GENETICALLY MODIFIED RECOMBINANT CELL LINES

Abstract

Glycoproteins that are transgenically produced in mammalian cells exhibit non-human glycan structures. As in humans, this can possibly lead to immune responses, the drug manufacturing potential of these drugs is limited. On the other hand, recombinant protein production in human cells is inefficient due to the cells' poor protein yields, proliferation potential and cellular density. The present application solves these issues by providing a recombinant vertebrate cell that is comprising a non-vertebrate and/or artificial phosphatidylethanolamine-binding protein (PEBP). Compared to a parent cell line, the recombinant cells of the invention exhibit improved cell growth, protein yield and excellent compatibility with other established protein production methods. Furthermore, methods, for producing a cell line with improved vitality, protein expression and cell growth characteristics by introducing a non-vertebrate and/or artificial PEBP is given. Moreover, both a nucleic acid construct that is suitable for regulating recombinant protein expression in a cell by coding for such a PEBP and a recombinant cell comprising such a nucleic acid construct is provided. Lastly, a method for the recombinant expression of a target protein by culturing such a recombinant vertebrate cell of the invention is given, wherein the cell is also comprising an expression construct encoding the target protein.

Claims

1. A recombinant vertebrate cell, comprising (i) a nucleic acid sequence encoding for a non-vertebrate and/or artificial phosphatidylethanolamine-binding protein (PEBP) and/or (ii) a protein consisting of an amino acid sequence of the non-vertebrate and/or artificial phosphatidylethanolamine-binding protein (PEBP).

2. The recombinant vertebrate cell of claim 1, wherein the vertebrate cell is a mammalian cell, such as a human cell, a mouse cell or a hamster cell.

3. The recombinant vertebrate cell of claim 1, wherein the non-vertebrate and/or artificial PEBP is a plant- or insect PEBP.

4. The recombinant vertebrate cell of claim 1, wherein the non-vertebrate and/or artificial PEBP consists of an amino acid sequence that when aligned with the sequence of SEQ ID NO: 623 (human PEBP1) does not comprise the most C terminal alpha helix, preferably does not comprise the sequence of amino acids 177 to 187 of SEQ ID NO: 623, nor a sequence that is 90% identical to such a sequence.

5. The recombinant vertebrate cell of claim 1, wherein the non-vertebrate and/or artificial PEBP is a protein that is heterologous to the species of the vertebrate cell, and/or wherein the nucleic acid sequence is for a heterologous expression of the non-vertebrate and/or artificial PEBP.

6. The recombinant vertebrate cell of claim 1, wherein the vertebrate cell expressing the non-vertebrate and/or artificial PEBP compared to the same vertebrate cell not expressing the non-vertebrate and/or artificial PEBP has a significantly increased vitality, growth and/or proliferation.

7. The recombinant vertebrate cell of claim 1, wherein the non-vertebrate and/or artificial PEBP comprises, and preferably consists of, an amino acid sequence having at least 80% sequence identity to a sequence shown in SEQ ID NO: 820 (CG10298), SEQ ID NO: 821 (CG18594) or SEQ ID NO: 1 (NtFT4).

8. A method producing a cell line with improved characteristics selected from cell vitality, cellular protein expression and cell growth, the method comprising the steps of providing a candidate vertebrate cell of a selected vertebrate species, introducing into the vertebrate cell a non-vertebrate and/or artificial phosphatidylethanolamine-binding protein (PEBP) to obtain a cell with improved characteristics, and culturing the cell with improved characteristics to obtain the cell line with improved characteristic.

9. The method of claim 8, further comprising a step of selection of one cell clone with improved characteristics, and subsequence culturing to obtain a clonal cell-line with improved characteristic.

10. The method of claim 8, wherein the candidate vertebrate cell is a mammalian cell such as a human cell, a mouse cell or a hamster cell.

11. The method of claim 8, wherein the non-vertebrate and/or artificial PEBP consists of an amino acid sequence that when aligned with the sequence of SEQ ID NO: 623 (human PEBP1) does not comprise the most C terminal alpha helix, preferably does not comprise the sequence of amino acids 177 to 187 of SEQ ID NO: 623, nor a sequence that is 90% identical to such a sequence.

12. An isolated nucleic acid construct suitable for recombinant expression in a vertebrate cell or cell line, comprising a coding element operatively linked with one or more expression elements which is suitable for regulating protein expression in the vertebrate cell, wherein the coding element comprises a nucleic acid sequence encoding a non-vertebrate and/or artificial phosphatidylethanolamine-binding protein (PEBP).

13. The isolated nucleic acid construct of claim 12, suitable for transient or stable transfection of the vertebrate cell.

14. A recombinant vertebrate cell comprising the nucleic acid construct of claim 12.

15. A method for recombinant expression of a target protein, comprising a step of culturing under suitable conditions a recombinant vertebrate cell of claim 1 or a cell line with improved characteristics of claim 1, wherein the recombinant vertebrate cell or cell line with improved characteristics further comprises an expression construct encoding the target protein.

Description

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0075] The figures show:

[0076] FIGS. 1(A)-1(F): Drosophila CG18594 PEBP induces mesenchymal traits in MCF-7 cells. Effects of CG18594 on the induction of apoptosis, cell proliferation, migration and the expression and localization of epithelial or mesenchymal markers. FIG. 1A: Apoptosis was induced by 10 ng/mL TNFα (TOP) or 500 nM staurosporine (BOTTOM) for 24 h in MCF-7 cells expressing PEBP1, PEBP4, CG18594, CG10298, NtFT2 or NtFT4, and in MCF-7 control cells. Quantification of apoptotic cells in all cell lines induced by TNFα (TOP) or staurosporine (BOTTOM) (mean±s.e.m., n=9 biologically independent samples (MCF-7 TNFα-induced n=6), p value from Welch's t-test, ***p<0.001, **p<0.01, *p<0.05, N.S. not significant). FIG. 1B: (TOP): Proliferation of MCF-7, MCF-7.sub.CG18594 and MCF7.sub.NtFT4 cells. (BOTTOM): BrdU incorporation by proliferating cells after 2 h was quantified by fluorescence microscopy in relation to total cells stained with DAPI (representative image detail, right) (mean±s.e.m., n=6 random pictures with >200 cells, p value from Welch's t-test, **p<0.01, scale bar 50 μm). FIG. 1C: Migration of MCF-7, MCF-7.sub.CG18594 and MCF-7.sub.NtFT4 cells through the 8-μm pores of a Boyden chamber. Serum served as chemoattractant for 24 h and cells were stained with crystal violet for quantification (mean±s.e.m., n=10 random pictures, p value from Welch's t-test, ***p<0.001, *p<0.05). FIG. 1D: Mesenchymal-like morphological changes of MCF-7.sub.CG18594 cells. Immunofluorescent staining of E-cadherin in MCF-7 (TOP) and MCF-7.sub.CG18594 cells (BOTTOM) and quantification of fluorescence intensity across a linear ROI (white bar). Peak differences between intensities measured from membrane-bound (high values) and cytosolic E-cadherin (low values) (scale bar=25 μm). FIG. 1E: Immunodetection of total E-cadherin protein and its cleavage products in MCF-7 and MCF-7.sub.CG18594 cells. The full-length E-cadherin is indicated by a filled arrowhead and the two main cleavage products that increase in MCF-7.sub.CG18594 cells (˜50 kDa, ˜33 kDa) are marked by empty arrowheads. GAPDH was used as a reference. FIG. 1F: Gene expression analysis by qRT-PCR of epithelial and mesenchymal markers in MCF-7 and MCF-7.sub.CG18594 cells in relation to GAPDH (mean±s.e.m., n=3 biologically independent samples, p value from Welch's t-test, ***p<0.001, **p<0.01, *p<0.05, N.S. not significant).

[0077] FIGS. 2(A)-2(C): NtFT4 forms aggregates, recruits several human interaction partners and thereby amplifies the insulin response in human cells. FIG. 2A: Subcellular localization of HA-EGFP-tagged NtFT4 and CG18594 in HEK-293T cells. Cells were transiently transfected with pcDNA3-HA-EGFP (green=HA-EGFP-NtFT4 top line, HA-EGFP-CG18594 middle line and HA-EGFP bottom line) and pcDNA3-myc-mRFP-H2AZ (red) constructs and imaged 24 h post-transfection. Scale bar 10 μm FIG. 2B: Mediators of NtFT4 in human cells based on protein-protein interactions. Interaction partners of HA-EGFP-NtFT4 were identified by protein quantification in eluates. Protein extracts of cells transiently expressing HA-EGFP and HA-EGFP-NtFT4 were precipitated using anti-HA-labeled magnetic beads, digested with trypsin and analyzed by LC-MS/MS. Differential protein abundance was plotted as log.sub.2 fold changes in HA-EGFP-NtFT4 samples against corresponding p values. Enriched proteins in the HA-EGFP-NtFT4 samples are indicated by color coding from blue to red. Insulin-responsive adhesion proteins DSGi, DSCi and PKP1 are underlined (n=3 biologically independent samples, p values were calculated using the Bayes moderated t-test with default settings). FIG. 2C: Growth response of MCF-7, MCF-7NtFT4 and MCF-7.sub.CG18594 cells to insulin. Serum-starved cells were induced with 40 μg/mL recombinant insulin or with serum-free medium for 48 h and viable cells were quantified (mean ±s.e.m., n=9 biologically independent samples; MCF-7.sub.NtFT4 insulin-exposed n=8, MCF-7.sub.CG18594 insulin exposed and starved cells n=12), p value from Welch's t-test, ***p<0.001, **p <0.01).

