Methods for assembling large nucleic acids from short fragments

Abstract

Here, we report a modular and efficient method for cloning multigene plasmids that involves only a single cloning step without the use of a pre-existing single-gene vector. In the first step, PCR-generated DNA blocks carrying genes for different antibody chains are ligated with their respective adapters, which act as connectors for the different DNA blocks. In the second step, the fragments generated by PCR in the first step are assembled in the correct configuration via unique recombination sites, DNA blocks, and adapter fragments located at one end of each scaffold. This new strategy results in a modular and efficient method that allows expression cassettes to be cloned directly into each scaffold without an intermediate cloning step, enabling the rapid cloning of variable gene constructs for diverse antibody formats. This new cloning method according to the present invention offers considerable advantages in terms of time, labor, and cost. TIFF2023509538000019.tif79170

Description

[Technical Field]

[0001] Here, we report a modular and efficient method for cloning multigene plasmids that involves only a single cloning step without the use of a pre-existing single-gene vector. In the first step, PCR-generated DNA blocks carrying genes for different antibody chains are ligated with their respective adapters, which act as connectors for the different DNA blocks. In the second step, the fragments generated by PCR in the first step are assembled in the correct configuration via unique recombination sites, DNA blocks, and adapter fragments located at one end of each scaffold. This new strategy results in a modular and efficient method that allows expression cassettes to be cloned directly into each scaffold without an intermediate cloning step, enabling the rapid cloning of variable gene constructs for diverse antibody formats. This new cloning method according to the present invention offers considerable advantages in terms of time, labor, and cost. [Background technology]

[0002] Background of the Invention Therapeutic monoclonal antibodies are the standard of care for the treatment of many types of cancer. For the production of therapeutic antibodies, CHO cells are the most common producer cell line, capable of expressing complex glycoproteins that require post-translational modifications. A targeted integration (TI) system was developed to integrate the required DNA sequences encoding antibody components into a predetermined locus in the CHO genome.

[0003] Celie, PHN et al. reported a recombination cloning strategy for protein expression (Curr. Opin. Struct. Biol. 38 (2016) 145-154 (Non-Patent Document 1)).

[0004] Betton reported a high-throughput cloning and expression strategy for protein production (Biochimie 86 (2004) 601-605 (Non-Patent Document 2)).

[0005] Chaudhary, VK et al. reported a rapid restriction enzyme-free cloning of PCR products, a high-throughput method applicable to library construction (PLOS One 9 (2014) e111538 (Non-Patent Document 3)).

[0006] Tan, L. et al. reported on homologous alignment cloning, a rapid, flexible, and highly efficient general molecular cloning method (PEER J.6(2018)e5146 (Non-Patent Document 4)). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Celie, PHN et al., Curr.Opin.Struct.Biol.38(2016)145-154 [Non-patent document 2] Betton, Biochimie 86 (2004) 601-605 [Non-patent document 3] Chaudhary, VK et al., PLOS One 9(2014)e111538 [Non-patent document 4] Tan, L. et al., PEER J.6(2018)e5146 Summary of the Invention

[0008] When using the TI approach to generate production cell lines, front and back vectors carrying various gene expression cassettes for antibody chains are used. The cloning procedure for the TI front and back vectors is generally based on the preliminary cloning of a single-gene vector encoding a specific antibody chain. The corresponding gene expression cassettes for specific antibody chains are then cloned onto each multigene front or back vector, which are then transfected into host cells. Cloning single-gene vectors represents a time-consuming and labor-intensive process step in projects involving high resource investments.

[0009] This invention reports an improved method for creating multigene vector systems, a cloning strategy that saves time, effort, and resources. The method of the present invention no longer requires the use of a preliminary single-gene vector. This TI cloning method of the present invention is based on a modular system vector backbone and adapter fragments as predefined general parts, and DNA blocks (antibody chain sequences) as project-specific parts. It has been found to be advantageous to use adapter fragments containing the terminator of the previous DNA block and the promoter of the next one as connectors. This ensures the correct positioning of the DNA blocks. In the method of the present invention, the adapters and DNA blocks are adjusted by PCR to have unique recombination sites (R sites) at the 3' ends of the fragments and defined RE recognition sites at the 5' ends. The TI-front / back base vectors also contained the corresponding R sites and RE recognition sites flanking the backbone.

[0010] The present invention is based, at least in part, on the discovery that the generation of these common portions (adapters, backbone) and variable DNA blocks improves cloning of TI vectors because each fragment contains a specific R site at one end of the fragment and a defined RE recognition site at the other. Depending on the desired gene configuration, these portions are ligated and assembled into the final TI vector using restriction enzymes and assembly of the homologous R sites of the two fragments. Due to the ability to generate most of the required sequence by PCR, this method reduces the time and effort required for gene synthesis, as only the variable DNA blocks need to be supplied.

[0011] One aspect of the present invention is a linear expression vector backbone, - a promoter and a first single-stranded enzyme restriction site for a first restriction enzyme at its 3' end, - a single-stranded recombination site at its 5' end the linear expression vector backbone comprising a first DNA block, in the 5' to 3' direction, - a first single-stranded enzyme restriction site for a first restriction enzyme, - a nucleic acid encoding a protein of interest, - first single-strand recombination site the first DNA block comprising: a first adapter nucleic acid, in the 5' to 3' direction: - a first single-strand recombination site, - polyA signal sequence, - promoters, - a second single-stranded restriction site for a second restriction enzyme the first adaptor nucleic acid comprising a second DNA block, in the 5' to 3' direction: - a first adapter nucleic acid comprising a second single-stranded enzyme restriction site for a second restriction enzyme; - a nucleic acid encoding a second protein of interest, - second single-strand recombination site the second DNA block comprising with DNA ligase, The first enzyme restriction site is different from the second enzyme restriction site, the second enzyme restriction site is for a type IIB restriction enzyme, the first recombination site and the second recombination site are different; the second recombination site and the recombination site at the 5' end of the vector backbone are identical; Each recombination site is a nucleic acid sequence of 15 to 80 bp in length that is unique to the incubated nucleic acid; the first single-stranded enzyme restriction site of the linear vector backbone can specifically hybridize with the first single-stranded enzyme restriction site of the first DNA block, the first single-stranded recombination site of the first DNA block can specifically hybridize with the first single-stranded recombination site of the first adapter nucleic acid, the second single-stranded enzyme restriction site of the first adapter nucleic acid can specifically hybridize with the second single-stranded enzyme restriction site of the second DNA block, and the second single-stranded recombination site of the second DNA block can specifically hybridize with the single-stranded recombination site of the linear vector backbone.

[0012] In one embodiment, the second enzyme restriction site is a BsaXI restriction site.

[0013] In one embodiment, the proteins of interest are antibody chains. In one embodiment, a first protein of interest is an antibody light chain and a second protein of interest is an antibody heavy chain, or vice versa.

[0014] One aspect of the present invention is a method for manufacturing a computer-implemented system comprising the following elements: - Enzyme restriction sites - a first expression cassette for a first protein of interest; - first recombination site - Enzyme restriction site for type IIB restriction enzymes - a second expression cassette for a second protein of interest - second recombination site is a nucleic acid comprising the first recombination site is different from the second recombination site; The enzyme restriction site may be different from or identical to the enzyme restriction site for a type IIB restriction enzyme.

[0015] In one embodiment, the second enzyme restriction site is a BsaXI restriction site.

[0016] In one embodiment, the proteins of interest are antibody chains. In one embodiment, a first protein of interest is an antibody light chain and a second protein of interest is an antibody heavy chain, or vice versa.

[0017] One aspect according to the invention is a cell comprising a nucleic acid according to the invention.

[0018] In one embodiment, the cell is a mammalian cell. In one embodiment, the mammalian cell is a CHO cell.

[0019] One aspect of the invention is a method for producing an antibody, comprising the steps of: a) culturing mammalian cells containing the nucleic acid according to the invention; b) recovering the antibody from the cells or culture medium; c) optionally purifying the antibody using one or more chromatography steps. Including, This is the method for producing antibodies.

[0020] General Explanations and Definitions definition Useful methods and techniques for practicing the present invention are described, for example, in Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones, NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); and Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0021] The use of recombinant DNA technology makes it possible to produce derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion, or insertion, at individual or several nucleotide positions. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).

[0022] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0023] The term "about" refers to a range of ±20% of the preceding numerical value. In one embodiment, the term "about" refers to a range of ±10% of the preceding numerical value. In one embodiment, the term "about" refers to a range of ±5% of the preceding numerical value.

[0024] The term "Cre-recombinase" refers to a tyrosine recombinase that catalyzes site-specific recombination between LoxP sites using a topoisomerase I-like mechanism. The enzyme has a molecular weight of approximately 38 kDa and consists of 343 amino acid residues. It is a member of the integrase family.

[0025] The term "comprising" also includes the term "consisting of."

