expressing specific antibody A was constructed, named A-LFHL, and was used to express the same three-chain bispecific bivalent antibody as in Example 1.

[0165] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a light chain expression cassette [LC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0166] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0167] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LFHL:

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] Comparative Example 4

[0173] In this comparative example, an expression vector for expressing specific antibody A was constructed, named A-LFHF, and was used to express the same three-chain bispecific bivalent antibody as in Example 1.

[0174] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0175] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0176] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to LFHL:

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] Comparative Example 5

[0182] In this comparative example, an expression vector for expressing specific antibody A, named A-FLHL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 1. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC], a heavy chain expression cassette [HC], and a light chain expression cassette [LC] arranged in sequence, each expression cassette containing a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0183] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0184] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to FLHL:

[0185] The pDonor0.0-A-LC and the pLanding0.0-A-FLH plasmids prepared in Comparative Example 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, the fragments were ligated with T4 DNA ligase to transform 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 10 pLanding0.0-A-FLHL is shown.

[0186] Comparative Example 6

[0187] In this comparative example, an expression vector for expressing specific antibody A, named A-FHLL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 1. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC], a light chain expression cassette [LC], and a light chain expression cassette [LC] arranged in sequence. Each expression cassette contained a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0188] 1. The steps for constructing the expression cassette are the same as those in Example 1.

[0189] 2. Construction of recombinant expression plasmids with expression cassettes arranged according to FHLL:

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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 11 pLanding0.0-A-FHLL shown.

[0194] Comparative Example 7

[0195] In this comparative example, an expression vector for expressing specific antibody B was constructed, named B-LFH, and was used to express the same three-chain bispecific bivalent antibody as in Example 2.

[0196] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0197] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0198] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFH: The construction method is the same as that of Comparative Example 1, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0199] Comparative Example 8

[0200] In this comparative example, an expression vector for expressing specific antibody B was constructed, named B-FLH, and was used to express the same three-chain bispecific bivalent antibody as in Example 2.

[0201] The expression vector contains a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0202] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0203] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FLH: The construction method is the same as that of Comparative Example 2, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0204] Comparative Example 9

[0205] In this comparative example, an expression vector for expressing specific antibody B was constructed, named B-LFHL, and was used to express the same three-chain bispecific bivalent antibody as in Example 2.

[0206] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a light chain expression cassette [LC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0207] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0208] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFHL: The construction method is the same as that of Comparative Example 3, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0209] Comparative Example 10

[0210] In this comparative example, an expression vector for expressing specific antibody B was constructed, named B-LFHF, and was used to express the same three-chain bispecific bivalent antibody as in Example 2.

[0211] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0212] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0213] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFHL: The construction method is the same as that of Comparative Example 4, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0214] Comparative Example 11

[0215] In this comparative example, an expression vector for expressing specific antibody B, named B-FLHL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 2. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC], a heavy chain expression cassette [HC], and a light chain expression cassette [LC] arranged in sequence, each expression cassette containing a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0216] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0217] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FLHL: The construction method is the same as that of Comparative Example 5, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0218] Comparative Example 12

[0219] In this comparative example, an expression vector for expressing specific antibody B, named B-FHLL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 2. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC], a light chain expression cassette [LC], and a light chain expression cassette [LC] arranged in sequence. Each expression cassette contained a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0220] 1. The steps for constructing the expression cassette are the same as those in Example 2.

[0221] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FHLL: The construction method is the same as that of Comparative Example 6, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 2.

[0222] Comparative Example 13

[0223] In this comparative example, an expression vector for expressing specific antibody C was constructed, named C-LFH, and was used to express the same three-chain bispecific bivalent antibody as in Example 3.

[0224] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0225] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0226] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFH: The construction method is the same as that of Comparative Example 1, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0227] Comparative Example 14

[0228] In this comparative example, an expression vector for expressing specific antibody C was constructed, named C-FLH, and was used to express the same three-chain bispecific bivalent antibody as in Example 3.

[0229] The expression vector contains a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC] and a heavy chain expression cassette [HC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0230] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0231] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FLH: The construction method is the same as that of Comparative Example 2, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0232] Comparative Example 15

[0233] In this comparative example, an expression vector for expressing specific antibody C was constructed, named C-LFHL, and was used to express the same three-chain bispecific bivalent antibody as in Example 3.