[0078] FIG. 3: Transcriptome analysis of MCF-7.sub.NtFT4 cells. MCF-7 and MCF-7.sub.NtFT4 cells were exposed to 40 μg/mL recombinant insulin or starved in serum-free medium, followed by whole-transcriptome MACE analysis and pairwise comparisons. The figure gives shows an overview of deregulated genes in MCF-7 and MCF-7.sub.NtFT4 cells. Pairwise comparisons between insulin-induced and starved cells. Venn diagrams show common responses in the overlap and differential responses in gene expression to insulin exposure. Genes with an FDR <0.05 were included (n=3 biologically independent samples).

[0079] FIG. 4: Specific PEBP signaling pathway mediated by NtFT4. Overview of PEBP signaling altered by NtFT4 expression. Human PEBPs regulate cell proliferation and apoptosis by crosstalk among various signaling pathways (RTK-MAPK, GPCR, NFK(B) Notch and Shh). PEBP1 acts as switch between GPCR and MAPK signaling in a phosphorylation-dependent manner, binding and thereby inhibiting the phosphorylation/activation of either Raf or GRK2. PEBP1 can also bind IKK subunits to inhibit NF-KB signaling and it can inhibit the cleavage and translocation of NCID to the nucleus downstream of Notchi. PEBP4 also intervenes in the MAPK cascade, possibly modulating between MAPK signaling and AKT1. NtFT4 was shown to specifically induce cell proliferation and survival without reducing protein synthesis. A specific function of NtFT4 combines cellular responses that affect cell adhesion, proliferation and differentiation. This involves integrins, desmosomes, insulin-AKTi and NF-KB signaling. The interaction of HA-EGFP-NtFT4 with desmosomal cadherins 963 and cytosolic PKP1 and JUP suggests a specific role in cell adhesion via desmosomes. The enhanced induction of proliferation following insulin exposure is likely to be mediated by the translocation of desmosomal components to the nucleus where they have been shown to induce proliferation. Additionally, transcriptome analysis revealed enhanced expression of genes associated with cell adhesion and interaction with the extracellular matrix (integrins, collagens and ECM-modifying MMP13, MMP15, MMP17 and TIMP2, TIMP3) linking cell adhesion to the observed increase in the proliferation of MCF-7.sub.NtFT4 cells. In the cytosol, deregulated genes indicate specific changes in signaling via insulin and Akti by an increase of BMPs (BMP1, BMP4, BMP8B) and IL18. Other key regulators in this pathway are not affected specifically by NtFT4. In the signaling cascade involving the translocation of NF-κB the nucleus, the expression of Nfkbiz is specifically increased in MCF7-7.sub.NtFT4 cells. The transcription factor SOX9 is upregulated in the nucleus of MCF7-7.sub.NtFT4 cells, as well as slight increases in FOXO1 and FOXO3. Insulin exposure did not downregulate 4EBP3 in MCF7-7.sub.NtFT4 cells (in contrast to MCF-.sub.7 cells) and had differing effects on the transcription of various ribosomal S6 kinases. Red arrows indicate transcriptional upregulation. Blue arrows indicate interaction with HA-EGFP-NtFT4. Dashed arrows indicate protein translocation.

[0080] FIGS. 5(A)-5(C): Improved growth and transfection efficiency in HEK-293T.sub.NtFT4 cells. FIG. 5A: Viable cell density and cell viability of HEK-293T (squares), HEK-293T.sub.CG18594 (triangles) and HEK-293T.sub.NtFT4 (circles) cells adapted to RPMI-1640+1% FCS in 40-mL suspension cultures. Viable cell density (solid lines) and cell viability (dashed lines) were measured in batch cultures for 12 days until all lines surpassed their VCD peaks. Representatively shown for n=1. FIG. 5B: Viable cell density and cell viability of HEK-293T (squares) and HEK-293T.sub.NtFT4 (circles) cells grown in 250 mL ExCELL medium in Optimum Growth flasks (Thomson) were measured in batch cultures of three different cell passages of HEK-293T and HEK-293T.sub.NtFT4 cells (passage ranging between P2-8). n=3 FIG. 5C: Transient transfection efficiency of HEK-293T (TOP) cells and HEK-293T.sub.NtFT4 (BOTTOM) cells with pcDNA3Lifeact-mRFP was quantified by FACS analysis. Aliquots of cells were harvested after 1-4 days and after gating for viable, single cells (FIG. 9), cells with a distinct mRFP signal were quantified according to the indicated gate (grey). Data were analyzed using Flowing Software v2.5.1.

[0081] FIGS. 6(A)-6(F): Improved protein expression in HEK-293T.sub.NtFT4 cells. HEK-293T and HEK-293T.sub.NtFT4 cells were transiently transfected with pAPT-1E4IgG1 and antibody yield was quantified in the supernatants. FIG. 6A Immunodetection of IgG1 4 days after transfection with different plasmid and PEI concentrations (plasmid=10-30 μg/mL, PEI=30-90 μg/mL). Quantification of band intensities using ImageJ v1.501 revealed highest yield using 20 μg/mL plasmid and 60 μg/mL PEI (arrowhead). FIG. 6B: Viable cell density (solid lines) and cell viability (dashed lines) of HEK-293T (squares) cells and HEK-293T.sub.NtFT4 (circles) cells after transient transfection with pAPT-1E4-IgG1. Aliquots of cells were analyzed on each day after transfection until protein expression reached a steady state (mean±s.e.m., n=3 biologically independent samples). FIG. 6C: Yield of human IgG1 in the supernatants of HEK-293T (circles) cells and HEK-293T.sub.NtFT4 (squares) cells analyzed by ELISA (mean±s.e.m., n=3 biologically independent samples). FIG. 6D: Quantification of IgGi protein in the supernatants of HEK-293T and HEK-293T.sub.NtFT4 cells of days 2-6 by western blot using anti-human κ light chain antibody of reduced samples and measuring relative band intensities using ImageJ v1.50i. FIG. 6E: Immunodetection of IgG1 after transient transfection in HEK-293T.sub.NtFT4 cells and Avastin using non-reducing (left lanes) and reducing (right lanes) loading buffer to analyze correct antibody assembly of heavy and light chains. Under reducing conditions only the kappa-light chains are detected for transiently expressed IgGi (left) and Avastin (right), empty arrowheads. Under non-reducing conditions the assembled antibodies (filled arrowheads) are detected for both the transiently expressed IgG1 and Avastin. FIG. 6F: Expression of NtFT4 in HEK-293T.sub.NtFT4 cells. Prior to experiments, NtFT4 expression was verified by sqRT-PCR using GAPDH as the reference gene.

[0082] FIGS. 7(A)—7(B): Peptide sequence alignment and 3D protein models of PEBP1, PEBP4, NtFT2, NtFT4, CG18594 and CG10298. FIG. 7A: Sequences of human PEBP1 (NP_002558.1) and PEBP4 (NP_659399.2), Nicotiana tabacum NtFT2 (AFY06688.1) and NtFT4 (AFS17372), and Drosophila melanogaster CG18594 (NP_651051.1) and CG10298 (NP_649643.1) were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). Identical amino acids are indicated by black boxes, and similar amino acids by gray boxes. The highly conserved region of PEBPs (binding pocket) is marked by a blue line. Phosphorylation sites in PEBP1 (T42 and 5.sup.153) are marked by red asterisks and are responsible for the interaction and thus function switches of PEBP1. The decisive motifs of NtFT2 and NtFT4 (NAPDIIDS and YAPGW, respectively) are marked by the green line. In PEBP1, this loop region is affected by phosphorylation of S153. The region of the C-terminal α-helices of animal PEBPs is marked by an orange line. FIG. 7B: Protein models of PEBPs were created using SWISS-MODEL (https://swissmodel.expasy.org/). Human, bovine, mouse and rat RKIP served as the main templates for human and fly PEBPs. These were supplemented with fly PEBP CG7054 and TM16 from Trichuris muris for CG10298 and CG18594. Protein models for NtFT2 and NtFT4 were created using FT from Arabidopsis thaliana under different conditions as template. The α-helices and β-strands are highlighted in blue and green, respectively. The loop region, which appears to be critical for protein interaction, was turned facing the top right and is flanked by the α-helix containing S153 (PEBP1) and the sequence motifs defining plant NtFT proteins (red arrow). The C-terminal α-helices of animal PEBPs are indicated by gray arrows.

