method

Abstract

The present invention relates to site-specific DNA recombinase enzymes comprising a nuclear localisation signal (NLS). More specifically, the present invention relates to a Bxb1 serine integrase comprising an NLS and methods of use thereof, in particular for antibody display and expression

Description

[0001] METHOD

[0002] Field of the Invention

[0003] The present invention relates to site-specific DNA recombinase enzymes comprising a nuclear localisation signal (NLS). More specifically, the present invention relates to a Bxbl serine integrase comprising an NLS and methods of use thereof, in particular for antibody display and expression.

[0004] Background to the Invention

[0005] Different display platforms have become the cornerstone of modem antibody and protein engineering campaigns. There are many different platforms, such as phage display, mammalian display, yeast display and bacterial display, which are based on the display of a protein on the surface of the cell or bacteriophage. Among various display platforms, phage display is still the most widely used for antibody and protein engineering due to its simplicity, speed and ability to display large libraries. However, therapeutic antibodies generated with phage display often show poor biophysical properties compared to antibodies generated from immunizations. Mammalian display, on the other hand, possesses characteristics that gives it the ability to produce antibodies with good developability profiles, as has been demonstrated by improving the poor biophysical properties of a phage display-matured antibody. In addition, mammalian display has the ability to display full-length IgGs with mammalian glycosylation, thus making it more attractive for therapeutic antibody discovery than phage, bacterial, and yeast display. Genes can be stably transfected into mammalian cells using virus vectors. However, such methods will insert the gene into a random location and risk insertion of multiple gene copies in a single cell.

[0006] For sound construction of mammalian display libraries, each library variant should be stably integrated into the genome of the cell to defined locus so that each cell harbors only one library variant in the same genomic locus. This makes it possible to perform multiple selection rounds for the libraries, compare expression levels of the library variants and to avoid enrichment of unwanted “passenger” variants during selection. One way to achieve stable, single copy integration of a gene is to use so called “landing pad”, which is first integrated into the genome of an organism, and then the gene of interest is integrated into the landing pad by using serine or tyrosine integrases, which are site-specific DNA recombinase enzymes. Of the different integrases, Mycobacteriophage Bxbl serine integrase is one of the most used integrases for stable integration of genes due to its high accuracy and efficiency. Bxbl serine integrase catalyses site-specific recombination between attP and attB sites, which can be utilized for stable integration of a gene of interest into the genome when a vector carrying the gene of interest contains an attP site and the landing pad contains an attB site. If there is only one attB site in the genome and one attP site in the vector, the whole plasmid will be integrated into the genome. It is also possible to integrate only the gene of interest without the vector through recombinase-mediated cassette exchange (RMCE) when a segment in the genome is flanked by two different attB sites and the gene of interest in the vector is flanked by two different attP sites.

[0007] However, stable integration of a gene into the genome of an organism is an inefficient process and many different approaches have been explored in order to enhance the efficiency of Bxbl -mediated stable integration of genes, including continuous expression of Bxbl by integrating it into the landing pad, optimal use of restriction sites, and optimization of transfection. In order to carry out its function, Bxbl has to get to the nucleus of the cell. Proteins less than 40 kDa can passively diffuse into the nucleus through nuclear pore complexes (NPCs) but larger proteins - such as Bxbl which is around 56 kDa - need so-called nuclear localization signal (NLS) in order to efficiently enter the nucleus.

[0008] Despite the central role of the translocation process in Bxbl function, no systematic study has been conducted to examine the effect of different nuclear translocation pathway elements on Bxbl -mediated stable integration efficiency.

[0009] Therefore, there is a need to provide a site-specific DNA recombinase enzyme which can stably integrate a gene into the genome of mammalian cells with high efficiency.

[0010] Summary of the Invention

[0011] The present inventors have investigated the effect of inclusion of an NLS and of use of proteins involved in the nuclear translocation process on Bxbl -mediated stable integration efficiency for a target gene. The present inventors have demonstrated that fusing an NLS to a Bxbl serine integrase results in increased stable integration efficiency. In this way, Bxbl -mediated gene integration may be improved.

[0012] In addition, the present inventors have identified various optimal NLSs and other components that may be used to enhance integration efficiency. For example, it has been found that use of Nucleoplasmin (abbreviated as NPL) NLS from Xenopus laevis together with the Bxbl serine integrase results in a surprisingly large increase in stable integration efficiency. In addition, it was found that co-expression of nuclear translocation component Ran GTPase-activating protein 1 (RanGAP) with Bxbl-NPLCx2 serine integrase increased the stable integration efficiency further.

[0013] Accordingly, the invention provides a site-specific DNA recombinase enzyme which comprises a first nuclear localisation signal (NLS). As explained above, the NLS enhances the integration efficiency of the site-specific DNA recombinase enzyme.

[0014] In one embodiment, the enzyme disclosed herein is a serine integrase. In a preferred embodiment, the enzyme disclosed herein is a Bxbl serine integrase. In one embodiment, the first NLS of the enzyme disclosed herein is a monopartite NLS. In an alternative embodiment, the first NLS of the enzyme disclosed herein is a bipartite NLS. In one embodiment, the first NLS of the enzyme disclosed herein has a net charge of greater than about 4.6 at pH 7.5. In a preferred embodiment, the first NLS of the enzyme disclosed herein has a net charge of about 7.6 at pH 7.5. In one embodiment, the first NLS is positioned at the C-terminus of the enzyme disclosed herein. In an alternative embodiment, the first NLS is positioned at the N-terminus of the enzyme disclosed herein. In one embodiment, the first NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL), a NLS from Simian Virus 40 or a NLS from the transcription factor EGL-13. In one embodiment, the first NLS of the enzyme disclosed herein comprises or consists of the amino acid sequence of one or more of SEQ ID NOs: 11-13, or a variant thereof. In one embodiment, the enzyme disclosed herein comprises a second NLS. In one embodiment, the second NLS of the enzyme disclosed herein is a monopartite NLS. In an alternative embodiment, the second NLS of the enzyme disclosed herein is a bipartite NLS. In one embodiment, the second NLS of the enzyme disclosed herein has a net charge of greater than about 4.6 at pH 7.5. In a preferred embodiment, the second NLS of the enzyme disclosed herein has a net charge of about 7.6 at pH 7.5. In one embodiment, the second NLS is positioned at the C-terminus of the enzyme disclosed herein. In an alternative embodiment, the second NLS is positioned at the N-terminus of the enzyme disclosed herein. In one embodiment, the first NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL) and the second NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL). In another embodiment, the first NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL) and the second NLS of the enzyme disclosed herein is a NLS from Simian Virus 40. In another embodiment, the first NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL) and the second NLS of the enzyme disclosed herein is a NLS from the transcription factor EGL-13. In another embodiment, the first NLS of the enzyme disclosed herein is a NLS from Xenopus laevis Nucleoplasmin (NPL) and the second NLS of the enzyme disclosed herein is a NLS from c-myc. In one embodiment, the second NLS of the enzyme disclosed herein comprises or consists of the amino acid sequence of one or more of SEQ ID NOs: 11-14, or a variant thereof

[0015] The invention also provides a polynucleotide encoding the enzyme disclosed herein.

[0016] The invention also provides an expression vector comprising the polynucleotide disclosed herein. In one embodiment, the expression vector disclosed herein comprises a polynucleotide encoding a nuclear translocation protein. In a preferred embodiment, the expression vector disclosed herein comprises a polynucleotide encoding Ran GTPase- activating protein 1 (RanGAP).

[0017] The invention also provides a cell comprising the enzyme, polynucleotide or expression vector disclosed herein. In one embodiment, the cell disclosed herein further comprises a targeting vector comprising a first recombinase recognition site and a second recombinase recognition site. In a preferred embodiment, the targeting vector disclosed herein comprises attP sites. In one embodiment, the first recombinase recognition site disclosed herein comprises or consists of the nucleotide sequence of SEQ ID NO: 5, or a variant thereof, and the second recombinase recognition site disclosed herein comprises or consists of the nucleotide sequence of SEQ ID NO: 6, or a variant thereof. In one embodiment, the targeting vector disclosed herein further comprises a selectable marker. In one embodiment, the first and second recombinase recognition sites disclosed herein flank a target sequence of interest in the targeting vector. In a preferred embodiment, the target sequence of interest disclosed herein is a DNA sequence encoding an antibody or fragment thereof. In one embodiment, the genome of the cell disclosed herein comprises first and second recombinase recognition sites compatible with the first and second recombinase recognition sites of the targeting vector disclosed herein. In a preferred embodiment, the first and second compatible recombinase recognition sites disclosed herein are attB sites. In one embodiment, the first and second compatible recombinase recognition sites disclosed herein respectively comprise or consist of the nucleotide sequence of SEQ ID NO: 7, or a variant thereof, and the nucleotide sequence of SEQ ID NO: 8, or a variant thereof. In one embodiment, the cell disclosed herein is a mammalian cell. In a preferred embodiment, the cell disclosed herein is a Chinese hamster ovary cell. The invention also provides a method of making the cell disclosed herein, wherein the method comprises: (i) transfecting a cell disclosed herein with a targeting vector comprising a first recombinase recognition site and a second recombinase recognition site which flank a target sequence of interest, and a selectable marker, and (ii) selecting transfected cells using the selectable marker. In one embodiment, the method disclosed herein comprises transfecting the cell disclosed with an expression vector disclosed herein.