[0234] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a light chain expression cassette [LC] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0235] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0236] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFHL: The construction method is the same as that of Comparative Example 3, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0237] Comparative Example 16

[0238] In this comparative example, an expression vector for expressing specific antibody C was constructed and named C-LFHF, which was used to express the same three-chain bispecific bivalent antibody as in Example 3.

[0239] The expression vector contains a light chain expression cassette [LC], a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC] and a recombinant heavy chain expression cassette [scFv-Fc] arranged in sequence, and each expression cassette contains a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene and a polynucleotide gene in sequence.

[0240] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0241] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to LFHL: The construction method is the same as that of Comparative Example 4, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0242] Comparative Example 17

[0243] In this comparative example, an expression vector for expressing specific antibody C, named C-FLHL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 3. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a light chain expression cassette [LC], a heavy chain expression cassette [HC], and a light chain expression cassette [LC] arranged in sequence, each expression cassette containing a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0244] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0245] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FLHL: The construction method is the same as that of Comparative Example 5, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0246] Comparative Example 18

[0247] In this comparative example, an expression vector for expressing specific antibody C, named C-FHLL, was constructed to express the same three-chain bispecific bivalent antibody as in Example 3. The expression vector contained a recombinant heavy chain expression cassette [scFv-Fc], a heavy chain expression cassette [HC], a light chain expression cassette [LC], and a light chain expression cassette [LC] arranged in sequence. Each expression cassette contained a promoter, a Kozak sequence, a signal peptide encoding gene, a target protein encoding gene, and a polynucleotide gene.

[0248] 1. The steps for constructing the expression cassette are the same as those in Example 3.

[0249] 2. Construction of a recombinant expression plasmid with expression cassettes arranged according to FHLL: The construction method is the same as that of Comparative Example 6, except that the expression cassette replaces the corresponding expression cassette in Step 1 of Example 3.

[0250] Effect Examples

[0251] In the bispecific bivalent antibodies formed after expression of each molecule constructed in the above-mentioned embodiments and comparative examples, 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 IgG-like antibodies and scFv-Fc are combined through disulfide bonds to form target proteins. There are multiple mismatching modes for the combination of light chain sequences, heavy chain sequences and scFv-Fc structures. Therefore, different construction methods of target genes in 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 yield of specific antibody A, specific antibody B, and specific antibody C.

[0252] (1) Transient expression verification

[0253] Stable high-fluorescence cells GBB003 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 step (1) above 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 specific antibodies A, B, and C in the supernatant were measured. The expression levels of specific antibodies A, B, and C were the highest when the target genes were arranged in the LFH manner. The transient transfection results are shown in Table 2.

[0254] Table 2. Statistical results of expression levels in transiently transfected cell pools on day 7

[0255]

[0256] (2) Select the stable high fluorescence cell GBB003 from (1) for transfection and integration verification

[0257] 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 12 As shown;

[0258] b) One day after transfection, the cell pool was used to construct a minipool and pressure screening was performed using pressure medium 2;

[0259] 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:

[0260] Table 3 Statistical results of expression levels in six-well batch culture of cell pools

[0261]

[0262]

[0263] 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 counting was performed 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.

[0264] Table 4: Statistical results of shake flask feeding expression

[0265]

[0266] 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.

[0267] Table 5 SDS-PAGE purity statistics of cell pools

[0268]

[0269]

[0270] f) Select FLHL and LHFF arrays for monoclonal

[0271] 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.

[0272] Table 6 Statistical results of expression levels of monoclonal 6-well batch culture

[0273]

[0274] 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.

[0275] Table 7: Statistical results of monoclonal shake flask feeding expression

[0276]

[0277] 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%.

[0278] Table 8: Monoclonal shake flask feed SDS-PAGE purity statistics

[0279]

[0280] 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 13 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 14 Show.