[0083] FIGS. 8(A)-8(B): Expression analysis of survival and cell cycle related genes. Analysis of gene expression by qRT-PCR in relation to GAPDH. FIG. 8A: Genes related to cell survival or FIG. 8B cell cycle regulation in MCF-7 and MCF-7.sub.CG18594 cells (mean ±s.e.m., n=3 biologically independent samples, p value from Welch's t-test, ***p<0.001, **p<0.01, *p<0.05, N.S. not significant).

[0084] FIGS. 9(A)-9(B): Gating strategy for FACS analysis and quantification of transfection efficiency in HEK-293T and HEK-293T.sub.NtFT4 cells. FIG. 9A: Intact cells were initially gated, excluding cell debris and larger cell aggregates (dot plot TOP LEFT, grey population). For subsequent analysis, only single cells (dark grey population, TOP RIGHT) were selected, plotting the population of intact cells against the area and height of FSC signals (FSC-A and FSC-H, respectively). For the analysis of transiently transfected cells with pcDNA3-LifeActmRFP, histograms of events detected at 610±20 nm were plotted (BOTTOM). The gate was set using non-transfected control cells with a maximum of 0.1% of the single cells in this gate (grey gate, BOTTOM RIGHT). The percentage of cells in this gate was quantified using Flowing Software v2.5.1 (BOTTOM LEFT, representative data for HEK-293T.sub.NtFT4 cells 3 days after transfection) FIGS. 9B: Transient transfection efficiency (using 20 μg/mL pcDNA3-LifeAct-mRFP and 60 μg/mL PEI) of HEK-293T (black line) and HEK-293T.sub.NtFT4 (grey line) cells up to 4 days post-transfection.

[0085] FIGS. 10(A)-10(D): Gene integration and expression in MDA-MB231 and MCF-7 cell lines. FIG. 10A and FIG. 10C: Verification of correct integration into the AAVS1 safe harbor locus by 5′ and/or 3′ border PCR and agarose gel electrophoresis. A primer pair for each border was used, one primer binding to the adjacent chromosomal region and the other to the integrated sequence. MDAMB231 (FIG. 10A) and MCF-7 (FIG. 10C) lines carrying the expression cassettes for human PEBP1 and PEBP4, tobacco NtFT2 and NtFT4, and fruit fly CG10298 and CG18594 were verified. FIG. 10B and FIG. 10D: Verification of gene expression in clonal lines expressing the different PEBPs by sqRT-PCR. GAPDH expression was included as reference and GFP expression to verify expression of the selection marker GFP-2A-Puromycin. FIG. 10B: Expression of PEBPs in MDA-MB231 and MDAMB231.sub.PEBP cells. FIG. 10D: Expression of PEBPs in MCF-7 and MCF-7.sub.PEBP cells.

[0086] And in the sequences:

[0087] The following sequences are of particular exemplary use in context of the invention:

[0088] SEQ ID NO: 623 shows sp|P30086|PEBP1_HUMAN Phosphatidylethanolamine-binding protein 1 OS=Homo sapiens OX=9606 GN=PEBP1 PE=1 SV=3

TABLE-US-00001 MPVDLSKWSGPLSLQEVDEQPQHPLHVTYAGAAVDELGKVLTPTQVKNRP TSISWDGLDSGKLYTLVLTDPDAPSRKDPKYREWHHFLVVNMKGNDISSG TVLSDYVGSGPPKGTGLHRYVWLVYEQDRPLKCDEPILSNRSGDHRGKFK VASFRKKYELRAPVAGTCYQAEWDDYVPKLYEQLSGK

[0089] SEQ ID NO: 624 shows >sp|Q96S96|PEBP4_HUMAN Phosphatidylethanolamine-binding protein 4 OS=Homo sapiens OX=9606 GN=PEBP4 PE=1 SV=3

TABLE-US-00002 MGWTMRLVTAALLLGLMMVVTGDEDENSPCAHEALLDEDTLFCQGLEVFY PELGNIGCKVVPDCNNYRQKITSWMEPIVKFPGAVDGATYILVMVDPDAP SRAEPRQRFWRHWLVTDIKGADLKKGKIQGQELSAYQAPSPPAHSGFHRY QFFVYLQEGKVISLLPKENKTRGSWKMDRFLNRFHLGEPEASTQFMTQNY QDSPTLQAPRERASEPKHKNQAEIAAC

[0090] SEQ ID NO: 822 shows >tr|Q9VD02|Q9VD02_DROME GH14779P OS=Drosophila melanogaster OX=7227 GN=Dmel\CG7054 PE=1 SV=1

TABLE-US-00003 MDDIVPDVLDAVPAGTIKVIYGDDLEVKQGNELTPTQVKDQPIVSWSGLE GKSNLLTLLMVDPDAPTRQDPKYREILHWSVVNIPGSNENPSGGHSLADY VGSGPPKDTGLHRYIFLLYRQENKIEETPTISNTTRTGRLNFNARDFAAK HGLGEPIAANYYQAQYDDYVPIRNKTIVG

[0091] SEQ ID NO: 820 shows >tr|Q9VI08|Q9VI08_DROME GH28351P OS=Drosophila melanogaster OX=7227 GN=BcDNA:GH28351 PE=2 SV=1

TABLE-US-00004 MSDSTVCFSKHKIVPDILKTCPATLLTVTYGGGQVVDVGGELTPTQVQSQ PKVKWDADPNAFYTLLLTDPDAPSRKEPKFREWHHWLVVNIPGNQVENGV VLTEYVGAGPPQGTGLHRYVFLVFKQPQKLTCNEPKIPKTSGDKRANFST SKFMSKYKLGDPIAGNFFQAQWDDYVPKLYKQLSGKK

[0092] SEQ ID NO: 821 shows >tr|Q9VD01|Q9VD01_DROME LP12095P OS=Drosophila melanogaster OX=7227 GN=Pebpi PE=1 SV=1

TABLE-US-00005 MDTAGIIPDIIDVKPASKATITYPSGVQVELGKELTPTQVKDQPTVVFDA EPNSLYTILLVDPDAPSREDPKFRELLHWLVINIPGNKVSEGQTIAEYIG AGPREGTGLHRYVFLVFKQNDKITTEKFVSKTSRTGRINVKARDYIQKYS FGGPVAGNFFQAQYDDYVKTLIETVQ

[0093] SEQ ID NO: 82 shows TOBAC Flowering locus T-like protein FT2 (Nicotiana tabacum)

TABLE-US-00006 MLRANPLVVSGVIGDVLDPFTKSVDFDVVYNNNVQVYNGCGLRPSQIVNQ PRVDIAGDDFRTFYTLVMVDPDAPTPSNPNLREYLHWLVTDIPATTEATF GNEIVSYERPQPSLGIHRYIFVLFRQLDREVVNAPDIIDSREIFNTRDFA RFHGLNLPVAAVYFNCNREGGTGGRHL

[0094] SEQ ID NO: 1 shows >tr|J9WPJ0|J9WPJ0_TOBAC Flowering locus T OS=Nicotiana tabacum OX=4097 GN=FT4 PE=2 SV=1

TABLE-US-00007 MPRIDPLIVGRVVGDVLDPFTRSVDLRVVYNNREVNNACGLKPSQIVTQP RVQIGGDDLRNFYTLVMVDPDAPSPSNPNLREYLHWLVTDIPATTDTSFG NEVICYENPQPSLGIHRFVFVLFRQLGRETVYAPGWRQNFSTRDFAEVYN LGLPVSAVYFNCHRESGTGGRRAY

[0095] The sequence protocol comprises the following sequence:

[0096] SEQ ID Nos: 1-622 show plant PEBP amino acid sequences,

[0097] SEQ ID Nos: 623-624 show homo sapiens amino acid sequences,

[0098] SEQ ID Nos: 625-819 show mammalian amino acid sequences,

[0099] SEQ ID Nos: 820-928 show insect PEBPs amino acid sequences,

[0100] SEQ ID Nos: 929-1127 show prokaryote PEBP amino acid sequences,

[0101] SEQ ID Nos: 1128-1270 show fungi PEBP amino acid sequences,

[0102] SEQ ID Nos: 1271-1368 show primer nucleic acid sequences of table 1,

[0103] SEQ ID Nos: 1369-1370 show amino-acid sequences of protein interaction motifs.

