Modvec system: platform for constructing network generation expression vectors

Abstract

The present invention relates to a method for optimizing the expression level of a multi-chain protein, wherein the protein comprises at least two different polypeptide chains. The production of multi-chain proteins brings a huge challenge due to pairing / folding of a new quaternary structure consisting of multiple polypeptide chains, especially when counterheavy and light chains are paired in a multispecific form. The effective and appropriate assembly of multiple polypeptide chain molecules is crucial to that these different chains are expressed in appropriate ratios within the cell. The invention solves the problems.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 317,949, filed on March 8, 2022, the entire contents of which are hereby incorporated by reference.

[0003] Incorporated by Reference

[0004] The XML-formatted Sequence Listing is incorporated by reference in its entirety and can be identified by the following file attributes: File name: A-2671-WO01-SEC_Sequence_Listing.XML; File size: 8,686 bytes; Created on: March 6, 2023. Technical Field

[0005] The present invention relates to methods for preparing standardized expression cassettes. The present invention also relates to methods for recombining such standardized expression cassettes in vivo in host cells. Background Art

[0006] Multispecific antibodies and antibody-like constructs have several attractive features for people developing therapeutic molecules. Multispecific antibodies that target multiple targets simultaneously, such as the clinical potential of bispecific and trispecific antibodies, show a broad prospect for targeting complex diseases. However, due to the pairing / folding of the new quaternary structure consisting of multiple polypeptide chains (especially when pairing antibody heavy and light chains) after transfection into individual cells, there are huge challenges in the generation of those molecules. It is crucial for the effective and appropriate assembly of multiple polypeptide chain molecules that different chains are expressed in appropriate ratios within the cell.

[0007] The present invention uses a modular vector (ModVec) system to solve this problem to quickly construct different expression vectors and vector libraries for recombinant protein production. ModVec enables rapid and high-throughput construction of both simple and complex vector designs, enabling optimization of expression vectors for single molecules, for specific macromolecular forms, and / or for different expression hosts.

[0008] A major challenge in producing multi-chain molecules is the different product qualities caused by unbalanced chain expression ratios, and the ratios are molecule-specific and specific to the cell-host developmental process including the expression vector elements. Currently, there is no method for determining the optimal molecule-specific chain expression ratio with high throughput. The ModVec vector library provides a platform for rapidly determining the optimal expression configuration of a single multi-chain molecule. ModVec can be used in more general applications to assemble complex large DNA molecules for purposes other than expression vectors for antibody therapy. Summary of the invention

[0009] In one aspect, the present invention relates to a method for expressing a multi-chain protein comprising at least two different polypeptide chains, the method comprising

[0010] a. providing a polynucleotide sequence encoding the at least two different polypeptides, wherein the polynucleotide sequence is flanked on both 5' and 3' sides by a type IIS restriction endonuclease cleavage site, followed by its recognition site;

[0011] b. providing two or more sets of element sequences; wherein each set of element sequences together comprises at least one functional expression cassette,

[0012] Each element sequence is flanked on both the 5' and 3' sides by a type IIS restriction endonuclease cleavage site followed by its recognition site,

[0013] wherein these Type IIS restriction endonuclease recognition sites and cleavage sites are selected so that the element sequences of these groups can be assembled into a functional expression cassette;

[0014] c. providing at least one backbone entry vector, wherein the backbone entry vector comprises a plasmid comprising at least one type IIS restriction endonuclease cleavage site followed by its recognition site, and a sequence for maintaining the vector in a bacterial cell;

[0015] d. assembling the polynucleotide sequence encoding the at least two different polypeptides with the two or more sets of element sequences to produce a functional expression cassette in the backbone entry vector using a method based on digestion with a type IIS restriction enzyme and ligation via the cleavage sites and overhangs, thereby producing a connected vector comprising at least two functional expression cassettes capable of expressing the at least two different polypeptide chains;

[0016] e. transfecting mammalian cells with the ligated vector;

[0017] f. comparing the level of the preferred product produced and the level of impurities in each of the at least two polypeptides; and

[0018] g. Identifying the optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying the transfected mammalian cells having the optimal expression ratio of the at least two polypeptide chains.

[0019] In one embodiment, the method further comprises:

[0020] h. Identifying the arrangement of vector elements that provides the optimal expression ratio of the at least two polypeptide chains.

[0021] In one aspect, the invention relates to a vector comprising an arrangement of vector elements providing for an optimal expression ratio of the at least two polypeptide chains.

[0022] In one embodiment, the element sequence comprises at least two promoter sequences and at least two polyadenylic acid (polyA) sequences.

[0023] In one embodiment, the level of the preferred product produced is measured by a method selected from the group consisting of cation exchange chromatography (reducing or non-reducing), mass spectrometry, any other chromatographic separation, or a combination thereof.

[0024] In one embodiment, none of the Type IIS restriction endonuclease cleavage sites generates a 5' overhang of four nucleotides selected from the group consisting of GTAA, TCCA, and CACA upon cleavage by the Type IIS restriction endonuclease.

[0025] In one embodiment, the Type IIS restriction endonuclease cleavage site is selected from the group consisting of:

[0026] AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG, and CCAC.

[0027] In one embodiment, the mammalian cells are selected from the group consisting of CHO cells, CHOK1 cells, DXB-11, DG-44, COS-7, HEK293, BHK, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT060562, TRI cells, MRC 5 cells, FS4 cells, and mammalian myeloma cells.

[0028] In one embodiment, the optimal expression ratio of the at least two polypeptides is selected from the group consisting of: 1:1, 1:2, and 1:3.

[0029] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain, and a second antibody light chain, wherein the first antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain is 1:1:1:1.

[0030] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain, and a common antibody light chain, wherein the first antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the common antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the second antibody heavy chain, and the common antibody light chain is 1:1:2.

[0031] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain, and a second antibody light chain.

[0032] Wherein the modified antibody heavy chain comprises one of the following structures selected from the group consisting of:

[0033] VH-CH1-binding domain (BD)-hinge-CH2-CH3;

[0034] BD-VH-CH1-hinge-CH2-CH3; and

[0035] VH-CH1-hinge-CH2-CH3-BD;

[0036] wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab);

[0037] wherein the antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the BD binds to a third antigen or epitope, and

[0038] The optimal expression ratio of the first antibody heavy chain, the first light chain, the modified antibody heavy chain, and the second antibody light chain is 1:1:1:1.

[0039] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a modified antibody heavy chain, and a common antibody light chain,

[0040] Wherein the modified antibody heavy chain comprises one of the following structures selected from the group consisting of:

[0041] VH-CH1-binding domain (BD)-hinge-CH2-CH3;

[0042] BD-VH-CH1-hinge-CH2-CH3; and

[0043] VH-CH1-hinge-CH2-CH3-BD;

[0044] wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab);

[0045] wherein the antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the common antibody light chain to bind to the first antigen or epitope, wherein the BD binds to a second antigen or epitope, and

[0046] The optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain, and the common antibody light chain is 1:1:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figures 1A-1E Depicted is a modular vector platform ("ModVec") for high-throughput vector engineering. Figure 1A ) Example sequences of DNA modules. Each module (depicted with N and grey shading) is flanked by Golden Gate adapters (boxed regions) containing recognition sites for BsmBI (highlighted in bold). The BsmBI cleavage site is marked with a ^, and the overhangs generated by BsmI digestion are underlined. Figure 1B ) Assembly of the expression vector with the vector elements flanked by predefined complementary overhangs plus the expression vector backbone was achieved by Golden Gate reaction using BsmBI. Figure 1C ) Schematic diagram of a ModVec assembly of an 11 kb 11 segment (including the vector backbone). ITR: inverted terminal repeat of the piggyBac transposon. CDS: coding sequence. Figure 1D ) All 19 constructs were successfully assembled using ModVec in one round of cloning. 96% of colonies (109 out of 114) had correctly assembled constructs. Figure 1E ) Schematic diagram of single-tube vector library assembly. Figure 1F ) Efficient one-tube vector library construction using ModVec; 69 of 72 possible constructs were obtained by evaluating 350 colonies.

[0048] Figures 2A-2D It is depicted that the choice of promoter has a significant impact on manufacturability. Figure 2A ) Schematic diagram of the vector configuration. Both LC and HC genes are under the control of the same promoter (Promoter 1 or Promoter 2). The effect of promoter on the manufacturability of standard mAbA, bsAb B and bsAb C is shown in Figure 2B , Figure 2C and Figure 2D middle.