[0026] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses a cell into which one or more exogenous nucleic acids have been introduced and which is intended to be the starting point for subsequent genetic modification, including the progeny of such a cell. Thus, the term "mammalian cell comprising an exogenous nucleotide sequence" encompasses a cell comprising an exogenous nucleotide sequence integrated at a single site within a genomic locus of the mammalian cell, the exogenous nucleotide sequence comprising at least one first and at least one second recombinase recognition sequence (the recombinase recognition sequences are different) flanked by at least one first selectable marker. In one embodiment, a mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence integrated at a single site within a genomic locus of the host cell, the exogenous nucleotide sequence comprising first and second recombination recognition sequences flanked by at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and the recombination recognition sequences are all different.

[0027] As used herein, the term "recombinant cell" refers to a cell after final genetic modification, such as a cell that expresses a polypeptide of interest and can be used for the production of the polypeptide of interest on any scale. For example, a "mammalian cell containing an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), thereby introducing the coding sequence of the polypeptide of interest into the genome of the host cell, is a "recombinant cell". Although this cell can still undergo further RMCE reactions, it is not intended to do so.

[0028] The term "LoxP site" refers to a 34-bp-long nucleotide sequence consisting of two palindromic 13-bp sequences at the ends (ATAACTTCGTATA (SEQ ID NO: 35) and TATACGAAGTTAT (SEQ ID NO: 36), respectively) and a central 8-bp core (asymmetric) spacer sequence. The core spacer sequence determines the orientation of the LoxP site. Depending on the relative orientation and position of the LoxP sites with respect to each other, the intervening DNA is either excised (LoxP sites oriented in the same direction) or inverted (LoxP sites oriented in the opposite direction). The term "floxed" refers to the DNA sequence located between two LoxP sites. When two floxed sequences, i.e., a target floxed sequence in the genome and a floxed sequence in the donor nucleic acid, are present, both sequences can be exchanged for each other. This is called "recombinase-mediated cassette exchange."

[0029] Exemplary LoxP sites are shown in the table below: TIFF0007801226000001.tif53128

[0030] Both "mammalian cells containing an exogenous nucleotide sequence" and "recombinant cells" are "transformed cells." This term includes the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell in nucleic acid content, for example, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0031] An "isolated" composition is one that has been separated from a component of its natural environment. In some embodiments, the composition is purified to greater than 95% or 99% purity, as measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0032] An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0033] An "isolated" polypeptide or antibody means a polypeptide or antibody molecule that has been separated from a component of its natural environment.

[0034] The term "integration site" refers to a nucleic acid sequence within a cell's genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides in the cell's genome. In certain embodiments, the integration site comprises a stretch of nucleotide sequence. In certain embodiments, the integration site is located within a specific locus in the genome of a mammalian cell. In certain embodiments, the integration site is within an endogenous gene of a mammalian cell.

[0035] The terms "vector" or "plasmid" can be used interchangeably and, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector as an autonomously replicating nucleic acid structure and a vector that has integrated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0036] As used herein, the term "adjacent" means that a first nucleotide sequence is located at either the 5' or 3' end of a second nucleotide sequence, or at both ends. The adjacent nucleotide sequence may be located next to the second nucleotide sequence or at a predetermined distance from it. There is no particular limitation on the length of the adjacent nucleotide sequence. For example, the adjacent sequence may be a few base pairs or several thousand base pairs.

[0037] Deoxyribonucleic acid comprises a coding strand and a non-coding strand. The terms "5'" and "3'" as used herein refer to positions on the coding strand.

[0038] As used herein, the term "exogenous" refers to a nucleotide sequence that is not native to a particular cell and is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. Therefore, an exogenous nucleotide sequence is an artificial sequence, which may result, for example, from the combination of subsequences of different origins (e.g., the combination of a recombinase recognition sequence with an SV40 promoter and a green fluorescent protein coding sequence is an artificial nucleic acid), or from partial deletion or nucleic acid base mutation of a sequence (e.g., a sequence or cDNA encoding only the extracellular domain of a membrane-bound receptor). The term "endogenous" refers to a nucleotide sequence that originates from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but the "exogenous" sequence has been introduced into the cell by, for example, recombinant DNA technology.

[0039] Random Embedding The traditional method for inserting plasmid DNA into the genome of mammalian cells is the use of random plasmid integration (RI) followed by clonal selection and screening. When mammalian cells are transfected with an exogenous plasmid, integration occurs at one or a few chromosomal sites. While this approach has been successfully used by industrial cell line development (CLD) manufacturers, it has inherent drawbacks [1], [2]. With random integration, expression levels are highly dependent on the local chromosomal context and are therefore unpredictable. For example, insertions can occur at sites in the genome that are transcriptionally inactive or weakly transcriptionally active, resulting in low or no product expression. Screening a large number of clones to identify high-yield clones is time-consuming and labor-intensive. Furthermore, productive clones may be inconsistent and harbor a larger number of integrated transgenes. This can induce chromosomal rearrangements, leading to repeat-induced promoter methylation and thus gene silencing [3], [4]. As a result, some clones become unstable under selective pressure. As the number of integration sites increases, the analysis of microheterogeneity, e.g., sequence variants, becomes more difficult. Taking all issues into consideration, targeted integration offers a valuable alternative for integrating transgenes into the genome of host cells [5].

[0040] Targeted embedding Targeted integration (TI) results in the integration of a transgene at a single, predetermined locus within the genome of mammalian cells [6]. Therefore, host cell lines require or are engineered to contain known specific recombination sites located at loci called "hotspots," which ensure reliable and stable production of transgenes with low or even single copy numbers [1]. In their paper, Crawford et al. described how they established stable host cell lines through the combination of two technologies: ΦC31 integrase and CRE-Lox recombinase technology. Using this system, they integrated a "platform plasmid" into the CHO genome. This platform provides a base cassette containing flanking CRE-Lox recombination sites (L3 and 2L) that enable recombinase-mediated cassette exchange (RMCE) with plasmids encoding antibody chains and different resistance genes.

[0041] Therefore, targeted integration allows exogenous nucleotide sequences to be integrated into a predetermined site in the genome of a mammalian cell. In certain embodiments, targeted integration is mediated by a recombinase that recognizes one or more recombination recognition sequences (RRS). In certain embodiments, targeted integration is mediated by homologous recombination.

[0042] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. RRSs can be used to define locations in a nucleotide sequence where recombination events are expected to occur.

[0043] In certain embodiments, the RRS is selected from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence. If multiple RRSs are to be present, the selection of each sequence is dependent on the other, provided that non-identical RRSs are selected.

[0044] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0045] In certain embodiments, where the RRS is a LoxP site, the cells require Cre recombinase to effect recombination. In certain embodiments, where the RRS is an FRT site, the cells require FLP recombinase to effect recombination. In certain embodiments, where the RRS is a Bxb1 attP site or a Bxb1 attB site, the cells require Bxb1 integrase to effect recombination. In certain embodiments, where the RRS is a φC31 attP site or a φC31 attB site, the cells require φC31 integrase to effect recombination. These recombinases can be introduced into cells using expression vectors containing the coding sequences for these enzymes.

[0046] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre is a 38-kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp nonpalindromic core region flanked by two 13-bp inverted repeats. Cre recombinase binds to the 13-bp repeats, thereby mediating recombination within the 8-bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. When two LoxP sequences are located in the same nucleotide sequence and in the same orientation, Cre-mediated recombination excises the DNA sequence located between the two LoxP sequences into a covalently closed circle. If two LoxP sequences are located in opposite positions on the same nucleotide sequence, Cre-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are located on two different DNA molecules and one of the DNA molecules is circular, Cre-mediated recombination will result in the integration of a circular DNA sequence.

[0047] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. Mutant LoxP sequences have been developed to increase the efficiency of Cre-mediated integration or replacement. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of LoxP L3, LoxP 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, and Lox66 sequences. For example, the Lox71 sequence has a 5-bp mutation in the left 13-bp repeat. The Lox66 sequence has a 5-bp mutation in the right 13-bp repeat. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0048] The term "matched RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matched RRSs are different sequences from each other but can be recognized by the same recombinase. In certain embodiments, the first matched RRS is a Bxb1 attP sequence and the second matched RRS is a Bxb1 attB sequence. In certain embodiments, the first matched RRS is a φC31 attB sequence and the second matched RRS is a φC31 attB sequence.

[0049] Establishment of a cloning method for expression vectors Expression vectors that are subsequently transfected into mammalian cells can be cloned using different methods. Generally, for antibodies, the complete gene expression cassettes for each antibody chain are placed on a preliminary single-gene vector. These are then cloned into the respective multigene vectors. An exemplary functional gene expression cassette consists of a CMV promoter, intron A, a 5'-untranslated region (5'-UTR), followed by the initiation codon ATG flanked by Kozak sequences, and a gene of interest (GOI) ending with a polyadenylation signal (polyA) [7], [8]. Cloning of vectors is performed using restriction enzymes and ligation and / or DNA assembly reactions.

