WO2023051972A1

WO2023051972A1 - Method for the generation and selection of a producer cell

Info

Publication number: WO2023051972A1
Application number: PCT/EP2022/070483
Authority: WO
Inventors: Dominic GÄTJEN
Original assignee: Miltenyi Biotec B.V. & Co. KG
Priority date: 2021-09-29
Filing date: 2022-07-21
Publication date: 2023-04-06

Abstract

The present invention provides a method for expressing and displaying desired proteins of interest (POI) on the surface of a lower eukaryote in a form that is accessible for detection and isolation of desired cell clones by introducing two kinds of nucleic acids into the lower eukaryotic host cell: I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety domain, and ii) an antimicrobial resistant marker encoding a protein that provides resistance to a chemical, wherein said nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) elements, and II) a second nucleic acid sequence comprising i) a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety that is capable of specifically interacting with said first binding moiety and ii) a second antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug.

Description

Title

Method for the generation and selection of a producer cell

Field of the Invention

The present invention generally relates to the field of the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, in particular to the transient expression of a capture matrix expressed by said power eukaryotic cell for capturing the desired POI and loss of said capture matrix after selection of one or more producer cells.

Background of the invention

Selection of highly productive hosts for protein expression is a significant component of bioprocess design. Particularly in the case of biopharmaceuticals and industrial enzymes, Pichia pastoris, a methylotrophic yeast, has shown its remarkable capacity for production of heterologous proteins. To this date, screening of high producer clones is carried out in a very time consuming and labor intensive plate based fashion, whereas the most suitable method for screening the ‘best producer’ within a population is fluorescence activated cells sorting (FACS), since it enables simultaneous analysis of extensive cell numbers in a very quick and easy manner. Previous efforts to enable surface display of proteins of interest have been achieved by chemically modifying yeast cells and applying a capture matrix targeting the protein of interest (POI). However, even though they facilitate a non-covalent display, additional steps in the screening process are necessary.

The classical Yeast Surface Display system in Saccharomyces cerevisiae, which was disclosed in 1997 (Boder, E. T., & Wittrup, K. D. (1997). Nature biotechnology, 75(6), 553-557; US6300065; US6699658), relies on the covalent attachment or incorporation of the protein of interest to the yeast’s cell wall by fusing an anchor protein or anchor protein subunit to the POI. A Pichia Surface Display system for selection of displayed antibodies through incorporation into the cell wall of Pichia pastoris has also been successfully established. The POI was tethered to the surface by fusing the antibodies to the Alpha- agglutinin (SAG1) cell wall anchor protein of S. cerevisiae (Ryckaert, S., Pardon, E., Steyaert, J., & Callewaert, N. (2010). Journal of biotechnology, 145(2), 93-98; WO2011089527).

W02009111183 discloses an adapter based lower eukaryotic display system. A first adapter molecule is fused to an outer surface anchoring molecule and a second adapter molecule is fused to the POI which is capable of pairwise interaction with the first adapter molecule, thereby enabling surface display of the POI. As adapter molecules interacting peptides especially coiled-coil peptides are disclosed. However, those approaches only permit a permanent location of the antibodies on the cell surface.

Switchable, transient or dual-mode surface display systems make use of different technological methodologies. A so-called secretion and capture assay initially developed by Rakestraw and colleagues relies on the intracellular in vivo biotinylation of the POI and its subsequent capture on the cell surface through prior avidin coating of the host cell. (Rakestraw, J. A., Aird, D., Aha, P. M., Baynes, B. M., & Lipovsek, D. (2011). Protein Engineering, Design & Selection, 24(6), 525-530; US20170261506).

Another example for such a non-covalent display of IgG molecules was developed by Rhiel et al. It is based on the capture of secreted native full-length antibodies on the cell surface through binding to an immobilized ZZ domain, which is targeted against the antibody Fc domain. After biotinylation of the cell, the streptavidin-ZZ fusion protein is applied to the biotinylated cell surface. This approach is independent of the genetic background of the antibody producing host and was originally demonstrated in S. cerevisiae. (Rhiel, L., Krah, S., Gunther, R., Becker, S., Kolmar, H., & Hock, B. (2014). PloS one, 9(12), el 14887; W02016066260A1). W02018041740 proposes the establishment of a switchable display system, based on coating of the cell surface with a coating agent and re-capturing of the secreted protein. Unlike previously described methods, this approach utilizes different, non-streptavidin or ZZ domain based coating agent. In this disclosure the protein pair FimGT/DsF are described as a new coating/recapturing pair. FimGT is derived from type 1 pili FimG of E. coli and DsF is a short peptide (15 aa) corresponding to the to the binding site (termed donor strand, Ds) of the neighboring subunit FimF (DsF) of FimG in the pilus formation. The interaction of these two peptides is among the strongest reported for noncovalent molecular interactions and is capable of enabling a strong genotype phenotype coupling for successful library screening against antigens by yeast display.

Another possibility to efficiently switch between surface-displayed and solubly secreted proteins was described by van Deventer and colleagues (Van Deventer, J. A., Kelly, R. L., Rajan, S., Wittrup, K. D., & Sidhu, S. S. (2015). Protein Engineering, Design and Selection, 28(10), 317-325). To establish a switchable system for the display and secretion of antibodies and antibody-like reagents in yeast, they introduced an amber stop codon between POI and cell wall anchor protein and supplemented the non-canonical amino acid O-methyl-L- tyrosine (OmeY) that suppresses the amber stop, and thereby they could switch between the two different protein species. The advantage of those described switchable systems is the availability of two protein states at different time points. For screening and isolation of clones the POI is displayed on the cell surface and amenable for FACS sorting without the need for any modification on the POI itself. After a successful screening process this system enables immediate production of the protein of interest in a soluble form.

Simultaneous display and secretion of POIs can be achieved by a dual mode expression. The POI is present as surface displaced protein as well as soluble protein in the supernatant. Shaheen et al. (2013. PloS one, 8(7), e70190) designed a surface-anchored “bait” Fc portion by fusion to the GPI anchor protein Sedl. After homo-dimerization of the Fc portion of an IgG molecule to the “bait” Fc, functional “half’ IgGs are displayed on the cell surface of P. pastoris. The secretion of full length mAbs is not affected resulting in parallel display and secretion of IgGs. US9365846 discloses the bait/pray system described by Shaheen and colleagues using a heavy Fc immunoglobulin domain fused to a surface anchor polypeptide as bait and full length antibodies and monovalent antibody fragments comprising an Fc moiety as pray.

US9890378 discloses a slightly altered bait/pray antibody display system with a light immunoglobulin chain or functional fragment thereof fused to a surface anchor polypeptide as bait molecule.

Rather than using a “bait” peptide, Cruz-Teran et al. (2017. ACS synthetic biology, 6(11), 2096- 2107) describe a method relying on inefficient ribosomal skipping mediated by “self-cleaving” 2 A peptides. For simultaneous cell surface display and soluble secretion of proteins in S. cerevisiae, F2A peptide sequences are incorporated between the protein of interest and the yeast cell wall protein Aga2. With a described efficiency of approximately 50 %, ribosomal skipping produces two proteins from a single open reading frame in an equivalent ratio. Therefore, both the solubly secreted protein and the protein- Aga2 fusion that is tethered to the yeast cell surface are simultaneously expressed without any additional genetic modifications. Besides switchable systems and dual-mode expression, there is also the possibility of a transient display of proteins. The cold capture as described by Brezinsky et al. (2003. Journal of immunological methods, 277(1-2), 141-155) shows that labeling of secreted proteins on the cell surface using fluorescent antibodies is feasible at low temperatures, since the protein release from transport vesicles is assumed to be delayed due to slowed vesicle fusion and product release.. However, due to their rather thick cell wall, this approach has not been described for yeasts so far.

The above mentioned bait/pray systems disclosed by Shaheen et al. (US9890378; US9365846; 2013. PloS one, 8(1), e70190) have the disadvantage that isolated clones are not suitable for subsequent large scale production of the POI in a soluble form since a significant portion of the protein will be retained on the cell wall.

Elements conferring initiation of replication independent from the genome are termed autonomously replicating sequences (ARS) and were initially identified in the budding yeast Saccharomyces cerevisiae. There are -400 ARS elements in the yeast genome which generally consist of a few hundred base pairs, but not all of them are active initiators of replication (Dhar et al., 2012, Res Microbiol: 163(4):243-53). Different yeast species that harbor active ARS elements display a high sequence diversity of determinants for successful initiation of replication. (Liacho et al., 2010, PLoS Genet.; 6:el000946). Therefore, ARSs are usually only functional in a few yeast species. Liachko, I., & Dunham, M. J. (2014. FEMS yeast research, 14(2), 364-367; US9670495) describe a short ARS sequence that is functionally active in at least 10 different species of budding yeast. This ARS and an additionally optimized derivative confer improved plasmid stability in comparison to other currently used ARS modules.

There is a need in the art for an alternative or improved method for the generation and selection of a lower eukaryotic producer cell expressing and secreting a desired protein of interest (POI).

Brief description of the invention

The present invention provides a method for expressing and displaying desired proteins of interest on the surface of a lower eukaryote in a form that is accessible for detection and isolation of desired cell clones (producer cells) by introducing two kinds of nucleic acids into the lower eukaryotic host cell:

I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety domain (the capture matrix), and ii) an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy, if the producer cell is an auxotrophic mutant cell and the expressed selection marker complements the deficiency of said auxotrophic mutant cell, wherein said nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) element, and

II) a second nucleic acid sequence comprising a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety (domain) that is capable of specifically interacting with said first binding moiety (domain). Regularly said second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug. Normally said antimicrobial resistant marker of said second nucleic acid sequence should be a different antimicrobial resistant marker as compared to said antimicrobial resistant marker of said first nucleic sequence.