[0018] The invention also provides a method of displaying an antibody library comprising: (i) providing a plurality of different targeting vectors as disclosed herein, wherein for each different targeting vector the target DNA sequence of interest encodes a different antibody or fragment thereof; (ii) transfecting a plurality of cells with the plurality of targeting vectors to generate a plurality of display cells; and (iii) selecting transfected display cells using the selectable marker. In one embodiment, the method comprises (i) providing a plurality of different targeting vectors as disclosed herein, wherein for each different targeting vector the target DNA sequence of interest encodes a different antibody or fragment thereof; (ii) transfecting a plurality of cells comprising the plurality of targeting vectors disclosed herein with an expression vector disclosed herein to generate a plurality of display cells; and (iii) selecting transfected display cells using the selectable marker. In one embodiment, the method comprises making a plurality of cells by the method disclosed herein and then transfecting the plurality of cells with the plurality of targeting vectors. In one embodiment, the step of making the plurality of cells by the method disclosed herein is carried out about 1 day before the step of transfecting the plurality of cells with the plurality of targeting vectors.

[0019] The invention also provides an antibody display library comprising a plurality of targeting vectors as disclosed herein, wherein the target sequence of interest is a DNA sequence encoding an antibody or fragment thereof. In one embodiment, the antibody expression library comprises a plurality of display cells as disclosed herein.

[0020] Brief Description of the Figures

[0021] Figure 1 is a diagram illustrating constructs encoding Mycobacteriophage Bxbl serine integrase with N-terminal and C-terminal nuclear localisation signal fusions (NLS). The upper construct illustrates Mycobacteriophage Bxbl serine integrase with N-terminal NLS fusion. N-terminal NLS and Mycobacteriophage Bxbl serine integrase gene was separated by a GS-linker and human influenza hemagglutinin (HA) epitope tag. The lower construct illustrates Mycobacteriophage Bxbl serine integrase with C-terminal NLS fusion. Amino acid sequences of the NLSs are shown below the constructs.

[0022] Figure 2 shows the normalised stable integration efficiencies (nSIE) of different Bxbl -NLS variants. Figure 2A shows the average nSIEs of Bxbl -NLS variants carrying either N-terminal (columns denoted by “N”) or C-terminal (columns denoted by “C”) NLS fusion. The error bars represent standard deviation of three independent experiments. Asterisks denote statistically significant difference to Bxbl without NLS (column denoted by “Bxbl -NLS”). Figure 2B shows Bxbl -NLS variants sorted according to nSIE. Asterisks denote statistically significant difference to Bxbl-NLSC. Asterisks denote P- values in Eigure 2A and Eigure 2B: (**) p < 0.01; (***) p < 0.001.

[0023] Figure 3 is a diagram illustrating constructs encoding double and triplet NLS variants of Bxbl. The construct at the top shows the configuration of the double NLS variants with NLSs at N-terminus and C-terminus. The constructs in the middle and at the bottom illustrate the configuration of Bxbl-NPLCx2 and Bxbl-NPLCx3 variants, respectively. NLS sequences of Bxbl-NPLCx2 and Bxbl-NPLCx3 are separated by a flexible GGGGSGGGGSGS linker.

[0024] Figure 4 A shows a comparison of Bxbl -NLS variants containing two or three NLS sequences to Bxbl -NLS variants with only one NLS sequence. Average normalised stable integration efficiencies (nSIE) of double and triple NLS variants are represented to the right of the graph (“Double and triple NLS”). Lor comparison, the nSIE of single NLS variants from Eigure 2A are represented to the left of the graph (“Single NLS”). Error bars represent the standard deviation of three independent experiments. Asterisks denote p- value in Eigure 4A: (****) p < 0.0001. Figure 4B is a heatmap showing statistical differences between individual single NLS variants and double / triple NLS variants.

[0025] Figure 5A is a schematic illustration of the genetic construct expressing both Bxbl-NPLCx2 and Importin a, Importin P or RanGTP. The two proteins were separated by GS linker, Furin cleavage site and T2A peptide. Figure 5B is a diagram illustrating the translocation of proteins into the nucleus. Importin a interacts with Importin P through IBB (Importin P-binding) domain. Then, NLS sequence has access to the binding cavity of Importin a. The ternary complex is then translocated into the nucleus where binding of RanGTP to Importin P causes dissociation of the ternary complex. Subsequently, Importin a binds to exportin complex composed of CAS and RanGTP, and the complex is transported out from the nucleus. The exportin complex is dissociated in the cytoplasm by Ran GAP. Figure 6 shows the effect of co-expression of transport proteins. The graph shows the average normalised stable integration efficiencies (nSIEs) of each Bxbl-NPLCx2- transport protein construct. The error bars represent standard deviation of three replicate transfections. Asterisks denote statistically significant difference to NPLCx2. Asterisks denote p-values in Figure 6: (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001.

[0026] Figure 7 is a comparison of the stable integration efficiency of Bxbl-NPLCx2 with and without RanGAP co-expression using an antibody construct (abrilumab) and GFP. The stable integration efficiencies are normalized to Bxbl without NLS fusion.

[0027] Brief Description of the Sequence Listing

[0028] SEQ ID NOs: 1-4 correspond to the nucleotide sequences encoding the NLS from Xenopus laevis Nucleoplasmin (abbreviated as NPL), the NLS from Simian Virus 40 (SV40), the NLS from transcription factor EGL-13 and the NLS from c-Myc, respectively.

[0029] SEQ ID NOs: 5-8 correspond to the recombinase recognition sequences for attP, attPm, attB and attBm, respectively.

[0030] SEQ ID NO: 9 corresponds to the nucleotide sequence encoding the Bxbl serine integrase.

[0031] SEQ ID NO: 10 corresponds to the nucleotide sequence encoding Mouse RanGAP SEQ ID NOs: 11-14 correspond to the amino acid sequences of the NLS from Xenopus laevis Nucleoplasmin (abbreviated as NPL), the NLS from Simian Virus 40 (SV40), the NLS from transcription factor EGL-13 and the NLS from c-Myc, respectively.

[0032] SEQ ID NO: 15 corresponds to the amino acid sequence of the Bxbl serine integrase.

[0033] SEQ ID NO: 16 corresponds to the amino acid sequence of Mouse RanGAP.

[0034] SEQ ID NO: 17 corresponds to the amino acid sequence of the GS linker.

[0035] SEQ ID NO: 18 corresponds to the amino acid sequence Mouse Importin alpha.

[0036] SEQ ID NO: 19 corresponds to the amino acid sequence Mouse Importin beta. SEQ ID NO: 20 corresponds to the amino acid sequence Mouse RanGTP.

[0037] SEQ ID NO: 21 corresponds to the amino acid sequence Mouse RCC1.

[0038] Detailed Description of the Invention

[0039] It is to be understood that different applications of the disclosed methods and products may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0040] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0041] General Definitions

[0042] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0043] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes two or more such cells, and the like.

[0044] In general, the term “comprising ” is intended to mean including but not limited to. For example, the phrase a method is one “comprising ” particular steps, should be interpreted to mean that the method includes those steps, but the method may comprise further steps.

[0045] In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of’ is intended to be limiting.

[0046] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0047] The terms “Nucleoplasmin” and “NPL” are used interchangeably herein, and are intended to refer to the Nucleoplasmin protein from Xenopus laevis.

[0048] The terms “nucleic acid” “nucleic acid sequence”, “polynucleotide” and “nucleotide sequence” are used interchangeably herein, and are intended to refer to a polymeric chain of nucleotides of any length e.g. deoxyribonucleotides, ribonucleotides, or analogues thereof. For example, the polynucleotide may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The polynucleotide may consist of DNA. The polynucleotide may be mRNA. Since the polynucleotide may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0049] For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides or amino acids at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the amino acids or nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0050] Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: X, SEQ ID NO: X would be the reference sequence. To assess whether a sequence is at least 80% identical to SEQ ID NO: X (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: X, and identify how many positions in the test sequence were identical to those of SEQ ID NO: X. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: X. If the sequence is shorter than SEQ ID NO: X, the gaps or missing positions should be considered to be non-identical positions.

[0051] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0052] Herein, the term “plasmid” is intended to refer to a nucleic acid molecule that can replicate independently of a cell chromosome. The term “plasmid” is intended to cover circular nucleic acid molecules and linear nucleic acid molecules. Furthermore, the term “plasmid” is intended to cover bacterial plasmids, but also cosmids, minicircles (Nehlsen, K., Broil S., Bode, J. (2006), Gene Ther. Mol. Biol., 10: 233-244; Kay, M.A., He, C.-Y, Chen, Z.-H. (2010), Nature Biotechnology, 28: 1287-1289) and ministrings (Nafissi N, Alqawlaq S, Lee EA, Foldvari M, Spagnuolo PA, Slavcev RA. (2014), Mol Ther Nucleic 15 Acids, 3:el65). Optionally, the plasmid is a circular nucleic acid molecule. Optionally, the plasmid is a nucleic acid molecule that is of bacterial origin.