[0049] Figures 3A-3H Described is the use of vector configuration library screening to improve the productivity of four-chain iso-IgG. Figure 3A ) Schematic diagram of the vector configuration of the three expansion pools. Figure 3B) LC1:LC2 ratios of ProA purification samples from pools 905, 910, and 911 before expansion for further analysis. Figure 3C ) Expand the ProA production of pools 905, 910 and 911. Figure 3D ) Cation exchange purification recoveries of the three pools. Figure 3E ) The final yield of correctly assembled iso-IgG-D after cation exchange. The cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F , Figure 3G and Figure 3H The identity of each peak was confirmed by mass spectrometry.

[0050] Figures 4A-4H Depicted is the use of vector configuration library screening to improve the productivity of asymmetric trispecific molecules. Figure 4A ) Schematic diagram of vector configuration library design. Three different sets of configurations were tested, single LC, dual LC A and dual LC B. In each configuration, three different promoters, promoter 1, promoter 2 and promoter 3, were used to control the transcription of the polypeptide chains. Figure 4B ) ProA yields of all vector configurations. The average ProA yields of the vector configurations in the dual LC A, dual LC B, and single LC groups were 117.2 ± 43.4 mg / L, 112.6 ± 39.2 mg / L, and 40.7 ± 20.73 mg / L, respectively. Values ​​are expressed as mean ± standard deviation. Figure 4C ) Product mass of ProA purified samples from all vector configurations. The average nrMCE%MP for vector configurations in dual LC A, dual LC B, and single LC were 75.6±14.9%, 72.4±16.6%, and 71.7±21.6%, respectively. Figure 4D ) Final yields of correctly assembled tsAb E for all carrier configurations. The average final yields of the carrier configurations in the dual LC A, dual LC B, and single LC groups were 88.7±33.6 mg / L, 81.4±35.6 mg / L, and 28.5±16.8 mg / L, respectively. Figure 4E ) ProA production of stable pools generated from default and optimal configurations using a standard cell line development protocol. The optimal pool had a 124% increase in ProA production compared to the default pool. Figure 4F ) The product quality of ProA samples from the default and optimal pools is comparable. Figure 4G ) The final yield of tsAb E from the default pool was 114 mg / L and the final yield from the best pool was 257 mg / L. The final yield of the best pool increased by 125% compared to the default pool. Figure 4H ) Schematic representation of the optimal vector configuration identified by library screening and our platform vector configuration.

[0051] Figures 5A-5D Screening of a library of vector configurations for difficult-to-express iso-IgG is depicted. Figure 5A ) Schematic diagram of the vector configurations used. Expression of iso-IgG D involves co-transfection of two bicistronic vectors containing genes for all four polypeptide chains. The Config 1 vector has the LC and HC genes from the same mAb in the same bicistronic vector. The Config 2 vector has the LC and HC genes in separate bicistronic vectors. Figure 5B ) ProA yields of all recovered stable CHO cell pools; 29 out of 44 pools were recovered. Figure 5C ) LC1:LC2 ratios of ProA purified samples from all pools. Pools marked with asterisks were enlarged for further analysis. Figure 5D ) Product mass of all pools measured by non-reducing MCE main peak and SEC main peak.

[0052] Figures 6A-6G It is depicted that gene position in an expression vector can have a significant impact on manufacturability. Fig. 6A ) Schematic diagram of the DA2 and DB2 configurations. Both configurations use promoter 2 to drive expression of all polypeptide chains. Figure 6B )Pool DA2 has a higher ProA yield than pool DB2. Figure 6C ) The product quality of ProA purified samples from pools DA2 and DB2 was comparable. Fig.6D ) Pool DA2 had a higher final yield of tsAb E than pool DB2. Fig. 6E ) Electropherogram of ProA purified sample from pool DA2 (non-reducing microcapillary electrophoresis). For the ProA purified sample, the relative peak area of ​​half mAb1 is 10.1 and that of half mAb2 is 7.4. Fig. 6F ) Electropherogram of the ProA purification sample from pool DB2 (non-reducing microcapillary electrophoresis). The relative peak area of ​​half mAb1 is 5.8, and the relative peak area of ​​half mAb2 is 9.8. Figure 6G ) The half-mAb1:half-mAb2 relative peak ratio for the ProA purified sample from pool DA2 was 1.4, while this value was 0.6 for the ProA purified sample from pool DB2. DETAILED DESCRIPTION

[0053] The method of the present invention allows to produce the target expression cassette from the element sequence group by assembling the nucleic acid fragment construct via the single-stranded overhang formed at the two ends of the fragment using a II type restriction endonuclease. In the present invention, a II type restriction endonuclease can be used. The II type restriction endonuclease recognition site is a recognition site of a restriction endonuclease that recognizes double-stranded DNA and cuts the double-stranded DNA at a cleavage site outside the recognition site on the double-stranded DNA. The II type restriction endonuclease cuts so that an overhang of 3 to 6 nucleotides is produced, depending on a specific II type restriction endonuclease. Typically, in the method of the present invention, an enzyme that produces 4 nucleotide overhangs can be used. However, the II type endonuclease that produces a longer single-stranded overhang can also be used. The nucleotide range that forms the overhang after cutting is referred to as a cleavage site in this article. Since the nucleotides of the cleavage site are not part of the recognition site, they can be selected as needed without destroying the cleavage activity of the II type restriction endonuclease.

[0054] For practicing the present invention, any Type II restriction endonuclease may be used that provides "sticky" ends sufficient for effective ligation at its cleavage site. Selection of such enzymes is provided on the REBASE webpage (rebase.neb.com / cgi-bin / asymmlist) and in the review by Szybalsky et al. (1991, Gene [Gene], 100: 13-26). Most preferred are the following Type II restriction endonucleases: Bsal, Bbsl, BsmBI, Sapl, BspMI, Aarl, Esp3l, Bpil, and Hgal. Many of the cited restriction endonucleases are available from New England Biolabs. The sources of these enzymes can also be found on the above-mentioned REBASE webpage. Type II restriction endonucleases with asymmetric recognition sites (such as those shown in this webpage) can be used in the present invention, which have a cleavage site outside the recognition site and provide at least three, preferably 4 or more nucleotide residues overhangs when cleaved (such as BsmBI, BN736I; BpuAI, VpaK321, SfaNI, etc.). In one embodiment, the Type II restriction endonuclease is BsmBI.

[0055] It is recommended that the recognition site contains at least 4, more preferably at least 6 or more base pairs, so as to minimize the chance of finding this site in the target sequence portion. It is also possible to use a type II restriction nuclease (e.g., SfaNI) with a 5bp recognition site. The type II restriction endonuclease that produces a 4nt single-stranded overhang at the end of the digested fragment can theoretically produce an end with 256 possible sequences. The type II restriction endonuclease with even longer recognition sites (e.g., comprising ten or more base pairs) has been engineered. In one embodiment, the recognition site is 5'-CGTCTC-3'. In one embodiment, after being cut by the IIS restriction endonuclease, the 5' overhang length is four nucleotides, but is not selected from the group consisting of GTAA, TCCA, and CACA. In one embodiment, after cleavage by the IIS restriction endonuclease, the 5' overhang is four nucleotides in length and is selected from the group consisting of AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG, and CCAC.

[0056] Examples of ligases to be used in the present invention include T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, all of which are commercially available from New England Biolabs.

[0057] In one aspect, the invention relates to a method for expressing a multi-chain protein comprising at least two different polypeptide chains, the method comprising

[0058] a. providing a polynucleotide sequence encoding the at least two different polypeptides, wherein the polynucleotide sequence is flanked on both 5' and 3' sides by a type IIS restriction endonuclease cleavage site, followed by its recognition site;

[0059] b. providing two or more sets of element sequences; wherein each set of element sequences together comprises at least one functional expression cassette,

[0060] Each element sequence is flanked on both the 5' and 3' sides by a type IIS restriction endonuclease cleavage site followed by its recognition site,

[0061] wherein the type IIS restriction endonuclease recognition sites and cleavage sites are selected so that the element sequences of these groups can be assembled into a functional expression cassette;

[0062] c. providing at least one backbone entry vector, wherein the backbone entry vector comprises a plasmid comprising at least one type IIS restriction endonuclease cleavage site followed by its recognition site, and a sequence for maintaining the vector in a bacterial cell;

[0063] d. assembling the polynucleotide sequence encoding the at least two different polypeptides with the two or more sets of element sequences to produce a functional expression cassette in a backbone entry vector using a method based on digestion with a type IIS restriction enzyme and ligation via cleavage sites and overhangs, thereby producing a connected vector comprising at least two functional expression cassettes capable of expressing the at least two different polypeptide chains;

[0064] e. transfecting mammalian cells with the ligated vector;

[0065] f. comparing the level of the preferred product produced and the level of impurities in each of the at least two polypeptides; and

[0066] g. Identifying the optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying the transfected mammalian cells having the optimal expression ratio of the at least two polypeptide chains.