EXAMPLES

[0104] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0105] The examples show:

[0106] Example 1: CG18594 induces mesenchymal traits, promoting apoptotic resistance and proliferation in MCF-7 cells. Initially, the pro-apoptotic activity of PEBP1 and the anti-apoptotic activity of PEBP4 in the human breast cancer cell line MCF-7 were investigated, confirming their reported properties (FIG. 1A). Among the non-human PEBPs, CG18594 in particular conferred pronounced apoptotic resistance (FIG. 1A), increasing the ability of MCF-7 cells to inhibit responses to TNFα, in a similar manner to PEBP4.

[0107] The ability of MCF-7.sub.CG18594 cells to resist apoptosis was surprising because CG18594 sequence alignments and protein models indicated higher similarity to PEBP1 than PEBP4 (42.6% vs. 27.0%, FIG. 7). In the control line, the ratio of apoptotic MCF-7 cells increased by 26.1% from 8.0% (±1.5%) without treatment to 34.1% (±2.7%) in the presence of TNFα. In the two most resistant lines, TNFα increased the proportion of apoptotic cells by only 8.5% (MCF-7.sub.CG18954) and 12.3% (MCF-7PEBP.sub.4). In contrast, NtFT4 and NtFT2 acted in a similar manner to PEBP1, increasing TNFα sensitivity (MCF-7NtFT4+43.7%; MCF-7NtFT2+30.1%; MCF-7.sub.PREP1+33.0%). Finally, CG10298 showed no significant effect on the induction of apoptosis by TNFα (FIG. 1A).

[0108] CG18594 also induced the proliferation of MCF-7 cells (+25.5% BrdU incorporation in MCF-7.sub.CG18594 vs. MCF-7, FIG. 1B). This was confirmed by analyzing the doubling times of MCF-7 cells in monolayer cultures. In T25 flasks, the doubling time of MCF-7.sub.CG18594 cells declined by 7.6% compared to MCF-7 controls. A more pronounced decline when cells were visualized by live imaging in 384-well plates was observed. Here, the doubling time of the MCF-7.sub.CG18fflcells (determined by measuring the confluence) was reduced by 61.4%.

[0109] The analysis of MCF-7.sub.CG18594 cell growth also revealed a partial change toward a mesenchymal-like morphology, which was confirmed by the slight loss of apical-basal polarity during MCF-7.sub.CG18594 cell migration (FIG. 1C). This reflected the altered distribution of E-cadherin in MCF-7.sub.CG18594 cells, with less distinct localization to the membrane (FIG. 1D). According to qRT-PCR data, the E-cadherin gene (CDH1) was expressed normally, but the abundance of the full-length protein (135 kDa) declined due to enhanced cleavage, which caused the accumulation of cytoplasmic fragments and thus altered E-cadherin signaling (FIG. 1E, FIG. 1F). The mesenchymal markers N-cadherin, Snai1, Snai2 and vimentin were barely detectable whereas the epithelial markers E-cadherin and β-catenin, as well as the cytokeratins Krt18 and Krt19, remained strongly expressed in MCF-7.sub.CG18594 cells (FIG. 1F). Even so, various survival genes and cell cycle regulators were slightly induced in the MCF-7.sub.CG18594 cells (FIG. 8). CG18594 thus appears to confer some mesenchymal traits (apoptotic resistance, migratory capacity, partial loss of polarity) but does not induce a full epithelial to mesenchymal transition in MCF-7 cells.

[0110] Example 2: NtFT4 amplifies the insulin response in MCF-7 cells. NtFT4 and NtFT2 increased the sensitivity of MCF-7 cells toward TNFα in a similar manner to PEBP1. However, PEBP1 had no effect on growth and NtFT2 reduced the growth rate of MCF-7 and MDA-MB231 cells, whereas NtFT4 was the only PEBP to induce proliferation in both MCF-7 (+2.03%) and MDA-MB231 cells (+5.67%). The analysis of BrdU incorporation revealed an even greater increase in the proliferation of MCF-7.sub.NtFT4 cells (+20.7% vs. MCF-7, FIG. 1B), and live-cell imaging produced a similar result (−22.7% doubling time,). The two latter techniques are likely to be more accurate because they do not require cell detachment prior to counting, which was influenced by the effect of NtFT4 on cell adhesion. The proliferation of MCF-7.sub.CG18594 was associated with the partial expression of mesenchymal traits, whereas NtFT4 appeared to interfere directly with epithelial cell junction proteins and their cell signaling pathways. The transient expression of HA-EGFP-NtFT4 together with myc-mRFP-H2AZ revealed the different subcellular localization of NtFT4 and CG18594 (FIG. 2A). Whereas HA-EGFP-CG18594 was located uniformly throughout the cell, like HA-EGFP, HA-EGFP-NtFT4 accumulated in the nucleus and also appeared to form aggregates in the cytoplasm.

[0111] Immunoprecipitation of HA-EGFP-NtFT4 interaction complexes followed by LC-MS/MS revealed that multiple NtFT4 interaction partners were associated with epithelial cell adhesion, especially components of desmosomes. These not only stabilize cell junctions but also act as components in the insulin signal transduction pathway, translocating from the membrane to the nucleus to induce proliferation (see FIG. 2B). Therefore MCF-7.sub.NtFT4 and MCF-7.sub.CG18594 cells were investigated for their response to insulin. Although cell numbers also increased in untreated cultures, insulin exposure triggered the rapid proliferation of MCF-7.sub.NtFT4 cells (+243.3%) over a period of 48 h compared to MCF-7 (+121.8%) and MCF-7.sub.CG18594 cells (+57.7%; FIG. 2C).

[0112] Transcriptome analysis of MCF-7 and MCF-7.sub.NtFT4 cells, either starved in serum free medium or exposed to insulin, revealed 4811 differentially regulated genes (FDR<0.05) in MCF-7 cells and 4026 differentially regulated genes in MCF-7.sub.NtFT4 cells following insulin treatment (FIG. 3). 2023 genes were identified that were specifically deregulated in MCF-7 cells and 1238 specifically deregulated in MCF-7.sub.NtFT4 cells, indicating differing responses to insulin induction.

[0113] The differential gene expression in MCF-7.sub.NtFT4 and MCF-7 cells cultivated under starvation conditions or in the presence of insulin was also analyzed. These data support the previous assumption as 3300 (exposure to insulin) and 1794 (starved cells) genes were observed with significant differential expression (FDR<0.05) between MCF-7.sub.NtFT4 and MCF-7 cells. But only genes with a normalized read count >5 and a |log.sub.2FC|>1 were included for further analysis. These genes were analyzed using STRING to identify differentially affected processes. The comparison of MCF-7.sub.NtFT4 and MCF-7 cells grown in medium containing insulin revealed several significantly enriched processes among the 471 deregulated genes that were included in the query. The GO terms of special interest to narrow down the function of NtFT4 in human cells were regulation of cell differentiation (84 genes deregulated in MCF-7.sub.NtFT4 cells, FDR=4.98×10.sup.−9), regulation of cell population proliferation (75 genes deregulated in MCF-7.sub.NtFT4 cells, FDR=3.62×10.sup.−7) and cell adhesion (45 genes deregulated in MCF-7.sub.NtFT4 cells, FDR=1.23×10.sup.−5). Many of these genes were upregulated in MCF-7.sub.NtFT4 cells, whereas no enriched processes were identified among the downregulated genes. In starved cells, the overall number of deregulated genes was lower (318 genes included in the query), but the GO terms regulation of cell differentiation (61 genes deregulated in MCF7.sub.NtFT4 cells, FDR=7.89×10.sup.−7, regulation of cell population proliferation (49 genes deregulated in MCF-7.sub.NtFT4 cells, FDR=1.20×10.sup.−4) and cell adhesion (32 genes deregulated in MCF-7.sub.NtFT4 cells, FDR=1.60×10.sup.−4) were again significantly enriched and also mainly found among the upregulated genes.

[0114] Significant upregulation of several genes encoding members of the integrin (ITGA6, ITGAV, ITGA2 and ITGB1), collagen (COL4A6 and COL5A1) and galectin (LGALS7B, LGALS3 and LGALS1) families revealed that cell-cell and cell-matrix interactions are affected by the expression of NtFT4. Some individual metalloprotease genes were influenced by NtFT4 (MMP13, ADAM29) but there was no major transcriptional reprogramming of the MMP or ADAM families. The comparison of insulin-exposed and starved cells revealed the upregulation of several genes in the cell cycle and small molecule metabolic process categories in both MCF-7 and MCF-7.sub.NtFT4 cells. In MCF-7.sub.NtFT4 cells, 108 of the 386 genes upregulated by insulin exposure were related to the cell cycle, compared to 76 of 478 insulin-induced genes in MCF-7 cells. These 108 genes included 48 that were specifically or more strongly upregulated in MCF-7.sub.NtFT4 cells. Several of these genes are involved in the DNA damage response or cell cycle signaling and regulation, whereas others play a role in the assembly of the kinetochore and centrioles or are directly involved in cytokinesis.