[0053] The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x.

[0054] The term “between” in relation to a pair of reference numerical values and its grammatical equivalents as used herein can include the numerical values themselves and the range of values between the reference numerical values. The term “antibody” as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, single domain antibodies and epitope-binding fragments of any of the above.

[0055] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0056] Site-specific DNA recombinase enzyme

[0057] The invention relates to a site-specific DNA recombinase enzyme comprising a nuclear localisation signal (NLS). The site-specific DNA recombinase enzyme disclosed herein may be an enzyme which facilitates the insertion of a nucleotide sequence into the genome of a eukaryotic cell by site-specific recombination. In some aspects, the sitespecific DNA recombinase enzyme is endogenous to the cell, i.e. encoded by an endogenous gene. In some aspects, the site-specific DNA recombinase enzyme is exogenous to the cell (i.e. the site-specific DNA recombinase is heterologous and is not naturally expressed in the cell). According to the invention, the site-specific DNA recombinase enzyme is modified so that it is operatively linked to a nuclear localisation sequence (NLS) as defined herein. In some aspects, the site-specific DNA recombinase enzyme is modified so that it is further operatively linked to a second NLS as defined herein. In some aspects, the site-specific DNA recombinase enzyme is modified so that it is operatively linked to the first and second NLS as defined herein.

[0058] In some aspects, the site-specific DNA recombinase enzyme is an integrase, for example a serine integrase or a tyrosine integrase. The terms “integrase” and “recombinase” are equivalent and are used interchangeably herein. Preferably, the sitespecific DNA recombinase enzyme is a serine integrase, and may be any serine integrase. Serine integrases are well known in the art and include, without limitation, EcoYBCK, OC31, SCH10.38c, SCC88.14, SC8F4.15c, SCD12A.23, Bxbl, WwK, Sau CerB, Bsu CisB, TP901-1, 0370.1, 0105, OFC1, Al 18, Cacl956, Cacl951, Sau CerA, Spin, TnpX, TndX, SPBc2, SC3C8.24, SC2E1.37, SCD78.04c, R4, ORvl, Y4bA and Bja serine integrases. Preferably, the site-specific DNA recombinase enzyme is a Bxbl serine integrase from My cobacteriophage. In one aspect, the site-specific DNA recombinase enzyme comprises the amino acid sequence of SEQ ID NO: 15, or a variant thereof. In another aspect, the site-specific DNA recombinase enzyme is Bxbl or a variant thereof and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 15. In one aspect, the site-specific DNA recombinase enzyme is a tyrosine integrase, and may be any tyrosine integrase. Tyrosine integrases are well known in the art and include, without limitation, BS codV, BS ripX, BS ydcL, CB tnpA, CollD, CP4 E.coli, Cre E.coli, D29, DLP12, DN int, EC FimB, EC FimE, EC orf, EC xerC, EC xerD, (pl 1, cpl3, (p80, cpadh, cpCTX, cpLC3, FLP, cpR73, HI orf, HI rci, HI xerC, HI xerD, HK22, HP1, L2, L5, L54, X, LL orf, LL xerC, LO L5, MJ orf, ML orf, MP int, MT int, MT orf, MV4, P186, P2, P21, P22, P4, P434, PA sss, PM fimB, pAEl, pCLl, pKDl, pMEA, pSAM2, pSB2, pSB3, pSDL2, pSElOl, pSB2, pSB3, pSDL2, pSElOl, pSE211, pSMl, pSRl, pWS58, R721, Rci, SF6, SLP1, SM orf, SsrA, SSV1, T12, Tn21, Tn4430, Tn554a, Tn554b, Tn7, Tn916, Tuc, WZ int, XisA, and XisC.

[0059] Site-specific DNA recombinase enzymes recognise unique nucleic acid sequences known as recombinase recognition sites. For example, serine integrases such as Bxbl are able to catalyse site-specific recombination between one or more recombinase recognition sites in the eukaryotic nuclear genome and one or more recombinase recognition sites on, for example, a targeting vector. A targeting vector is a vector that comprises at least one recombinase recognition site and a target nucleotide sequence of interest. In a preferred embodiment, the targeting vector comprises two recombinase recognition sites flanking a target sequence of interest. The site-specific DNA recombinase enzyme of the invention catalyses site-specific recombination between one or more recombinase recognition sites in the targeting vector and one or more recombinase recognition sites present in the nuclear genome of a cell, thereby integrating the transgene into the genome. A person skilled in the art is aware of appropriate recombinase recognition sites that are recognised by a given site-specific DNA recombinase enzyme such as Bxbl, based on the common general knowledge. Furthermore, a person skilled in the art is familiar with methods to design suitable recombinase recognition sites that are recognised by a given site-specific DNA recombinase enzyme such as Bxbl, based on the common general knowledge. The recombinase recognition site(s) may thus be any such site(s) recognised by the site-specific recombinase enzyme.

[0060] The recombinase recognition sites in the genome may be one or more bacterial attachment (attB) sites. Heterologous wild-type attB sites can be integrated into a eukaryotic nuclear genome to create a transgenic cell line. The recombinase recognition sites on the targeting vector may be one or more phage attachment (attP) sites. A serine integrase and an attP -bearing targeting vector may then be introduced into eukaryotic cells harboring the heterologous attB sites in the genome. In one aspect, the targeting vector comprises one attP site and the eukaryotic genome comprises one attB site. Preferably, the targeting vector comprises two different attP sites, and the genome comprises two different attB sites, such that the serine integrase integrates only the target sequence of interest into the genome, without the rest of the targeting vector, through recombinase-mediated cassette exchange (RMCE). The two different attP sites may be referred to herein as attP and attPm sites. The two different attB sites may be referred to herein as attB and attBm sites. The locations of attP sites and attB sites may be reversed such that the attP sites are present in the genome and the attB sites are present in a targeting vector.

[0061] The site-specific DNA recombinase enzyme disclosed herein may have a normalised stable integration efficiency value of greater than 1.0. Stable integration efficiency is a measure of the percentage of cells expressing the target sequence of interest after introducing the site-specific DNA recombinase enzyme of the invention and the targeting vector comprising the target sequence of interest into the cells. Stable integration efficiency may be normalised to the stable integration efficiency of the site-specific DNA recombinase enzyme which does not comprise an NLS, i.e. normalised to the percentage of cells expressing the target sequence of interest after introducing a site-specific DNA recombinase enzyme lacking an NLS and the targeting vector comprising the target sequence of interest into the cells. The percentage of cells expressing the target sequence of interest may be measured using flow cytometry or any other suitable technique for the measurement of physical and chemical characteristics of a population of cells. In one aspect, the site-specific DNA recombinase enzyme disclosed herein has a normalised stable integration efficiency value of at least about 1.6, at least about 1.8, at least about 2.0, at least about 2.2, at least about 2.4, at least about 2.6, at least about 2.8, at least about 3.0, at least about 3.2, at least about 3.4, at least about 3.6, at least about 3.8, at least about 4.0, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, at least about 5.0, at least about 5.2, at least about 5.4, at least about 5.6, at least about 5.8, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10.0, at least about 15.0, or at least about 20.0. The stable integration efficiency of a site-specific DNA recombinase enzyme disclosed herein may also be expressed as a percentage. For example, the site-specific DNA recombinase enzyme disclosed herein has a stable integration efficiency of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10%.

[0062] For example, the stable integration efficiency of a site-specific DNA recombinase enzyme disclosed herein may be measured using a targeting vector that comprises a target sequence of interest that encodes for a fluorescent protein, such as green fluorescent protein or a variant thereof After introduction of the site-specific DNA recombinase enzyme disclosed herein into the cell, the target sequence of interest is integrated into the nuclear genome of the cell, provided the one or more recombinase recognition sites in the genome are compatible with the one or more recombinase recognition sites in the targeting vector. The percentage of GFP positive cells is then measured using flow cytometry. For normalisation, the flow cytometry data are compared to the flow cytometry data obtained when the experiment is performed using a site-specific DNA recombinase enzyme that does not comprise an NLS.

[0063] Nuclear localisation signal (NLS)

[0064] The invention relates to a site-specific DNA recombinase enzyme comprising a nuclear localisation signal (NLS). The site-specific DNA recombinase enzyme may comprise at least one NLS. The at least one NLS included in the site-specific DNA recombinase enzyme may be referred to herein as a “first NLS”. In one aspect, the sitespecific DNA recombinase enzyme may comprise an additional NLS. In a further aspect, the site-specific DNA recombinase enzyme may further comprise one or more additional NLSs. The additional NLS included in the site-specific DNA recombinase enzyme may be referred to herein as a “second NLS”. NLSs are signal sequences that direct proteins into the nucleus via an active translocation pathway. NLSs comprise amino acid sequences which mediate nuclear transport into the nucleus. NLSs may be cationic peptides, for example, highly cationic peptides. NLSs may comprise one or more importin a binding sequence. The importin a binding sequence is capable of binding to ten tandem armadillo (ARM) repeats in importin a.