[0067] In one embodiment, the method further comprises:

[0068] h. Identifying the arrangement of vector elements that provides the optimal expression ratio of the at least two polypeptide chains.

[0069] In one aspect, the invention relates to a vector comprising an arrangement of vector elements providing for an optimal expression ratio of the at least two polypeptide chains.

[0070] Each group of element sequences will typically be able to be assembled into an expression cassette. In the context of the present invention, an expression cassette is intended to indicate a nucleic acid sequence that instructs the cell machinery to prepare RNA and protein. Typically, an expression cassette will contain a coding sequence and a sequence that controls the expression of the coding sequence. Typically, an expression cassette may contain at least a promoter, an open reading frame, and a terminator sequence. Other element sequences include control sequences, insulator sequences, barcode DNA sequences, primer sequences, promoter sequences, polyadenylic acid sequences, and IRES sequences, and mammalian selectable marker sequences. In one embodiment, the element sequence comprises at least two promoter sequences and at least two polyadenylic acid sequences.

[0071] The term "control sequence" is defined herein as including all components necessary or advantageous for producing mRNA or polypeptides in vitro or in host cells. Each control sequence may be natural or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, Shine-Delgarno sequences, optimal translation initiation sequences (as described in Kozak, 1991, J.Biol.Chem. [Journal of Biological Chemistry] 266: 19867-19870), polyadenylation sequences, propeptide sequences, pre-propeptide sequences, promoters, signal sequences, and transcription termination signals. At least, control sequences typically include promoters and transcription stop signals (terminators or termination signals). Typically, translation start signals and stop signals may also be present. Control sequences may be optimized for their specific purposes.

[0072] The term "promoter" is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcription start site of the nucleic acid sequence encoding the biological compound to initiate transcription. RNA polymerase efficiently catalyzes the assembly of a messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" will also be understood to include a 5'-noncoding region (between the promoter and the start of translation) for translation after transcription into mRNA, cis-acting transcription control elements (such as enhancers), and other nucleotide sequences that can interact with transcription factors.

[0073] The method of the present invention is typically performed so that the elements of the expression cassette are assembled in the backbone entry vector so that they are in operable connection. The term "operable connection" or "operably linked" and the like are defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence so that the control sequence directs the production of mRNA or polypeptide.

[0074] An "insulator sequence" or "insulator" is a nucleic acid segment that reduces or eliminates the transcriptional influence of adjacent regions to which the insulator is associated. Insulators are preferably placed upstream of other control sequences and / or downstream of genes. Insulators are preferably placed between different genes, transcription units or genetic domains to reduce or prevent interference with adjacent expressed sequences.

[0075] The function of an "enhancer sequence" or "enhancer" is to increase transcription from a promoter near the enhancer. Enhancers can be both upstream and downstream of a gene and act in either direction.

[0076] A "barcode DNA sequence" or "barcode" can be used to identify nucleic acid molecules, for example, where sequencing can reveal a certain barcode coupled to a target nucleic acid molecule. In some cases, sequence-specific events can be used to identify nucleic acid molecules, where at least a portion of a barcode is recognized in a sequence-specific event, for example, at least a portion of a barcode can participate in a ligation or extension reaction. Thus, a barcode can allow identification, selection, or amplification of a DNA molecule coupled thereto.

[0077] "IRES" or "internal ribosome entry site" means a site that allows internal ribosome entry sufficient to initiate translation in an assay for cap-independent translation (such as the bicistronic reporter assay described herein). The presence of an IRES allows cap-independent translation of a linked protein coding sequence that would otherwise not be translated.

[0078] A "selectable marker gene" or "selectable marker" encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) complement auxotrophic deficiencies of the cell; or (c) provide important nutrients that are not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used to select in both prokaryotic and eukaryotic host cells.

[0079] Other selectable genes can be used to amplify genes to be expressed. Amplification is the process of replicating genes required for the production of proteins necessary for growth or cell survival in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure, wherein only transformants are uniquely suitable for survival due to the presence of selectable genes in the vector. Selection pressure is applied in the following manner: culture transformed cells under conditions of continuously increasing the concentration of the selector in the culture medium, thereby simultaneously amplifying the selectable gene and the DNA encoding another gene, such as one or more components of the multi-chain protein construct described herein. Therefore, the polypeptide of the increased amount is synthesized by the amplified DNA.

[0080] Therefore, an element in the context of the present invention is any component of an expression cassette. A group of elements is a group of elements that can together produce an expression cassette. The method of the present invention requires providing two sets of element sequences. This means that enough elements are provided so that at least two different expression cassettes can be produced. This means that at least two different species of at least one element provided must be present. That is, for the purposes of the present invention, a promoter combined with two ORFs and two termination signals constitutes two sets of element sequences.

[0081] In the methods of the present invention, typically, at least two of these groups of element sequences contain a promoter element, an open reading frame element, and a termination signal element.

[0082] In the methods according to the invention, one or more of these sets of elements may comprise "partial" element sequences, such as UTRs, signal peptides and split open reading frames.

[0083] Each group of element sequences is provided in a form such that the group can be assembled into a functional expression cassette in a backbone entry vector. Then, typically, each element is flanked on both sides by a Type II restriction endonuclease cleavage site, followed by its recognition site, and the Type II restriction endonuclease recognition site and cleavage site are selected so that the element sequences of these groups can be assembled into a functional expression cassette. Therefore, each element sequence and flanking sequence typically comprise, in order from one end to the other: a Type II restriction endonuclease recognition site; its cleavage site; an element sequence; a Type II restriction endonuclease cleavage site; its recognition site.

[0084] Therefore, these sets of elements are prepared or provided in a suitable vector with type II restriction endonuclease recognition sites and a standardized cleavage site (preferably 4-bp), which are selected so that after assembly, for example using a one-pot method (such as Golden gate cloning), a functional expression cassette is formed.

[0085] A set of backbone entry vectors are prepared or provided. These vectors contain left and right connector sequences suitable for assembly using sequence homology.

[0086] A subset of element sequences is selected together with a backbone (bbn) entry vector. These can be assembled, for example using GoldenGate cloning, to generate a functional expression cassette contained within the backbone entry vector.

[0087] Exemplary host cells include prokaryotic cells, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, and Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are in common use and can be used herein, such as Pichia, e.g., P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0088] Host cells for expressing glycosylated antigen binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from the following hosts, such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. Multiple virus strains for transfecting such cells are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0089] Vertebrate host cells are also suitable hosts, and recombinant production of antigen-binding proteins by such cells has become routine. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 77:4216, 1980); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or subcloned 293 cells grown in suspension culture) (Graham et al., J. Gen Virol. [Journal of General Virology] 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. [Reproduction Biology] 23:243-251, 1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce the multi-chain protein of the present invention. In another embodiment, a cell line that does not produce autoantibodies but can produce and secrete heterologous antibodies can be selected from the B cell lineage. CHO cells are host cells for expressing the multi-chain proteins of the present invention.

[0090] The host cells are transformed or transfected with the above nucleic acids or vectors to produce multi-chain proteins, and cultured in conventional nutrient media appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In addition, novel vectors and transfected cell lines having multiple copies of transcription units separated by selective markers are particularly suitable for expressing antigen binding proteins. Therefore, the present invention also provides a method for preparing the multi-chain proteins described herein, which method includes culturing host cells containing one or more expression vectors in a culture medium under conditions that allow expression of the multi-chain proteins encoded by one or more expression vectors described herein; and recovering the multi-chain protein from the culture medium.