[0115] Taken together, these data show that the expression of NtFT4 in human cells accelerated their growth via the specific deregulation of genes linking cell adhesion (or rather cell surface signaling) with intrinsic cues controlling cell proliferation and differentiation. The growth-promoting effects of insulin were enhanced in MCF-7.sub.NtFT4 cells, but proliferation was also induced in the absence of this growth factor by the upregulation of genes involved in intercellular signaling and interactions with the extracellular matrix (ITGA6, ITGAV, COL5A 1, LGALS7, SPOCK1), signal transduction (TRIB2, BMP4, CASK) and transcriptional regulation (SOX9) (FIG. 4). These findings suggest two major application routes for NtFT4, namely the development of novel therapies targeting PEBPs and the utilization of plant PEBPs to improve cell growth in human cell lines used for recombinant protein production.

[0116] Example 3: Improved growth of HEK-293T cell suspension cultures expressing NtFT4. A major drawback of mammalian (particularly human) cells for heterologous protein expression is their susceptibility to loss of vigor under unfavorable conditions, resulting in poor growth. This often occurs during scale-up, because the optimal density or medium composition achieved at lower scales can be disrupted. Cells growing in suspension are also more susceptible to growth suppression due to anoikis and mechanical stress. Therefore, human HEK-293T cells were used, which can be grown as monolayers or suspension cultures, to investigate the effect of NtFT4 and CG18594 on growth and thus determine whether these proteins can improve growth and performance in a biotechnologically relevant human cell line.

[0117] HEK-293T cells were established that stably express NtFT4 or CG18594 before converting them to suspension cultures and adapting them to animal component-free Ex-CELL 293 medium. Then the viable cell density (VCD) was analyzed in different culture conditions.

[0118] HEK-293T.sub.CG18594 cells showed impaired growth and viability, whereas the growth of HEK-293T.sub.NtFT4 cells improved at all stages. Therefore, these cells were selected for further analysis (FIG. 5). The maximum VCD increased by 13.5% in serum-containing RPMI medium (from 2.15×10.sup.6 to 2.44×10.sup.6 cells/mL, FIG. 5A) and by 30.1% in the Ex-CELL 293 medium (from 2.59×10.sup.6 to 3.37×10.sup.6 cells/mL; FIG. 5B). In the latter, the VCDs of three different passages were measured to also take passage number into account. Passage number had an effect on VCDs, as shown by variations in the highest VCDs across the three passages of HEK-293T and HEK-293T.sub.NtFT4 cells (VCD.sub.max HEK-293T: 1.89×10.sup.6-2.50×10.sup.6 cells/mL; VCD.sub.max HEK-293T.sub.NtFT4 2.31×10.sup.6-3.81×10.sup.6 cells/mL). However, the HEK-293T.sub.NtFT4 cells always achieved a higher VCD compared to the parallel HEK-293T cultures (VCD.sub.max HEK-293T.sub.NtFT4+49.9%, +21.7%, and +52.6%, respectively). This increase in VCD matched the response of MCF-7.sub.NtFT4 cells to insulin, reflecting the presence of recombinant insulin as a major growth factor in complete ExCELL medium.

[0119] In theory, accelerated growth should lead to shorter cultivation times and higher cell densities, which should in turn achieve higher productivity in terms of protein expression. HEK-293T.sub.NtFT4 cells thus meet the primary criterion for an optimized expression platform. Next, the impact of NtFT4 expression on transfection efficiency was determined by monitoring transfection over 4 days using a non-secreted version of the marker protein mRFP. Multiple transfection parameters were tested (such as different reagents and DNA concentrations, durations and media) but generally observed no differences in transfection efficiency between HEK-293T.sub.NtFT4 and HEK-293T cells (FIG. 5C). In each case, the peak of 85% mRFP-positive cells was reached after 3 days based on our threshold gates (FIG. 9). A slightly higher frequency of mRFP-positive HEK-293T cells was observed compared to HEK-293T.sub.NtFT4 cells on day 1, but no difference or a slightly higher frequency in HEK-293T.sub.NtFT4 cells thereafter. The HEK-293T.sub.NtFT4 cells were also found to be more tolerant toward the transfection procedure. Using high-cell densities (20×10.sup.6 cells/mL) during transfection as previously recommended for Ex-CELL medium, it was possible to apply 20 μg/mL of plasmid DNA and 60 μg/mL linear polyethylenimine (PEI) without affecting the viability of HEK-293T.sub.NtFT4 cells (Table 1).

TABLE-US-00008 TABLE 1 Viability of HEK-293T.sub.NtFT4 cells after transfection at high cell densities. HEK-293T.sub.NtFT4 cells were transiently transfected with pcDNA3-Lifeact-mRFP and the impact of different plasmid and PEI concentrations on cell viability and viable cell densities was analyzed for 6 days after transfection. Viability and viable cell density were measured using a Vi-Cell XR cell counter. Viability VCD 10 μg/mL 20 μg/mL 10 μg/mL 20 μg/mL plasmid 30 plasmid 60 plasmid 30 plasmid 60 μg/mL PEI μg/mL PEI μg/mL PEI μg/mL PEI Day 2 94.9% 95.6% 1.63 × 10.sup.6 1.45 × 10.sup.6 Day 3 95.2% 96.5% 1.89 × 10.sup.6 1.82 × 10.sup.6 Day 6 96.4% 95.4% 4.22 × 10.sup.6 3.75 × 10.sup.6

[0120] The VCD tends to decrease over time in highly-productive transient expression experiments (particularly in batch cultures) but the viability of the HEK-293T.sub.NtFT4 cells returned to >90% by the second day after transfection and, in contrast to HEK-293T cells, the VCD increased throughout the experiments. Recombinant protein production was quantified by expressing a human IgG1 antibody in HEK-293T and HEK-293T.sub.NtFT4 cells. The expression level was optimized by adapting plasmid and PEI concentrations in HEK-293T.sub.NtFT4 cells (FIG. 6A), and then compared the two most promising concentrations of plasmid and PEI in both cell lines and used 20 μg/mL plasmid and 60 μg/mL PEI for subsequent comparisons of the two cell lines (FIG. 6 B-D). The VCD of the HEK-293T.sub.NtFT4 cells again increased rapidly after transfection from 1×10.sup.6 to 6.36×10.sup.6 cells/mL (±3.25×105) after 6 days, whereas that of the HEK-293T cells increased from 1×10.sup.6 to 2.69×10.sup.6 cells/mL (±1.31×10.sup.5) under the same conditions (FIG. 6B). Importantly, the enhanced proliferation of HEK-293T.sub.NtFT4 cells did not come at the expense of lower protein yields. By day 2, the concentration of IgG1 in the HEK-293T.sub.NtFT4 cell medium was already 15.67% higher than in the corresponding HEK-293T cultures, and by day 5 the HEK-293T.sub.NtFT4 cells surpassed the HEK-293T cells by 46.75% (FIG. 6C, D). After 7 days, the secreted IgG1 concentration reached a steady state (7.51±0.07 mg/L in the HEK293T.sub.NtFT4 cultures, compared to 5.64±0.29 mg/L for the HEK-293T cells). By day 8, the IgG1 yield of the HEK-293T.sub.NtFT4 cells (7.52±0.43 mg/L) exceeded the HEK-293T cells (5.70±0.24 mg/L) by 31.95%. The HEK-293T cells never reached the level that HEK293T.sub.NtFT4 cells had already achieved by day 5 (6.11±0.15 mg/L). Also, the assembly of the heavy and light chains of the secreted IgG1 antibody was confirmed by immunodetection of the κ-light chain under non-reducing conditions (FIG. 6E).

[0121] The Materials and Methods show:

[0122] Experimental Design: The functionality of non-mammalian PEBPs was analysed using human breast cancer cell lines because the functions of the human proteins PEBP1 and PEBP4 have previously been characterized in detail using these models. Two PEBPs were selected each from tobacco (Nicotiana tabacum) and fruit fly (Drosophila melanogaster). The two FT-like proteins NtFT2 and NtFT4 are representative of one subclade of plant PEBPs (the other two subclades found in angiosperms are the TFL1-like and MFT-like proteins), and were selected to see whether their functional specificity would transfer to a non-plant host. Furthermore, two poorly-characterized fruit fly PEBPs were also selected (CG18594 and CG10298) which are similar to human PEBP1. Analysis of cell line growth and exposure to apoptosis-inducing agents led us to focus on NtFT4 and CG18594, the two PEBPs with the strongest and most consistent effects in the human cells. HEK-293T rather than HEK-293 or either of the commercially used HEK-293 derivatives were selected because the former is a major model cell line for biomedical research and is also used as an expression platform for recombinant proteins and viral particles.