[0065] In one aspect, the first NLS is a monopartite NLS. In another aspect, the first NLS is a bipartite NLS. A bipartite NLS typically contains two basic amino acid clusters separated by a spacer sequence, whereas a monopartite NLS contains a single basic amino acid cluster. The basic amino acid clusters typically comprise lysine and / or arginine residues. The basic amino acid clusters typically comprise from about 2 to about 4 amino acids, for example nucleoplasmin NLS has a sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 11), wherein the basic amino acid clusters are shown in bold. The spacer sequence in a bipartite NLS may be of any appropriate length. Typically, the spacer sequence in a bipartite NLS has a length of from about 9 amino acid residues to about 30 amino acid residues. Nucleoplasmin NLS has a spacer sequence of 10 amino acid residues.

[0066] In one aspect, the first NLS has a net charge of greater than about 4.6 at pH 7.5. Preferably, the first NLS has a net charge of about 7.6 at pH 7.5. In one aspect, the first NLS has a net charge of at least about 4.6 and at most about 7.6 at pH 7.5. In one aspect, the first NLS has a net charge of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5 or about 7.6, at pH 7.5. Net charge can be predicted computationally, for example, using computer programs such as SnapGene™.The skilled person is aware of various methods to predict net charge in polypeptides based on the common general knowledge. Lor example, net charge in polypeptides may be predicted according to the method disclosed in Kozlowski, L.P. IPC - Isoelectric Point Calculator. Biol Direct 11, 55 (2016). https: / / doi.org / 10.1186 / sl3062-016-0159-9.

[0067] In one aspect, the first NLS comprises more than 3 positive amino acid residues. Positive amino acid residues include arginine, lysine and histidine. In a further aspect, the NLS comprises at least 4 positive amino acid residues, at least 5 positive amino acid residues, at least 6 positive amino acid residues, at least 7 positive amino acid residues, or at least 8 positive amino acid residues.

[0068] In one aspect, the first NLS is positioned at the C-terminus of the site-specific DNA recombinase enzyme. In another aspect, the first NLS is positioned at the N-terminus of the site-specific DNA recombinase enzyme.

[0069] In the invention, the first NLS is operatively linked to the site-specific DNA recombinase enzyme. The first NLS may be directly linked to the site-specific DNA recombinase enzyme or indirectly linked to the site-specific DNA recombinase enzyme. The first NLS may be operatively linked to the site-specific DNA recombinase enzyme by chemical bonds, for example covalent bonds. In one aspect, the first NLS is linked to the site-specific DNA recombinase enzyme via a peptide bond. Preferably, the first NLS is linked to the site-specific DNA recombinase enzyme via a linker molecule, such as a covalent linker molecule, for example a GlySer (GS) linker. In one aspect, the GS linker comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof. Preferably, the first NLS used herein is that of the Xenopus laevis Nucleoplasmin protein. In this aspect, the first NLS typically comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof In one aspect, the first NLS is that of the SV40 large T antigen. In this aspect, the first NLS typically comprises the amino acid sequence of SEQ ID NO: 12, or a variant thereof. In one aspect, the first NLS is that of the transcription factor EGL-13. In this aspect, the first NLS typically comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof.

[0070] In another aspect, the site-specific DNA recombinase enzyme comprising a first NLS further comprises a second NLS. The second NLS may be selected from any NLS defined herein.

[0071] In one aspect, the second NLS is a monopartite NLS. In another aspect, the second NLS is a bipartite NLS.

[0072] In one aspect, the second NLS has a net charge of greater than about 4.6 at pH 7.5. Preferably, the second NLS has a net charge of about 7.6 at pH 7.5. In one aspect, the second NLS has a net charge of at least about 4.6 and at most about 7.6 at pH 7.5. In one aspect, the second NLS has a net charge of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5 or about 7.6, at pH 7.5. The net charge may be as predicted by any suitable method known to the skilled person. Lor example, the net charge can be predicted computationally, for example, using computer programs such as SnapGene™. The skilled person is aware of various methods to predict net charge in polypeptides based on the common general knowledge.

[0073] In one aspect, the second NLS is positioned at the C-terminus of the site-specific DNA recombinase enzyme. In another aspect, the second NLS is positioned at the N- terminus of the site-specific DNA recombinase enzyme. In one aspect, the first NLS is positioned at the C-terminus of the site-specific DNA recombinase enzyme and the second NLS is positioned at the N-terminus of the site-specific DNA recombinase enzyme. In another aspect, the first NLS is positioned at the N-terminus of the site-specific DNA recombinase enzyme and the second NLS is positioned at the C-terminus of the sitespecific DNA recombinase enzyme. In another aspect, the first NLS is positioned at the C- terminus of the site-specific DNA recombinase enzyme and the second NLS is positioned at the C-terminus of the first NLS. In another aspect, the first NLS is positioned at the N- terminus of the site-specific DNA recombinase enzyme and the second NLS is positioned at the N-terminus of the first NLS.

[0074] In one aspect, a second NLS is operatively linked to the site-specific DNA recombinase enzyme. The second NLS may be directly linked to the site-specific DNA recombinase enzyme or indirectly linked to the site-specific DNA recombinase enzyme. The second NLS may be operatively linked to the site-specific DNA recombinase enzyme by chemical bonds, for example covalent bonds. In one aspect, the second NLS is linked to the site-specific DNA recombinase enzyme via a peptide bond. Preferably, the second NLS is linked to the site-specific DNA recombinase enzyme via a linker molecule, such as a covalent linker molecule, for example a GlySer (GS) linker. In one aspect, the GS linker comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof.

[0075] In another aspect, the second NLS is operatively linked to the first NLS. The second NLS may be directly linked to the first NLS or indirectly linked to the first NLS. The second NLS may be operatively linked to the first NLS by chemical bonds, for example covalent bonds. In one aspect, the second NLS is linked to the first NLS via a peptide bond. Preferably, the second NLS is linked to the first NLS via a linker molecule, such as a covalent linker molecule, for example a GlySer (GS) linker. In one aspect, the GS linker comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof.

[0076] Preferably, the second NLS is that of the Xenopus laevis Nucleoplasmin protein. In this aspect, the second NLS comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof. In one aspect, the second NLS is that of the SV40 large T antigen. In this aspect, the second NLS comprises the amino acid sequence of SEQ ID NO: 12, or a variant thereof. In one aspect, the second NLS is that of the transcription factor EGL-13. In this aspect, the second NLS comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof. In one aspect, the second NLS is that of the proto-oncogene product c-Myc. In this aspect, the second NLS comprises the amino acid sequence of SEQ ID NO: 14, or a variant thereof.

[0077] In one aspect, the first NLS is that of the Xenopus laevis Nucleoplasmin protein and the second NLS is that of the Xenopus laevis Nucleoplasmin protein. In one aspect, the first NLS is that of the Xenopus laevis Nucleoplasmin protein and the second NLS is that of the transcription factor EGL-13. In one aspect, the first NLS is that of the Xenopus laevis Nucleoplasmin protein and the second NLS is that of the SV40 large T antigen. In one aspect, the first NLS is that of the Xenopus laevis Nucleoplasmin protein and the second NLS is that of the proto-oncogene product c-Myc. In one aspect, the first NLS comprises the amino acid sequence of SEQ ID NO: 11 and the second NLS comprises the amino acid sequence of SEQ ID NO: 11. In one aspect, the first NLS comprises the amino acid sequence of SEQ ID NO: 11 and the second NLS comprises the amino acid sequence of SEQ ID NO: 13. In one aspect, the first NLS comprises the amino acid sequence of SEQ ID NO: 11 and the second NLS comprises the amino acid sequence of SEQ ID NO: 12. In one aspect, the first NLS comprises the amino acid sequence of SEQ ID NO: 11 and the second NLS comprises the amino acid sequence of SEQ ID NO: 14.

[0078] Polynucleotides and expression vectors

[0079] The invention also provides a polynucleotide encoding the site-specific DNA recombinase enzyme disclosed herein. The terms “polynucleotide” and “nucleic acid” are used interchangeably herein.

[0080] In one aspect, the site-specific DNA recombinase enzyme is encoded by a nucleic acid sequence. In one aspect, the site-specific DNA recombinase enzyme is Bxbl or a variant thereof and is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 9, or a variant thereof. The nucleic acid sequence encoding the site-specific DNA recombinase enzyme may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9.

[0081] In one aspect, the nucleic acid sequence is a DNA construct. Preferably, the DNA construct is an expression vector.

[0082] An expression vector is typically any nucleic acid vector that comprises a transcription regulatory region operably linked to a nucleotide sequence to be transcribed and optionally, to be expressed, for instance, but not limited to, a sequence encoding at least one polypeptide. Expression vectors can comprise synthetic or cDNA-derived DNA fragments encoding a protein, operably linked to a suitable transcriptional and / or translational regulatory element derived from mammalian, viral or insect genes. Such regulatory elements include transcriptional promoters, enhancers, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Mammalian expression vectors can also comprise nontranscribed elements such as an origin of replication, other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences such as splice donor and acceptor sites. A selectable marker gene to facilitate recognition of transfectants may also be incorporated (e.g. AmpR). In certain aspects, expression of the site-specific DNA recombinase enzyme from the expression vector is induced by an exogenous agent in an inducible expression vector system. Transcriptional and translational control sequences in expression vectors useful for transfecting mammalian cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from viruses such as polyoma, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). Viral genomic promoters, control and / or signal sequences may be utilized to drive expression, provided such control sequences are compatible with the host cell chosen. Non-viral cellular promoters can also be used (e.g., the P-globin and the EF-la promoters), depending on the cell type in which the site-specific DNA recombinase enzyme is to be expressed. DNA sequences derived from the SV40 viral genome, for example, the SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements useful for expression of a heterologous DNA sequence. Early and late promoters are particularly useful because both are obtained easily from the SV40 virus as a fragment that also comprises the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used.