[0091] Host cells used to produce antigen-binding proteins of the invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. [Methods in Enzymology] 58:44, 1979; Barnes et al., Anal. Biochem. [Analytical Biochemistry] 102:255, 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90103430; WO 87 / 00195; or U.S. Pat. No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor); salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates); buffers (such as HEPES); nucleotides (such as adenosine and thymidine); antibiotics (such as gentamycin TM drugs); trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0092] When culturing host cells, multi-chain proteins can be produced in the cell, periplasmic space or directly secreted into the culture medium. If antigen-binding proteins are produced in the cell, then as a first step, the granular fragments of the host cells or dissolved fragments are removed, for example, by centrifugation or ultrafiltration. Bispecific antigen-binding proteins can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or affinity chromatography, using one or more target antigens or protein A or protein G as affinity ligands. Protein A can be used for purifying proteins including polypeptides based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. [Journal of Immunology Methods] 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. [Journal of the European Molecular Biology Association] 5: 15671575, 1986). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can increase flow rates and reduce processing times compared to agarose. In the case of proteins containing CH3 domains, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques, such as ethanol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible, depending on the specific multi-chain protein to be recovered.

[0093] As used herein, the term "antibody" refers to a tetrameric immunoglobulin comprising two light chain polypeptides (each about 25 kDa) and two heavy chain polypeptides (each about 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) can be κ (kappa) or λ (lambda). The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3) and optionally an immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε) chains, and they define antibody isotypes as IgM, IgD, IgG, IgA and IgE, respectively. Antibodies of IgG and IgA categories are further subdivided into several subclasses, namely IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2, respectively. The heavy chains in IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), while the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3 and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chain is linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain) and between the hinge region of the antibody heavy chain.

[0094] In human antibodies, CH1 means the region having the amino acid sequence at positions 118-215 of the EU index. A highly flexible amino acid region called a "hinge region" exists between CH1 and CH2. CH2 represents the region having the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341 to 446 of the EU index.

[0095] "CL" means the constant region of the light chain. In the case of the κ chain of a human antibody, CL means the region having the amino acid sequence at positions 108 to 214 of the EU index. In the λ chain, CL means the region having the amino acid sequence at positions 108 to 215.

[0096] The EU index (as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and the AHo numbering scheme (Honegger A. and Plückthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) may be used in the present invention. Amino acid positions and the complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody may be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399, and 409 are equivalent to AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535, and 551, respectively.

[0097] In one embodiment, the optimal expression ratio of the at least two polypeptides is selected from the group consisting of: 1:1, 1:2, and 1:3.

[0098] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain, and a second antibody light chain, wherein the first antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain is 1:1:1:1.

[0099] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain, and a common antibody light chain, wherein the first antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the common antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the second antibody heavy chain, and the common antibody light chain is 1:1:2.

[0100] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain, and a second antibody light chain.

[0101] The modified antibody heavy chain comprises one of the following structures selected from the group consisting of:

[0102] VH-CH1-binding domain (BD)-hinge-CH2-CH3;

[0103] BD-VH-CH1-hinge-CH2-CH3; and

[0104] VH-CH1-hinge-CH2-CH3-BD;

[0105] wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab);

[0106] wherein the antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the BD binds to a third antigen or epitope, and

[0107] The optimal expression ratio of the first antibody heavy chain, the first light chain, the modified antibody heavy chain, and the second antibody light chain is 1:1:1:1.

[0108] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a modified antibody heavy chain, and a common antibody light chain,

[0109] The modified antibody heavy chain comprises one of the following structures selected from the group consisting of:

[0110] VH-CH1-binding domain (BD)-hinge-CH2-CH3;

[0111] BD-VH-CH1-hinge-CH2-CH3; and

[0112] VH-CH1-hinge-CH2-CH3-BD;

[0113] wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab);

[0114] wherein the antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the common antibody light chain to bind to the first antigen or epitope, wherein the BD binds to a second antigen or epitope, and

[0115] The optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain, and the common antibody light chain is 1:1:2.

[0116] A "binding domain" or "BD" may typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. For example, a Fd fragment has two VH regions and generally retains some antigen binding function of a complete antigen binding domain. Additional examples of antibody fragments, antibody variants, or binding domain formats include (1) a Fab fragment, a monovalent fragment having VL, VH, CL, and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) a Fd fragment having two VH and CH1 domains; (4) a Fv fragment having the VL and VH domains of a single arm of an antibody; (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs), and (7) single-chain Fv (scFv), the latter being preferred (e.g., derived from a scFV library). Cation exchange chromatography ("CEX") is a form of ion exchange chromatography (IEX) that is used to separate molecules based on their net surface charge. More precisely, cation exchange chromatography uses a negatively charged ion exchange resin that has an affinity for molecules with a net positive surface charge. Cation exchange chromatography is used for both preparative and analytical purposes and can separate a wide range of molecules from amino acids and nucleotides to large proteins. Here, we focus on preparative cation exchange chromatography of proteins. CEX can be performed under reducing and non-reducing conditions.

[0117] Mass spectrometry ("MS") is an analytical technique used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a graph of intensity as a function of mass-to-charge ratio.

[0118] The disclosure of each reference set forth herein is incorporated by reference in its entirety.

[0119] The present invention is further illustrated by the following examples:

[0120] Examples

[0121] ModVec is designed to be extremely flexible, using a modular "slot-based" approach to assemble one or more expression cassettes in a variety of possible arrangements while allowing combinatorial exploration of sequence diversity within each slot. The Modvec system is divided into multiple slots, each of which can contain a DNA sequence with a unique Golden Gate overhang. The DNA sequence can be a vector element, including elements required for vector replication (ColE Ori, pMB1 Ori...), antibiotic resistance (Amp, Kan, Crm...), elements required for viral infection (LTR), transposition (ITR), episomal replication (OriP), targeted integration (loxP, Frt, attB / P), and elements for mammalian gene expression (e.g., promoters, enhancers, internal ribosome entry sites (IRES), self-processing viral 2A peptides, polyadenylation signals, control sequences, insulator sequences including MAR and UCOE, etc.).

[0122] As a proof of principle, ModVec was first used to assemble complex mammalian expression vectors. Two ModVec backbones were generated, including pMVP5, which can be used in common mammalian cell lines (HEK, CHO) used in research, and a vector backbone designed specifically to adapt to Amgen's CHO cell line. These vector backbones contain only the minimum vector elements required for replication in E. coli and antibiotic selection to maintain the plasmid.

[0123] In the first experiment, the ModVec concept was tested by assembling a vector with a functional DNA sequence in each slot: 14-slot (including the vector backbone), 8kb DNA assembly. It contains three ECs; EC1 and EC2 are bicistronic and EC3 is monocistronic. It has three different promoters to drive the expression of 5 coding sequences. It has been shown that using circular plasmids as input to the GG reaction increases efficiency, especially for multi-segment golden gate assembly (Potapov, Ong et al. 2018). Therefore, a "parts" vector was generated, in which the vector elements expected to be frequently reused in each slot were first cloned into the pGG_Cm_BsaI vector using GG together with BsaI and confirmed by Sanger sequencing. In this process, a "parts library" of vector elements (such as promoters, IRES, polyadenylation, control sequences, insulators, etc.) with ModVec-appropriate overhangs was established in a format that would be easy to store and refill when needed. Using a combination of circular "parts" and synthetic linear DNA, the first 14-well assembly used 20ul GG reaction and 1ul of each vector element (concentration between 40-70ng / ul). pMVP5 was used as the vector backbone. The GG reaction was transformed into E. coli and 32 colonies were picked for Sanger sequencing. The designed 8kB DNA vector was successfully obtained; however, the efficiency of correct assembly was only 3.125% (1 out of 32 picked colonies was correct). Internal and external data (Kanigowska, Shen et al. 2016) show that GG efficiency may be higher with smaller reaction volumes (micro-GG). The 14-well assembly was then performed at smaller reaction volumes of 2ul, 1ul, and 0.5ul using the ECHO acoustic liquid handler, which is capable of precise liquid transfer at these very small volumes. These reactions were scaled down proportionally to the initial 20ul reaction. Micro-GG increased the efficiency of this 14-segment ModVec assembly to 16% (13 out of 78 colonies were correct).

[0124] By carefully analyzing the successes and failures of the first PoC ModVec assembly, it was determined that a specific set of 11GG overhangs always ligated correctly in this context. It was then tested whether using only this set of overhangs could improve ModVec efficiency. ModVec was used to assemble a 10-trough assembly with these optimized overhangs in a 1 ul GG reaction. It was found that more than 95% of the clones contained correctly assembled vectors (548 of 576 colonies were correct).