Using for example Fluorescence-activated cell sorting (FACS) as a tool for identifying and isolating cells provides a means for selection of cell clones that highly express the desired POI (producer cells) through a phenotype-genotype linkage. The method is based on the expression of said first binding moiety such as a scFv on the cell surface targeting said second binding moiety on the secreted POI such as the human Fd-region of IgGl -antibodies present on e.g. a Fab. The desired POI, e.g. a Fab, that should be detected is captured by the first binding moiety, e.g. as scFv, and retained on the surface for detection and quantification through immunofluorescence (FIG. 1 A-D). After successful isolation of high producer clones, the clones are grown in non-selective medium to promote the plasmid loss of the capture matrix (“switch-off’ of capture matrix expression) and subsequently may be further verified with regards to their protein production capacity e.g. in a bioreactor run. Due to the loss of this plasmid, the metabolic burden of the producer cell is reduced, which may lead to an increase of the secreted amount of the desired POI.

Since most surface display based screening approaches rely on the covalent incorporation of the POI to the cell surface or a chemical modification of the host cell, the advantage of the presented invention may be that the cell expresses its own capture matrix simultaneously but in a “switch-on/off ’ manner. This system remains switchable without the need for further steps in the screening process for producer cells or the addition of externally produced capture proteins. This simplification leads to a streamlined process that can be seamlessly integrated into the classical screening approach but with the advantage of a several fold higher through-put and an earlier timepoint for screening, e.g. it may be possible to screen up to 1E08 clones in a FACS- based method instead of several hundred clones only. The method disclosed herein depends on two vectors within the lower eukaryotic host cell instead of one vector. It is a surprising finding of the present method that this “two-vector-system” is beneficial as compared to a “one-vector- system” as it yields in higher titers of the desired POI. The two antimicrobial resistant markers used in the disclosed method that encodes proteins that provides resistance to a chemical or anti-microbial drug yield in a lower rate of false positive producer cells, especially when using Pichia as a lower eukaryote cell that is known to have a high rate of false positive producer cells. Brief description of the drawings

FIG.l: Schematic overview of a FACS-based method for isolation of high producing Pichia clones using non-covalent display of antibodies (Fabs) and subsequent verification of secretion capacity. (A) Pichia cells transformed with plasmids for expression of an antibody capture matrix and Fab antibody fragment were cultivated in minimal medium supplemented with methanol. Cells were induced for capture matrix and Fab antibody fragment expression while kept under constant selective pressure (“switch-on”). Secreted Fab fragments were bound to the antibody capture matrix and displayed on the cell surface. Antibodies can be either specific for the His tag, Igl light chain K or IgG Fd-region of the Fab, respectively. (B) Surface-displayed Fabs can be detected and quantified by indirect staining of cells with highest fluorescence signal intensity by FACS. Biotinylated antibodies either specific for the His tag, Igl light chain K or IgG Fd-region of the Fab, respectively, and a secondary antibody specific for biotin or streptavidin both conjugated with a fluorophore were used for Fab labelling. Antibody capture matrix was labelled using PE-conjugated antibodies specific for an HA or c-myc tag incorporated between the cell well anchor and capture antibody. (C) Induced P. pastoris cells were simultaneously labelled for antibody expression and Fab display. Cells displaying highest Fab amounts were sorted with the MACSQuant Tyto using a stringent sorting gate of 0.1-1 %. (D) After isolation of cells displaying higher amounts of Fab antibody fragments, those cells were grown on non-selective agar plates for episomal plasmid clearance (“switch-off’) and loss of capture matrix expression. Isolated clones were further analyzed for their secretion capacity in an ELISA assay to determine the highest producer clone which could be used for subsequent production in a bioreactor.

FIG.2: Schematic drawing. P. pastoris cells expressing a capture matrix as a fusion protein, comprised of a cell wall anchor protein, protein tags and an antibody specific for the POI (Fab antibody fragment), while simultaneously secreting Fab antibody fragments that are displayed on the cell surface. The Fab display is mediated through binding of the capture antibody specific to constant parts of the Fab. Fluorophore-conjugated antibodies specific for the protein tags or specific for the Fab allow for the detection and quantification of expressed capture matrix and secreted and displayed Fabs.

FIGG: Illustration of an exemplary PSD plasmid pPIC6a A::PSD for recombinant expression of an antibody capture matrix in P. pastoris. The antibody capture matrix is encoded by a gene coding for a fusion protein, comprising an antibody (“Antibody capture protein”), GPI or PIR anchor protein (“cell wall protein”), respectively, and an HA and a c-myc tag (“affinity tags”). The ARS element is located downstream of the ORF. nat'. Nourseothricin resistance gene. FIG.4: Normalized relative cell surface expression of tested PSD plasmid constructs in P. pastoris. Up to 100 PSD constructs were transformed into P. pastoris and after induction of the cells for surface protein expression, approximately 1 x 10⁶ cells were labeled with fluorophore conjugated antibodies specific for the affinity tags of the fusion protein. The relative fractions of cells expressing the fusion protein normalized to the construct with the highest expression are plotted in the graph. Error bars represent the standard deviation.

FIG.5: Confocal laser scanning microscopy images of Pichia cells expressing an anti-human Fd scFv antibody capture matrix. (A) Immunofluorescence of cells labeled with an antibody specific for the c-myc tag (FITC conjugated) under BP 515-565 filter (20x magnification). (B) Immunofluorescence of cells labeled with an antibody specific for the HA tag (APC conjugated) under BP 620/60 filter (40x magnification).

FIG.6: Flow cytometric analysis of several scFv-derived capture matrices and different primary antibodies for their applicability in the detection and quantification of surface displayed Fab antibody fragments that were spiked in. For all immunofluorescence stainings, an APC- conjugated antibody specific for the HA-tag of the scFv fusion protein and a VioBlue- conjugated secondary antibody specific for biotin were used. Fabs were spiked in at a concentration of 1 pM. Relative fraction of the population double positive for scFv expression and Fab display are shown in the dot plots. (A) As a negative control for the biotinylated anti- his tag primary detection antibody, an anti-human scFv in N-term-Vn-VL-C-term orientation was expressed and no Fab was added to the sample. A biotin-conjugated anti-his tag antibody was used as primary detection antibody. With 0.9 % of cells shifting into the Fab positive channel, the background signal is relatively low. (B) An anti-human Fab scFv (N-term-VL-Vn- C-term) was expressed as capture matrix and a biotin-conjugated anti-his tag antibody was used as primary detection antibody. (C) An anti-human Igl light chain K SCFV (N-term-Vn-VL-C- term) was expressed as capture matrix and a biotin-conjugated anti-his tag antibody was used as primary detection antibody. (D) An anti-human IgG Fd-region scFv (N-term-VL-Vn-C-term) was expressed as capture matrix and a biotin-conjugated anti-his tag antibody was used as primary detection antibody. (E) An anti-human IgG Fd-region scFv (N-term-Vn-VL-C-term) was expressed as capture matrix and a biotin-conjugated anti-his tag antibody was used as primary detection antibody. (F) As a negative control for the biotinylated anti-human Fab primary detection antibody, an anti-human scFv in N-term-Vn-VL-C-term orientation was expressed and no Fab was added to the sample. With 1.0 % of cells shifting into the Fab positive channel, the background signal is relatively low. (G) An anti-human IgG Fd-region scFv (N- tcrm-Vi.-Vii-C-lcrm) was expressed as capture matrix and a biotin-conjugated anti-human IgG Fd-region antibody was used as primary detection antibody. (H) An anti-human IgG Fd-region scFv (N-term-Vn-VL-C-term) was expressed as capture matrix and a biotin-conjugated antihuman IgG Fd-region antibody was used as primary detection antibody. (I) As a negative control for the biotinylated anti-human Igl light chain K primary detection antibody, an antihuman scFv in N-term-Vn-VL-C-term orientation was expressed and no Fab was added to the sample. With 1.0 % of cells shifting into the Fab positive channel, the background signal is relatively low. (J) An anti-human IgG Fd-region scFv (N-term-VL-Vn-C-term) was expressed as capture matrix and a biotin-conjugated anti-human Igl light chain K antibody was used as primary detection antibody. (K) An anti-human IgG Fd-region scFv (N-term-Vn-VL-C-term) was expressed as capture matrix and a biotin-conjugated anti-human Igl light chain K antibody was used as primary detection antibody.

FIG.7: Titration curves of surface displayed Fab fragments analyzed for different scFv capture matrix variants. An anti-human Fd region scFv expressed with alternating N- and C-terminal orientation of the VH and VL and an anti-human Fab scFv were expressed on the surface of P. pastoris. Display of Fab antibody fragments was detected by biotinylated anti-histidine antibodies, followed by labeling with VioBlue conjugated anti-biotin antibodies. For both antihuman Fd and anti-human Fab scFvs, 10- or 12-point curves were obtained on separate days and were plotted using different symbols for each construct. Values show total fractions of cells expressing scFvs and binding Fab fragments.

FIG.8: Quantification of expressed anti-human Fd scFv (N-term-Vn-VL-C-term orientation) molecules per cell. (A) Linear regression of Logio PE molecules per bead determined with the MACSQuant X. FL: Fluorescence. (B) Histogram plot of untransformed Pichia cells not expressing an anti-human scFv capture matrix. A gate is drawn for scFv positive cells. (C) Histogram plot of Pichia cells expressing an anti-human Fd scFv matrix. A gate is drawn for scFv positive cells. ScFv negative cells were excluded from the plot.