[0281] Table 9 Monoclonal non-reduced CE-SDS purity statistics

[0282]

[0283] The purified samples were analyzed by reduced CE-SDS method. The analysis results are shown in Table 9. 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 15 As shown. Taking the monoclonal B6 of C-FLHL-2 as an example, the CE-SDS detection image is as follows Figure 16 shown.

[0284] Table 9 Monoclonal reduced CE-SDS analysis results

[0285]

[0286] Through the above examples and comparative examples, a bispecific antibody expression cell line consisting of one heavy chain (HC), one light chain (LC), and a recombinant heavy chain (scFv-Fc, single-chain variable fragment (scFv)-based (Fc fragment) antibody) was screened as expected. The preparation process of the above-mentioned three-chain antibody is controllable, the workload is greatly reduced compared to the traditional method, and the antibody mismatch rate of the three-chain antibody combination is lower. Since the expression of the heavy chain in conventional antibody expression depends on the correct folding of the light chain, and the light chain can assist the secretion of the heavy chain, in the bispecific bivalent antibody formed after the expression of the seven polynucleotide molecules (LFH, FLH, LFHL, LFHF, LHFF, FLHL, FHLL), placing the light chain expression cassette at the front end of the plasmid design can produce more light chain antibody fragments, thereby further improving the yield of the target molecule. Therefore, it explains why the light chain is placed in the first expression cassette and the light chain expression level is increased in the molecular design of the present invention, resulting in a higher expression level of the target protein and a lower mismatch rate. The cell line constructed with the target gene arrangement in LHFF showed the highest expression level and the lowest mismatch rate. That is, the specific arrangement of the heavy chain, light chain, and recombinant heavy chain in the polynucleotide provided by the present invention is conducive to host cell expression, resulting in high expression of the three-chain antibody and low mismatch rate.

[0287] The preparation method of the starting cells GBB003 cells used in the above specific embodiments is as follows:

[0288] 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).

[0289] The operation process of this embodiment is as follows Figure 17 As shown, it mainly includes the following steps:

[0290] (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 18 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.

[0291] (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.

[0292] (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.

[0293] (4) Observe the growth of enriched cells at any time and expand them when the coverage rate reaches more than 50%.

[0294] (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 17 The specific steps are shown in a~h.

[0295] 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.

[0296] 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;

[0297] 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.

[0298] 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.

[0299] 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:

[0300]

[0301]

[0302] 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.

[0303] d) The coding and location information of the screened protein high-expressing cell lines are returned to the robot control software;

[0304] 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).

[0305] 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.

[0306] 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.

[0307] 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;

[0308] 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.

[0309] 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.

[0310] Step g: Photographing Monoclonal Cells In this example, a Thermo Fisher Scientific M7000 electron microscope was used to photograph the cells.

[0311] 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.

[0312] 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.

[0313] Wherein, step d and step e can be completed manually. In this embodiment, they are completed automatically by a robotic arm.

[0314] (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.

[0315] (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 high-fluorescence cell line was subjected to a stability study 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 CHO is: NW_003616785.1:83044.

[0316] 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 polynucleotide encoding a three-chain antibody, characterized in that: The three-chain antibody comprises a heavy chain, a light chain and a recombinant heavy chain; the heavy chain and the light chain constitute a half antibody; the recombinant heavy chain contains a heavy chain Fc region and a single-chain antibody scFv; The polynucleotide encoding the three-chain antibody contains the light chain encoding gene, the heavy chain encoding gene, the recombinant heavy chain encoding gene and the recombinant heavy chain encoding gene arranged in sequence.

2. The polynucleotide according to claim 1, wherein Also contains a polynucleotide segment 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 amino acid sequence of the signal peptide is selected from the sequence shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11; Optionally, the signal peptide amino acid sequence of the light chain is shown as SEQ ID NO.9; the signal peptide amino acid sequence of the heavy chain is shown as SEQ ID NO.10; and the signal peptide amino acid sequence of the recombinant heavy chain is shown as SEQ ID NO.11; Optionally, the polynucleotide comprises polyA.