[0125] The 95% efficiency of assembly obtained under the optimized ModVec design and conditions enables the single-tube construction of expression vector libraries. Functionally similar vector elements with the same overhangs are mixed together to produce a "trough library". Two different promoters used to drive the 12 CDS in EC1 are combined with three different promoters to drive the expression of the selection marker EC3, including the elimination of the bridge of EC2, which can produce 72 different expression vectors through combinatorial assembly. After transformation, 350 colonies were picked, and after sequencing verification, a total of 312 contigs were generated. These 312 contigs matched 69 of the 72 possible vectors, demonstrating the low sequence-dependent bias in Modvec assembly and indicating that the library strategy of this system is possible.

[0126] Materials and Methods

[0127] Vector construction

[0128] All DNA fragments including vector elements and antibody LC and HC were synthesized by Twist Bioscience or PCR amplified from existing templates and cloned into the vector backbone using the standard Golden Gate assembly protocol (Engler et al., 2008). To generate the component vectors, each Golden Gate reaction contained 2 μL DNA fragments (5 ng / μL) and 2 μL pGG vector (20 ng / μL), 1 μL of 5 μM ATP (Thermo Fisher, R0441), 0.5 μL T4 ligase (Thermo Fisher, EL0014) and 0.5 μL BsaI (Thermo Fisher, ER0291) in a fast digestion buffer (Thermo Fisher, B64), and 3 μL H2O. The Golden Gate reaction was run on a thermal cycler at 37°C for 2 min and at 16°C for 3 min for 15 cycles. The final 5 min incubation was performed at 85°C to inactivate all enzymes. Miniaturized Golden Gate reactions and one-pot vector library cloning reactions were set up using an ECHO525 liquid handler (Labcyte) for nanoliter-scale liquid transfer. The volume of each DNA fragment and vector backbone was scaled down for miniaturized GoldenGate reactions. For ModVec assembly, a TECAN liquid handler was used to mix 15 μL of Golden Gate reaction mixture, which contained 2 μL of pGG parts vector (40-70 ng / μL), 2 μL of expression vector backbone (50 ng / μL), 1.5 μL of fast digestion buffer (Thermo Fisher Scientific, B64) containing 5 μM ATP (Thermo Fisher Scientific, R0441), 0.75 μL of T4 ligase (Thermo Fisher Scientific, EL0014) and 0.75 μL of BsmBI (Thermo Fisher Scientific, FD0454), and variable amounts of H2O to make up to 15 μL. For each Golden Gate cycle, the 37°C incubation was extended to 3 min and the 16°C incubation was extended to 5 min, and for ModVec assembly, the number of cycles was extended to 20. Plasmid DNA was prepared using the Qiagen miniprep kit (Qiagen, 27104). After sequencing confirmation, the plasmid DNA encoding HC and LC were mixed at a defined ratio.

[0129] Cell culture and protein expression

[0130] To generate stable cell pools by random genomic integration, Bio-Rad Gene The Xcell electroporation system electroporates 25 μg of DNA into our in-house proprietary suspension Chinese Hamster Ovary (CHO) cells. After electroporation, the entire transfection is plated in a proprietary recovery medium. Every 2-3 days until recovery, use Live cell density and viability were monitored using a counter (Beckman Coulter), and the medium was replaced. Recovery was defined as >85% viability. Recovered cells were used to inoculate 50-mL fed-batch production in shake flasks and harvested after 10 days. To generate stable cell pools using piggyBac transposase-based expression vectors, Lipofectamine LTX (Thermo Fisher Scientific, 11668030) was used at 4 x 10 6 4 μg of DNA was used to transfect proprietary suspension CHO cells knocked out for glutamine synthetase (CHO GS KO) at 10 viable cells / mL. 48 to 72 hours after transfection, transfected cells were transferred to selection medium with methionine sulfoximine (MSX). Recovered cells were used to culture at 1 x 10 6 4-mL fed-batch production was carried out by inoculating 10 cells / mL and harvesting after 10 days. Live cell density and viability were monitored by a Biotin® counter (Beckman Coulter), and the medium was changed on days 3, 6, and 8. On day 10, live cell density and viability were measured and the culture medium was replaced by a ForteBio RT-PCR instrument equipped with a protein A biosensor. Red, conditioned media from these production batches were used to determine titers.

[0131] High-throughput protein purification

[0132] As previously described (Gong et al., 2021), magnetic ProA beads (GE Life Sciences) were added to The Flex system (Thermo Fisher Scientific) was used to purify proteins. Briefly, 100 μL of magnetic ProA beads were added to 4 mL of CHO GS KO culture one day before harvest. The beads were collected and Kingfisher purified with a 24-probe magnetic head. After washing 3 times with phosphate buffered saline and 2 times with Milli-Q water, the protein was eluted with 500 μL of 100 mM sodium acetate at pH 3.6 for 10 min, and then neutralized by adding 10 μL of 3M Tris (pH 11.0).

[0133] Large-scale ProA purification followed by cation exchange chromatography

[0134] Proteins expressed in 50-mL CHO cultures were purified as previously described (Gong et al., 2021; Yoo et al., 2014) using ProA affinity capture (1 mL HiTrapMabSelect SuRe, GE Life Sciences, catalog number GE11-0034-93), eluted with 100 mM sodium acetate (pH 3.6), and immediately buffer exchanged to 10 mM sodium acetate, 150 mM NaCl (pH 5.2) using a 5 mL HiTrap desalting column (GE Life Sciences, catalog number GE17-1408-01).

[0135] Cation exchange chromatography was performed as previously described (Gong et al., 2021). In brief, 1.5-1.8mL ProA purified samples were diluted with 20mL of 20mM MES (pH 6.2) and loaded onto a 1-mL cation exchange column (SP-HP HiTrap, GE Life Sciences, catalog number GE29-0513-24) at 1mL / min. After washing the column with 8 column volumes of the same buffer at 1mL / min, the protein was eluted with a linear 0-400mM NaCl gradient at 0.4mL / min for 40 column volumes. Fractions with a purity of 90% or more as determined by size exclusion chromatography (SEC) were merged, and their concentrations were determined as previously described (Winters et al., 2015) using a Multiscan GO microplate reader (Thermo Fisher Scientific). The final merged samples were analyzed by SEC, non-reduced microcapillary electrophoresis (nrMCE) and liquid chromatography-mass spectrometry (LC-MS).

[0136] Non-reducing microcapillary electrophoresis and size exclusion chromatography

[0137] The product quality of the purified material was analyzed using reducing and non-reducing microcapillary electrophoresis and SEC as previously described (Gong et al., 2021; Guo et al., 2021). For nrMCE, 6 μL of protein was mixed with 21 μL of sample buffer (8.4 mM TrisHCl pH 7.0, 7.98% glycerol, 2.38 mM EDTA, 2.8% SDS, and 2.4 mM iodoacetamide), heated at 85 °C for 10 min, and then analyzed using a Caliper LabChip GXII Touch instrument (PerkinElmer). For analytical SEC, a BEH column ( 1.7 μm, 4.7×300 mm), with 100 mM sodium phosphate pH 6.9, 50 mM NaCl, 7.5% ethanol as running buffer, at a flow rate of 0.45 mL / min. Protein samples were analyzed on a HPLC instrument (Waters).

[0138] Liquid chromatography-mass spectrometry and RapidFire-mass spectrometry

[0139] Complete non-reducing LC-MS was performed to determine the MW of purified samples as previously reported in (Campuzano et al., 2019; Spahr et al., 2018). A 30 μL aliquot of 1% trifluoroacetic acid (TFA) was added to 30 μL of each purified sample. Next, approximately 15 μg of each sample was injected onto an Agilent 1290 UPLC, with the column effluent directly coupled to an Agilent 6224 electrospray time-of-flight (ESI-TOF) mass spectrometer (Agilent Technologies). Chromatographic separations were performed using RRHD 300SB-C8 2.1 × 50 mm, 1.8 μm particle size ultra-performance liquid chromatography (UPLC) column (Agilent Technologies). The column was heated to 70 °C at a flow rate of 0.5 mL / min. Chromatographic solvents of aqueous "A" (0.1% TFA in H2O) and organic "B" (0.1% TFA in 90% n-propanol) were used. The gradient used was isocratic with 80% A / 20% B for 4 min, 28% A / 72% B for 2 min, 10% A / 90% B for 0.5 min, and finally 5% A / 95% B for 0.5 min. The MS method scanned m / z [1000–7000], acquiring 0.7 spectra / sec. The resulting spectra were summed and then deconvoluted using Agilent Mass Hunter Qualitative Analysis Software (version B.07.00) or the complete program module from Protein Metrics (PMIIntact).