[0123] Reagents, plasmids and cloning: Reagents were purchased from Thermo Fisher Scientific or Sigma-Aldrich if not otherwise stated. Incubations were carried out at room temperature if no temperature is specified. Primers used for cloningTo create stable cell lines expressing different PEBPs, the Genome-TALER human AAVS1 safe harbor gene knock-in kit (GeneCopoeia) was used. For stable transfection of MCF-7, MDA-MB231 and HEK-239T cells with PEBP1, PEBP4, NtFT2, NtFT4, CG18594 and CG10298 constructs, the coding sequces were amplified by PCR using primers with attached restriction sites and were transferred to vector DC-DON-SH1 by restriction and ligation. For the immunoprecipitation of PEBPs, the codon-optimized NtFT4 coding sequence was cloned in frame with HA-EGFP and transferred to vector pcDNA3 by amplifying each segment, digesting the products with restriction enzymes SpeI/XhoI (HA-EGFP), XhoI/ApaI (NtFT4) and SpeI/ApaI (pcDNA3) and ligating them (all restriction enzymes were from New England Biolabs). Vectors pcDNA3-HA-EGFP-CG18594 or pcDNA3-HA-EGFP-NtFT4 and pcDNA3-myc-mFRP-H2AZ were used for colocalization studies, and were prepared by restriction ligation using XhoI/XbaI. The vector pcDNA3-LifeAct-mRFP was used to measure transfection efficiency in the stable cell lines already expressing cytoplasmic GFP. Therefore, the coding sequences for LifeAct (Ibidi) and mRFP were attached by PCR and transferred the product to pcDNA3 using KpnI/XbaI. All plasmids were verified by sequencing. Vector pAPT-1E4-IgG1, encoding a human IgG1 with a κ light chain, was a kind gift from Dr. Nicole Raven (Fraunhofer IME, Aachen, Germany).

[0124] Cell Culture: Adherent MCF-7, MDA-MB231 and HEK-293T cells were grown in RPMI-1640 GlutaMAX medium with 5% fetal calf serum (FCS) and a 1% antibiotic-antimycotic mix in six-well plates for transfection and in T25 and T75 flasks. HEK-293T cells and their derivatives were converted to shaking suspension cultures either in complete RPMI-1640 medium by reducing the serum to 1% in steps or by immediate transfer to Ex-CELL-293 medium with 10 mM HEPES and 6 mM L-glutamine. Suspension cultures were incubated on an orbital shaker at 110 rpm in a 5% CO.sub.2 atmosphere at 37° C. with a relative humidity of ˜93%. The cells were cultivated in 250-mL Erlenmeyer flasks containing 40 mL medium or in 500-mL Optimum Growth flasks (Thomson) containing 250 mL medium.

[0125] Establishing stable cell lines: Cells were transferred to six-well plates in Opti-MEM for transfections using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. DC-DON plasmids with the expression cassette for different PEBPs were co-transfected with the two TALEN plasmids targeting the AAVS1 safe harbor locus (GeneCopoeia). Successful transfection was verified by fluorescence microscopy to detect expression of the integrated selection marker GFP 2A puromycin. Subsequently, positive cells were selected in complete medium containing 1 μg/mL puromycin dihydrochloride. Stable clonal lines were established by limiting dilution in 96-well plates followed by the selection of colonies grown from single cells. Established MCF-7, MDA-MB231 (FIG. 10) and HEK-293T cells expressing different PEBPs were named according to the convention MCF-7.sub.PEBP, MDA-MB231.sub.PEBP and HEK-293T.sub.PEBP (where ‘PEBP’ refers to the lines collectively or is replaced by the name of a specific protein).

[0126] Verification of single-gene integration and gene expression: RNA was isolated from each established line using the NucleoSpin RNA kit (MachereyNagel) followed by reverse transcription using PrimeScript RT master mix (Takara) and sqRT-PCR with gene-specific and GAPDH primers. To confirm gene integration, genomic DNA from each cell line was isolated using the NuceloSpin tissue kit for genomic DNA purification (Macherey-Nagel) and specific knock-in was verified by PCR using the primer mixes for the 5′ and 3′ junction regions (GeneCopoeia).

[0127] Apoptosis assay: MCF-7.sub.PEBP and MDA-MB231.sub.PEBP cell lines (PEBP1, PEBP4, NtFT2, NtFT4, CG18594 and CG1o298) were seeded at a density of 2.5×10.sup.5 cells/mL in six-well plates. After 24 h the cells were starved for another 24 h by replacing the medium with RPMI plus 1% antibiotic-antimycotic mix but without FCS. Apoptosis was induced by adding 500 nM staurosporine or 10 ng/mL TNFα (100 ng/mL was used for the MDA-MB231 cells) for 24 h. Cells were then harvested with a cell scraper, washed with PBS, filtered through a 35-μm cell strainer (Falcon, Corning) and stained with 7-amino-actinomycin (7-AAD) and Annexin V-BV421 (BD Biosciences) in Annexin V binding buffer (0.01 M HEPES/NaOH (pH 7.4), 0.14 M NaCl, 2.5 M CaCl.sub.2). A FACSCelesta (BD Biosciences) was used for subsequent analysis with the following parameters: 7-AAD excitation=561 nm, detection=670/30 nm; Annexin V-BV421 excitation=405 nm, detection=450/40 nm. Gating and assignment of populations was carried out as shown in FIG. S2. Data were quantified using FACSDiva v8.0.1.1 (BD Biosciences). Populations containing distinguishable late apoptotic and dead cells (7-AAD and Annexin-V positive) were compared.

[0128] Proliferation assay: Cells were not uniform in their ability to settle after seeding so several methods to analyze the proliferation of adherent MCF-7PEBP and MDA-MB231.sub.PEBP cell lines (PEBP1, PEBP4, NtFT2, NtFT4, CG18594, CG10298) have been used. First, cells were counted for 4 days after seeding 3×10.sup.5 cells in three replicates for each time point into T25 flasks in complete medium. At each time point, the cells were harvested, carefully resuspended in 1 mL complete medium and counted using a Vi-Cell XR cell counter (Beckmann Coulter). Using the initial seeding density and the density after 4 days, average doubling times for each replicate were calculated. Cells were also seeded at 500 or 1000 cells per well into 384-well plates (16 wells per cell line, three replicates) and confluency was monitored every 2 h for 48 h using the IncuCyte ZOOM system (Sartorius) allowing doubling times to be calculated. Finally, cell proliferation was measured incorporating 5-bromo-2′deoxyuridine (BrdU). 20,000 cells were seeded into eight-well chamber slides in complete RPMI-1640 medium, added 50 μL 0.3 mg/mL BrdU after 24 h and incubated for 2 h. The cells were then fixed with ice-cold ethanol (70%), treated with 1.5 M HCl and blocked with PBS containing 5% (v/v) FBS with intermediate washing steps in PBS. The cells were incubated with anti-BrdU-Alexa Fluor 488 at 4° C. overnight. For microscopy, cells were washed with PBS+5% (v/v) FBS, nuclei were counterstained with 4′,6-diamidino2-phenylindole (DAPI) and the cells were mounted with anti-fade. BrdU-labeled cells were counted under a DMi8 fluorescence microscope (Leica Microsystems) at six random positions containing at least 300 cells to ensure comparable confluency. To analyze the growth response to insulin, 5×10.sup.5 MCF-7.sub.PEBP cells (NtFT4 and CG18594) were seeded in complete medium in T-25 flasks. After settling, the cells were starved for 24 h in serum-free medium before adding 40 μg/mL recombinant human insulin. After 48 h the cells were harvested and counted using a Vi-CELL XR cell counter.

[0129] Migration assay: The MCF-7.sub.PEBP cell lines (NtFT4 and CG18594) were seeded (1×10.sup.5 cells in 100 μL serum-free medium) in 24-transwell Boyden chambers with a pore size of 8 μm (Corning) and complete medium in the lower chamber, and were incubated for 24 h. Medium was then removed from the upper chamber and the non-migrated cells were carefully removed using a cotton swab. The inlet was transferred to a new 24-well chamber containing 4% (w/v) formaldehyde for 30 min at 4° C. After drying, fixed cells were stained in a 0.1% (w/v) crystal violet for 5 min and washed with water. Random images of migrated cells were captured using a MZ 16 F fluorescence stereomicroscope (Leica Microsystems) and counted using ImageJ v1.501.