FIG.9: Determination of loss of capture matrix on DNA and protein level. For magnetic labelling of scFv expressing cells, scFv fusion proteins were labelled with antibodies specific for HA tag (PE conjugated) and incubated with anti-PE microbeads. MACS enriched cells expressing anti-human Fd region scFv (N-term-Vn-VL-C-term orientation) were cultivated in selective and non-selective YPD medium, respectively, for five days. On each day, an aliquot of cells cultivated in selective and cells cultivated in non-selective medium was additionally induced in buffered minimal medium supplemented with 1 % methanol and labeled with antibodies specific for affinity tags (fluorophore conjugated) to determine scFv expression and plated on selective and non-selective YPD agar to determine plasmid DNA stability. Relative fractions of cells of the total population expressing scFvs or exhibiting antibiotic resistance were plotted in the graph. Error bars represent the standard deviation.

FIG.10: Illustration of the plasmid pD902:Fab for recombinant expression of Fab fragments in P. pastoris. The heavy and light chain (“IgGl-FAB-EC”) of the Fab fragment are encoded by separate open reading frames whereas the heavy chain is encoded by a gene encoding for an affinity tag-fusion protein (“Tag-IgGl-FAB-HC)”. Zeo^R: zeocin resistance gene. SmiP. Linearization site for genomic integration.

FIG.11: Analysis of Fab displaying Pichia cells with varying Fab secretion capacities. Producers strains with different product titers were transformed with pPIC6a A::scFv for coexpression of the scFv capture and enabling of Fab surface display. Cells were analyzed for Fab display 2 and 24 h after induction with methanol. Cells were double-labeled with biotinylated anti-histidine and anti-bio tin- VioBlue antibodies for Fab display and with anti-HA APC antibody for scFv capture matrix expression. Relative fraction of population being double positive for Fab display and scFv expression are displayed in the top right corner of each dot plot. Additionally, secreted Fab in culture supernatant was determined with ELISA and values are displayed in the bottom right corner of each dot plot. N. d.: Not detectable.

FIG.12: Assay for determination of potential errant diffusion and binding of secreted Fabs from high producing clones and masking of low producers. Cells expressing scFvs and displaying Fab antibody fragments were labeled with biotinylated anti-histidine and anti-biotin-VioBlue antibodies and an anti-HA APC antibody. Relative fraction of the population double positive for scFv expression and Fab display are shown in the dot plots. Cultivation of the cells was carried out in static conditions. (A) A constitutively eGFP expressing Pichia clone was transformed with the anti-human Fd scFv capture matrix and induced for scFv expression. Afterwards cells were labelled for scFv expression and Fab display. Since they harbor no Fab expression construct, cells were not positive for Fab display and used as a negative control. (B) A previously generated high Fab secreting Pichia clone was transformed with the anti-human Fd scFv capture matrix and induced for scFv and Fab expression. Afterwards cells were labelled for scFv expression and Fab display. (C) A constitutively eGFP expressing Pichia clone and previously generated high Fab secreting Pichia clone, both transformed with the anti-human Fd scFv capture matrix, were mixed together in an equivalent ratio. Cells were induced for scFv and Fab expression followed by labelling of scFv expression and Fab display. 95 % of cells that were double positive were also eGFP negative, indicating absence of spill over from high producers and potential masking of non-producer cells.

FIG.13: FACS-based isolation of scFv capture expressing and Fab displaying Pichia cells. Cells transformed with the scFv capture and Fab expression plasmid were scraped from selective YPD agar plates and induced with methanol for induction of scFv and Fab expression. After 24 h of cultivation, cells were labelled for scFv expression and Fab display and sorted with the MACSQuant Tyto cell sorter. Sort performance was evaluated with the MACSQuant X. (A) Input fraction for cell sorting was labelled with anti-HA PE and biotinylated anti-His and streptavidin- V421 for scFv expression and Fab display. Initially, approximately 8 % of cells were double positive for scFv expression and Fab display. (B) Cells from the positive chamber of the cell sorting were analyzed for sort performance. Double positive cells were enriched up to 10-fold through sorting and made up >80 % of total population.

FIG.14: Microscale screening of individual clones from FACS input and sort fraction. (A) Up to 92 single clones from either previously on YPD agar grown FACS input or sort fraction were evaluated for Fab antibody secretion capacity via microscale screening. Cells were cultivated for 72 h in deep well plates and in buffered minimal media supplemented with methanol. Titer of secreted Fabs in the cell free supernatant was analyzed in a quantitative sandwich ELISA and plotted in a graph. (B) One-Way ANOVA of Fab titer means from of sorted Picha clones and unsorted FACS input fraction.

FIG.15: Determination of residual scFv expression of positively sorted cells after growth on non- selective medium. (A) After methanol induction for 24 h cells were labelled with antibodies specific for HA (APC conjugated) and c-myc tag (FITC conjugated) to determine expression of scFv capture matrix. Left'. Untransformed Pichia as negative control. Middle'. Pichia transformed with pPIC6a A::scFv as positive control. Right'. Exemplary dot plot for a sorted clone of the FACS positive fraction. (B) Overview of residual scFv expression for all 30 tested clones including additional controls. Values were normalized to Pichia cells transformed with pPIC6a A::scFv. Error bars represent the standard deviation. Stars above the columns indicate statistical significance.

FIG.16: Microscale screening of individual P. pastoris clones either co-transformed with a linearized anti-CD20 Fab expression vector and the circular scFv capture matrix expression plasmid or transformed with a linearized anti-CD20 Fab expression vector only. (A) Up to 92 single clones either co-transformed with a linearized anti-CD20 Fab expression vector and the circular scFv capture matrix expression plasmid (left panel) or transformed with a linearized anti-CD20 Fab expression vector only (right panel) were evaluated for Fab antibody secretion capacity via microscale screening. Cells were cultivated for 72 h in deep well plates and in buffered minimal media supplemented with methanol. Titers of secreted Fabs in the cell free supernatant were analyzed in a quantitative sandwich ELISA and plotted in a graph. (B) One- Way ANOVA of Fab titer means of co-transformed P. pastoris clones (left panel) or P. pastoris clones transformed with the Fab expression vector only (right panel) is shown.

Detailed description of the invention

In a first aspect the present invention provides a method for the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, the method comprising: a) transforming lower eukaryote host cells with

I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety (domain), and ii) an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy, if the producer cell is an auxotrophic mutant cell and the expressed selection marker complements the deficiency of said auxotrophic mutant cell, wherein said first nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) element, thereby expressing said polypeptide on the cell surface of said lower eukaryote host cells and said protein that provides resistance to said chemical or antimicrobial drug or said selection marker, and

II) a second nucleic acid sequence comprising a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety (domain) that is capable of specifically interacting with said first binding moiety (domain); thereby producing lower eukaryote host cells that express the desired POI and display said desired POI on said cell surfaces due to the interaction of said first and said second binding moiety, b) culturing the transformed lower eukaryote host cells in a selective medium, wherein said selective medium comprises said chemical or anti-microbial drug, or wherein said selective medium does not comprise the substance that complements the deficiency of the auxotrophic mutant cell, c) contacting the transformed lower eukaryotic host cells of step b) with a detection means that specifically binds to said desired POI that is displayed on the cell surface, d) identifying and isolating one or more transformed lower eukaryotic host cells with which the detection means is bound that display a higher amount of desired protein on their cell surfaces as compared to other lower eukaryotic host cells of step c), e) culturing one or more of the lower eukaryote host cells identified and isolated in step d) that display said higher amounts of desired protein on their cell surface in a non-selective medium, wherein said non-selective medium does not comprise said chemical or anti-microbial drug and/or wherein said non-selective medium comprises the substance that complements the deficiency of the auxotrophic mutant cell, thereby inducing loss of the expression of said polypeptide comprising said cell surface anchoring protein fused to said first binding moiety (domain) due to loss of said vector comprising said ARS elements, and thereby generating one or more producer cells.

Said method, wherein said second nucleic acid sequence may comprise an second antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug, wherein said second antimicrobial resistant marker (gene) may be different from said antimicrobial resistant marker (gene) of said first nucleic acid sequence.

Said antimicrobial resistant marker of said first nucleic acid sequence and/or said antimicrobial resistant marker of said second nucleic acid sequence may provide resistance to a drug (a chemical or antimicrobial/antibiotic drug), including, but not limited to, G418/Geneticin, Nourseothricin (Nat), Zeocin, Blasticidin, Hygromycin, fluoroacetamide, and 2-deoxyglucose. Said method, wherein said transforming of lower eukaryote host cells with said first nucleic acid sequence and said second nucleic acid sequence may be a co-transformation.

Said method, wherein said lower eukaryote host cells may be transformed with said first nucleic acid sequence before said lower eukaryote host cells may be transformed with said second nucleic acid sequence, or vice versa, i.e. the transformation of said first and said second nucleic acids may be two separated transformation processes.

Said method, wherein during said step of transformation in step a) the following chemical or anti-microbial drugs are present (are present in the cell medium that comprises the lower eukaryote host cells): a) a chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance, and b) a chemical or anti-microbial drug for that said second nucleic acid sequence provides resistance, and wherein in steps b) to d) said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance is present (is present in the cell medium that comprises the lower eukaryote host cells).

It is self-explaining that the presence of said chemical or anti-microbial drug for that said second nucleic acid sequence provides resistance after step a) (i.e. after integration of said second nucleic acid sequence into the genome of the cell) is not required anymore.

Said method, wherein said steps b) to e) may not comprise (in the cell medium that comprises the lower eukaryote host cells) said chemical or anti-microbial drug for that said second nucleic acid sequence provides resistance.