[0140] The RapidFire-MS system was used for samples from the high-throughput vector configuration library screen. An equal volume of 0.1% w / w formic acid was added to 50 uL of the provided sample, and 20 uL of this solution was injected onto the RapidFire-MS for analysis. The SPE cartridge was a 4-uL PLRP Cartridge / column. The mobile phases were 10% n-propanol with 0.1% formic acid and 90% n-propanol with 0.1% formic acid. All data were processed using PMIIntact.

[0141] result

[0142] Modular Vector (ModVec) Platform for High-Throughput Assembly of Complex Vectors

[0143] To achieve high-throughput vector engineering, the GG assembly-based mammalian modular vector (ModVec) system was developed to construct different expression vectors and vector libraries for recombinant protein production. ModVec enables high-throughput construction of simple and complex vector designs to support the optimization of expression vectors for single molecules, specific macromolecular patterns and / or different expression hosts.

[0144] ModVec is designed to be extremely flexible so that one or more expression cassettes can be assembled in a variety of possible arrangements, while allowing combinatorial exploration of sequence diversity within each module. Each module contains a DNA sequence or a library of DNA sequences with carefully designed GG overhangs ( Figure 1B ). These DNA sequences can be vector elements commonly used for recombinant expression, such as sequences required for vector replication (origin of replication) and antibiotic resistance, elements required for viral infection, transposition, episomal replication, targeted integration, or elements commonly used for mammalian gene expression. These DNA sequences are flanked by GG adapters ( Figure 1A The boxed regions in the PCR product contain, for example, BsmBI restriction sites, and they can be generated by DNA synthesis or PCR amplification. A set of complementary, non-promiscuous four-bp overhangs flanked by BsmBI restriction sites was designed to enable seamless and unidirectional assembly of DNA elements into a reasonable expression cassette ( Figure 1B ).

[0145] The ModVec concept was first tested by assembling an 11 kb, 11-module bicistronic construct in a 15 μL GG reaction ( Figure 1C ). 19 constructs were assembled and six colonies of each construct were plated for sequence verification. All 19 bicistronic constructs were successfully assembled by one round of cloning. More than 95% (109 / 114) of the clones contained correctly assembled vectors ( Figure 1D ). These results demonstrate that ModVec is an efficient method for assembling large, complex DNA fragments when the sets of GG overhangs are compatible (i.e., do not promiscuous with each other). The 95% efficiency of correct assembly obtained with the optimized ModVec enables the single-tube construction of expression vector libraries. Functionally similar vector elements with the same overhangs are mixed together to generate a "modular library" ( Figure 1E). As a demonstration of a single-tube vector engineering library, two different promoters driving 12 coding sequences (CDS1 to CDS12) were combined with three different promoters driving CDS13 expression, which can generate 72 different expression vectors through combinatorial assembly. A miniaturized GG reaction was set up using an ECHO liquid handler, which has been shown to increase GG assembly efficiency (Kanigowska et al., 2016). Each of the 25 portions of 80nL was mixed in a 2.5μL GG assembly reaction. After 30 cycles of GG assembly, the full volume was transformed and plated. After incubation overnight, approximately 1000 colonies grew on agar plates, of which 350 colonies were inoculated in liquid culture for sequence verification by Sanger sequencing. A total of 312 contigs were generated, which matched 69 of the 72 possible vectors ( Figure 1F ).

[0146] These results demonstrate that ModVec is an effective cloning platform for HT assembly of complex expression vectors and expression configuration libraries. Therefore, it is now possible to identify vector configurations with balanced expression levels of polypeptide chains in bsAbs by high-throughput vector configuration screening, thereby improving bsAb productivity and product quality. The ModVec cloning system will next be used to explore different transcriptional regulation strategies to improve the productivity and product quality of complex multi-chain, multi-specific antibodies produced recombinantly in mammalian cells.

[0147] Effects of promoters on the productivity of double-chain monoclonal antibodies (mAbs) and symmetrical bispecific antibodies (bsAbs)

[0148] Effect of the tested promoters on the productivity of symmetrical, double-stranded molecules A, B and C ( Figure 2B-2D Molecule A is a typical monoclonal antibody, and molecules B and C are symmetrical bispecific antibodies. A bicistronic vector was constructed in which the LC and HC genes of each molecule were driven by the same promoter (promoter 1 or promoter 2). Figure 2A ). Both Promoter 1 and Promoter 2 are derived from the huCMV promoter, but Promoter 2 is stronger than Promoter 1. The piggyBac transposase-based expression vector was used for efficient genomic integration and rapid generation of stable cell pools. Internal observations showed that the piggyBac transposase-based expression vector had very high reproducibility, so a stable Chinese hamster ovary (CHO) cell pool was generated for each configuration. Regardless of the molecule type, the pool transfected with the construct using Promoter 2 had a higher yield after protein A (ProA) affinity chromatography than the pool transfected with the construct using Promoter 1 ( Figure 2B-2D). The ProA production of molecule A was 403 mg / L in the pool transfected with the construct using promoter 1 and 521 mg / L in the pool transfected with the construct using promoter 2, a 30% increase in production by switching promoter 1 to promoter 2. More significant increases were observed for molecules B and C, where switching promoter 1 to promoter 2 resulted in an 83% and 100% increase in ProA production, respectively. Cell recovery and growth characteristics were also comparable between pools transfected with all constructs. For the three symmetrical molecules, altering the promoter strength of both the HC and LC genes together had no significant effect on product quality, as measured by size exclusion chromatography (SEC % main peak, Figure 2B-2D , middle figure) as measured by the percentage of the main substance in. Thus, by using promoter 2, the final yield of molecules A, B, and C was increased by 28%, 122%, and 96%. A more significant increase in productivity was observed for molecules that initially had lower yields (molecules B and C). The results show that regulating promoter strength is an effective method to increase the productivity of mAbs and bsAbs, and further indicate that it may be most effectively applied to proteins that are difficult to express.

[0149] The vector engineering strategy was next extended to improve both productivity and product quality of iso-IgG, a more complex four-chain bsAb.

[0150] Improving product quality and productivity of hetero-IgG by chain balancing

[0151] Hetero-IgG is the most common bispecific format due to its antibody-like structure (Labrijn et al., 2019), but its manufacture is also more challenging than mAbs due to the possibility of multiple product-derived impurities caused by incorrect LC-HC and HC-HC pairing (Brinkmann and Kontermann, 2017). Many protein engineering strategies such as charge pair mutation (CPM) (Dillon et al., 2017; Gunasekaran et al., 2010), knob-in-hole structure (KiH) (Ridgway et al., 1996), chain exchange engineered domain (SEED) (Davis et al., 2010) and common light chain (Krah et al., 2017; Shiraiwa et al., 2019) have been used to promote correct chain pairing and prevent incorrect chain pairing, thereby improving the manufacturability of hetero-IgG. However, differences in the expression levels of each polypeptide chain comprising hetero-IgG in mammalian cells can lead to the formation of product-related impurities and reduce the yield of the correct bsAb. For example, in a stable CHO pool of the molecule hetero-IgG-D transfected with our default expression vector, a 60-fold higher yield of LC1 relative to LC2 was observed, and a correspondingly high amount of mispaired material with two copies of LC1 was secreted (Guo et al., 2021). It is hypothesized that the yield of correctly paired hetero-IgG relative to mispaired product-related impurities can be improved by balancing the expression of each polypeptide chain to a near 1:1:1:1 ratio through vector engineering. Through our success with double-chain molecules, a vector engineering approach was adopted to balance the expression of polypeptide chains using promoters of different strengths.