[0050] Therefore, expression vectors are cloned by digesting gene cassettes from a preliminary single-gene vector and then ligating them into the multiple cloning site (MCS) of the backbone vector to obtain a multigene vector. Therefore, appropriate restriction enzymes are selected according to the MCS. Restriction enzymes (REs) are catalytic enzymes that hydrolyze dsDNA at specific sequences. They cleave dsDNA strands along the palindromic pattern of their recognition sites, generating single-stranded overhangs called sticky ends [9]. When two fragments are cleaved with the same enzyme, the overhangs result in complementary single strands that can be attached by ligation. Enzymes that cleave at the MCS are used and selected to ensure that individual fragments are ligated only in the correct orientation.

[0051] Cloning by assembly reaction is also known. This cloning method allows for the seamless assembly of multiple DNA fragments due to homologous sequences at the ends of each fragment. Recombination sites (R sites) are 15-80 bp long sequences surrounding the gene cassette of the target gene (GOI). Depending on the sequence, each fragment has a specific R site at the beginning and end of the sequence, which is identical to the R site of the preceding or following fragment. Therefore, cloning of a single gene, specifically a pre-vector containing appropriate R sites, is required. The gene flanked by the R sites is then excised using a restriction enzyme that cuts next to the R site. Finally, assembly of all fragments occurs in a single step. An exonuclease generates single-stranded 3' overhangs that facilitate annealing of fragments that share complementarity at the R site. A polymerase fills gaps within each annealed fragment, and a ligase fills gaps in the assembled DNA

[10] ,

[11] . Figure 2 provides a schematic diagram of the assembly reaction. Compared to RE cloning, assembly of individual fragments via R sites already offers improved efficiency and labor. [The present invention 1001] a linear expression vector backbone, - a promoter and a first single-stranded enzyme restriction site for a first restriction enzyme at its 3' end, - a single-stranded recombination site at its 5' end the linear expression vector backbone comprising a first DNA block, in the 5' to 3' direction, - a first single-stranded enzyme restriction site for a first restriction enzyme, - a nucleic acid encoding a protein of interest, - first single-strand recombination site the first DNA block comprising: a first adapter nucleic acid, in the 5' to 3' direction: - a first single-strand recombination site, - polyA signal sequence, - promoters, - a second single-stranded restriction site for a second restriction enzyme the first adaptor nucleic acid comprising a second DNA block, in the 5' to 3' direction: - a second single-stranded enzyme restriction site for a second restriction enzyme, - a nucleic acid encoding a second protein of interest, - second single-strand recombination site the second DNA block comprising with DNA ligase, the first enzyme restriction site is different from the second enzyme restriction site; the second enzyme restriction site is for a type IIB restriction enzyme; the first recombination site and the second recombination site are different; the second recombination site and the recombination site at the 3' end of the vector backbone are identical; each recombination site is a nucleic acid sequence of 15 to 80 bp in length that is unique to the incubated nucleic acid; the first single-stranded enzyme restriction site of the linear vector backbone is capable of specifically hybridizing with the first single-stranded enzyme restriction site of the first DNA block, the first single-stranded recombination site of the first DNA block is capable of specifically hybridizing with the first single-stranded recombination site of the first adaptor nucleic acid, the second single-stranded enzyme restriction site of the first adaptor nucleic acid is capable of specifically hybridizing with the second single-stranded enzyme restriction site of the second DNA block, and the second single-stranded recombination site of the second DNA block is capable of specifically hybridizing with the single-stranded recombination site of the linear vector backbone. [The present invention 1002] 1001. The method of claim 1001, wherein said second enzyme restriction site is a BsaXI restriction site. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the target protein is an antibody chain. [The present invention 1004] From 5' to 3', the following elements: - a first promoter nucleic acid sequence, - a restriction site for a first restriction enzyme, - a first nucleic acid encoding a first protein of interest; - a first recombination site, - a first polyadenylation signal sequence, - a second promoter nucleic acid sequence, - a second enzyme restriction site for a type IIB restriction enzyme, - a second nucleic acid encoding a second protein of interest; - a second recombination site, and - second polyadenylation signal sequence A linear or circular nucleic acid comprising: the first recombination site is different from the second recombination site; the first enzyme restriction site is different from the enzyme restriction site for a type IIB restriction enzyme; The linear or circular nucleic acid, wherein each recombination site is a unique nucleic acid sequence of 15 to 80 bp in length. [The present invention 1005] The nucleic acid of the present invention 1004, obtained by the method of any one of the present inventions 1001 to 1003. [The present invention 1006] The nucleic acid of any one of 1004 to 1005 of the present invention, wherein the enzyme restriction site for a type IIB restriction enzyme is a BsaXI restriction site. [The present invention 1007] The nucleic acid of any one of claims 1004 to 1006, wherein the target protein is an antibody chain. [The present invention 1008] A cell containing any one of the nucleic acids of the present invention 1004 to 1007. [The present invention 1009] 1. A method for producing an antibody, comprising: a) culturing mammalian cells containing the nucleic acid of the present invention; b) recovering the antibody from the cells or culture medium; c) optionally purifying said antibody using one or more chromatography steps. Including, This method results in the production of the antibody. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description of Embodiments of the Invention All currently used methods for cloning expression vectors rely on the cloning of single-gene vectors that provide either specific restriction enzymes or specific R sites flanking the gene expression cassette of interest. Therefore, for highly complex molecules, a huge number of individual vectors, e.g., more than 20 single-gene vectors, are required to generate the final multigene expression vector. This represents a time-consuming and labor-intensive process. As the complexity of the molecules to be expressed increases, new time-, labor-, and resource-saving cloning strategies must be provided, particularly for creating multicomponent vector systems. This problem has been solved in the present invention!

[0053] In this invention, we report an efficient cloning system for multigene expression vectors that involves only a single cloning step without the need for the generation of preliminary single-gene vectors. The cloning method according to the invention is based on PCR-produced components (vector backbone, adapters) and supplied antibody chains, as well as synthesized DNA blocks.

[0054] The present invention is based, at least in part, on the discovery that an adapter spanning the termination sequence of a previous expression cassette and the promoter of a next expression cassette can be advantageously used to link different expression cassettes in a single step without the intermediate provision of a single gene vector.

[0055] Thus, each nucleic acid fragment used in the method according to the invention is flanked by restriction enzyme recognition sites and specific unique recombination sites (R sites).

[0056] The present invention is based, at least in part, on the discovery that flanking restriction enzyme recognition sites and specific unique recombination sites allows for the direct cloning of multiple gene cassettes into their respective vector backbones. This is done without the intermediate step of single-gene vector cloning, allowing for the rapid cloning of different gene constructs. A further advantage of the modification of nucleic acid fragments according to the present invention is the unique feature of the R-site sequence, which provides new binding sites for primers in the case of sequence variant analysis (SVA). A further advantage of the method according to the present invention is that it reduces the time and effort required for gene synthesis. This is achieved by generating and modifying the nucleic acid fragments used via PCR.

[0057] In one example, the method according to the invention was used to generate targeted integration front and back vectors that together encode the different chains of a bispecific antibody.

[0058] Exemplary Description of the Method According to the Invention The following is an exemplary description of the method according to the invention, which is chosen purely as an example, as the method according to the invention is generally applicable, so that any other vector system can be produced as well.

[0059] Below, the method according to the present invention is exemplified with a two-vector targeted integration system in which part of the antibody gene is located on the front vector and the other part is located on the back vector, and the front and back vectors carry all the elements necessary to withstand selection pressure after integration into the landing site in the genome of the TI host cell.

[0060] Depending on the intended construction of the expression cassette, DNA blocks encoding each chain of the antibody are assembled by methods according to the invention and incorporated into a basic vector backbone.

[0061] In the method of the present invention, a nucleic acid sequence adapter (or short adapter) containing the terminator sequence of the preceding DNA block and the promoter sequence of the following DNA block is used, thereby positioning the promoter / terminator sequences of the first / last DNA block on the vector backbone. This ensures the correct positioning of the DNA blocks. Each adapter and DNA block has a restriction enzyme recognition sequence at its 5' end and a unique R site at its 3' end. This allows for the insertion of individual gene expression cassettes according to the intended configuration. In Figure 3, the method is schematically shown for a bsAb with the xLC-knob-LC-hole configuration. A linearized backbone was generated by digesting each base vector with the restriction enzyme PspOMI. Similarly, any other restriction enzyme that cuts the backbone vector only once in the intended region can be used. Ligation of the cut sites (represented by scissors) of each fragment pair then occurs in separate reactions. Thus, the first DNA block is part of the backbone, and the first adapter is ligated to the second DNA block, resulting in longer fragment lengths. In the assembly reaction (represented by the cross lines), these ligated fragments are finally recombined with their corresponding fragments that share the same R site.

[0062] Thus, the cloning method according to the present invention is based on a modular system that uses predefined general parts (adapters and front and back vector backbones) and molecule-specific parts (i.e., in this example, antibody chain sequences) that are adjusted by PCR to contain the necessary R sites at the 3' end. Depending on the desired gene organization, these parts are ligated and assembled into the final vector using restriction enzymes and assembly cloning methods.