In one embodiment of the invention, said method may provide a method for the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, wherein the lower eukaryote host cell is a yeast, and wherein the yeast is Pichia pastoris, the method comprising: a) transforming lower eukaryote host cells with

I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety, and ii) an antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy, if the producer cell is an auxotrophic mutant cell and the expressed selection marker complements the deficiency of said auxotrophic mutant cell, wherein said first nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) element, thereby expressing said polypeptide on the cell surface of said lower eukaryote host cells and said protein that provides resistance to said chemical or antimicrobial drug or said selection marker, and

II) a second nucleic acid sequence comprising i) a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety that is capable of specifically interacting with said first binding moiety, ii) a second antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug; thereby producing lower eukaryote host cells that express the desired POI and display said desired POI on said cell surfaces due to the interaction of said first and said second binding moiety, b) culturing the transformed lower eukaryote host cells in a selective medium, wherein said selective medium comprises said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance, or wherein said selective medium does not comprise the substance that complements the deficiency of the auxotrophic mutant cell c) contacting the transformed lower eukaryotic host cells of step b) with a detection means that specifically binds to said desired POI that is displayed on the cell surface, d) identifying and isolating one or more transformed lower eukaryotic host cells with which the detection means is bound that display a higher amount of desired protein on their cell surfaces as compared to other lower eukaryotic host cells of step c) e) culturing one or more of the lower eukaryote host cells identified and isolated in step d) that display said higher amounts of desired protein on their cell surface in a non-selective medium, wherein said non-selective medium does not comprise said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance and/or wherein said non-selective medium comprises the substance that complements the deficiency of the auxotrophic mutant cell, thereby inducing loss of the expression of said polypeptide comprising said cell surface anchoring protein fused to said first binding moiety due to loss of said vector comprising said ARS elements, and thereby generating one or more producer cells.

Said embodiment of the invention, wherein said second antimicrobial resistant marker (gene) may be different from said antimicrobial resistant marker (gene) of said first nucleic acid sequence.

Said embodiment of the invention, wherein during said step of transformation in step a) the following chemical or anti-microbial drugs are present (are present in the cell medium that comprises the lower eukaryote host cells): a) a chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance, and b) a chemical or anti-microbial drug for that said second nucleic acid sequence provides resistance, and wherein in steps b) to d) said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance is present (is present in the cell medium that comprises the lower eukaryote host cells). Said desired POI may be a polypeptide, protein or a complex of proteins such as a heterodimeric or hetero-multimeric protein, e.g. an antibody or antigen binding fragment thereof.

Said POI may be for example an antibody or antigen binding fragment thereof, an enzyme, a therapeutically effective protein/polypeptide such as IFN-gamma, albumin, or Botulinum toxin serotype F.

Said first nucleic acid sequence may be a circular plasmid comprising a cell surface anchoring protein (in FIG 3: a cell wall protein) fused to a first binding moiety/domain (in FIG 3 a capture protein) operably linked to a constitutive or inducible promoter of eukaryotic cells, an ARS element and an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy (in FIG 3 the “nat” sequence (Nours eothricin resistance gene)) operably linked to a promoter of eukaryotic cells.

Typical plasmids usable as first nucleic acid sequence as disclosed herein may be e.g. pPICalpha A::PSD, pFLD, pGAPZ and pGAPZa A, B, and C, pPICZ and pPICZa A, B, and C. Said second nucleic acid sequence may comprise a nucleic sequence encoding a signal peptide that directs for secretion of the desired POI. Said second nucleic acid sequence may be a linearized plasmid/vector and may comprise flanking sites of about 1000 nucleotides that may be homologous to the genome of the host cell and may allow integration of a gene or genes encoding said desired POI into said genome. Said second nucleic acid sequence may be a nucleic acid sequence capable of being integrated into the genome of the lower eukaryotic cell. Vectors that may be used are well-known in the art and may comprise Yep vectors (yeast episomal plasmids), YCp vectors, YRp vectors or linearized YAC vectors (yeast artificial chromosome).

The FIG 10 shows a typical structure of a plasmid usable as second nucleic acid sequence as disclosed herein.

Said ARS element may be a functionally active ARS.

Functionally active ARS are well-known in the art or ARS activity may be determined by the methods well-known in the art and described e.g. in WO2017055436A1 or Liachko, I. and Dunham, M. J. (FEMS Yeast Res. 2014 Mar;14(2):364-7). Typically, ARS function in yeast can be easily tested by transforming circular plasmids.

Said ARS element may be selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:5, when said lower eukaryotic cell is a yeast such as Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces bayanus, Pichia pastoris, Lachancea waltii Kluyveromyces lactis, and Kluyveromyces wickerhamii.

Said ARS element may be selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:25 and SEQ ID NO:27 to SEQ ID NO:30, when said lower eukaryotic cell is the yeast Pichia pastoris.

More preferentially, said ARS element may be SEQ ID NO: 1 or SEQ ID NO:6, when said lower eukaryotic cell is the yeast Pichia pastoris. The sequences of SEQ ID NO:1 to SEQ ID NO:30 as displayed in the sequence listing protocol refer to the standard IUPAC nucleotide code:

A = adenine; C = cytosine; G = guanine; T = thymine; R = A or G; Y = C or T; S = G or C; W = A or T; K = G or T; M = A or C; B = C or G or T; D = A or G or T; H = A or C or T; V = A or C or G; N = A or C or G or T.

Said method, wherein said method comprises the steps a) transforming lower eukaryote host cells with

I) said first nucleic acid sequence

II) said second nucleic acid sequence comprising a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety that is capable of specifically interacting with said first binding moiety, wherein mutagenesis is used to generate a plurality of host cells encoding a variegated population of mutants of the desired POI in addition to nonmutated desired POI, thereby producing a plurality of lower eukaryote host cells that express a variegated population of mutants of the desired POI and display said variegated population of mutants of the desired POI on said cell surfaces due to the interaction of said first and said second binding moiety, b) culturing the plurality of transformed lower eukaryote host cells in a selective medium, wherein said selective medium comprises said chemical or anti-microbial drug, and/or wherein said selective medium does not comprise the substance that complements the deficiency of the auxotrophic mutant cell c) contacting the plurality of transformed lower eukaryotic host cells of step b) with a detection means that specifically binds to said desired POI that is displayed on the cell surface, d) identifying and isolating one or more transformed lower eukaryotic host cells with which the detection means is bound that display a higher amount of desired protein on their cell surfaces as compared to other lower eukaryotic host cells of step c) and additionally identifying an amended characteristics of the desired protein of said one or more isolated lower host cells due to the mutagenesis as compared to the desired protein that was not mutated, e) culturing said one or more of the lower eukaryote host cells identified and isolated in step d) in a non-selective medium, wherein said non-selective medium does not comprise said chemical or anti-microbial drug and/or wherein said non-selective medium comprises the substance that complements the deficiency of the auxotrophic mutant cell, thereby inducing loss of the expression of said polypeptide comprising said cell surface anchoring protein fused to said first binding moiety due to loss of said vector comprising said ARS elements, and thereby generating one or more producer cells.

Said method, wherein said second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug.

Said method, wherein the cell surface anchoring protein may be a GPI, a PIR or a FL/FS protein. Said method, wherein the cell surface anchoring protein may be selected from the group consisting of a-agglutinin (ScSAGl), ScCWPl (Cwplp), ScCWP2 (Cwp2p), ScGASl (Gaslp), ScYAP3 (Yap3p), ScFLOl (Flolp), ScCRH2 (Crh2p), PpPIRl, PpPIR2 (PpPirl-2p), , ScSEDl (Sedlp), ScTIPl (Tiplp), CriHWPl (Hwplp), CYALS3 (Als3p), C2/RBT5 (Rbt5p), PpFLO9, PpSPIl, ScTIRl (Tirlp), ScYCR89w (YCR89w) PpGCW21 PpGCW51 and PpGCW61 (last 3 proteins are PpGcw)

Preferentially, said cell surface anchoring protein may be a-agglutinin (ScSAGl), PpPIRl (Pirlp) or ScSEDl (Sedlp).

Said method, wherein the lower eukaryote host cell may be a yeast.

Said method, wherein the yeast may be Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces bayanus, Pichia pastoris, Lachancea waltii Kluyveromyces lactis, or Kluyveromyces wickerhamii.

Said method, wherein the lower eukaryote host cell may be Pichia pastoris.

Said method, wherein said chemical or anti-microbial drug may be Nours eothricin and said antimicrobial resistant marker may be the nourseothricin N-acetyl transferase (NAT) from Streptomyces noursei, or said chemical or anti-microbial drug may be Blasticidin and said antimicrobial resistant marker may be Blasticidin resistance gene ( hsd) from Aspergillus terreus, or said chemical or anti-microbial drug may be Zeocin and said antimicrobial resistant marker may be Sh hie gene (bleomycin-resistance gene) from Streptoalloteichus hindustanus, or said chemical or anti-microbial drug may be G418 and said antimicrobial resistant marker may be neo gene from Tn5 encoding an aminoglycoside 3'-phosphotransferase, or said auxotrophy may be a histidin deficiency and said selection marker that is based on said auxotrophy may be the HIS4/HIS6 gene, or said auxotrophy may be a tryptophan deficiency and said selection marker that is based on said auxotrophy may be the TRP1 gene, or said auxotrophy may be an uracil deficiency and said selection marker that is based on said auxotrophy may be the URA3 gene, or said auxotrophy may be an adenine deficiency and said selection marker that is based on said auxotrophy may be the ADE1/ADE2 gene, or said auxotrophy may be an arginine deficiency and said selection marker that is based on said auxotrophy may be the ARG4 gene.

In one embodiment of the invention, said antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug of said first nucleic acid sequence may be Nours eothricin and said antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug of said second nucleic acid sequence may be Zeocin.