[0152] Eleven vector configurations of hetero-IgG-D were constructed using ModVec. Each vector configuration contained two co-transfected bicistronic vectors, in which the two polypeptide CDSs in the vector were driven by the same or different promoters (Promoter 1, Promoter 2, and Promoter 3, Figure 5A In some vector configurations, the position of the LC or HC coding sequences is swapped within the bicistronic cassette ( Figure 5A Configuration 2). Four transfections / vector configurations were performed in CHO cells and a total of 44 pools were generated. Stable pools were generated by random genomic integration with metabolic selection. Pools from different vector configurations had different recovery rates; of the 44 pools transfected, 15 did not recover. Those that failed to recover tended to be transfected with the configurations of LC1 / LC2 and HC1 / HC2 in the same vector ( Figure 5A In addition to the differences in the recovery of the stable CHO pool, the yield of the vector configuration library after ProA affinity purification ( Figure 5B ), product mass measured by LC1:LC2 ratio ( Figure 5C), as well as the percentage of major substance peaks (%MP) in non-reducing microcapillary electrophoresis (nrMCE) and size exclusion chromatography (SEC) showed a wide range ( Figure 5D ).

[0153] Three pools were selected for scale-up production and further analysis: two pools had LC1:LC2 ratios close to the ideal value of 1 ( Figure 3B , pools 910 and 911) and one pool had a skewed LC1:LC2 ratio >5 ( Figure 3B , pool 905). The vector configuration used to generate these stable pools is Figure 3A Conditioned media from these three amplified pools were purified using ProA affinity capture followed by cation exchange (CEX) using Sepharose SP resin. All three pools had similar ProA yields ( Figure 3C After cation exchange, the recovery of the major iso-IgG-D species from pool 905 was 12%, while pools 910 and 911 had recoveries of 46% and 40%, respectively ( Figure 3D ). Thus, the yield of correctly assembled iso-IgG-D after cation exchange was higher for pools 910 and 911 (115 and 75 mg / mL, respectively) than for pool 905 (34 mg / L) ( Figure 3E ).

[0154] In addition to producing different relative levels of the major species, pools transfected with different vector configurations also had different profiles of product-related impurities, as shown in the cation exchange chromatograms ( Figure 3F-3H The major species in the ProA purified sample of pool 905 was identified by mass spectrometry as having 2 copies of LC1 (2x LC1, i.e., LC1:HC1:LC1:HC2, Figure 3F The mispaired heterotetramer is the main peak in the IgG-D matrix, while the correctly assembled hetero-IgG-D is a minor species ( Figure 3F This explains the low purification yield and recovery of pool 905 ( Figure 3D-3E ), and is consistent with a high LC1:LC2 ratio ( Figure 3B ). This also suggests that when promoter 1 is used to drive expression of both LCs, the expression levels of LC1 and LC2 are unbalanced. We did not observe antibody-like homodimers from pool 905, which would be caused by HC1:HC2 mispairing; the main problem with CHO pools transfected with this vector configuration is LC-HC mispairing. When the promoter driving both LC1 and LC2 was switched to promoter 2 (pool 910, Figure 3A ), 2x LC1 became a minor species, and correctly assembled iso-IgG-D became the major species in the ProA purified material ( Figure 3G ), which resulted in higher recovery of the main species after cation exchange ( Figure 3D-3E ). Interestingly, changing both LC promoters to promoter 2 brought the expression of LC1 and LC2 into equilibrium. We previously reported that LC2 is poorly secreted from mammalian cells (Guo et al., 2021). It is possible that switching both LC promoters to promoter 2 has a greater effect on “expression-defective” LC2, resulting in a more balanced LC1:LC2 ratio. We have transfected >10 replicate CHO pools with the promoter permutations used to generate pool 905 and consistently observed high LC1:LC2 ratios (5–60) and high levels of 2x LC1 material. Furthermore, in pool 911, when the promoter of LC1 was changed to promoter 3, its transcriptional strength was between promoter 1 and promoter 2, while the LC2 promoter was unchanged (promoter 2), and correctly assembled hetero-IgG-D was still the major species. However, the major impurity became half antibody 1 (LC1:HC1) instead of 2xLC1 ( Figure 3H ). The change of the major impurity from 2X LC1 in pools 905 and 910 to half antibody 1 in pool 911 may be the result of changes in the LC gene promoter affecting the expression of both LC and HC genes from the bicistronic cassette.

[0155] High-throughput vector engineering for asymmetric, three-chain hetero-IgG-based trispecific molecules

[0156] Next, we tested whether a high-productivity vector of the challenging three-chain asymmetric trispecific antibody E (tsAb E) could be engineered. In transient transfection tests, tsAb E was produced with titers 5 to 10 times lower than the expected level of mAb. Inspired by the successful screening of 11 vector configurations for hetero-IgG, we decided to apply vector engineering methods to improve the productivity of tsAb E, but include more vector configurations in the library design. It is speculated that testing more strategies to regulate the expression of polypeptide chains will increase the possibility of finding a configuration in which all three polypeptide chains of the asymmetric tsAb E have a stoichiometrically optimal ratio. In addition to promoters of different strengths, gene dosage (LC gene copy number) was also changed, and the cistron arrangement of the LC and HC genes was used as a variable in the library design. The final library contained 189 vector configurations, including 27 monocistronic vectors with a single LC gene copy (Single LC), 81 bicistronic vectors with two LC gene copies in the first cistron of each vector (Bi-LC A), and 81 bicistronic vectors with two LC gene copies in the second cistron of each vector (Bi-LC B) ( Figure 4A ). Three promoters (Promoter 1, Promoter 2, and Promoter 3) are used to drive expression of each polypeptide chain in a combinatorial manner; therefore, in this vector library design, genes in a bicistronic vector can be optionally driven by the same or different promoters.

[0157] CHO cells were transfected in 24-well culture plates using a proprietary high-throughput expression system that relies on transposase-mediated integration with metabolic selection. One pool was generated for each vector configuration. All pools had comparable recovery and growth profiles (data not shown), which is common for this expression system. Figure 4B As shown, there are orders of magnitude differences in the productivity of this tsAb, depending on the vector configuration; the ProA yield of all pools ranged from 20 mg / L to 220 mg / L, which clearly demonstrates the ability of the vector configuration to affect the yield. Pools transfected with dual LC A (117.2 ± 43.4 mg / L) or dual LC B (112.6 ± 39.2 mg / L) configurations had higher average ProA yields than pools transfected with a single LC configuration (40.7 ± 20.73 mg / L) (Student's t test, dual LC A vs. single LC p < 0.0001, dual LC B vs. single LC p < 0.0001; values ​​are expressed as mean ± standard deviation). Dual LC A pools and dual LC B pools had comparable ProA yields (Student's t test, dual LC vs. dual LC B p = 0.48). An initial assessment of product quality was performed by running non-reducing microcapillary electrophoresis (nrMCE) on each ProA pool; the mean peak percentage of major species (%MP) between pools generated from the three sets of support configurations was comparable ( Figure 4C , dual LC A MP: 75.6±14.9%, dual LC B MP: 72.4±16.6%, single LC MP: 71.7±21.6%; dual LC A vs. dual LC B p=0.19, dual LC A vs. single LC p=0.34, dual LC B vs. single LC p=0.88, Student's t test). Figure 4D As shown, the average yield of tsAb E correctly assembled in the pools produced from double LC A (88.7 ± 33.6 mg / L) or double LC B (81.4 ± 35.6 mg / L) configuration is higher than the average yield of the pools (28.5 ± 16.8 mg / L) produced from the single LC configuration. The yield of the correct material is comparable between the pools produced from double LC A and double LC B (double LC A compared to double LC B p=0.48, double LC A compared to single LC p<0.0001, double LC B compared to single LC p<0.0001, Student's t test). These results show the impact of LC gene copy number on the productivity of complex therapeutic molecules. This is consistent with previous findings, i.e., the gene dosage determined by gene copy number can have a great impact on productivity, and excess LC is generally beneficial to productivity (González et al., 2002).

[0158] Since the performance of the dual LC A group and the dual LC B group were comparable, the performance of individual configurations from these two groups was compared to understand the effect of gene position on productivity and product quality. Configurations DA2 (dual LC A configuration with promoter 2 driving expression of all polypeptide chains) and DB2 (dual LC B configuration with promoter 2 driving expression of all polypeptide chains) were selected for further analysis ( Fig. 6A ). In DA2, the LC gene is in the first cistron and the HC gene is in the second cistron, while the positions of the LC gene and the HC gene are swapped in DB2. Figure 6B As shown, the ProA yield of pool DA2 was 155 mg / L. Exchanging the position of the LC and HC genes by placing the HC gene in the first cistron and the LC gene in the second cistron reduced the overall productivity. The ProA yield of pool DB2 was 113 mg / L. This is consistent with gene position effects in multicistronic expression vectors, where the expression level of genes downstream of the first gene is generally reduced relative to the expression level of the first gene (Eszterhas et al., 2002; Patel et al., 2021). In this case, reducing the expression of the LC gene while increasing the expression of the HC gene resulted in an overall decrease in productivity, which is again consistent with the expectation that high levels of LC expression are beneficial to overall productivity (González et al., 2002). The product quality measured by nrMCE MP and pre-MP was comparable between ProA samples purified from pools DA2 and DB2 (nrMCE MP, 80% for DA2 and 81% for DB2; nrMCE pre-MP, 18% for DA2 and 19% for DB2; Figure 6C Mass spectrometry confirmed that nrMCE MPs correctly assembled tsAb E and that nrMCE pre-MPs were half mAb1 (LC:HC1) and half mAb2 (LC:HC2, Fig. 6E and 6F ).