[0063] The vector backbone used in the method according to the invention requires the sequence of the first expression cassette (promoter-5'-UTR) followed by a restriction enzyme recognition site. These sequences are located immediately at the beginning of the integration site in the vector. In addition, an R site is located in the vector backbone before the final polyA sequence at the end of the integration site. Recognition sequences for different restriction enzymes are located between the restriction enzyme recognition site and the final polyA sequence. The cleavage sites are used to close the backbone, forming a circular vector.

[0064] Any vector can be employed for use in the method according to the present invention. A vector backbone is used as the basic structure of a vector for use in the method according to the present invention. This backbone is extended with the required sequences (two DNA fragments synthesized via PCR) and then ligated into the backbone vector. This procedure is shown diagrammatically for the front vector backbone in Figure 4. Compared to the front vector, the back vector backbone encodes Lox sites (LoxFAS and L2), respectively, which, after ligation with fragment 1 and fragment 3 (via RE BamHI, PspOMI, and PacI), form the targeted integration back vector backbone.

[0065] Generally, for the generation of vector backbones used in the methods of the present invention, primer pairs with 3'-overhang sequences containing their unique binding sites as well as an overhang sequence according to the inventive concept were used. For example, to generate fragment 1 in Figure 4, a template containing the sequence CMV-intron A-5'-UTR-RE was used and amplified with the respective primer pairs. Fragments 2 and 3 were similarly amplified from a plasmid containing a polyA sequence. The PCR-generated fragments were analyzed using analytical agarose gel electrophoresis (AGE).

[0066] All PCR-amplified fragments were flanked by RE recognition sites and could then be integrated into the correct location of the vector backbone. Therefore, PCR samples were digested with the respective restriction enzymes (BamHI / PspOMI, PspOMI / PacI) and subsequently purified. To prevent self-ligation of the vector, the backbone was digested with the REs BamHI and PacI and then dephosphorylated. All digested inserts were then ligated into the respective backbone vectors. Chemically competent cells were transformed with the ligation products and plated on agar plates containing selective pressure. Sequencing of each sequence was used to verify the sequence identity of one of the clones with the correct backbone vector. Finally, the vectors were enriched in a plasmid maxi-prep and used as the base vector backbone from here on.

[0067] The present invention is based, at least in part, on the discovery that type IIB REs must be used for efficient assembly of gene fragments. Type IIB REs include BsaXI, CspCI, AloI, PpiI, PsrI, FaII, Bsp24I, BcgI, BpII, HaeIV, CjeI, CjePI, Hin4I, BaeI, AlfI, and BslFI (see, e.g., Tengs, T., et al., Nucl. Acids Res. 32 (2004) e121). These REs cleave both DNA strands at specific locations away from their recognition sequences; i.e., these enzymes cleave both strands on either side of their recognition sequences, thereby creating any cleavage site that is not palindromic / symmetrical.

[12] The complete cleavage site / recognition sequence of BsaXI, an exemplary type IIB RE, is displayed below in compact form with the recognition sequence highlighted by shading. TIFF0007801226000002.tif16132

[0068] To ligate the fragments in the first cloning step, the type IIB enzyme BsaXI was used as the RE.

[0069] In one example of the method of the present invention, front and back expression vectors containing expression cassettes for producing T cell bispecific antibodies (TCBs) were constructed. Each vector had the expression cassette configuration kHC-xLC-xLC (front vector) and hHC-LC (back vector). Thus, the front vector encodes gene expression cassettes for three antibody chains (knob heavy chain and two crossover light chains), and the back vector contains gene expression cassettes for hole heavy and light chains. The cloning strategy for this example of the method of the present invention is outlined in Figure 5.

[0070] More specifically, in the first step, primers were designed to provide, via PCR, a DNA block fragment carrying an adapter and a BsaXI recognition site.

[0071] A single gene vector was used as a template for the generation of the different DNA blocks. It should be noted that any other source, such as genomic DNA or cDNA, is equally suitable. To extend the antibody chain genes with the appropriate R sites and BsaXI recognition sequences, primers with the respective additional sequences in the overhangs were used. Figure 6 shows the positions of the primers (solid lines) for generating the various DNA blocks. Gray arrows represent the primers with their binding sites and overhang sequences. Below the template sequences, depicted in solid lines, the final PCR products are displayed with their expected lengths.

[0072] To ensure that the PCR-amplified fragment would not self-ligate, the sequence of RE BsaXI was designed to have either a symmetric or palindromic cleavage site. The BsaXI cleavage site at the 3' end of the sequence was designed as three consecutive guanine bases, as shown below. TIFF0007801226000003.tif13136

[0073] When the PCR-generated DNA block was subsequently digested with BsaXI RE, the enzyme cleaved its recognition sequence from the entire fragment. This resulted in a CCC overhang sequence at the 3' end (antisense strand) of the DNA block in the form of a sticky end. Any remaining base pairs, represented by the letter N, were randomly occupied to prevent the formation of secondary structures in the primer sequence. The use of a PCR temperature of 61°C resulted in the most efficient amplification.

[0074] Cloning of the back vector was performed using the back base vector backbone, HindIII-hHC-R11, R11-adapter-BsaXI, and BsaXI-LC-R19. Cloning of the TI front vector was performed using three DNA blocks (HindIII-knob-R7, BsaXI-xLC-R9, and BsaXI-xLC-R1) connected via adapters (R7-adapter-BsaXI and R9-adapter-BsaXI). Therefore, DNA blocks for both vectors were generated by PCR. Preparative AGE followed by gel extraction was performed. Figure 7 shows the various PCR-amplified DNA blocks in an agarose gel after electrophoresis. Bands migrating at the expected size were extracted and purified from the agarose gel. The DNA blocks were then digested with the respective REs (HindIII, BsaXI) and purified.

[0075] To generate the adapter fragment, a template was created by isolating the CMV-intron A-5'-UTR sequence from an existing vector by restriction digestion, followed by gel AGE and finally gel extraction. The respective primer pair was used to generate the adapter fragment with a BsaXI recognition site.

[0076] According to the design of the BsaXI cleavage site of the DNA block for ligating various DNA blocks, a sticky end was introduced at the 3' end of the adapter sequence complementary to the CCC sticky end of the DNA block. The design of the BsaXI sequence in the adapter fragment is shown below, where the letter N represents any base A, T, G, or C. TIFF0007801226000004.tif15144

[0077] Subsequent digestion of the PCR-generated adapter with the RE BsaXI causes the enzyme to cleave its recognition sequence from the fragment, leaving a GGG overhang sequence at the 3' end of the adapter sequence, which allows for highly efficient ligation of the adapter and DNA block without the formation of by-products, since neither the adapter nor the DNA block can self-ligate.

[0078] Two different template concentrations per adapter were tested. Therefore, PCR reactions to generate three adapters were performed in duplicate, one containing 20 ng of template DNA and the other containing 10 ng of template DNA. As detectable in each agarose gel, the PCR reactions resulted in inefficient production of adapter fragments (2069, 2072, and 2067 bp) and amplification of nonspecific fragments beyond the target band. Furthermore, hybridized primers, generated by the approximately 100 bp long forward primer, were visible at the bottom of the agarose gel.

[0079] It was found that PCR resulted in adequate amplification when larger amounts of template DNA and different forward and backward primer concentrations were used.

[0080] Figure 8 shows the PCR results for 30 ng of template DNA and two different primer concentrations for each adapter fragment. A fixed amount of forward primer was used in the first chamber within each adapter (Figure 8A, B, C), while half the amount of forward primer was used in the second gel chamber. Increasing the template DNA and decreasing the forward primer eliminated hybridizing primers, reducing nonspecific fragments and improving the efficiency of adapter amplification. Further improvement was achieved by using lower primer concentrations, which resulted in thicker bands in each adapter PCR (second gel chamber for each sample A, B, C).

[0081] Therefore, it was found that using 30 ng of template DNA in combination with a forward primer used in half the amount of the reverse primer to generate the adapter fragments allowed for the amplification of suitable quantities and quality of adapters. It was further found that the suitable annealing temperature was approximately 64.6°C.

[0082] Two PCR reactions per adapter were separated by preparative AGE, yielding bright, isolated bands for each PCR-amplified adapter fragment at the expected sizes of 2069 bp for R7-adapter-BsaXI, 2072 bp for R9-adapter-BsaXI, and 2062 bp for R11-adapter-BsaXI. PCR-generated adapter fragments migrating at the expected sizes were excised and extracted.

[0083] Multiple ligations of linear fragments are the first step in the method according to the present invention. For the illustrative antibody format (knob-xLC-xLC-hole-LC), three parallel ligations (labeled as scissors) result in the final front vector, and two parallel ligations result in the final back vector. The final front vector requires ligation of the linear front base vector backbone to the first DNA block (HindIII-knob-R7), the first adapter (R7-adapter-BsaXI) to the second DNA block (BsaXI-xLC-R9), and the second adapter (R9-adapter-BsaXI) to the final DNA block (BsaXI-xLC-R1). The final back vector requires ligation of the digested back base vector backbone to the first DNA block (HindIII-knob-R7) and the R11-adapter-BsaXI to the second DNA block (BsaXI-LC-R19). For all ligations, the mass of the larger fragment was used to calculate the mass of the second fragment at a 1:1 molar ratio.