Said method, wherein said detection means may be at least one antibody or antigen binding fragment thereof.

Said method, wherein said detection means may be an antibody or antigen binding fragment thereof coupled to a fluorophore.

Said method, wherein said detection means may be a first antibody or antigen binding fragment thereof that may be haptenylated, e.g. biotinylated, and specific for an epitope of the desired POI, and a second antibody or antigen binding fragment thereof that may be specific for a hapten, e.g. biotin and may be coupled to a fluorophore or said detection means may be an antibody or antigen binding fragment thereof that may be biotinylated and specific for an epitope of the desired POI, and a biotinylated streptavidin that may be coupled to a fluorophore. Said identifying and isolating one or more transformed lower eukaryotic host cells may be performed with flow cytometric sorting, e.g. fluorescence-activated cell sorting (FACS).

Said method, wherein said desired POI may comprise a tag, and wherein said detection means may bind to or may be specific for said tag.

Said tag may be a peptide such as His6x, HA, c-myc, FLAG, or Strep-Tag.

In one embodiment of the invention said second binding moiety of said desired POI that may be capable of specifically interacting with said first binding moiety may be part of the desired POI itself.

Said method, wherein i) said first binding moiety may be a receptor and said second binding moiety may be its cognitive ligand, or vice versa, or ii) said first binding moiety may be a non-antibody scaffold and said second binding domain may be its cognitive target, or iii) said first binding moiety may an antibody or antigen binding fragment thereof specific for said second binding moiety of said desired POI.

Non-antibody scaffolds for yeast display are well-known in the art, see e.g. Konning and Kolmar (2018, Microbial Cell Factories, 17: 32)..

Said non-antibody scaffold may be a Z-domain of protein A and said its cognitive target may be an antibody or antigen binding fragment thereof comprising an Fc portion of an immunoglobin IgG.

Said non-antibody scaffold may be affitins (nanofitins) specific for human immunoglobulin G (hlgG), if said desired POI may be an antibody.

Said method, wherein i) said first binding moiety may be an antibody or antigen binding fragment thereof specific for the kappa chain of the immunoglobulin light chain and said second binding moiety may be an antibody or antigen binding fragment thereof comprising the kappa chain of the immunoglobulin light chain, or ii) said first binding moiety may be an antibody or antigen binding fragment thereof specific for the Fd region of an immunoglobulin IgG and said second binding moiety may be an antibody or antigen binding fragment thereof comprising the Fd region of an immunoglobulin IgG.

Said method, wherein said first binding domain may be a scFv specific for the Fd region of an immunoglobulin IgG, wherein said second nucleic acid sequence may comprise the gene encoding the VL and CH domains of a light chain of an immunoglobulin and the gene coding the VH and CHI domains of a heavy chain of an immunoglobulin, and said desired POI may be a Fab, and wherein the second binding domain may be the Fd region of the Fab.

In another embodiment of the invention said second binding moiety of said desired POI that may be capable of specifically interacting with said first binding moiety may be a peptide/polypeptide fused to the desired POI.

Said method, wherein the first binding moiety may be a first adapter peptide and the second binding moiety may be a second adapter peptide and wherein the first and second adapter peptides may be capable of a specific pairwise interaction. Said method, wherein the first and second binding moieties may be coiled-coil peptides that may be capable of the specific pairwise interaction.

Coiled-coil peptides that are capable of the specific pairwise interactions are well-known in the art, e.g. pairs such as FimGt/DsF, Agal-Aga2 and Im7/E7.

In another aspect the present invention provides a method for producing a desired protein (POI), the method comprising: a) transforming lower eukaryote host cells with

II) a second nucleic acid sequence comprising a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety (domain) that is capable of specifically interacting with said first binding moiety (domain); thereby producing lower eukaryote host cells that express the desired POI and display said desired POI on said cell surfaces due to the interaction of said first and said second binding moiety, b) culturing the transformed lower eukaryote host cells in a selective medium, wherein said selective medium comprises said chemical or anti-microbial drug, or wherein said selective medium does not comprise the substance that complements the deficiency of the auxotrophic mutant cell c) contacting the transformed lower eukaryotic host cells of step b) with a detection means that specifically binds to said desired POI that is displayed on the cell surface, d) identifying and isolating one or more transformed lower eukaryotic host cells with which the detection means is bound that display a higher amount of desired protein on their cell surfaces as compared to other lower eukaryotic host cells of step c) e) culturing one or more of the lower eukaryote host cells identified and isolated in step d) that display said higher amounts of desired protein on their cell surface in a non-selective medium, wherein said non-selective medium does not comprise said chemical or anti-microbial drug and/or wherein said non-selective medium comprises the substance that complements the deficiency of the auxotrophic mutant cell, thereby inducing loss of the expression of said polypeptide comprising said cell surface anchoring protein fused to said first binding moiety (domain) due to loss of said vector comprising said ARS elements, and thereby generating one or more producer cells that produce the desired POI.

In a further aspect the present invention provides a system (a combination of nucleic acids to be transformed into a lower eukaryotic host cell; a kit) for the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, the system comprising:

I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety (domain), and ii) an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy, if the producer cell is an auxotrophic mutant cell and the expressed selection marker complements the deficiency of said auxotrophic mutant cell, wherein said first nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) element, and

II) a second nucleic acid sequence comprising a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety (domain) that is capable of specifically interacting with said first binding moiety (domain). Said method, wherein said second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug.

All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

The term "host cell" as used herein is intended to refer to a cell into which a recombinant vector and/or plasmid has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences (e.g. the pressure of a selective medium or the loss of the pressure by using a non- selective medium), such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "eukaryotic" refers to a nucleated cell or organism, and includes insect cells, plant cells, mammalian cells, animal cells and lower eukaryotic cells.

The term "lower eukaryotic cells" includes yeast and filamentous fungi. Yeast and filamentous fungi include, but are not limited to Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum,

Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp. and Neurospora crassa. Lower eukaryotic cells have systems of GPI proteins that are involved in anchoring or tethering expressed proteins to the cell wall so that they are effectively displayed on the cell wall of the cell from which they were expressed. GPI proteins which may be used in the methods herein include, for example Saccharomyces cerevisiae CWP1; CWP2; SED1; GAS1; Pichia pastoris SPI1; GCW21; GCW51; GCW61; and H. polymorpha TIPI. Additional GPI proteins may also be useful. Suitable GPI proteins can be identified using the methods and materials of the invention described and exemplified herein.

In general, the GPI protein used in the methods disclosed herein may be a chimeric protein or fusion protein comprising the GPI protein fused at its N-terminus to the C-terminus of a binding moiety. The N-terminus of the binding moiety may be fused to the C-terminus of a signal sequence that enables the GPI fusion protein to be transported through the secretory pathway to the cell surface where the GPI fusion protein is secreted and then bound to the cell surface. In some aspects, the GPI fusion protein comprises the entire GPI protein and in other aspects, the GPI fusion protein comprises the portion of the GPI protein that is capable of binding to the cell surface.

GPI, PIR, FL/FS anchor proteins

GPI

Glycosylphosphatidylinositol (GPI) is a glicolipid structure that can be incorporated into the C- terminal hydrophobic region of a protein during posttranslational modification. GPI anchored proteins are bound to the cell membrane as N-terminal fusions by the insertion of the phosphatidylinositol lipid part into the hydrophobic lipid bilayer.

FL/FS

Flol (flocculation protein 1) is a cell wall protein mainly involved in flocculation. FS and FL consist of two different regions of Flol: FS (Flol short, amino acids 1 to 1099) and FL (Flol long, positions 1 to 1447). Both FS and FL lack the GPI attachment site, but contain the secretion signal domain, the flocculation functional domain and some segments of the central region. The Flol system can be used for displaying C-terminal fusions.

PIR

Proteins with internal repeats (PIR) in yeast are located on the cell wall and contain several tandemly repeats with highly conserved amino acids. PIR proteins are not attached to the cell wall by GPI, but bind to it either by an ester linkage between the P-l,3-glucan and the carboxyl group of a glutamine in the internal repeats or through disulfide bonds among cysteine residues and particular cell wall components. With this system, up to three different options for protein display are available: N-terminal, C-terminal and internal fusion.

The terms “producer cell” or producer cell line” or “producer clone” as used herein may be used interchangeable and refer to a cell (a clone) such as a lower eukaryotic cell that is able to produce stably the desired POI as disclosed herein.

A high producer cell may produce more desired POI as compared to most producer cells in a cell sample of producer cells comprising said high producer cell, e.g. at least 1.5 fold more desired POI, at least 2 fold more desired POI, at least 3 fold more desired POI, at least 5 fold more desired POI, or at least 10 fold more desired POI as compared to other producer cells of said sample or as compared to the average production of desired POI produced by one cell of said sample.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.

The terms “(genetically) engineered cell” and “(genetically) modified cell” as used herein can be used interchangeably. The terms mean containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny

The terms “introducing a nucleic acid sequence into a lower eukaryotic cell” or “transforming a lower eukaryotic cell with a nucleic acid sequence” may be used interchangeably and means that nucleic acids such as DNA and/or RNA are introduced into a cell by methods well-known in the art for allowing the cell to uptake nucleic acids. Such methods are e.g. transformation, transfection, transduction, magnetofection and electroporation.

The terms “specifically binds to” or “specific for” with respect to an antigen-binding domain of an antibody or fragment thereof (e.g. a scFv) refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other antigens in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to the homologous antigen from another species. This cross-species reactivity is typical to many antibodies and therefore not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.) or homologous variants of this antigen from the same gene family. These cross reactivities are typical to many antibodies and therefore not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to a limited number of completely different structures, known as mimo topes. This reactivity is typical to many antibodies and therefore not contrary to the definition of that antigen-binding domain as specific.