3. The polynucleotide according to claim 1, wherein The half-antibody targets EpCAM, CD38 or HER2; Optionally, the half antibody targets EpCAM, the gene encoding the light chain is shown as SEQ ID NO.2, and / or the gene encoding the heavy chain is shown as SEQ ID NO.3; Optionally, the half antibody targets CD38, the gene encoding the light chain is shown as SEQ ID NO.4, and / or the gene encoding the heavy chain is shown as SEQ ID NO.5; Optionally, the half antibody targets HER2, the gene encoding the light chain is shown as SEQ ID NO.6, and / or the gene encoding the heavy chain is shown as SEQ ID NO.

7.

4. The polynucleotide according to claim 1, wherein The recombinant heavy chain targets CD3; Optionally, the gene encoding the recombinant heavy chain is shown as SEQ ID NO.

8.

5. The polynucleotide according to claim 1, wherein The three-chain antibody is a bispecific antibody, in which the half antibody and the recombinant heavy chain target different antigens.

6. The polynucleotide according to any one of claims 1 to 5, characterized in that Containing 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 light chain; The second expression cassette contains the gene encoding the heavy chain; The third expression cassette and the fourth expression cassette contain the coding gene of the recombinant heavy chain; Optionally, the first expression cassette contains a promoter, a kozak sequence, a signal peptide encoding gene, the 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 heavy chain encoding gene 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.

7. The polynucleotide according to claim 6, characterized in that Select any one of (I) to (III): (I) The half-antibody targets EpCAM; the gene encoding the light chain is shown in SEQ ID NO.2, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.9; the gene encoding the heavy chain is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain signal peptide 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; (II) the half-antibody targets CD38; the gene encoding the light chain is shown in SEQ ID NO.4, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.9; the gene encoding the heavy chain is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain signal peptide 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; (III) the half-antibody targets HER2; the gene encoding the light chain is shown in SEQ ID NO.6, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.9; the gene encoding the heavy chain is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain signal peptide 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.

8. A carrier, characterized in that The vector carries the polynucleotide encoding the three-chain antibody according to any one of claims 1 to 7.

9. The carrier according to claim 8, characterized in that The vector is a plasmid; Optionally, the plasmid also contains an integration site for integrase.

10. A recombinant cell, characterized in that The genetic material of the recombinant cell has the polynucleotide encoding the three-chain antibody according to any one of claims 1 to 7 integrated therein, and the recombinant cell expresses the three-chain antibody.

11. The recombinant cell according to claim 10, characterized in that The recombinant cell is a prokaryotic cell or a eukaryotic cell; 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.

12. The recombinant cell according to claim 10 or 11, characterized in that The polynucleotide encoding the three-chain antibody is site-specifically integrated into the genome of the recombinant cell; Optionally, the recombinant cell is a CHO-K1 cell, and the integration site of the polynucleotide encoding the three-chain antibody is within a highly expressed fragment of the starting cell, and 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 11th to 430th base interval 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.

13. The method for preparing a recombinant cell according to any one of claims 10 to 12, characterized in that: The method comprises introducing the polynucleotide encoding the triabody according to any one of claims 1 to 7 or the vector according to claim 8 or 9 into a starting cell of the recombinant cell.

14. The preparation method according to claim 13, characterized in that The method comprises integrating the polynucleotide encoding the three-chain antibody into the genome of the starting cell; Optionally, the starting cell contains a marker gene, and the marker gene is knocked out after the polynucleotide encoding the three-chain antibody is integrated into the genome of the cell, and the preparation method includes sorting recombinant cells that do not contain the marker gene; Optionally, the starting cell is a CHO-K1 cell, the genome of the CHO-K1 cell contains a marker gene, and the marker gene is integrated into the highly expressed fragment according to claim 12; Optionally, the marker gene includes a fluorescent marker gene.

15. The preparation method according to claim 14, 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, φC31, RDF, FLP, φBT1, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, 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 φRV, or a mutant of any one 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.

16. Use of the polynucleotide encoding a three-chain antibody according to any one of claims 1 to 7, or the vector according to claim 8 or 9, or the recombinant cell according to any one of claims 10 to 12, or the preparation method according to any one of claims 13 to 15 in preparing a three-chain antibody.

17. A method for preparing a three-chain antibody, characterized in that: The method comprises using cells to express the polynucleotide encoding the triabody according to any one of claims 1 to 7.

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