[0159] Although nrMCE pre-MP levels were comparable between ProA samples from pools DA2 and DB2, the relative abundance of mAb1 and mAb2 was different ( Fig. 6E and 6F In the nrMCE electropherogram of the ProA-purified sample from pool DA2, the relative peak area of ​​half mAb1 was 10.1 and the relative peak area of ​​half mAb2 was 7.4 ( Fig. 6E For the ProA purified sample from pool DB2, the relative peak area of ​​half mAb1 was 5.8, and the relative peak area of ​​half mAb2 was 9.8 ( Fig. 6F The relative peak ratios of half mAb1:half mAb2 were calculated and the results are shown in Figure 6GThe half mAb1: half mAb2 relative peak ratio was 1.4 in the ProA purified sample from pool DA2 and 0.6 in the ProA purified sample from pool DB2. These results further demonstrate that the LC or HC gene position in the expression vector can significantly affect both the productivity of the main substance and the spectrum of impurities produced from CHO cells. These results also support our hypothesis that combining promoters of different strengths, gene copy numbers, and gene positions is an effective method to regulate the expression level of polypeptide chains and can increase the possibility of identifying vector configurations that produce high productivity and product quality of multi-chain protein therapeutics.

[0160] The top vector configurations were selected from the vector library screen to advance cell line development (CLD) of tsAb E and compared with the default platform vector (shown in Figure 4H The default vector configuration uses promoter 1 to drive expression of all polypeptide chains and combines a bicistronic vector with the LC gene in the first cistron and HC1 in the second cistron with a monocistronic vector with HC2. In contrast, the best configuration identified from the vector library consisted of two bicistronic vectors, each expressing a copy of the LC gene in the first cistron and either HC1 or HC2 in the second cistron, with promoter 2 driving expression of both LC genes and promoter 1 driving expression of both HC1 and HC2 genes ( Figure 4H The stable CHO pool generated from the default configuration produced tsAb E with a ProA yield of 119 mg / L, while the best configuration from the vector library produced 267 mg / L after ProA, an increase of 124% ( Figure 4E The product quality measured by nrMCE was comparable between the ProA purification samples from the two pools ( Figure 4F ). Therefore, the final yield of correctly assembled tsAb E was 114 mg / L in the default pool and 257 mg / L in the optimal pool ( Figure 4G For this exciting and difficult to express tsAb therapeutic, the optimal vector configuration identified through vector configuration library screening resulted in a 125% increase in productivity over our default vector configuration.

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Claims

1. A method for expressing a multi-chain protein comprising at least two different polypeptide chains, the method comprising a. providing a polynucleotide sequence encoding the at least two different polypeptides, wherein the polynucleotide sequence is flanked on both 5' and 3' sides by a type IIS restriction endonuclease cleavage site, followed by its recognition site; b. providing two or more sets of element sequences; wherein each set of element sequences together comprises at least one functional expression cassette, Each element sequence is flanked on both the 5' and 3' sides by a type IIS restriction endonuclease cleavage site followed by its recognition site, wherein these Type IIS restriction endonuclease recognition sites and cleavage sites are selected so that the element sequences of these groups can be assembled into a functional expression cassette; c. providing at least one backbone entry vector, wherein the backbone entry vector comprises a plasmid comprising at least one type IIS restriction endonuclease cleavage site followed by its recognition site, and a sequence for maintaining the vector in a bacterial cell; d. assembling the polynucleotide sequence encoding the at least two different polypeptides with the two or more sets of element sequences to produce a functional expression cassette in the backbone entry vector using a method based on digestion with a type IIS restriction enzyme and ligation via the cleavage sites and overhangs, thereby producing a connected vector comprising at least two functional expression cassettes capable of expressing the at least two different polypeptide chains; e. transfecting mammalian cells with these ligated vectors; f. comparing the level of the preferred product produced and the level of impurities in each of the at least two polypeptides; and g. Identifying the optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying the transfected mammalian cells having the optimal expression ratio of the at least two polypeptide chains.

2. The method according to claim 1, further comprising: h. Identifying the arrangement of vector elements that provides the optimal expression ratio of the at least two polypeptide chains.

3. The method according to any preceding claim, wherein the element sequences are selected from the group consisting of: control sequences, insulator sequences, barcode DNA sequences, primer sequences, promoter sequences, polyadenylation sequences, and IRES sequences, and mammalian selectable marker sequences.

4. The method according to claim 3, wherein the element sequences comprise at least two promoter sequences and at least two polyadenylation sequences.

5. A method according to any preceding claim, wherein the level of the preferred product produced is measured by a method selected from the group consisting of: Cation exchange chromatography (reducing or non-reducing), mass spectrometry, or a combination thereof.

6. The method according to any preceding claim, wherein none of the Type IIS restriction endonuclease cleavage sites produce a 5' overhang of four nucleotides selected from the group consisting of GTAA, TCCA and CACA upon cleavage by the Type IIS restriction endonucleases.

7. The method according to any preceding claim, wherein the Type IIS restriction endonuclease cleavage sites are selected from the group consisting of: AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG, and CCAC.

8. The method of any preceding claim, wherein the mammalian cells are selected from the group consisting of CHO cells, CHOK1 cells, DXB-11, DG-44, COS-7, HEK293, BHK, TM4, CV1, VERO-76, HELA, MDCK, BRL3A, W138, Hep G2, MMT 060562, TRI cells, MRC 5 cells, FS4 cells, and mammalian myeloma cells.

9. The method according to any preceding claim, wherein the optimal expression ratio of the at least two polypeptides is selected from the group consisting of: 1:1, 1:2, and 1:

3.

10. The method of claim 1, wherein the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain, and a second antibody light chain, wherein the first antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain is 1:1:1:

1.

11. The method of claim 1, wherein the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain, and a common antibody light chain, wherein the first antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the second antibody heavy chain associates with the common antibody light chain to bind to a second antigen or epitope, wherein the optimal expression ratio of the first antibody heavy chain, the second antibody heavy chain, and the common antibody light chain is 1:1:

2.

12. The method according to claim 1, wherein the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain, and a second antibody light chain, Wherein the modified antibody heavy chain comprises one of the following structures selected from the group consisting of: a. VH-CH1-binding domain (BD)-hinge-CH2-CH3; b. BD-VH-CH1-hinge-CH2-CH3; and c.VH-CH1-hinge-CH2-CH3-BD; wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab); wherein the antibody heavy chain associates with the first antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the second antibody light chain to bind to a second antigen or epitope, wherein the BD binds to a third antigen or epitope, and The optimal expression ratio of the first antibody heavy chain, the first light chain, the modified antibody heavy chain, and the second antibody light chain is 1:1:1:

1.

13. The method according to claim 1, wherein the multi-chain protein comprises an antibody heavy chain, a modified antibody heavy chain, and a common antibody light chain, Wherein the modified antibody heavy chain comprises one of the following structures selected from the group consisting of: a. VH-CH1-binding domain (BD)-hinge-CH2-CH3; b. BD-VH-CH1-hinge-CH2-CH3; and c.VH-CH1-hinge-CH2-CH3-BD; wherein the BD is selected from single-chain Fv (scFv) or single-chain Fab (scFab); wherein the antibody heavy chain associates with the common antibody light chain to bind to a first antigen or epitope, and the VH of the modified antibody heavy chain associates with the common antibody light chain to bind to the first antigen or epitope, wherein the BD binds to a second antigen or epitope, and The optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain, and the common antibody light chain is 1:1:

2.

14. A carrier comprising an arrangement of carrier elements according to claim 2, step h.

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