The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody- immunoadhesin chimeras, that specifically recognize (i.e. bind) a target antigen. "Antibody fragments" comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antibody fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (VHH, nanobodies), diabodies, dsFv, Fab’, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains.

The Fd region is the heavy chain of the Fab, i.e. approximately the first 220 amino acids from the N-terminus of the heavy chain comprised of the VH and CHI regions.

As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one-letter or three-letter codes for amino acid residues are used herein. Polypeptides may include disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, amidation or any other modifications.

The term "mutagenesis" as used in the context of the present invention shall refer to a method of providing mutants of a nucleotide sequence, e.g. through insertion, deletion and/or substitution of one or more nucleotides, so to obtain variants thereof with at least one change in the non-coding or coding region. Mutagenesis may be through random, semi-random or site directed mutation. WO2017055436A1 discloses plasmids comprising ARS sequences that are similar in structure as used herein. A "plasmid" as used herein is defined as a vector which is a nucleic acid construct used to transform a host cell for expression of a protein, polypeptide, or peptide, and the vector is not found in nature in the host cell it transforms. A plasmid, also referred to as "plasmid vector" is specifically understood as an extrachromosomal nucleic acid which is particularly physically separated from a chromosomal DNA. A plasmid may or may not include DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e., of recombinant genes and the translation of their mRNA in a suitable host organism. Plasmid vectors usually comprise an origin for autonomous replication in the host cells, selectable markers, a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.

The ARS comprised in the plasmid described herein may be characterized by not being operably linked to the recombinant gene or any promoter that is operably linked to the recombinant gene in the plasmid.

An "Autonomously Replicating Sequence" or "ARS" or “ARS element” is a sequence that serves as an origin of DNA replication on eukaryotic chromosomes. An ARS, when incorporated into a DNA molecule, supports replication of the DNA molecule by binding a protein complex that unwinds and replicates the DNA. An ARS can be confirmed, i.e. functionally validated by incorporating the sequence into a DNA molecule that is not selfreplicating in a given host and demonstrating that the DNA molecule replicates autonomously in the host only when the ARS is present.

ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions.

The term "functionally active ARS" refers to an ARS that is capable of transforming a non-self replicating DNA construct into an autonomously replicating DNA construct upon insertion of the ARS into the DNA construct. ARS activity may be determined by the methods described e.g. in WO2017055436A1 or other assays known in the art. Typically ARS function in yeast can be easily tested by transforming circular plasmids as demonstrated by e.g. by Liachko, I. and Dunham, M. J. (FEMS Yeast Res. 2014 Mar;14(2):364-7). The term "operably linked" as used herein refers to the association of nucleotide sequences on a single nucleic acid molecule, e.g. a vector, in a way such that the function of one or more nucleotide sequences is affected by at least one other nucleotide sequence present on said nucleic acid molecule. For example, a promoter is operably linked with a coding sequence of a recombinant gene or GOI, when it is capable of effecting the expression of that coding sequence

A “selection marker" refers to a gene (or the encoded polypeptide) that confers a phenotype which allows the organism expressing the gene to survive under selective conditions. A selection marker generally is a molecule that, when present or expressed in a cell, provides a selective advantage (or disadvantage) to the cell containing the marker. For example, the genetic markers for selection of transformants can include the ability to grow in the presence of an agent that otherwise would kill the cell, the ability to grow in the absence of a particular nutrient, a selection marker that allows a transformed cell to grow on a medium devoid of a necessary nutrient that cannot be produced by a deficient and untransformed cell, a selection marker that allows a transformed cell to grow on medium, e.g., an energy source, that cannot be used/metabolized by a deficient and untransformed cell, or a selection marker that encodes an enzyme for which chromogenic substrates are known.

In some embodiments, the selection marker provides resistance to a drug (a chemical or antimicrobial/ antibio tic drug), including, but not limited to, G418/Geneticin, Nours eothricin (Nat), Zeocin, Blasticidin, Hygromycin, fluoroacetamide, and 2-deoxyglucose. Then the selection marker may be termed as an antimicrobial resistant marker or antibiotic resistant marker.

The selectable marker system may include an auxotrophic mutant of y yeast strain such as P. pastoris host strain and a wild type gene which complements the host’s defect, herein referred to as selection marker based on auxotrophy. Examples of such selectable marker systems include, but are not limited to amino acid auxotrophy such as arginine, methionine or histidine auxotrophy or nucleotide biosynthesis auxotrophy such as uracil auxotrophy or thymidine auxotrophy.

Example 1: Cloning of library constructs for the expression of the capture matrix and transformation of E. coli

DNA sequences of the genetic elements required for the expression of a surface displayed fusion protein that serves as a capture matrix in a Pichia surface display (PSD) system were obtained from previously published data (Tab. 1). Genes were synthesized as codon optimized sequences for the expression in Pichia pastoris (ATUM, Inc.) and cloned via Golden Gate Assembly into the modified pPIC6a A vector (Invitrogen) for a total amount of individual 100 PSD constructs. Besides generic elements that are required for the expression and attachment on the cell surface and an autonomously replicating sequence (ARS) element to maintain the episomal plasmid in Pichia cells, two C-terminal affinity tags that allow for detection and quantification of the surface expressed fusion protein were added (FIG. 2). Competent 10-beta E. coli cells (New England Biolabs) were transformed with the respective pPIC6a A::PSD plasmids by heat shock (FIG. 3). Therefore, cells were thawed on ice for 10 minutes and supplemented with 10-100 ng of plasmid DNA. After further incubation on ice for 5 minutes, a heat shock at 42 °C was applied for 30 seconds. Afterwards, cells were incubated on ice for 5 minutes and mixed with SOC medium and incubated in a shaker at 37 °C for 45 min. Cells were then plated on nourseothricin containing (50 pg/ml) agar plates to select for transformants after overnight incubation.

Tab. 1: Individual genetic elements for designing PSD expression vectors in P. pastoris.

Modular parts are flexible and interchangeable and accompanied by flanking overhangs for use of type IIS assembly strategy.

Example 2: Transformation of PSD library constructs in P. pastoris and expression of surface displayed capture matrix

For the expression of surface displayed fusion proteins in yeast, P. pastoris cells were transformed with the appropriate expression plasmid using electroporation. Single colonies were picked and transferred into either BMD1 medium (200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin, 10 g/1 dextrose) or BMGy (200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin, 20 g/1 glycerol) and cells were cultivated in 96 deep-well plates at 28 °C, 80 % humidity and 320 rpm for 60 hours under selective pressure using appropriate antibiotics. Afterwards, the medium was exchanged with BMM medium (200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin) supplemented with methanol at a final concentration of 0.5 % to induce protein expression. Cells were further cultivated at 24 °C, 80 % humidity and 320 rpm for up to 72 hours with additional methanol feeding spikes and under selective pressure using appropriate antibiotics. For each construct, approximately 1 x 10⁶ cells were analyzed for the expression of surface protein using flow cytometry and stained as described in example 3. Antibodies specific for the HA tag (APC conjugated, Miltenyi Biotec) and the c-myc tag (FITC conjugated, Miltenyi Biotec) were used to label the two C-terminal affinity tags of the fusion protein for determination of total surface displayed protein. Relative fraction of cells expressing the surface displayed fusion protein were determined for each construct and plotted on a graph (FIG 4). Main effectors for maximized capture molecule expression were determined using a statistical analysis software (SAS Institute).

Example 3: Immunofluorescence staining of surface expressed scFv capture matrix and determination of homogeneous cell surface expression via confocal laser scanning microscopy

Cells of example 2 were stained using antibodies targeted against the protein tags and conjugated with fluorochromes suitable for imaging detection with the laser scanning microscope LSM 710 (Carl Zeiss AG). Therefore, approximately 1 x 10⁶ cells were washed with ice-cold PBS-F buffer (8 g/1 NaCl, 0.2 g/1 KC1, 1.44 g/1 Na₂HPO₄, 0.24 g/1 KH₂PO₄, 1 g/1 BSA, pH 7.4) and after centrifugation (5 min, 1500 x g) resuspended in 100 pl PBS-F. For immunofluorescence staining of the expressed scFv capture matrix, an antibody specific for the HA tag (APC conjugated, Miltenyi Biotec) and an antibody specific for the c-myc tag (FITC conjugated, Miltenyi Biotec) were added to the cells in a working dilution of 1:50, followed by an incubation on ice in the absence of light for 10 minutes. Cells were washed again in ice-cold PBS-F buffer and finally resuspended in 100 pl PBS-F. For imaging purposes, cells were transferred to a 96-well imaging plate (Greiner Bio-One) and fluorescence images collected with the LSM 710. All tested constructs show a homogenous and uniform expression of the capture matrix on the cell surface (FIG. 5).