[0084] For ligation, the front-based vector backbone was digested with RE PspOMI and HindIII, subsequently dephosphorylated in the same reaction, and finally purified using a purification kit. After ligation, all samples were separated by preparative AGE, followed by extraction of the correctly ligated fragments. The target ligation product, backbone-front-knob-R7, migrated at 8215 bp, while the unligated linear backbone-front migrated at less than 6009 bp. The band of the unligated single DNA block (HindIII-knob-R7) migrated at 2210 bp, and the self-ligated DNA block (R7-knob-knob-R7) migrated at 4420 bp. Ligation of R7-adapter-BsaXI with the DNA block (BsaXI-xLC-R9) resulted in a bright, discrete band (R7-adapter-xLC-R9) migrating at the correct size of 2913 bp. The lower band is the single unligated fragment R7-adapter-BsaXI (2038 bp) and the DNA block BsaXI-xLC-R9 (878 bp), without any self-ligated fragments as by-products. The same is shown in Figure 9C, where the framed band migrating at 2914 bp represents the target ligation product R9-adapter-xLC-R1, the product of ligation of R9-adapter-BsaXI (2041 bp) and the DNA block BsaXI-xLC-R1 (876 bp) without any self-ligation. In each gel chamber, two identical ligation mixtures were combined and separated.

[0085] For ligation, the backbone base vector backbone was digested with REs PspOMI and HindIII, subsequently dephosphorylated in the same reaction, and finally purified via a purification kit. After ligating the backbone to the first DNA block (HindIII-hole-R11) and the adapter (R11-adapter-BsaXI) to the second DNA block (BsaXI-LC-R19), the reaction was analyzed by preparative AGE, and the target ligation product was finally purified by gel extraction. As seen in Figure 10A, ligation resulted in a bright, isolated band at the expected size of 7,587 bp for the target ligation product (backbone-backbone-hole-R11). The unligated linear backbone backbone is also represented by a bright, isolated band at 6,062 bp. A single unligated DNA block (HindIII-hole-R11) appears at a size of 1528 bp, and the self-ligated fragment appears at 3056 bp (R11-hole-hole-R11). In the second ligation (Figure 10B), a bright, isolated band appeared at the expected size of 2842 bp for the target ligation product R11-adapter-LC-R19. The unligated single fragment band migrates at 2031 bp for R11-adapter-BsaXI and 814 bp for BsaXI-LC-R19. The bands of the target ligation products (backbone-back-hole-R11 and R11-adapter-LC-R19) migrating at the expected sizes were excised and finally extracted and purified from the gel slice.

[0086] In summary, ligation of the newly generated fragment with the BsaXI cleavage site is efficient and results in high yields of the targeted ligation product.

[0087] In the assembly reaction, the various ligated fragments are finally recombined with their corresponding fragments that share the same R site. Thus, all extracted and purified ligation products for the corresponding vectors were assembled in one reaction.

[0088] The amount of DNA in each assembly reaction was calculated to give a total DNA amount of 0.2 pmol for all fragments, taking into account a vector:insert molar ratio of 1:2 for this calculation.

[0089] After assembly, chemically competent bacterial cells were transformed with the TI vector, followed by plasmid preparation of clones found on selective plates. The final vectors isolated were then tested for accuracy by control digestion and Sanger sequencing of plasmids containing the correct band pattern after AGE.

[0090] For the final front vector assembly reaction, three fragments were required according to the TCB configuration. The ligation product was backbone-front-knob-R7, R7-adapter-xLC-R9, and finally R9-adapter-xLC-R1. Each fragment terminated with the first R site of the next fragment. All ligation fragments were combined in a single assembly reaction. Two types of chemically competent cells were then transformed with the assembly products and spread onto agar plates containing selective pressure. As a positive control, a predetermined assembly control mixture was assembled in parallel and transformed into cells, while assembly and subsequent transformation with water served as a negative control. After overnight incubation, clones carrying the circular plasmid encoding the selectable marker were grown. The positive control plate overgrown more than 1,000 colonies, while no colonies were observed on the negative control plate. Selection plates following transformation of 5-alphaF'Iq competent E. coli bacteria yielded 18 isolated single colony-forming units (CFUs). Fewer but thicker colonies were seen on the plates after transformation of 10-beta competent E. coli bacteria.

[0091] A total of 10 clones from the transformation containing the final assembled front vector were picked and cultured for plasmid preparation. Test digestion of the isolated plasmids with three enzymes (HindIII, PvuI, and AfeI) followed by AGE was performed to determine which clones contained the desired TI front vector containing all three assembled fragments in the correct arrangement. From plate 1 (5-alpha F'Iq competent E. coli bacteria), we were able to isolate clones that displayed five bands with the expected sizes of 4612 bp, 3346 bp, 2836 bp, 1642 bp, and 1374 bp. From plate 2 (10-beta competent E. coli bacteria), none of the three grown colonies displayed the correct band pattern.

[0092] The sequence identity of one of the correct final front clones was verified by sequencing each sequence. The sequencing results confirmed that the correct final front vector was generated by the new cloning method according to the present invention.

[0093] The assembly reaction for the final back vector required two ligation fragments according to the TCB configuration. The ligation products were backbone-back-hole-R11 and R11-adapter-LC-R19. These were combined in a single assembly reaction. Two different bacterial strains were transformed with the assembly products and spread onto plates containing selective pressure. The same positive and negative controls as for the final front vector were used. As expected, after incubation, the positive control plate showed over 1,000 colonies, while the negative control plate showed none. The transformed 10-beta-competent E. coli bacteria again produced fewer but thicker growing colonies. A total of 20 clones were picked for plasmid preparation.

[0094] To verify which clones contained correctly assembled plasmids, the concentrated plasmids were digested with the enzymes HindIII, EcoRI, and PacI and analyzed by preparative AGE. A clone was considered correct if four bands appeared at sizes of 5668 bp, 2776 bp, 1433 bp, and 422 bp. Nearly 90% of the tested transformants could have the correct vector based on the resulting band pattern. For the transformation of 5-alpha F'Iq competent E. coli bacteria, control digestion of 7 out of 10 clones yielded the correct band pattern at the correct size. For the transformation of 10-beta competent E. coli, 9 out of 10 clones were found to be likely correct.

[0095] The sequences of selected plasmids were then determined by Sanger sequencing and compared to the reference sequence. Alignment of the sequenced vector regions with the reference sequence resulted in 100% coverage of both sequences.

[0096] Compared to 10-beta competent E. coli bacteria, 5-alpha F'Iq competent E. coli bacteria are more efficient in the transformation process and result in more colonies on plates. This is particularly advantageous when more than one insert is involved in the assembly reaction. Therefore, it is recommended to use 5-alpha F'Iq competent E. coli for the subsequent cloning of TI vectors consisting of three or more genes for antibody chains in the form of DNA blocks.

[0097] Overview and Opinion Herein we report a modular and efficient method for cloning multigene plasmids that involves only a single cloning step without the use of a pre-existing single-gene vector.

[0098] In the first cloning step, the DNA blocks produced by PCR carrying the genes for the different antibody chains are ligated with their respective adapters, which act as connectors for the different DNA blocks.

[0099] This method is based on the ligation of individual fragments, i.e., backbone, DNA block, and adapter, via an RE cleavage site located at one end of each fragment. It has been found that an RE that does not generate symmetric or palindromic cleavage sites must be used, thereby reducing or even eliminating the generation of self-ligation by-products.

[0100] In the second step, the ligated fragments from the first step are assembled in the correct orientation via a unique R site, DNA block, and adapter fragment located at one end of each scaffold.

[0101] This new strategy provides a modular and efficient method, allowing expression cassettes to be cloned directly into the respective backbones without intermediate cloning steps, enabling rapid cloning of the variable gene constructs of diverse antibody formats. This new cloning method according to the present invention offers considerable advantages in terms of time, labor, and cost.

[0102] Comparative Example In a comparative example, front and back vectors for targeted integration encoding bispecific antibodies were cloned by the cloning method according to the present invention, but using a type IIP RE with symmetric target and cleavage sites, i.e., HindIII. The BsAb consists of two heavy chains (knob and hole) and two light chains, one of which is crossed. The front vector encodes the crossed light chain (xLC) and knob heavy chain (kHC), and the back vector carries the genes for the light chain (LC) and hole heavy chain (hHC) (xLC-kHC-LC-hHC).

[0103] In the first step, a primer pair with a 3'-overhang sequence is used, thereby adding an overhang sequence to the unique binding site. To generate fragment 1 in Figure 4, a template with the sequence CMV-intron A-5'-UTR-HindIII was used and amplified with primer pair oSA105 / 106. Fragments 2 and 3 were amplified from a DNA source with a polyA sequence by using primer pairs oSA107 / 108 and oSA109 / 108, respectively. Figure 11 shows the binding sites and sequence overhangs of the primers, templates, and amplified PCR products, along with their expected lengths.