[0083] In a further aspect, the expression vector is a plasmid. A plasmid can be transfected or transformed into cells and replicate independently of, or within, the host cell genome.

[0084] The expression vector may further comprise a polynucleotide encoding a nuclear translocation protein. Preferably, the expression vector further comprises a polynucleotide encoding Ran GTPase-activating protein 1 (RanGAP). In a further aspect, the expression vector comprises a polynucleotide having the sequence of SEQ ID NO: 10, or a variant thereof. Alternatively, the nuclear translocation protein may be encoded by a different expression vector to the expression vector which encodes the site-specific DNA recombinase enzyme.

[0085] Expression vectors comprising the polynucleotides disclosed herein can be used to express proteins transiently, or can be integrated into a mammalian genome by random or targeted recombination such as, for example, homologous recombination or recombination mediated by recombinases that recognize specific recombination sites (e.g., Cre-lox- mediated recombination).

[0086] Expression vectors comprising polynucleotides disclosed herein may further comprise a selectable marker for the identification of successful transformants. This is useful when transfecting cells with expression vectors disclosed herein. For example, the selectable marker may be a fluorescent marker. Fluorescent markers are suitable selectable marker genes in mammalian expression vectors. Examples of fluorescent markers are well- known in the art, including, but not limited to Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (e.g. mKate, mKate2, mPlum, mRaspberry or E2-crimson). Alternatively or additionally, the selectable marker may be a gene conferring resistance an antibiotic. For example, the selectable marker may be a gene conferring resistance to G418 / geneticin, blasticidin, hygromycin B, puromycin, or zeocin.

[0087] Cells

[0088] The invention relates to cells comprising the site-specific DNA recombinase enzyme disclosed herein, or the polynucleotide or expression vector encoding the same. In one aspect, the site-specific DNA recombinase enzyme disclosed herein is introduced into a cell. In some aspects, the cell is a eukaryotic cell. Preferably, the eukaryotic cell is a mammalian cell, for example a Chinese Hamster ovary (CHO) cell. In some aspects, the cell is an isolated cell.

[0089] Preferably, introducing the site-specific DNA recombinase enzyme of the invention into the cell involves introducing any of the expression vector disclosed herein into any of the cells disclosed herein. Various methods can be used to introduce the expression vector encoding the site-specific DNA recombinase enzyme into the cell. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, microinjection, microparticle bombardment, infection with recombinant viral vectors. Preferably, the expression vector disclosed herein is introduced into the cell disclosed herein by transfection. The expression vector may be introduced into a cell by any method known to those of skill in the art. Other methods of transfection include the use of transfection reagents, dendrimers, cell penetrating peptides or polyamines. In certain aspects, introducing the site-specific DNA recombinase enzyme into mammalian cells involves using expression vectors which comprise a selectable marker for the positive identification of successfully transformed cells, as described above.

[0090] The cell disclosed herein may further comprise a targeting vector. The targeting vector may be selected from any targeting vector disclosed herein. A targeting vector is a vector that comprises one or more recombinase recognition sites positioned next to a target nucleotide sequence of interest. In a preferred embodiment, the targeting vector comprises two recombinase recognition sites flanking a target sequence of interest. A recombinase recognition site may be any such site described herein as being recognised by the sitespecific DNA recombinase of the invention. The recombinase recognition site may be any suitable recognition site which recognised by the site-specific DNA recombinase described herein. For example, the recombinase recognition site is recognised by the Bxbl serine integrase. The site-specific DNA recombinase enzyme catalyses site-specific recombination between one or more recombinase recognition sites in the targeting vector and one or more recombinase recognition sites present in the mammalian nuclear genome, thereby integrating the transgene into the genome. Preferably, the mammalian nuclear genome comprises two recombinase recognition sites which are compatible with the recombinase recognition sites in the targeting vector. In one aspect, the targeting vector comprises a first recombinase recognition site and a second recombinase recognition site. In one aspect, the first and second recombinase recognition sites are attP sites. In one aspect, the first recombinase recognition site comprises or consists of the sequence of SEQ ID NO: 5, or a variant thereof, and the second recombinase recognition site comprises or consists of the sequence of SEQ ID NO: 6, or a variant thereof.

[0091] A targeting vector disclosed herein may further comprise a selectable marker for the identification of successful transformants. For example, the selectable marker may be a fluorescent marker. Fluorescent markers are suitable selectable marker genes in mammalian expression vectors. Examples of fluorescent markers are well-known in the art, including, but not limited to Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (e.g. mKate, mKate2, mPlum, mRaspberry or E2-crimson). Preferably, the targeting vector comprises a membrane-anchored eGFP gene. Alternatively or additionally, the selectable marker may be a gene conferring resistance to an antibiotic. For example, the selectable marker may be a gene conferring resistance to G418 / geneticin, blasticidin, hygromycin B, puromycin, or zeocin. Preferably, the targeting vector comprises a puromycin N-acetyltransferase gene.

[0092] The target sequence of interest may be any target sequence desired to be integrated into a cell. Preferably, the target sequence of interest is a nucleotide sequence encoding an antibody or fragment thereof. The antibody or fragment thereof (typically an antigenbinding fragment) may be selected from any known format of antibody or antibody fragment. In another aspect, the target sequence of interest is a nucleotide sequence encoding a T-cell receptor or fragment thereof. The T-cell receptor or fragment thereof may be selected from any known format of T-cell receptor.

[0093] The nuclear genome of the cell disclosed herein may comprise first and second recombinase recognition sites which are compatible with first and second recombinase recognition sites of the targeting vector. This means that the site-specific DNA recombinase enzyme disclosed herein is able to recognise the recombinase recognition sites in the nuclear genome and the recombinase recognition sites of the targeting vector and catalyse site-specific recombination between these sites.

[0094] In one aspect, the compatible recombinase recognition sites in the nuclear genome of the cell disclosed herein are different to the recombinase recognition sites in the targeting vector. In one aspect, the first and second compatible recombinase recognition sites are attB sites. In one aspect, the first compatible recombinase recognition site in the nuclear genome comprises or consists of the sequence of SEQ ID NO: 7, or a variant thereof, and the second compatible recombinase recognition site in the nuclear genome comprises or consists of the sequence of SEQ ID NO: 8, or a variant thereof.

[0095] The nuclear genome of the cell disclosed herein may comprise one or more endogenous compatible recombinase recognition sites, i.e. compatible recombinase recognition sites which are naturally occurring in the nuclear genome of the cell disclosed herein. In one aspect, the nuclear genome of the cell disclosed herein comprises a first endogenous compatible recombinase recognition site and a second endogenous compatible recombinase recognition site.

[0096] The nuclear genome of the cell disclosed herein may comprise heterologous first and second compatible recombinase recognition sites, i.e. compatible recombinase recognition sites which are not naturally present in the nuclear genome of the cell disclosed herein. Various methods can be used to introduce the heterologous recombinase recognition sites into the mammalian nuclear genome and such methods are well known in the art. In one aspect, the nuclear genome of the cell disclosed herein comprises a first compatible recombinase recognition site which is endogenous to the cell (i.e. naturally occurring in the nuclear genome of the cell disclosed herein), and a second compatible recombinase recognition site which is heterologous to the cell (i.e. not naturally occurring in the nuclear genome of the cell disclosed herein).

[0097] One method of introducing heterologous recombinase recognition sites into the mammalian nuclear genome is by generating a mammalian display cell line. A mammalian display cell line may be generated by integrating a landing pad into the genome of a mammalian cell. The landing pad typically comprises a nucleotide sequence encoding a selectable marker flanked by recombinase recognition sites. In one aspect, the selectable marker contained within the landing pad is the blasticidin S deaminase gene. Methods for generating a mammalian display cell line by integrating a landing pad into the genome are described in the art, for example in Huhtinen et al., Front Bioeng Biotechnol.

[0098] 2023;l 1 :1170081.

[0099] In one aspect, a plurality of different landing pad is integrated into the genome of a mammalian cell. In a further aspect, two different landing pads, comprising different pairs of recombinase recognition sites, are integrated into the genome of a mammalian cell. According to these aspects, different target sequences of interest on different targeting vectors can be integrated at different landing pads in the mammalian genome. In this aspect, one of the targeting vectors may comprise recombinase recognition sites which are compatible with the recombinase recogntion sites on one of the landing pads, and the other targeting vector may comprise different recombinase recogntion sites which are compatible with the recombinase recogntion sites on the other landing pad. For example, a target sequence of interest encoding the heavy chain of an antibody of interest can be integrated at one landing pad present in the mammalian genome and a different target sequence of interest encoding the light chain of the same antibody of interest can be integrated at a different landing pad present in the mammalian genome.