[0130] Immunofluorescence: MCF-7 and MCF-7.sub.CG18594 cells were fixed with 4% formaldehyde for 30 min at 4° C., washed three times with PBS and blocked for 60 min with blocking solution (PBS, 5% FBS, 0.3% Triton X-100) before incubating overnight at 4° C. with Anti-E-cadherin (24E10) (Cell Signaling; #3195) in PBS containing 1% BSA and 0.3% Triton X-100. Cells were then washed three times with PBS and incubated with anti-rabbit IgG-Alexa Fluor 546 for 2 h in the dark. Cells were analyzed by fluorescence microscopy using a DMi8 inverted microscope (Leica Microsystems) and fluorescence intensities in linear regions of interest were measured using LAS X software (Leica Microsystems).

[0131] Live cell imaging for subcellular localization: Localization studies using the pcDNA3 vectors containing constructs HA-EGFP, HA-EGFP-NtFT4, HA-EGFP-CG18594 and myc-mRFP-H2AZ were carried out by co-transfecting HEK-293T cells with EGFP plasmids and pcDNA3-myc-mRFP-H2AZ using Lipofectamine 3000. Cells were transiently transfected in six-well plates in Opti-MEM medium and fluorescence was imaged in living cells 24 h post-transfection using a TCS SP5 X laser scanning microscope (Leica Microsystems).

[0132] Protein extraction, analysis and western blotting: Adherent cells were harvested with a cell scraper, washed with cold PBS, and proteins were extracted using 300 μL cold RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS containing protease and phosphatase inhibitor cocktails) for 30 min on ice with occasional shaking. Protein concentrations in the extract supernatant were measured using the Pierce Coomassie Plus Protein Assay (Thermo Fisher Scientific) according to the manufacturer's specifications. Proteins were separated by SDS-PAGE and stained using the PAGE Blue protein staining kit or transferred to a 0.02-μm nitrocellulose membrane using the wet Mini Trans-Blot Cell system (Bio-Rad Laboratories). Western blots were probed with the following antibodies: E-cadherin mAb rabbit 24E10 (Cell Signaling, #3195); GAPDH Di6H11 rabbit mAb (Cell Signaling, #5174); IGF-I receptor β D23H3 rabbit mAb (Cell Signaling, #9750); phospho-Akt (Ser473, D9E) rabbit mAb (Cell Signaling, #4060); phospho-mTOR (Ser2448, D9C2) rabbit mAb (Cell Signaling, #5536); HA tag rabbit pAb (MBL, #561); human IgG (H+L) alkaline phosphatase (AP) (Thermo Fisher Scientific, #62-8422). Primary antibodies were detected using either anti-rabbit IgG sAb AP and SigmaFast BCIP/NBT tablets or anti-rabbit IgG sAb horseradish peroxidase and the SuperSignal West dura kit, and the signals were detected using a G:Box Chemi (Syngene). Brightness and contrast were optimized using Adobe Photoshop CS6 v13.0.1 ×64 (Adobe Systems).

[0133] Quantitative PCR: Gene expression was analyzed by quantitative real-time PCR using Kapa SYBR Fast qPCR Master Mix and the CFX96 Real-Time System (Bio-Rad). Each reaction was carried out in technical triplicates and the primer sequences are provided in Table 2 Specificity was ensured by melt curve analysis and the sequencing of PCR products, and by including no-template and no-reverse-transcription controls. Individual PCR efficiency was determined using LinReg PCR v2017.0 and relative gene expression levels were normalized to GAPDH following the initial testing of GAPDH, ACTB, GUSB and MRPL32.

TABLE-US-00009 TABLE 2 Oligonucleotide Sequences used in context of the invention Name Sequence 5′ to 3′ Purpose PEBP1 for XbaI aaatctagaATGCCGGTGGACCTCAGC cloning of PEBP1 rev AscI agaggcgcgccCTACTTCCCAGACAGCTGCTCG donor PEBP4 for XbaI aaatctagaATGGGTTGGACAATGAGGC plasmids PEBP4 rev AscI agaggcgcgccCTAGCAGGCAGCTATCTCCG into NtFT2 for XbaI aaatctagaATGTTAAGAGCAAATCCTTTAG DC-DON- NtFT2 rev AscI agaggcgcgccTTATAGGTGACGGCCACC SH1 NtFT4 for XbaI aaatctagaATGCCAAGAATAGATCCTTTG backbone NtFT4 rev AscI agaggcgcgccTTAATATGCGCGGCGG CG18594 for XbaI aaatctagaATGGACACCGCCGGCATTAT CG18594 rev AscI agaggcgcgccTTACTGGACCGTCTCGATGAGG CG10298 for XbaI aaatctagaATGTCCGATTCCACCGTG CG10298 rev AscI agaggcgcgccCTACTTCTTGCCAGATAGTTGC HA-EGFP for SpeI aaaactagtatatacccctacaatatccctaac cloning of tacactATGGTGAGCAAGGGC pcDNA3- EGFP rev XbaI aaaatctagaTTACTTGTACAGCTCGTCCATG HA-EGFP EGFP-rev XhoI aaaatctagaCTTGTACAGCTCGTCCATG cloning of NtFT4 for XhoI aaactgagATGCCAAGAATAGATCCTTTG pcDNA3- NtFT4 rev ApaI aaagggcccTTAATATGCGCGGCGG HA-EGFP- LifeAct-mRFP for acaggtaccatgggcgtggccgacctgatcaag NtFT4 KpnI aagttcgagagcatcagcaaggaagagggcgac ccaccggtcgccaccATGGCCTCCTCCGAG mRFP rev XbaI aaatctagaTTAGGCGCCGGTGGA DC-DON-SH1 seq CGGTGGGAGGTCTATATAAGCAG sequencing for DC-DON-SH1 seq GACAGTGGGAGTGGCACCTTC rev CMV for CGCAAATGGGCGGTAGGCGTG T7 for TAATACGACTCACTATAGGGAGA bGH rev TAGAAGGCACAGTCGAGG 5’ Junction PCR CCGGAACTCTGCCCTCTAAC verification Primer for of genomic integration 5’ Junction PCR CCCGTGAGTCAAACCGCTAT into the Primer rev aavs1 safe 3’ Junction PCR AGCTATCTGGTCTCCCTTCC harbor Primer for locus 3’ Junction PCR TCCTGGGATACCCCGAAGAG Primer rev GFP for ATCGAGAAGTACGAGGACG transgene GFP rev CACCACGAAGCTGTAGTAGC expression - reference gene PEBP1 for AGACCCACCAGCATTTCGTG transgene PEBP1 rev GGTGGTCTCCAGATCGGTTG expression - PEBP4 for TCCTGGATGGAGCCGATAGT PEBP PEBP4 rev AACTGGTAGCGATGGAAGCC expression NtFT2 for AGATATCCCTGCAACCACAGAAGCAAC NtFT2 rev AAACAGCGGCAACAGGCAAATTGAGAC NtFT4 for GATATCCCAGCAACTACAGATACAAG NtFT4 rev GAAACGGGCAAACCAAGATTGTAAAC CG18594 for CCACCATCACCTATCCTTCC CG18594 rev GAACTTCTCGGTGGTGATCT CG10298 for AGCCAGCCAAAGGTGAAA CG10198 rev GGACCTGCACCCACATATT GAPDH for CTCTGCTCCTCCTGTTCGAC gene GAPDH rev TTCCCGTTCTCAGCCTTGAC expression - β-ACTIN for CACAGAGCCTCGCCTTTGC reference β-ACTIN rev AATCCTTCTGACCCATGCCC gene RpL32 for GGTTACGACCCATCAGCCC RpL32 rev TCAATGCCTCTGGGTTTCCG GUSB for GCTCCGAATCACTATCGCCA GUSB rev CGCACTTCCAACTTGAACAGG CDH1 for TTCTGCTGATCCTGTCTGAT gene CDH1 rev AGGTGGTCACTTGGTCTTTA expression - CTNNB1 for AAGGAGCTAAAATGGCAGTG epithelial CTNNB1 rev TCCTAAAGCTTGCATTCCAC markers KRT18 for TGACACCAATATCACACGAC KRT18 rev TCAATCTGCTGAGACCAGTA KRT19 for CACTACTACACGACCATCCA KRT19 rev CTCAGCGTACTGATTTCCTC CDH2 for ATGTGCCGGATAGCGG gene CDH2 rev GGCTCACTGCTCTCATATTG expression - SNAI1 rev CTTGACATCTGAGTGGGTCTG mesenchymal SNAIl for CTCGAAAGGCCTTCAACTG markers SNAI2 for ATTCGGACCCACACATTAC SNAI2 rev CAGAATGGGTCTGCAGAT VIM for AGGAGGAGATGCTTCAGAGA VIM rev AGCTCCTGGATTTCCTCTTC AKT1 for GACCATGAACGAGTTTGAGT gene AKT1 rev CGTACTCCATGACAAAGCAG expression - BCL2 for GAACTGGGGGAGGATTGTG cell BCL2 rev AGAAATCAAACAGAGGCCG survival BIRC5 for CGACCCCATAGAGGAACATA BIRC5 rev CTAAGACATTGCTAAGGGGC JUN for ACTGCAAAGATGGAAACGAC JUN rev TGCTCATCTGTCACGTTCT MAPK8 for GAGCTCATGGATGCAAATCT MAPK8 rev AGGCGTCATCATAAAACTCG MYCfor GAGACAGATCAGCAACAACC MYC rev TTTCAACTGTTCTCGTCGTT BRCA1 for CCAGAACAAAGCACATCAGA gene BRCA1 rev ATGTTTCCGTCAAATCGTGT expression - BRCA2 for TACATGAACAAATGGGCAGG proliferation BRCA2 rev ATTTCTGCCTTTTGGCTAGG CCND1 for TCGGTGTCCTACTTCAAATG CCND1 rev ATGGAGTTGTCGGTGTAGAT CDKN1A for CACTGTCTTGTACCCTTGTGC CDKN1A rev AAAATGCCCAGCACTCTTAG CDKN2A for GAGGGCTTCCTGGACAC CDKN2A rev TCAATCGGGGATGTCTGAG ID1 for GAATCATGAAAGTCGCCAGT ID1 rev TGAAGGTCCCTGATGTAGTC PTEN for CAAGATATACAATCTTTGTGCTGA PTEN rev TGGTCCTTACTTCCCCATAG