[0104] The PCR-generated fragments were analyzed using analytical agarose gel electrophoresis (AGE). Compared to DNA markers, all fragment sizes were consistent with the predicted fragment lengths of 1663 bp for fragment 1, 421 bp for fragment 2, and 413 bp for fragment 3.

[0105] All PCR-amplified fragments were flanked by RE recognition sites, potentially making them susceptible to subsequent integration into the vector backbone. Therefore, PCR samples were digested with the respective restriction enzymes (BamHI / PspOMI, PspOMI / PacI) and subsequently purified. To prevent self-ligation of the vector, the backbone was digested with the REs BamHI and PacI and then dephosphorylated. All digested inserts were then ligated into the respective backbone. Chemically competent cells were transformed with the ligation products and plated on agar plates containing selective pressure. Sequencing of each sequence was used to verify the sequence identity of one of the clones with the correct TI base vector. Finally, the vector was concentrated in a plasmid preparation and used as the vector backbone.

[0106] DNA blocks flanked by HindIII recognition sites and their respective R sites, as well as adapters serving as connectors for each DNA block, were then generated by PCR. According to this method, individual fragments—i.e., the vector backbone carrying the first DNA block and the adapter carrying the second DNA block—were ligated via HindIII cleavage site ligation. The ligated fragments were then assembled in a single reaction. To generate the individual DNA blocks, vectors carrying the respective antibody chain genes were used as PCR templates. It should be noted that any other source, such as genomic DNA or cDNA, is equally suitable. Specific primer pairs were used to extend the template sequence containing the HindIII cleavage site and the individual R site sequences. The positions of the primers (thick blue arrows) and the final amplification products (solid blue lines) are shown in Figure 12. It was confirmed that an annealing temperature of 61°C was the most efficient for all DNA blocks, resulting in isolated bright bands in AGE.

[0107] Thus, the DNA blocks HindIII-xLC-R15 and HindIII-knob-R1 were produced by PCR. In Figure 13, a single bright band appeared at the expected size of 1496 bp for the DNA block (HindIII-xLC-R1) (left gel in Figure 13) and 861 bp for the DNA block (HindIII-xLC-R15) (right gel in Figure 13).

[0108] To generate the adapter, a template was prepared by isolating the sequence CMV-intron A-5'-UTR from an existing vector using RE BstEII. It should be noted that any other source, such as genomic DNA or cDNA, is also suitable. The different fragments were separated by AGE and then extracted from the gel. A specific primer pair with one binding site in the template fragment was preferably used. The overhang sequence of the forward primer encodes the sequence for a unique R site, and the reverse primer encodes a HindIII cleavage site at the binding site. It was determined that the most efficient amplification of the fragment sequence occurred at 64.6°C.

[0109] In this way, the R15-adapter was generated by PCR. Analytical AGE was performed using a portion of the PCR reaction product (Figure 14). A bright band with no by-products appeared at the expected size of 2045 bp. The remaining PCR reaction mixture was digested with HindIII RE and purified.

[0110] The first cloning step was the ligation of the various fragments. Due to this antibody format and construction, two simultaneous ligations were required: the ligation of the front vector backbone with the first DNA block (ligation 1) and the ligation of the adapter and the last DNA block (ligation 2). In both ligations, 500 ng of the larger fragment was applied, and the mass of the second fragment was calculated at a 1:1 molar ratio.

[0111] First, for ligation 1, the front vector backbone was digested with PspOMI and HindIII, dephosphorylated in parallel in the same reaction, and then purified using a commercially available purification kit according to the manufacturer's instructions. After ligation, all samples were separated by preparative AGE to extract the correct ligated fragment. Four isolated bands appear in the first gel chamber (Figure 15). Two faint bands migrating at the bottom of the gel could be assigned to the unligated fragment R15-xLC-HindIII (856 bp) and the self-ligated fragment R15-xLC-xLC-R15 (1712 bp). The target ligation product (backbone-front-xLC-R15) is the largest fragment, thus the upper bright band at 6857 bp, with the unligated linear backbone front just below. The bands of Ligation 2 in the second gel chamber are as follows: the three major products are R15-adapter-HindIII (2038 bp) and HindIII-knob-R1 (2982 bp) as unligated single fragments and the self-ligated HindIII-knob-R1 fragment (R1-knob-knob-R1); the target ligation product (R15-adapter-knob-R1) is represented by a faint band at 3525 bp. Thus, Ligation 2 using a type IIP RE with symmetric target and cleavage sites, i.e., HindIII, is not efficient, as indicated by the large number of side products.

[0112] Without being bound by this theory, it is hypothesized that the rapid rate of self-ligation is caused by the symmetric cleavage site of the HindIII RE. After cleavage of the palindromic recognition site, a 5'AGCT overhang is generated that is complementary to the overhang of the same fragment in a doubly rotated orientation.

[0113] Furthermore, without being bound by this theory, a possible explanation for the bands appearing in ligation 2 (which cannot be assigned to defined fragments) is that in the PCR for fragment production, primers additionally bind to the R site, thus amplifying fragments of different sizes. In subsequent ligations, non-specific PCR fragments result in various ligated fragments of different sizes. This theory would also explain the smearing that appears along the gel chamber.

[0114] The ligation of the adapter and DNA block was optimized through several iterations (data not shown), whereby each ligation was inefficient and produced side products. Optimization experiments included different PCR templates, pre-PCR heating, different ligases, and ligation times.

[0115] To summarize the results of this comparative example, the use of type IIP endonucleases, namely the properties of the HindIII cleavage site, resulted in symmetric cleavage sites, which allowed the DNA blocks and adapters to self-ligate.

[0116] As a result, the amount of target ligation product required for the subsequent assembly reaction could not be achieved. ***

[0117] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Brief explanation of the drawings]