[0100] Methods

[0101] The invention relates to a method of making a cell disclosed herein, comprising introducing a targeting vector into a cell comprising the enzyme, polynucleotide or expression vector of the invention, wherein the targeting vector comprises first and second recombinase recognition sites which flank a target sequence of interest and a selectable marker, and selecting for targeted cells (i.e. the cells containing the targeting vector or the target sequence of interest) using the selectable marker. The method may further comprise integrating a landing pad into the genome of the cell, wherein the landing pad comprises a nucleotide sequence flanked by a first and second recombinase recognition site. In a further aspect, the first and second recombinase recognition sites are attP sites. In one embodiment of the method disclosed herein, introducing a targeting vector into the cell comprises transfecting the cell with the targeting vector.

[0102] Also disclosed is a method of making a cell disclosed herein comprising integrating a landing pad into the genome of the cell, wherein the landing pad comprises a nucleotide sequence flanked by one or more recombinase recognition sites. In one aspect, the landing pad comprises a nucleotide sequence flanked by a first and second recombinase recognition site. In one aspect, the first and second recombinase recognition sites in the landing pad are attB sites. In one aspect, the landing pad comprises a selectable marker. The method may further comprise selecting for display cells (i.e. the cells containing the landing pad) using the selectable marker.

[0103] Also disclosed herein is a method for integrating a target sequence of interest into the genome of a cell comprising introducing a targeting vector into a cell comprising the enzyme, polynucleotide or expression vector of the invention, wherein the targeting vector comprises first and second recombinase recognition sites which flank the target sequence of interest. Optionally, the targeting vector comprises a selectable marker. The method may further comprise selecting for targeted cells (i.e. the cells containing the targeting vector or the target sequence of interest) using the selectable marker. In a further aspect, the first and second recombinase recognition sites on the targeting vector are attP sites.

[0104] Also disclosed herein is a method of expressing a target sequence of interest, comprising introducing a targeting into a cell comprising the enzyme, polynucleotide or expression vector of the invention, wherein the targeting vector comprises first and second recombinase recognition sites which flank the target sequence of interest. Optionally, the targeting vector comprises a selectable marker. The method may further comprise selecting for targeted cells (i.e. the cells containing the targeting vector or the target sequence of interest) using the selectable marker. In a further aspect, the first and second recombinase recognition sites on the targeting vector are attP sites.

[0105] Also disclosed herein is a method for constructing a mammalian antibody display library comprising introducing a plurality of targeting vectors into a plurality of cells comprising the enzyme, polynucleotide or expression vector of the invention, wherein the targeting vector comprises a target sequence of interest encoding an antibody or fragment thereof. The plurality of targeting vectors typically each encode a different antibody library variant. Each antibody library variant may be stably integrated into a defined locus in the genome of a mammalian display cell, so that each display cell only carries one antibody library variant in the same genomic locus. Typically, an antibody display library contains in the range of 1,000,000 to 1,000,000,000 different antibody variants.

[0106] In some aspects, the cells of the present disclosure may be used for screening libraries of antibodies with desired activity or activities. The antibody library variants may be in any format described herein, e.g. may be whole antibody molecules or may be antibody fragments, including but not limited to single chain antibodies (e.g. scFv antibodies), Fv antibodies, Fab antibodies, Fab'2 fragments, diabodies. Preferably, the antibody library variants are full-length monospecific IgG antibodies. The antibody library variants may also be full-length bispecific IgG antibodies.

[0107] In one aspect, the cells of the present disclosure may be used to generate an antibody display library. In another aspect, the cells of the present disclosure may be used to generate an antibody expression library.

[0108] The invention also provides an antibody display library comprising a plurality of any of the targeting vectors disclosed herein, wherein the plurality of targeting vectors each comprise a target sequence of interest encoding a different antibody or fragment thereof.

[0109] The invention also provides an antibody expression library comprising a plurality of any of the cells disclosed herein.

[0110] The invention also provides a method of screening antibodies from an antibody expression library of the invention, comprising measuring one or more parameters of antibody binding, expression and / or function. For example, antibodies may be screened for target antigen binding affinity, optionally using a fluorescent binding partner and fluorescence-activated cell sorting. The method may comprise measuring antibody display cell binding using fluorescence-activated cell sorting. In one aspect, the method comprises selecting antibody display cells that have a desired binding affinity to the target antigen. In one aspect, the method comprises selecting antibody display cells that have desired biophysical properties. In one aspect, the method comprises selecting antibody display cells that have a desired activity or functionality.

[0111] Also disclosed herein is a method for constructing a mammalian T-cell receptor display library comprising introducing a plurality of targeting vectors into a plurality of cells comprising the enzyme, polynucleotide or expression vector of the invention, wherein the targeting vector comprises a target sequence of interest encoding a T-cell receptor or fragment thereof. The plurality of targeting vectors typically each encode a different T-cell receptor library variant. Each T-cell receptor library variant may be stably integrated into a defined locus in the genome of a mammalian display cell, so that each display cell only carries one T-cell receptor library variant in the same genomic

[0112] In some aspects, the cells of the present disclosure may be used for screening libraries of T-cell receptors with desired activity or activities.

[0113] In one aspect, the cells of the present disclosure may be used to generate a T-cell receptor display library. In another aspect, the cells of the present disclosure may be used to generate a T-cell receptor expression library. The disclosure also provides a T-cell receptor display library comprising a plurality of any of the targeting vectors disclosed herein, wherein the plurality of targeting vectors each comprise a target sequence of interest encoding a different T-cell receptor or fragment thereof.

[0114] Also disclosed herein is a T-cell receptor expression library comprising a plurality of any of the cells disclosed herein.

[0115] The following Examples illustrate the invention.

[0116] Example 1 - Methods and Materials

[0117] Mammalian display cell line and vector construction

[0118] Mammalian display CHO cell line, CHO-LP, was generated by integrating a Bxbl landing pad (LP) into the genome of Flp-In CHO cell line, as described previously (Huhtinen et al., Front Bioeng Biotechnol. 2023;l 1 :1170081). The Bxbl LP contained CMV promoter driven membrane-anchored mouse IgG2a Fc and blasticidin S deaminase (BSD), flanked by AttB and AttBm sites, enabling recombinase mediated cassette exchange (RMCE). A promoterless Bxbl targeting vector, pBxbl-TV-GFP, and a Bxbl integrase expression vector, pBxbl-EV, were used to evaluate the stable transfection efficiency, as described in Huhtinen et al., Front Bioeng Biotechnol. 2023;l 1 :1170081. The pBxbl-TV-GFP consisted of DNA encoding membrane anchored EGFP and puromycin N-acetyltransferase, flanked by AttP and AttPm sites for RMCE. The NLS variants were synthesized by GeneArt (Thermo Fisher) and cloned into the N-terminus and C-terminus of Bxbl by using BamHI-Aval and PshAI-SphI restriction sites in pBxbl-EV vector, respectively.

[0119] Cell culture and transfections

[0120] The CHO-LP cells were cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 2 mM GlutaMAX (Gibco) and 10% fetal bovine serum (Sigma- Aldrich) (hereafter referred as F-12 medium) at +37°C, 5% C02. The day before transfection, the wells of 6-well plates (Biocoat poly-D-Lysine cellware, Coming) containing 2 ml of F-12 medium were seeded with CHO-LP cells (400000 cells / well), and the cells were grown overnight. The next day the CHO-LP cells were co -transfected with Bxbl-NLS fusion variant in pBxbl-EV vector (1.25 pg) and pBxbl-TV-GFP vector (1.25 pg) using Lipofectamine 3000 reagent according to the manufacturer’s recommendations (Thermo Fisher Scientific). Used volume of Lipofectamine 3000 reagent was 3.75 pl. Nontransfected cells and cells transfected only with pBxbl-TV-GFP (1.25 pg) were used as negative and transient expression controls, respectively. Three days post-transfection, the cells were passaged. First, the old medium was discarded, and the cells were washed with 1 ml of DPBS (without calcium and magnesium) (Thermo Fisher Scientific) and 1 ml of 0.05% Trypsin-EDTA was added to the wells. The trypsinisation reaction was incubated at 37 °C with 5 % CO2 for 6 min. After the incubation, 1 ml of F-12 medium was added to the wells to stop the trypsination reaction. Subsequently, the cells were split in 1 :10 ratio to new 6-well plates containing fresh F-12 medium. The cells were then further cultured for three days before analysis.