[0134] Immunoprecipitation: HEK-293T cells were seeded in six-well plates and transfected with pcDNA3-HA-EGFP-NtFT4 and pcDNA3-HA-EGFP using Lipofectamine 3000. Immunoprecipitation was carried out using the Pierce Magnetic IP/Co-IP kit (Thermo Fisher Scientific) according to the manufacturer's specifications. Success was confirmed by silver staining of the eluate and input proteins using the Pierce Silver Stain for Mass Spectrometry (Thermo Fisher Scientific) according to the manufacturer's specifications, and by western blot.

[0135] LC-MS analysis: Immunoprecipitated proteins were reduced in 5 mM DTT for 30 min and alkylated with 14 mM chloracetamide for 30 min. Samples were digested with trypsin at 37° C. overnight. The digest was quenched with 1% formic acid and the peptides desalted with C18 stage tips prior to MS analysis. Dried peptides were redissolved in 2% acetonitrile supplemented with 0.1% trifluoroacetic acid (TFA) for analysis. Samples (0.5 μg) were analyzed using an EASY-nLC 1200 coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were separated on 20-cm frit-less silica emitters (New Objective, 0.75 μm inner diameter) packed in-house with reversed-phase ReproSil-Pur C18 AQ 1.9 μm resin (Dr. Maisch). The column was kept at 50° C. in a column oven throughout the run. Peptides were eluted for 115 min using a segmented linear gradient of 0-98% solvent B (solvent A=0.5% formic acid in water; solvent B=80% acetonitrile, 19.5% water and 0.5% formic acid) at a flowrate of 300 nL/min. Mass spectra were acquired in datadependent acquisition mode using a Top12 method in the Orbitrap analyzer with a mass range of 300-1759 m/z at a resolution of 120,000 FWHM, maximum IT of 55 ms, and a target value of 3×10.sup.6 ions. Precursors were selected with an isolation window of 1.2 m/z. HCD fragmentation was performed at a normalized collision energy of 25. MS/MS spectra were acquired with a target value of 5×10.sup.4 ions at a resolution of 15,000 FWHM, maximum IT of 150 ms and a fixed first mass of 100 m/z. Peptides with a charge of +1, >6, or with unassigned charge state were excluded from fragmentation for MS.sup.2, and dynamic exclusion for 30 s prevented repeated selection of precursors. Raw data were processed using MaxQuant software v1.6.3.4 MS/MS spectra were searched with the Andromeda search engine against the human UniProt database (15 May 2019) and were supplemented with sequences of the corresponding protein constructs of interest. Sequences of 248 common contaminant proteins and decoy sequences were automatically added during the search. Trypsin specificity was required and a maximum of two missed cleavages was allowed. Minimal peptide length was set to seven amino acids. Carbamidomethylation of cysteine residues was set as a fixed modification, whereas oxidation of methionine and protein N-terminal acetylation were set as variable modifications. MaxLFQ with ratio count 1 was enabled. Peptide-spectrum matches and proteins were retained if they were below a false discovery rate of 1%. Match between runs was enabled. Data were analyzed in R v3.5.3 combined with RStudio v1.1.463. The LFQ intensities were log.sub.2 transformed and reverse and contaminant hits removed. LIMMA v3.40.2 was used for differential expression analysis based on those values.

[0136] Mace Analysis: MCF-7 and MCF-7.sub.NtFT4 cells were treated with 40 μg/mL insulin in serum-free medium for 24 h. Cells were harvested and RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel). The isolated RNA was digested with TURBO DNase and analyzed by agarose gel electrophoresis. Massive Analysis of cDNA Ends (MACE), including sample processing, quality control, sequencing with 1×10.sup.6 raw reads per sample and data analysis, was carried out by GenXPro. The average raw count of each gene within a library was divided by the geometric mean of all counts in all samples and the median of the quotients was calculated per library. Each raw count was then divided by the libraryspecific median value. FDR values were calculated according to Benjamini-Hochberg, and p-values were calculated using the DEseq R package. For downstream data analysis, thresholds for genes included in differential gene expression and GO enrichment analysis between starved and insulin-exposed cells and between the two cell lines were set to a FDR <0.05 and a normalized count >0.1. For GO enrichment analysis, the PANTHER classification system was used. Interaction networks were analyzed using STRING v11 (62) and only genes with a log.sub.2 fold change >1 or <−1, p<0.001, and a mean value of normalized reads between the two samples >5 were included.

[0137] Transfection of suspension cultures: For transfection, HEK-293T and HEK-293T.sub.NtFT4 cells were collected from 40 mL cultures and resuspended in 2 mL complete Ex-CELL 293 medium at a density of 2×10.sup.7 cells/mL in six-well plates (42). Transfection efficiency was tested using 10-30 μg/mL DNA and 30-90 μg/mL 25 kDa linear polyethylenimine (PEI, Polysciences). Plasmid DNA was added to the cells followed by PEI, and cells were incubated for 4 h before dilution to 1×10.sup.6 cells/mL in 40 mL complete medium. Transfection efficiency was determined by measuring the expression of pcDNA3-LifeAct-mRFP in single cells using a FACSCelesta device (BD Biosciences) after 24, 48, 72 and 96 h (excitation=561 nm, detection=610/20 nm). Protein yields after transient transfection were determined by transfecting cells with pAPT-1E4-IgG1 and collecting secreted protein from the medium supernatants for up to 10 days. The expression of 1E4-IgG1 was measured by enzyme-linked immunosorbent assay (ELISA) or by western blotting using anti-human κ light chain IgGAP and bevacizumab/Avastin (kindly provided by Dr. Nicole Raven) as a control. Assembly of the antibody heavy and light chains was confirmed by electrophoresis using reducing and non-reducing loading buffers.

[0138] DAS-ELISA: For the quantification of human IgG1, 96-well plates were coated with anti-human IgG, Fc specific (Sigma-Aldrich, #I2136) in 50 mM carbonate buffer (pH 9.6) overnight at 4° C. The following steps include intermediate washes with PBS+0.1% Tween-20. Wells were blocked with PBS containing 5% nonfat milk for 1 h before diluted samples and standards (Avastin) were loaded and incubated for 2 h. The antibody was detected by incubation for 2 h with an anti-human kappa light chain-peroxidase secondary antibody (Sigma-Aldrich, #A7164) in blocking buffer, and subsequent staining with 3,3′, 5,5′-tetramethylbenzidine (TMB). The staining reaction was stopped with 1 M HCl and adsorption was measured at 450 nm using an Infinite 200 Pro plate reader (Tecan). All experiments were carried out as technical triplicates.

[0139] Statistical analysis: All boxplots in the figures were prepared using the default settings of OriginPro2020 (center line=median; box limits=upper and lower quartiles; whiskers=1.5×interquartile range; points=outliers). Statistical analysis, if not stated otherwise, was carried out using OriginPro2020 v9.7.5.184 (OriginLab). Equality of variances was analyzed by one-way ANOVA and Levene's test, and pairwise comparisons were carried out using Welch's t-test. FDRs (MACE and LC-MS/MS analysis) were calculated according to Benjamini-Hochberg.