[0118] [Figure 1] Scheme of the two-plasmid RMCE strategy involving the use of three RRS sites to simultaneously perform two independent RMCEs. [Figure 2] Schematic DNA assembly reaction. [Figure 3]Cloning strategy according to the present invention illustrated by the specific antibody xLC-knob-LC-hole. A) The TI-front vector backbone contains the initial sequence of the first DNA block and the terminal polyA sequence of the last DNA block. The vector is opened via PspOMI and the second RE, exposing the cleavage site of the second RE and the R1 site. In the first step, the cleavage site of the second RE (labeled with scissors) is ligated in a separate reaction. Second, the two ligated fragments are assembled together (labeled as a cross). B) The TI-back vector backbone also contains the initial and terminal sequences of the DNA blocks and is opened via PspOMI and the second RE, exposing the cleavage site of the second RE and the R1 site. The cloning procedure is the same for both vectors. [Figure 4] Vector backbone design and restriction cloning. Fragment 1 encodes the first sequence of the gene expression cassette from the first antibody chain and is used in both targeted integration vector backbone examples. Fragments 1 and 2 are distinguished only by their R site sequences. The front vector backbone is composed of the basic front vector backbone and PCR-generated fragments 1 and 2. The vector backbone carries a multiple cloning site (MCS) flanked by two Lox sites (L3 and LoxFAS, respectively) and RE recognition sites (BamHI and PacI) for subsequent ligation with fragments. [Figure 5] An exemplary TCB knob-xLC-xLC-hole-LC cloning strategy is shown. A) The front-based vector backbone, containing the initial sequence of the first DNA block and the terminal polyA sequence of the last DNA block, is opened via RE PspOMI, exposing a HindIII cleavage site and an R1 site. In the first step, the RE cleavage sites (labeled with scissors) are ligated in separate reactions. Second, the ligated three fragments are assembled together (labeled as crosses). B) The back-based vector backbone, containing the initial and terminal sequences of the DNA blocks, is opened via PspOMI, exposing a HindIII cleavage site and an R1 site. [Figure 6]Design and application of primers for DNA block production and corresponding binding sites, overhangs, templates, PCR products, and expected fragment lengths. A) HindIII-knob-R7 was amplified from the single gene vector using primer pair oSA110 / 159. B) HindIII-hole-R11 was amplified from the single gene vector using primer pair oSA110 / 156. C) BsaXI-xLC-R9 was amplified from the single gene vector using primer pair oSA172 / 158. D) BsaXI-xLC-R1 was also amplified from the single gene vector using primer pair oSA172 / 157. E) BsaXI-LC-R19 was amplified from the single gene vector using primer pair oSA172 / 163. [Figure 7] AGE of DNA blocks produced by PCR. Each PCR reaction was set up in a volume of 50 µL containing 10 ng of template and 2 µL of each primer (10 µM). All DNA blocks are represented as bright bands with the expected sizes: A) 2215 bp for DNA block HindIII-knob-R7; B) 1533 bp for DNA block HindIII-hole-R11; C) 907 bp for DNA block BsaXI-xLC-R1; D) 909 bp for DNA block BsaXI-xLC-R9; and E) 845 bp for DNA block BsaXI-LC-R19. The expected fragments were compared with a 1 kb-plus-DNA marker. [Figure 8]Preparative AGE of PCR-generated adapters amplified with two different forward primer concentrations. Each adapter was amplified using 30 ng of template DNA with 1 µL of forward primer (10 µM) in the first round (first chamber for each sample A, B, and C) and 0.5 µL of forward primer in the second round (second chamber for each sample A, B, and C). The reverse primer was kept constant at 1 µL. Expected fragment sizes were A) 2069 bp for R7-adapter-BsaXI; B) 2072 bp for R9-adapter-BsaXI; and C) 2062 bp for R11-adapter-BsaXI, compared to the 1 kb-plus-DNA marker on the left. [Figure 9] Ligation of linear DNA fragments of the front vector. Each ligation was performed using 500 ng of the larger fragment and the calculated amount of each second fragment at a 1:1 molar ratio. Two identical ligation mixtures were combined in each gel chamber. All bands were compared to the 1 kb-plus-DNA marker on the left: A) The target ligation product, marked with a blue frame, migrates at 8215 bp, the unligated backbone at 6009 bp, the DNA block HindIII-knob-R7 at 2210 bp, and the self-ligated fragment at 4420 bp. B) The target band, marked with a blue frame, migrates at 2913 bp, the single unligated R7-adapter-BsaXI at 2038 bp, and the unligated DNA block BsaXI-xLC-R9 at 878 bp. C) The target band runs at 2914 bp, the unligated fragment R9-adapter-BsaXI runs at 2041 bp, and the DNA block BsaXI-xLC-R1 runs at 876 bp. [Figure 10]Ligation of linear DNA fragments to back vectors. Each ligation was performed using 500 ng of the larger fragment and the calculated amount of each second fragment at a 1:1 molar ratio. All expected bands were compared to the 1 kb-plus-DNA marker on the left: A) The target ligation product (backbone-hole-R11), framed in blue, migrates at 7587 bp, while the unligated backbone migrates below 6062 bp. A single unligated DNA block (HindIII-hole-R11) migrates at 1528 bp, and the self-ligated fragment migrates at 3056 bp. B) The target ligation product R11-adapter-LC-R19, framed in blue, migrates at 2842 bp; the single unligated fragment R11-adapter-BsaXI migrates at 2031 bp, and BsaXI-LC-R19 migrates at 814 bp. [Figure 11] Design and application of primers for fragment production and corresponding binding sites, overhangs, templates, PCR products, and expected fragment lengths. A) Fragment 1 was amplified from a template encoding the CMV-intron A-5'-UTR sequence using primer pair oSA105 / 106. B) Fragment 2 was amplified from a template containing a polyA sequence by using primer pair oSA107 / 108. C) Fragment 3 was amplified from the same template as fragment 2 by using primer pair oSA109 / 108. [Figure 12]Design and application of primers for fragment production and corresponding binding sites, overhangs, templates, PCR products, and expected fragment lengths. A) HindIII-xLC-R15 was amplified from the single gene vector by using the primer pair oSA110 / 155. B) HindIII-knob-R1 was amplified from the single gene vector by using the primer pair oSA110 / 161. C) HindIII-LC-R9 was amplified from the single gene vector by using the primer pair oSA110 / 158. D) HindIII-hole-R19 was amplified from the single gene vector by using the primer pair oSA110 / 163. [Figure 13] Analytical AGE of DNA blocks produced by PCR. Expected fragments are compared to the 1 kb-plus-DNA marker on the left. Left gel: DNA block HindIII-knob-R1 migrates with the expected size of 1496 bp. Right gel) DNA block HindIII-xLC-R15 migrates with the expected size of 861 bp. [Figure 14] Analytical AGE of R15-adapter-HindIII PCR products. Each PCR reaction was set up in a 50 μL volume containing 10 ng of template and 2 μL of each primer (10 μM), allowing for a small volume of reaction mixture to be used for analytical AGE. All DNA blocks are represented as bright bands at the expected size of 2045 bp, as compared to the 1 kb-plus-DNA marker on the left. [Figure 15]Preparative AGE of ligations 1 and 2. Ligation 1 represents the ligation of the digested TI front base vector with the HindIII-xLC-R15 DNA block, and ligation 2 is the ligation of the R15-adapter-HindIII with the second DNA block, HindIII-knob-R1. Expected fragments are compared to the 1 kb-plus-DNA marker. A) The target ligation product of ligation is backbone front-xLC-R15 with the expected size of 6857 bp. The unligated linear TI front base vector migrates at 6009 bp. The unligated single HindIII-xLC-R15 fragment migrates at 856 bp, and the self-ligated fragment migrates at 1712 bp. B) The target ligation product R15-adapter-knob-R1 migrates at the expected size of 3525 bp, the unligated HindIII-knob-R1 migrates at 1491 bp, and the self-ligated fragment migrates at 2982 bp. The single unligated fragment R15-adapter-HindIII migrates at 2038 bp, and the self-ligated fragment migrates at 4076 bp.

[0119] Explanation of Abbreviations 5'-UTR 5'-untranslated region AGE agarose gel electrophoresis Amp Ampicillin att B Attachment site B BsAb bispecific antibody CLD cell line development dNTP deoxyribonucleotide triphosphate dsDNA double stranded DNA GFP Green Fluorescent Protein GOI Gene of Interest HCL host cell line HyTK Hygromycin-thymidine kinase KiH Knob-into-Hole MCS multiple cloning site LB Luria-Bertani RE restriction enzyme RMCE Recombinase-Mediated Cassette Exchange R site Recombination site TCB T cell bispecific antibody TCR T cell receptor TI Targeted Integration

[0120] Array Description TIFF0007801226000005.tif222142TIFF0007801226000006.tif236142TIFF0007801226000007.tif116142

[0121] Plasmid Description TIFF0007801226000008.tif145142

[0122] Explanation of cited references, etc. TIFF0007801226000009.tif138141

[0123] material TIFF0007801226000010.tif181162TIFF0007801226000011.tif141142All enzymes provided by New English Biolabs®, preferably purchased in high fidelity versions; *N=A / T / G / C. TIFF0007801226000012.tif42128

[0124] method Primer design Primers are single-stranded oligonucleotides approximately 18–30 nucleotides long. Because primer design is a critical parameter for PCR success, it follows several guidelines. To amplify a specific DNA sequence without generating any nonspecific by-products, it is important to create a primer pair that binds to only one specific site on the template DNA. A primer pair consists of a forward primer (5'-3' sense strand) and a reverse primer (5'-3' antisense strand). When the primer pair anneals to the template DNA, polymerase extends the sequence between them, amplifying the DNA strand between the annealed sites. A 40–60% GC content and a terminal C or G at the 3' end of the primer sequence enhance priming specificity and efficiency

[13] ,

[14] .

[0125] The use of specific primers generates DNA fragments that can be used in subsequent cloning steps. In addition to the binding site, these primers contain recognition sequences for restriction enzymes and / or recombination sites (R sites) in their overhangs.

[0126] Restriction digestion All restriction enzymes were purchased from NEB, and only high-fidelity variants were used according to the manufacturer's instructions.

[0127] Digestion of PCR-generated fragments Using designed PCR primers, restriction enzyme recognition sites were added to one end of the fragments. For subsequent ligation, the fragments required single-stranded overhangs, which were generated via digestion with the respective restriction enzymes. For this purpose, restriction digests were prepared directly in the PCR reaction mixture. The following table shows a standard protocol for this purpose. Exemplary PCR digestion conditions for PCR fragments TIFF0007801226000013.tif34128

[0128] After incubation at 37°C for 15 minutes to 1 hour, the samples were purified via AGE or directly via the Zymogene DNA Clean & Concentrator kit.

[0129] Preparative restriction digestion and AGE Digestion reactions were prepared as described in the table below. If several enzymes were used in a single digest, the volume of PCR-grade water was adjusted accordingly. Reactions were set up on ice and incubated in an Eppendorf thermal block at a constant temperature of 37°C for 15 minutes to 1 hour. Exemplary conditions for preparative restriction digestion mix TIFF0007801226000014.tif41128

[0130] Each sample was then mixed with purple loading dye and transferred to a 1% agarose electrophoresis gel.

[0131] Restriction digestion and AGE analysis Analytical restriction digestion was used to screen for correct clones after transformation. Therefore, enzymes that cleave DNA into fragments of different sizes were selected. A restriction digest mix was prepared according to the preparative digestion. After separating the samples on a 1% agarose gel, the lengths of the different fragments were analyzed and compared with DNA markers and the expected calculated lengths.

[0132] polymerase chain reaction PCR is used for the enzymatic in vitro amplification of specific DNA fragments from DNA templates. The reaction can be divided into three essential steps, which are repeated cyclically several times. The first step is "denaturation," which involves raising the temperature to 95-98°C to melt double-stranded DNA. The DNA is then available as two single strands. The second step is "annealing," which allows hybridization of the two fragment-flanking primers to their 3' ends by lowering the temperature to 55-65°C. Finally, enzymatic extension of the primers occurs in "extension," in which the polymerase synthesizes complementary strands in the 5'-3' direction at 72°C. After each cycle, the amplified product is again available as a template. Therefore, the growth is exponential

[14] .