[0121] Flow cytometry analysis

[0122] The culture medium was discarded, and the cells were washed with 1 ml of DPBS, and 1 ml of Coming Cellstripper Dissociation Reagent (Thermo Fisher Scientific) was added to the wells, and subsequently the cells were incubated at 37 °C with 5 % CO2 for 20 min. The dissociation reactions were stopped by adding 1 ml of F-12 medium. The dissociated cells were then pipetted to 15 ml Falcon tubes (Thermo Fisher Scientific). The cells were pelleted by centrifugation (1000 x g, 5 min, +4 °C), the supernatant was discarded, and the cells were resuspended in 200 pl of cold EasySep Buffer (Stemcell Technologies, Cambridge, UK). Subsequently, the resuspended cells were pipetted to U- bottom 96-well plate (Micro test plate 96 well, Sarstedt, Numbrecht, Germany), which was kept on ice. The stable transfection efficiency was assessed by measuring the percentage of GFP positive cells with BD Accuri Flow Cytometer (BD Biosciences). The obtained flow cytometry data was analyzed with FlowJo software (BD Biosciences).

[0123] Statistical analysis Statistical analyses were implemented by using GraphPad Prism 9 (GraphPad, San Diego, USA) and Microsoft Excel (Microsoft, Redmond, USA). The threshold for statistical significance (alpha level) was 0.05.

[0124] Example 2 - Nucleoplasmin NLS fusion in Bxbl substantially enhances stable integration efficiency

[0125] To examine the effects of different nuclear localization signals (NLS) on stable integration efficiency, Bxbl -NLS variants were created by cloning Myc proto-oncogene NLS (c-Myc NLS), simian virus 40 large T antigen NLS (SV40 NLS), transcription factor EGL-13 NLS (EGL-13 NLS), or nucleoplasmin NLS from Xenopus laevis (NPL NLS) to the N-terminus or C-terminus of Bxbl serine integrase (Figure 1).

[0126] The newly created single NLS variants were named Bxbl-c-MycN / C, Bxbl- SV40N / C, Bxbl-EGL-13N / C, and Bxbl-NPLN / C. CHO-LP cells were co -transfected with 1 :1 ratio of pBxbl-TV-GLP and pBxbl-EV with different Bxbl-NLS variants. Nontransfected cells and cells transfected only with pBxbl-TV-GLP vector (transient expression control) were used as controls. After transfection, cells were cultivated for six days and subsequently the stable integration efficiency of each Bxbl-NLS variant was assessed by measuring the percentage of GEP positive cells with flow cytometer. To reduce variation of the transfections, the stable integration efficiency of each Bxbl-NLS variant was normalized to the stable integration efficiency of Bxbl that lacked the NLS fusion. Of the tested Bxbl-NLS variants, Bxbl-NPLC exhibited the highest normalized stable integration efficiency (nSIE) (Figure 2). The Bxbl-NPLC showed over fivefold increase in nSIE compared to Bxbl lacking NLS fusion (T-test, equal variances assumed; p = 0.0004). The second-best variant was Bxbl-NPLN, which increased nSIE by 3.7-fold compared to Bxbl without NLS (T-test, equal variances assumed; p = 0.007). SV40 NLS and EGL-13 NLS also enhanced nSIE of Bxbl as both N-terminal (T-test, equal variances assumed; p = 0.0004) or C-terminal fusion (T-test, equal variances assumed; p = 0.003), respectively (Figure 2A). The worst NLS was c-Myc, of which neither N-terminal nor C- terminal fusion improved nSIE of Bxbl serine (Figure 2A).

[0127] Since positively charged amino acids are important for the function of NLS sequences, the number of positively charged amino acids of each NLS sequence was plotted against nSIE values. As expected, there was a strong and significant positive correlation between the two variables (Spearman correlation, r = 0.8487, p = 0.0143, n = 8). Furthermore, the net charge of each NLS at pH 7.5 was determined computationally using SnapGene software. The net charges for SV40 NLS, c-Myc NLS, EGL-13 NLS and NPL NLS were 4.61, 1.61, 4.61 and 7.61, respectively. When the net charge values were plotted against nSIE values, strong and significant correlation was observed between these two variables (Spearman correlation, r = 0.9258, p = 0.0048, n = 8). In fact, the correlation between net charge and nSIE was stronger and more significant than the correlation between the number of positively charged amino acids and nSIE.

[0128] Example 3 - Stable integration efficiency could be further enhanced by adding two nucleoplasmin nuclear localization signals to the C-terminus of Bxbl

[0129] After testing the Bxbl-NLS variants with one NLS fusion at N-terminus or C- terminus, it was investigated whether nSIE could be further increased by adding more NLS sequences to the Bxbl integrase. To this end, SV40 NLS, c-Myc NLS, EGL-13 NLS and NPL NLS were each cloned to the N-terminus of Bxbl-NPLC variant. In addition, Bxbl- NLS variants were created containing two and three NPL NLS sequences separated by a GlySer (GS) linker at the C-terminus of Bxbl serine integrase (Figure 3). The double and triple NLS variants were named SV40N-Bxbl-NPLC, c-MycN-Bxbl-NPLC, EGL-13N- Bxbl-NPLC, NPLN-Bxbl-NPLC, Bxbl-NPLCx2 and Bxbl-NPLCx3. The effect of the double and triple NLS variants on stable integration efficiency was then tested as described above. The results are presented in Figure 4, which shows the average nSIE values of double / triple NLS variants and average nSIE values of single NLS variants from Figure 2 as a comparison. When compared to the best single NLS variant, Bxbl-NPLC, the double NLS variants NPLN-Bxbl-NPLC and Bxbl-NPLCx2 moderately increased nSIE of Bxbl by 41 % (T-test, equal variances assumed; p = 0.0458) and 63 % (T-test, equal variances assumed; p = 0.0496). None of the other double or triple NLS variants showed significant increase in nSIE compared to Bxbl-NPLC, but in general all the double NLS variants had significantly higher nSIE values than most of the single NLS variants, Furthermore, the average nSIE of all the double and triple NLS variants was threefold higher than that of single NLS variants (T-test, equal variances assumed; p = 1.2x10-9). It was also interesting that adding three NPL NLS sequences to the C-terminus of Bxbl (Bxbl-NPLCx3) didn’t increase nSIE. Lastly, no significant differences were observed between the NLS variants that had more than one NLS sequence.

[0130] Example 4 - Co-expression of Ran GTPase-activating protein 1 (RanGAP) with

[0131] Bxbl-NPLCx2 serine integrase increases stable integration efficiency In addition to testing different NLS sequences and NLS sequence configurations, it was investigated whether co-expression of proteins that are involved in the nuclear translocation process have an effect on stable integration efficiency. To this end, mouse Importin a, Importin 13 and GTP -binding nuclear protein Ran (RanGTP), Ran guanine nucleotide exchange factor (RCC1) and Ran GTPase-activating protein 1 (RanGAP) were each co-expressed with Bxbl-NPLCx2. The translocation proteins were expressed from the same plasmid as Bxbl-NPLCx2. The genes were separated by a GS linker, Furin clevage site and T2A self-cleaving peptide, which enabled the individual expression of each protein (Figure 5A). The roles of Importin a, Importin 13 and RanGTP in the nuclear translocation process are illustrated in Figure 5B.

[0132] The effect of co-expression of transport proteins on stable integration efficiency was tested in the same manner as described above. The transfections were performed in three replicates. Of all the tested transport proteins, RanGAP was the only one showing statistically significant positive effects on stable integration efficiency (Figure 6). The coexpression of RanGAP with Bxbl-NPLCx2 increased the stable integration efficiency by 48 % compared to Bxbl-NPLCx2 expression alone. The rest of the co-expressed transport proteins had either insignificant or negative effects on stable integration efficiency. For example, co-expression of Importin P and RCC1 lowered stable integration efficiency by 61 % and 41 %, respectively, but RanGTP and Importin a didn’t have any effect.

[0133] Example 5 - Bxbl equipped with optimal NLS in combination with optimized transfection protocol produce up to 10 % stable integration efficiency

[0134] After finding the optimal NLS for Bxbl, Bxbl-NPLCx2, Bxbl-NPLCx2 with Importin a, Bxbl-NPLCx2 with Importin 13 and Bxbl-NPLCx2 with RanGTP were tested in combination with optimized transfection protocol in order to see how high stable integration efficiency percentage can be achieved. The main difference between the optimized transfection protocol and the transfection protocol used earlier was that in optimized protocol Bxbl was Bxbl is transfected a day before targeting vector. With this combination stable integration efficiencies of 9.97 %, 9.23 %, 7.93 % and 10.01 % were achieved for Bxbl-NPLCx2, Bxbl-NPLCx2 with Importin a, Bxbl-NPLCx2 with Importin 13 and Bxbl-NPLCx2 with RanGTP, respectively. The stable integration efficiency with Bxbl without NLS was only 2.16 %.

[0135] 6 - integration efficiency was not limited to GFP It was confirmed that the aforementioned findings, obtained with the small and readily expressed GFP protein, could be applied to more complex protein constructs such as antibodies. The comparison between the Bxbl (no NLS) and the Bxbl-NPLCx2 with and without the RanGAP co-expression was repeated using a DNA construct that allowed displaying abrilumab antibody on the cell surface. The nSIE of abrilumab was more than 7-fold higher using the Bxbl-NPLCx2 and more than 8-fold higher when also coexpressing RanGAP (Figure 7). These results suggest that the improved stable integration efficiency is also applicable to bigger gene constructs such as antibodies.