Example 4: Flow cytometric detection of scFv expression and successful Fab fragment surface capture

For detection of scFv expression in P. pastoris and successful Fab antibody surface capture, a set of different capture scFvs were tested. Tested scFv constructs included an anti-polyhistidine nanobody, an anti-polyhistidine scFv (N-term-VL-Vn-C-term orientation), a second anti- polyhistidine scFv (both N-term-VL-Vn-C-term and N-term-Vn-VL-C-term orientation), an anti-human IgG Fab-region scFv (both N-term-VL-Vn-C-term and N-term-Vn-VL-C-term orientation), an anti-human Ig light chains of K type scFv (both N-term-VL-Vn-C-term and N- term-Vn-VL-C-term orientation), and an anti-human IgG Fd-region scFv (both N-term-VL-Vn- C-term and N-term-Vn-VL-C-term orientation). Therefore, cells were transformed with individual capture scFvs episomal plasmids and treated as described in example 2. Cells were subsequently washed with ice-cold PBS-F buffer, resuspended in 100 pl PBS-F buffer and an anti-human CD19 or an anti-human CD33 Fab antibody fragment added in a final concentration of 1 pM. Incubation at room temperature for up to 1 hour was followed by a washing step with ice-cold PBS-F. Prior to addition of an antibody specific for a poly-histidine tag or the human Fab region of IgG or the human Ig light chains of K type (all biotin conjugated, Miltenyi Biotec) in a working concentration of 1:11, cells were resuspended in PBS-F buffer and incubated on ice for 10 minutes. Afterwards, cells were washed with PBS-F. For simultaneous labeling of the expressed scFv capture matrix and surface displayed Fab fragments, an antibody specific for HA tag (APC conjugated, Miltenyi Biotec) and an antibody specific for biotin or streptavidin (both VioBlue conjugated, Miltenyi Biotec) were added to the cells in a working concentration of 1:50. Incubation on ice and in the absence of light for 10 minutes, was followed by an additional washing step in PBS-F buffer. Cells were resuspended in PBS-F immediately before flow cytometry analysis with the MACSQuant X (Miltenyi Biotec) (FIG. 6). Example 5: Characterization of different scFv capture matrices by Fab titration

Binding efficiencies of different scFv capture matrices were evaluated by Fab titration and subsequent flow cytometry analysis. P. pastoris cells expressing an anti-human Fab scFv (N- term-VL-Vn-C-term) or an anti-human Fd scFv (both N-term-VL-Vn-C-term and N-term-Vn- VL-C-term orientation) capture matrix, treated as described in example 2, were incubated with different concentrations of spiked in Fab antibody fragment spanning several orders of magnitude. After two previous washing steps with ice-cold PBS-F buffer, approximately 1 x 10⁶ cells were resuspended in 100 pl PBS-F buffer and an anti-human CD19 recombinant Fab (Miltenyi Biotec) added to the cells, followed by an incubation at room temperature for up to 1 hour. Cells were then analyzed as described in example 4 at the following spiked in Fab concentrations: 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 10 nM, 100 nM, 200 nM, 250 nM, 500 nM, 1000 nM, 5000 nM. Relative fraction of scFv expressing cells binding Fab molecules were plotted against the Fab concentration in order to determine the scFv capture matrix candidate with the highest binding efficiency (FIG. 7).

Example 6: Quantification of cell surface displayed scFv molecules in P. pastoris

To estimate the number of scFv antibodies expressed per cell, BD Quantibrite™ Beads (BD Biosciences) for PE fluorescence quantification were used. Approximately 1 x 10⁶ cells of example 2 expressing the anti-human Fd-scFv (N-term-Vn-VL-C-term orientation) were washed with ice-cold PBS-F buffer and after centrifugation (5 min, 1500 x g) resuspended in 100 pl PBS-F. For immunofluorescence staining of the expressed scFv capture matrix, an antibody specific for the HA tag (PE conjugated, Miltenyi Biotec) was added to the cells in a working dilution of 1:50, followed by an incubation on ice, shielded from light for 10 minutes. Cells were washed again with ice-cold PBS-F buffer and finally resuspended in 100 pl PBS-F. Geometric mean fluorescence intensity for PE of treated cells was determined with the MACSQuant X (Miltenyi Biotec).

A linear regression of Logio PE molecules per bead against Logio fluorescence was plotted based on previously collected values from the BD Quantibrite™ Beads (see Tab. 2), using the following equation: y = mx + b (FIG. 8). To determine expressed scFv antibodies per cell, geometric mean fluorescence intensity was substituted in the equation and solved for Log scFv antibodies expressed per cell. A mean of approximately 5 x 10³ scFv molecules per cell was determined. Table 2: BD Quantibrite ™ Beads histogram statistics and calculated Logio values.

Example 7: Monitoring and determination of episomal plasmid loss and loss of surface expressed scFv capture matrix

To isolate scFv expressing cells, magnetic-activated cell sorting (MACS) was applied with magnetic cell separation columns that allow for positive selection cells. Cells of example 2 were labeled with a HA specific antibody (biotinylated; Miltenyi Biotec) and subsequently incubated with anti-biotin MicroBeads (Miltenyi Biotec) in order to select positive cells in a magnetic MS cell separation column (Miltenyi Biotec). In addition, an antibody specific for the c-myc tag (FITC conjugated, Miltenyi Biotec) was used to determine the efficiency of magnetic cell sorting.

For actual cell separation, up to 2 x 10⁸ cells were suspended in staining buffer (phosphate - buffered saline, pH 7.2, 5 g/1 BSA and 2 mM EDTA) and loaded onto the column. Isolated cells were used for the inoculation of non-selective YPD medium and selective YPD medium supplemented with the appropriate antibiotics and cultivated in a shaker at 30 °C and 250 rpm for up to 4 days with re-inoculation of fresh media every 24 h. To monitor the loss of the scFv capture matrix on protein and DNA level (FIG. 9), cells were either analyzed as described in example 3 or plated onto selective, containing the appropriate antibiotic, and non-selective YPD agar plates to determine plasmid stability. Before plating onto YPD plates, the samples were diluted based on the ODeoonm in order to reach about 100-1000 colonies per plate. After 3-4 days of incubation the colonies were counted to determine relative plasmid stability.

Example 8: Transformation of Fab antibody construct and scFv capture matrix plasmid

Fab antibody producing and scFv capture matrix expressing cells were generated by electroporation of P. pastoris cells. Therefore, linearized Fab expression plasmids (FIG.10) and circular scFv capture matrix expression plasmids were transformed into electrocompetent P. pastoris cells as described by Wu & Letchworth, 2004. Up to 10 pg of total DNA per 80 pl aliquot of electrocompetent cells were transformed at 1.5 kV, 25 pF and 200 > with the GenePulser® II (Bio-Rad Laboratories). Electroporation was followed by an outgrowth step in YPD and 1 M sorbitol for up to 2 hours at 28 °C for cells to recover and express the resistance genes conferring antibiotic resistance. Cells were then washed with ice-cold and sterile dIUC) and diluted to get single colonies on YPD agar plates supplemented with the appropriate antibiotics. Transformed yeast colonies appeared after 3-4 days of incubation at 28 °C.

Example 9: Flow cytometric analysis of antibody producing P. pastoris clones

Transformed yeast colonies from example 8 were transferred from agar plates into buffered minimal media supplemented with 1 % methanol and the appropriate antibiotic at an initial cell concentration of 1 x 10⁷ cells/ml. For capture and display of secreted Fab-molecules on yeast cells, the expression was performed in a static culture using deep-well plates for up to 30 hours and without shaking at 24 °C at an initial cell concentration of 1 x 10⁷ cells/ml. To specifically label surface displayed Fabs, 1 x 10⁶ cells were collected and treated as described in example 4 and analyzed in the MACSQuant X (Miltenyi Biotec). Dead cells were labelled with DAPI staining solution (Miltenyi Bio tec) and fluorescent cells excluded in the flow cytometric analysis. High expressing antibody clones show a stronger fluorescent signal intensity in the Fab displaying channel compared to non-producing clones (FIG. 11).

Example 10: ELISA assay for the detection of secreted Fab antibody fragments in the culture supernatant of antibody producing P. pastoris clones

For the quantification of secreted Fab antibody fragments by P. pastoris producing clones, Nunc-Immuno ™ 96-well plates (ThermoScientific) were coated with 150 ng per well of an antibody specific for poly -histidine (6x) tag (pure, Miltenyi Biotec). After blocking of the plates and a washing step, 100 pl of diluted culture supernatant of cells treated as described in example 9 were added to the plates, followed by an incubation for 1 hour at RT. Plates were washed with assay buffer (1 % BSA in PBS buffer) and an antibody specific for human Ig light chains of K type (HRP conjugated, Miltenyi Biotec) was added before the plates were washed again. Antibody concentration in the supernatant of producing cells was quantified trough addition of TMB substrate (ThermoFisher) and measuring of absorbance with the VersaMax (Molecular Devices) either in a kinetic ELISA at ODesonm or in an endpoint ELISA with addition of 1 % sulfuric acid at OD45011111.

Example 11: Assay for determination of potential masking of low or non-producing Pichia cells by errant diffusion and binding of secreted Fabs from high producing clones

Due to the close proximity of cultivated Pichia cells and inevitable saturation of scFv capture matrix, secreted Fabs of high producing antibody clones could potentially mask low or nonproducer cells as high producers. Therefore, a Pichia clone constitutively expressing eGFP and simultaneously expressing the scFV capture matrix, while not expressing any Fab fragment, was co-cultured with a high Fab producing clone in an equivalent ratio. For induction of scFv and Fab expression and to specifically label expression of scFv capture matrix and display of secreted Fab fragments, cells were treated as described in example 9. Despite scFv expression of non-producing Pichia cells, only Fab secreting cells were specifically displaying Fab fragments with a relative fraction of >95 % of the total double positive population (FIG.12).

Example 12: FACS Sorting of high producing antibody clones

To isolate scFv displaying and Fab antibody fragment displaying cells, multi-color based flow sorting was applied with the MACSQuant® Tyto® (Miltenyi Biotec) that allows sorting within a single-use, disposable cartridge. Cells were labeled with a HA tag specific antibody (PE conjugated, Miltenyi Biotec) and an biotin-conjugated anti-his tag antibody (Miltenyi Biotec) as a primary detection mean and streptavidin (Brilliant Violet421 conjugated, Biolegend) as a secondary detection mean to visualize cells, to adjust flow sorting speed and to enable gating on the desired target cell population. In addition, a staining solution specific for apoptotic Pichia cells were used (propidium iodide staining solution, Miltenyi Biotec) in order to exclude dead cells.