[0133] The table below shows the standard programming of the thermal cycler for this project.

[0134] Standard programming of the thermal cycler for PCR. TIFF0007801226000015.tif43128

[0135] PCR conditions vary depending on the length and interaction of the primers and template.

[0136] PCR using a temperature gradient To provide efficient PCR, the desired annealing temperature, T, of both primers must be A It is important to know the temperature. For this purpose, samples are tested at different temperatures by setting up a temperature gradient in the thermal cycler. The center of the gradient is usually the melting temperature T of both primers. M The table below shows the standard protocol for the temperature gradient PCR reaction used in this project. Programming the thermal cycler with a temperature gradient: TIFF0007801226000016.tif97142

[0137] For both PCR reactions, the extension time for each cycle was adjusted to the longest expected DNA fragment.

[0138] PCR purification Although the components of the PCR mix are essential for the PCR reaction, they can inhibit subsequent modification steps such as restriction digestion and ligation

[15] . Therefore, the amplified fragments must be isolated from the PCR reaction mixture. Purification was performed using the Zymogene DNA Clean & Concentrator Kit. The PCR reaction mixture was diluted with 5 volumes of binding buffer provided with the kit. In combination with guanidine salt, DNA selectively binds to a special glass fiber pre-packed in the column. The bound DNA was isolated by washing twice with wash buffer to remove short primers, dNTPs, enzymes, short failed PCR products, and salts. Finally, the DNA fragments were eluted using a low-salt solution

[16] . The purity and concentration of the eluted DNA were measured using a Nanodrop spectrophotometer.

[0139] Gel extraction DNA was extracted from agarose gels using the Zymoclean Gel DNA Recovery Kit. Gel slices containing the desired fragments were excised from the agarose gel and placed in 2 ml Eppendorf cups. The gel slices were dissolved in lysis buffer at 50°C in a thermal block. The solution was then pipetted onto the column in a tube and run at 13,000 rpm for 1 minute. The DNA fragments bound to the silica membrane, while other components, such as salts, enzymes, agarose, and other impurities, passed through the column and were discarded. The column was washed and centrifuged twice with ethanol-containing wash buffer. Finally, a variable volume of elution buffer was pipetted directly onto the column bed. A 1-minute incubation ensured higher concentrations. After the DNA was eluted from the matrix, the final concentration and quality were measured using a Nanodrop instrument.

[0140] Agarose gel electrophoresis Gel electrophoresis separates DNA by size for visualization or purification. Agarose MP was added to 1x TAE buffer at a final concentration of 0.7–1.2% and dissolved by heating in a microwave. Meanwhile, a well comb was placed in a gel tray. The addition of ethidium bromide to the agarose mixture allows for subsequent visualization of the DNA. The mixture was transferred to a tray and allowed to solidify completely. The solid gel was then placed in an electrophoresis chamber and filled with 1x TAE buffer until it reached the set line. A DNA ladder was pipetted into the first chamber of the gel by default. Samples containing loading dye were transferred to different wells, and electrophoresis was performed at 110 V for 1.5 hours. After electrophoresis, the gel was removed from the chamber and analyzed using a UV-Imager. The length of the separated DNA was estimated using DNA markers.

[0141] Assembly reaction and transformation for the final vector The assembly reaction was performed using the NEBuilder® HiFi DNA Assembly Cloning Kit. The appropriate amount of DNA for each fragment was calculated based on the equation provided in the supplier's protocol. For the calculation, a total of 0.2 pmol of DNA fragments and a vector:insert molar ratio of 1:2 were selected. TIFF0007801226000017.tif10128

[0142] The following table shows an exemplary pipetting scheme: Exemplary Assembly Reaction Mixtures TIFF0007801226000018.tif59134

[0143] After settling, the reaction mixture was incubated at 50°C for 1 h, and then 2 μL of the assembled fragment was transformed into NEB 10-beta competent E. coli cells.

[0144] Chemical transformation For chemical transformation, E. coli cells pretreated with calcium chloride were used. Cells stored in a -80°C freezer were thawed on ice for approximately 10 minutes. Then, 2 ml of the ligation or assembly mixture was pipetted directly onto the 50 μL cell mixture and placed on ice for 30 minutes. Positive and negative controls were also prepared simultaneously. After incubation, the cells were heat-shocked at 42°C for exactly 30 seconds and then placed on ice for 2 minutes. 250 ml of pre-warmed SOC medium was added to the transformation mixture and incubated at 37°C and 300 rpm for 1 hour to allow the cells to recover. Meanwhile, selection plates were warmed to 37°C in an incubator. Finally, 100–200 μL of the culture was pipetted onto LB-Amp agar plates and spread with plating beads. The plates were incubated overnight at 37°C. Only clones that had integrated the plasmid carrying the ampicillin resistance gene could withstand the selection pressure.

[0145] DNA (mini) preparation Plasmid DNA preparations were used to confirm that clones growing on the selection plates contained the correct plasmid. Purification was then performed using the QIAprep Spin Miniprep Kit. A single growing colony was picked with a pipette tip and inoculated into a 3 ml culture of ampicillin-containing LB medium, which was then incubated overnight at 37°C. The next day, 2 ml of the culture was pipetted into a tube and centrifuged. The bacterial pellet was resuspended in alkaline Tris buffer, and the suspension was clarified by adding neutralization buffer and centrifugation. The lysate was then transferred to a silica membrane column and placed in a tube. In the presence of high salt concentrations, DNA adsorbs to the silica membrane. Impurities were removed by washing with wash buffer. After discarding the flow-through, the column was emptied and centrifuged to remove any remaining buffer. Finally, the plasmid DNA was eluted with elution buffer or water, and its purity and concentration were measured using a Nanodrop instrument.

[0146] DNA (maxi) preparation After obtaining positive clones, the amount of DNA was expanded to ensure sufficient DNA was received in the transfection mixture. DNA maxi-preparation was performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF Kit. To do this, a small amount of the culture from the miniprep tube was transferred and inoculated into 300 ml of LB medium containing 0.5 g / L ampicillin. The suspension was incubated overnight at 37°C and 200 rpm. The next day, the LB-Amp medium appeared opaque due to the density of the grown bacteria. To separate the bacteria from the medium, the culture was centrifuged at 5600 x g in an SLC-3000 rotor for 15 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended and dissolved in alkaline lysis buffer. The bacterial lysate was clarified by adding denaturation buffer and loaded onto an equilibrated column where the plasmid DNA binds to the anion exchange resin. A first wash step using wash buffer was performed using an inserted filter to wash the remaining lysate from the column. After washing with buffer 1 to remove endotoxin and buffer 2 to remove contaminants, the plasmid DNA was eluted with high-salt elution buffer. Plasmids were precipitated by the addition of isopropanol and isolated as a pellet after a 30-minute centrifugation step at 4200 × g. The pellet was dissolved in 500 μL of the final buffer. DNA purity and concentration were measured using a Nanodrop spectrophotometer.

Claims

1. a linear expression vector backbone, a promoter and a first single-stranded enzyme restriction site for a first restriction enzyme at its 3' end, - a single-stranded recombination site at its 5' end the linear expression vector backbone comprising a first DNA block, in the 5' to 3' direction: a first single-stranded enzyme restriction site for a first restriction enzyme, - a nucleic acid encoding a protein of interest, - first single-stranded recombination site the first DNA block comprising: a first adapter nucleic acid, in the 5' to 3' direction: - a first single-strand recombination site, - poly A signal sequence, - promoters, - a second single-stranded enzyme restriction site for a second restriction enzyme the first adaptor nucleic acid comprising a second DNA block, in the 5' to 3' direction: a second single-stranded enzyme restriction site for a second restriction enzyme, - a nucleic acid encoding a second protein of interest, - second single-stranded recombination site the second DNA block comprising with DNA ligase, the first single-stranded enzyme restriction site is different from the second single-stranded enzyme restriction site; the second single-stranded enzyme restriction site is for a type IIB restriction enzyme; the first single-stranded recombination site and the second single-stranded recombination site are different; the second single-stranded recombination site and the recombination site at the 3' end of the vector backbone are identical; each recombination site is a nucleic acid sequence 15-80 bp in length that is unique to the incubated nucleic acid; the first single-stranded enzyme restriction site of the linear expression vector backbone can specifically hybridize with the first single-stranded enzyme restriction site of the first DNA block, the first single-stranded recombination site of the first DNA block can specifically hybridize with the first single-stranded recombination site of the first adaptor nucleic acid, the second single-stranded enzyme restriction site of the first adaptor nucleic acid can specifically hybridize with the second single-stranded enzyme restriction site of the second DNA block, and the second single-stranded recombination site of the second DNA block can specifically hybridize with the single-stranded recombination site of the linear expression vector backbone.

2. 2. The method of claim 1, wherein the second single-stranded enzyme restriction site is a BsaXI restriction site.

3. The method according to any one of claims 1 to 2, wherein the target protein is an antibody chain.

Images