[0136] Example 7 - Antibody discovery process using mammalian cell display

[0137] An antibody library (e.g., a library pre-selected with phage display, or a sitesaturation mutagenized antibody maturation library) is cloned in membrane-anchored IgG format into the mammalian display targeting vector, pBxbl-TV, which contains a selectable marker. Subsequently, the genes encoding the antibody library clones in the targeting vector are stably transfected into a predefined locus in the genome of a mammalian cell such as a CHO cell. The stable integration is catalyzed by Bxbl serine integrase, which inserts the genes of an antibody library clone to the landing pad in the CHO cell’s genome. The Bxbl serine integrase is fused with two copies of Xenopus laevis nucleoplasmin nuclear localization signal in the C-terminus to facilitate more efficient translocation of the Bxbl serine integrase to the nucleus. The landing pad is a DNA sequence flanked by Bxbl serine integrase recognition sites called AttB sites. The targeting vector contains reciprocal recognition sites, called AttP sites, flanking the DNA encoding the antibody genes and the selectable marker. The Bxbl serine integrase- catalyzed strand exchange between the AttB and AttP sites facilitates recombinase mediated cassette exchange (RMCE) and the cells with correct integration of the antibody genes can subsequently be selected with the selectable marker.

[0138] After the cells stably displaying antibodies have recovered from the drug selection, the cells are stained with fluorescently labeled target antigen and fluorescently labeled antibody binding to the Fc of the displayed antibody. This enables the selection of cells with high target antigen binding and high antibody display level using fluorescence- activated cell sorting (FACS). Selection based on surface expression and antigen binding makes it possible to select antibody clones that have good affinity and biophysical properties. The FACS selection is repeated 2-4 times to ensure proper enrichment of the clones of interest. After the collection of the cells of interest, the antibody genes are extracted from the genome using polymerase chain reaction (PCR) and sequenced using next generation sequencing (NGS). The NGS data is used to assess the enrichment of antibody clones during the FACS selection rounds. The most enriched antibody clones are then expressed as soluble antibodies and characterized to determine their antigen binding properties and other biophysical properties.

[0139] Example 8 - Conclusions

[0140] Different NLS types and different proteins involved in the nuclear translocation process affect the Bxbl -mediated stable integration efficiency in CHO cells harboring a landing pad for stable genomic integration of genes. It was observed that Nucleoplasmin NLS showed an increase in stable integration efficiency as N-terminal and C-terminal fusions to Bxbl. The effect was most prominent as a C-terminal fusion to Bxbl, which caused over fivefold higher stable transfection efficiency than Bxbl without NLS (Figure 1A).

[0141] Some proteins naturally have two independent nuclear localization signals, of which both have a role in nuclear transport. It was observed that that adding two Nucleoplasmin NLS sequences to the C-terminus of Bxbl slightly increased stable integration efficiency. Addition of three Nucleoplasmin NLS sequences did not increase stable integration efficiency (Figure 4). Rather, the addition of three Nucleoplasmin NLS sequences slightly lowers the stable integration efficiency relative to the addition of two Nucleoplasmin NLS, although the stable integration efficiency is still greater than using a single NLS. Co-expression of nuclear transport proteins had varying effects on stable integration efficiency. For example, co-expression of Ran GTPase-activating protein 1 (RanGAP) with Bxbl-NPLCx2 serine integrase increased the stable integration efficiency by 48 % but on the other hand co-expression of Importin P decreased the stable integration efficiency substantially. The combination of optimized transfection protocol and Bxbl with optimized NLS sequence, resulted in a stable integration efficiency of 10.01 %. For comparison, Bxbl without NLS produced stable integration efficiency of 2.16 %. All in all, it can be noted that by optimizing the type of NLS sequence fusion for Bxbl, the stable integration efficiency can be increased substantially.

[0142] Nucleotide sequences

[0143]

[0144] Amino acid sequences

[0145]

Claims

CLAIMS1. A site-specific DNA recombinase enzyme which comprises a first nuclear localisation signal (NLS).

2. The enzyme of claim 1, wherein the enzyme is a serine integrase, optionally wherein the enzyme is a Bxbl serine integrase.

3. The enzyme of claim 1 or 2, wherein the first NLS is a monopartite NLS or a bipartite NLS.

4. The enzyme of any one of the preceding claims, wherein the first NLS has a net charge of greater than about 4.6 at pH 7.5, optionally wherein the net charge is about 7.6 at pH 7.5.

5. The enzyme of any one of the preceding claims, wherein the first NLS is positioned at the C-terminus of the enzyme or at the N-terminus of the enzyme.

6. The enzyme of any one of the preceding claims, wherein the first NLS is a NLS from Xenopus laevis Nucleoplasmin, a NLS from Simian Virus 40 or a NLS from the transcription factor EGL-13.

7. The enzyme of any one of the preceding claims, wherein the first NLS comprises or consists of the amino acid sequence of one or more of SEQ ID NOs: 11-13, or a variant thereof.

8. The enzyme of any one of the preceding claims which comprises a second NLS.

9. The enzyme of claim 8, wherein the second NLS is a monopartite NLS or a bipartite NLS.

10. The enzyme of claim 8 or 9, wherein the second NLS has a net charge of greater than about 4.6 at pH 7.5, optionally wherein the net charge is about 7.6 at pH 7.5.

11. The enzyme of any one of claims 8-10, wherein the first NLS is positioned at theC-terminus of the enzyme and the second NLS is positioned at the N-terminus of the enzyme.

12. The enzyme of claim 11, wherein the first NLS is a NLS from Xenopus laevis Nucleoplasmin and / or wherein the second NLS is a NLS from Xenopus laevis Nucleoplasmin, a NLS from Simian Virus 40, a NLS from the transcription factor EGL-13, or a NLS from c-Myc.

13. The enzyme of any one of claims 8-12, wherein the second NLS comprises or consists of the amino acid sequence of one or more of SEQ ID NOs: 11-14, or a variant thereof.

14. A polynucleotide encoding the enzyme of any one of the preceding claims.

15. An expression vector comprising the polynucleotide of claim 14.

16. The expression vector of claim 15 which further comprises a polynucleotide encoding a nuclear translocation protein, optionally wherein the nuclear translocation protein is Ran GTPase-activating protein 1 (RanGAP).

17. A cell comprising the enzyme of any one of claims 1-13, the polynucleotide of claim 14 or the expression vector of claim 15 or 16.

18. The cell of claim 17, which further comprises a targeting vector comprising a first recombinase recognition site and a second recombinase recognition site, optionally wherein the first and second recombinase recognition sites are attP sites.

19. The cell of claim 18, wherein the first recombinase recognition site comprises or consists of the nucleotide sequence of SEQ ID NO: 5, or a variant thereof, and the second recombinase recognition site comprises or consists of the nucleotide sequence of SEQ ID NO: 6, or a variant thereof.

20. The cell of claim 18 or 19, wherein the targeting vector further comprises a selectable marker.

21. The cell of any one of claims 18-20, wherein the first and second recombinase recognition sites flank a target sequence of interest in the targeting vector, optionally wherein the target sequence of interest is a DNA sequence encoding an antibody or fragment thereof.

22. The cell of any one of claims 18-21, wherein the genome of the cell comprises first and second recombinase recognition sites compatible with the first and second recombinase recognition sites of the targeting vector, optionally wherein the first and second compatible recombinase recognition sites are attB sites.

23. The cell of claim 22, wherein the first and second compatible recombinase recognition sites respectively comprise or consist of the nucleotide sequence of SEQ ID NO: 7, or a variant thereof, and the nucleotide sequence of SEQ ID NO: 8, or a variant thereof.

24. The cell of any one of claims 17-23, wherein the cell is a mammalian cell, optionally wherein the cell is a Chinese hamster ovary cell.

25. A method of making the cell of any one of claims 18-24, wherein the method comprises:(i) transfecting a cell according to claim 17 with a targeting vector comprising a first recombinase recognition site and a second recombinase recognition site which flank a target sequence of interest, and a selectable marker; and(ii) selecting transfected cells using the selectable marker.

26. A method of making the cell of claim 17, wherein the method comprises transfecting the cell with an expression vector according to claim 15 or 16.

27. A method of displaying an antibody library, comprising:(i) providing a plurality of different targeting vectors as defined in claim 21, wherein for each different targeting vector the target DNA sequence of interest encodes adifferent antibody or fragment thereof, and optionally transfecting the plurality of targeting vectors into a plurality of cells;(ii) transfecting a plurality of cells with the plurality of targeting vectors to generate a plurality of display cells, or transfecting a plurality of cells comprising the plurality of targeting vectors with an expression vector according to claim 15 or 16 to generate a plurality of display cells; and(iii) selecting transfected display cells using the selectable marker.

28. The method of claim 27, comprising making a plurality of cells by the method of claim 26 and then transfecting the plurality of cells with the plurality of targeting vectors, optionally wherein the step of making the plurality of cells by the method of claim 26 is carried out about 1 day before the step of transfecting the plurality of cells with the plurality of targeting vectors.

29. An antibody display library comprising a plurality of targeting vectors as defined in claim 21, wherein the target sequence of interest is a DNA sequence encoding an antibody or fragment thereof.

30. An antibody expression library comprising a plurality of display cells as defined in claim 27.

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