For actual sorting, cells suspended in MACSQuant® Tyto® running buffer at a concentration of IxlO⁶ - IxlO⁷ cells/ml were transferred to a primed MACSQuant® Tyto® cartridge using a 10 ml syringe and a pre-separation filter (20 pm). Sorting was performed at 4 °C and the sort gate was set on scFv expressing and Fab displaying positive cells. Afterwards, the sorted cells were suspended in 500 pl PBS and plated on YPD agar plates to obtain single colonies. Sort performance was evaluated with the MACSQuant X (FIG. 13). Example 13: Microscale screening of FACS sorted and unsorted Pichia clones for Fab antibody secretion capacity

For cultivation and quantitative screening of secreted Fab fragments in microscale, >90 single colonies from sorted Pichia cells and unsorted input fraction of example 12 were incubated in a buffered minimal media supplemented with Glucose (BMD1) at 28 °C for 60 hours. Then, buffered minimal media supplemented with methanol (BMM2; 200 mM potassium phosphate buffer pH 6, 13,4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin, 1 % methanol) was added to the cells to induce protein expression. Cells were further cultivated at 24 °C for 72h with additional feeding spikes of methanol over the course of the cultivation. Afterwards, cells were harvested via centrifugation in order to determine the amount of secreted Fab fragment in cell-free culture supernatant as described in example 10. Prior to separation of culture supernatant and cells, an aliquot is collected and the optical density at 600nm determined to normalize for detected productivity. Statistical analysis of associated population means confirms a highly significant difference between the two populations in regards to Fab production (FIG.14).

Example 14: Determination of residual scFv capture matrix expression of sorted cells after FACS and removal of antibiotic selection pressure

Cells of example 12 that were sorted and subsequently plated on non-selective YPD agar were tested for residual expression of scFv-based antibody capture matrix. Therefore, single colonies were picked and cultivated in buffered minimal media supplemented with methanol (BMM2; 200 mM potassium phosphate buffer pH 6, 13,4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin, 1 % methanol) for 24 h at 24 °C and 320 rpm. 1 x 10⁶ cells were washed with ice-cold PBS-F buffer (8 g/1 NaCl, 0.2 g/1 KC1, 1.44 g/1 Na₂HPO₄, 0.24 g/1 KH₂PO₄, 1 g/1 BSA, pH adjusted to 7.4) and after centrifugation (5 min, 1500 x g) resuspended in 100 pl PBS-F. For immunofluorescence staining of the expressed scFv capture matrix, an antibody specific for the HA tag (APC conjugated, Miltenyi Biotec) and an antibody specific for the c-myc tag (FITC conjugated, Miltenyi Biotec) were added to the cells in a working dilution of 1:50, followed by an incubation on ice in the absence of light for 10 minutes. Cells were washed again in ice-cold PBS-F buffer and finally resuspended in 100 pl PBS-F. Cells were analyzed for scFv expression with the MACSQaunt X. ScFv expression was completely diminished for all 30 analyzed clones. No clone exceeded scFv expression greater than 2 % of the total population (FIG. 15). Example 15: Microscale screening for Fab antibody secretion capacity of P. pastoris clones co-transformed with the Fab expression and episomal scFv display vector and of P. pastoris clones transformed with the Fab expression vector only

Electrocompetent P. pastoris cells were either co-transformed with a linearized anti-CD20 Fab expression vector (FIG.10) and the circular scFv capture matrix expression plasmid or transformed with a linearized anti-CD20 Fab expression vector only. For cultivation and quantitative screening of secreted Fab fragments in microscale, >90 single colonies of transformed P. pastoris cells were incubated in a buffered minimal media supplemented with Glucose (BMD1) at 28 °C for 60 hours. Then, buffered minimal media supplemented with methanol (BMM2; 200 mM potassium phosphate buffer pH 6, 13,4 g/1 yeast nitrogen base, 4 x 10’⁴ g/1 biotin, 1 % methanol) was added to the cells to induce protein expression. Cells were further cultivated at 24 °C for 72h with additional feeding spikes of methanol over the course of the cultivation. Afterwards, cells were harvested via centrifugation in order to determine the amount of secreted Fab fragment in cell-free culture supernatant as described in example 10. Prior to separation of culture supernatant and cells, an aliquot is collected and the optical density at 600nm determined to normalize for detected productivity. Statistical analysis of associated population means confirmed a highly significant difference between the two populations in regards to Fab production (FIG.16).

Claims

1) A method for the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, wherein the lower eukaryote host cell is a yeast, and wherein the yeast is Pichia pastoris, the method comprising: a) transforming lower eukaryote host cells with

II) a second nucleic acid sequence comprising i) a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety that is capable of specifically interacting with said first binding moiety, ii) a second antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug; thereby producing lower eukaryote host cells that express the desired POI and display said desired POI on said cell surfaces due to the interaction of said first and said second binding moiety, b) culturing the transformed lower eukaryote host cells in a selective medium, wherein said selective medium comprises said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance, or wherein said selective medium does not comprise the substance that complements the deficiency of the auxotrophic mutant cell c) contacting the transformed lower eukaryotic host cells of step b) with a detection means that specifically binds to said desired POI that is displayed on the cell surface, d) identifying and isolating one or more transformed lower eukaryotic host cells with which the detection means is bound that display a higher amount of desired protein on their cell surfaces as compared to other lower eukaryotic host cells of step c) e) culturing one or more of the lower eukaryote host cells identified and isolated in step d) that display said higher amounts of desired protein on their cell surface in a non-selective medium, wherein said non-selective medium does not comprise said chemical or anti-microbial drug for that said first nucleic acid sequence provides resistance to and/or wherein said non-selective medium comprises the substance that complements the deficiency of the auxotrophic mutant cell, thereby inducing loss of the expression of said polypeptide comprising said cell surface anchoring protein fused to said first binding moiety due to loss of said vector comprising said ARS elements, and thereby generating one or more producer cells.

2) The method according to claim 1, wherein said second binding moiety of said desired POI that is capable of specifically interacting with said first binding moiety is part of the desired POI itself.

3) The method according to claim 1, wherein said second binding moiety of said desired POI that is capable of specifically interacting with said first binding moiety is a peptide/polypeptide fused to the desired POI.

4) The method according to claim 2, wherein i) said first binding moiety is a receptor and said second binding moiety is its cognitive ligand, or vice versa, or ii) said first binding moiety is a non-antibody scaffold and said second binding domain is its cognitive target, or iii) said first binding moiety is an antibody or antigen binding fragment thereof specific for said second binding moiety of said desired POI.

5) The method according to claim 4, wherein i) said first binding moiety is an antibody or antigen binding fragment thereof specific for the kappa chain of the immunoglobulin light chain and said second binding moiety is an antibody or antigen binding fragment thereof comprising the kappa chain of the immunoglobulin light chain, or ii) said first binding moiety is an antibody or antigen binding fragment thereof specific for the Fd region of an immunoglobulin IgG and said second binding moiety is an antibody or antigen binding fragment thereof comprising the Fd region of an immunoglobulin IgG.

6) The method according to claim 5, wherein said first binding domain is a scFv specific for the Fd region of an immunoglobulin IgG, wherein said second nucleic acid sequence comprises the gene encoding the VL and CL domains of a light chain of an immunoglobulin and the gene coding the VH and CHldomains of a heavy chain of an immunoglobulin, and said desired POI is a Fab, and wherein the second binding domain is the Fd region of the Fab.

7) The method according to claim 3, wherein the first binding moiety is a first adapter peptide and the second binding moiety is a second adapter peptide and wherein the first and second adapter peptides are capable of a specific pairwise interaction.

8) The method according to claim 7, wherein the first and second binding moieties are coiled- coil peptides that are capable of the specific pairwise interaction.

9) The method according to any one of claims 1 to 8, wherein the cell surface anchoring protein is a GPI, a PIR or a FL/FS protein.

10) The method according to claim 9, wherein the cell surface anchoring protein is selected from the group consisting of

ScSAGl, ScCWP2, ScGASl, ScFLOl, PpPIRl, ScSEDl, ScTIPl, PpPIR2, PpFLO9, PpSPIl, PpGCW21, PpGCW51 and PpGCW61.

11) The method according to any one of claims 1 to 10, wherein said ARS element is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:25 and SEQ ID NO:27 to SEQ ID NO:30.

12) The method according to claim 10, wherein said ARS element is SEQ ID NO:1 or SEQ ID NO:6.

13) The method according to any one of claims 1 to 12, wherein said detection means is at least one antibody or antigen binding fragment thereof. 14) The method according to claim 13, wherein said desired POI comprises a tag, and wherein said detection means binds to or is specific for said tag.

15) A system for the generation and selection of a producer cell expressing and secreting a desired protein of interest (POI), wherein the producer cell is a lower eukaryote host cell, wherein the lower eukaryote host cell is a yeast, and wherein the yeast is Pichia pastoris, the system comprising:

I) a first nucleic acid sequence comprising i) a gene encoding a polypeptide comprising a cell surface anchoring protein fused to a first binding moiety, and ii) an antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker that is based on auxotrophy, if the producer cell is an auxotrophic mutant cell and the expressed selection marker complements the deficiency of said auxotrophic mutant cell, wherein said first nucleic acid sequence is a plasmid comprising an autonomously replicating sequence (ARS) element, and

II) a second nucleic acid sequence comprising i) a gene or genes encoding said desired POI, wherein said desired POI comprises a second binding moiety that is capable of specifically interacting with said first binding moiety, ii) a second antimicrobial resistant marker encoding a protein that provides resistance to a chemical or anti-microbial drug; wherein said ARS element is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:25 and SEQ ID NO:27 to SEQ ID NO:30.