WO2023051972A1 - Procédé de génération et de sélection d'une cellule productrice - Google Patents

Procédé de génération et de sélection d'une cellule productrice Download PDF

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WO2023051972A1
WO2023051972A1 PCT/EP2022/070483 EP2022070483W WO2023051972A1 WO 2023051972 A1 WO2023051972 A1 WO 2023051972A1 EP 2022070483 W EP2022070483 W EP 2022070483W WO 2023051972 A1 WO2023051972 A1 WO 2023051972A1
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cell
protein
binding moiety
cells
antibody
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Dominic GÄTJEN
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Miltenyi Biotec B.V. & Co. KG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • 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.
  • POI protein of interest
  • 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.
  • adapter molecules interacting peptides especially coiled-coil peptides are disclosed.
  • those approaches only permit a permanent location of the antibodies on the cell surface.
  • 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.
  • 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.
  • DsF neighboring subunit FimF
  • 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.
  • ARS autonomously replicating sequences
  • 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.
  • POI protein of interest
  • 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:
  • 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
  • ARS autonomously replicating sequence
  • 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 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.
  • FACS Fluorescence-activated cell sorting
  • scFv scFv
  • FIG. 1 A-D immunofluorescence
  • 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.
  • 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.
  • 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.
  • Fabs antibodies
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 were cultivated in selective and non-selective YPD medium, respectively, for five days.
  • 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.
  • 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 constitutively eGFP expressing Pichia clone was transformed with the anti-human Fd scFv capture matrix and induced for scFv expression.
  • 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.
  • Input fraction for cell sorting was labelled with anti-HA PE and biotinylated anti-His and streptavidin- V421 for scFv expression and Fab display.
  • 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.
  • 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
  • ARS autonomously replicating sequence
  • 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
  • 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.
  • a drug a chemical or antimicrobial/antibiotic drug
  • G418/Geneticin 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 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.
  • step a) 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).
  • 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.
  • 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
  • ARS autonomously replicating sequence
  • 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)
  • said second antimicrobial resistant marker (gene) may be different from said antimicrobial resistant marker (gene) of said first nucleic acid sequence.
  • step a) 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.
  • a cell surface anchoring protein in FIG 3: a cell wall protein
  • a capture protein operably linked to a constitutive or inducible promoter of eukaryotic cells
  • an ARS element an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or anti-microbial drug and/or a selection marker
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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:
  • 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
  • said second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug.
  • gene antimicrobial resistant marker
  • the cell surface anchoring protein may be a GPI, a PIR or a FL/FS protein.
  • 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
  • said cell surface anchoring protein may be a-agglutinin (ScSAGl), PpPIRl (Pirlp) or ScSEDl (Sedlp).
  • the lower eukaryote host cell may be a yeast.
  • yeast may be Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces bayanus, Pichia pastoris, Lachancea waltii Kluyveromyces lactis, or Kluyveromyces wickerhamii.
  • the lower eukaryote host cell may be Pichia pastoris.
  • 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
  • NAT no
  • 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 detection means may be at least one antibody or antigen binding fragment thereof.
  • said detection means may be an antibody or antigen binding fragment thereof coupled to a fluorophore.
  • 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).
  • flow cytometric sorting e.g. fluorescence-activated cell sorting (FACS).
  • said desired POI may comprise a tag
  • 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.
  • 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 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 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 first binding domain may be a scFv specific for the Fd region of an immunoglobulin IgG
  • 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
  • said desired POI may be a Fab
  • the second binding domain may be the Fd region of the Fab.
  • 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.
  • 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.
  • 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.
  • the present invention provides a method for producing a desired protein (POI), the method comprising: a) transforming lower eukaryote host cells with
  • 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
  • ARS autonomously replicating sequence
  • 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
  • said second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug.
  • gene antimicrobial resistant marker
  • 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:
  • 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
  • ARS autonomously replicating sequence
  • 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 second nucleic acid sequence may comprise an antimicrobial resistant marker (gene) encoding a protein that provides resistance to a chemical or antimicrobial drug.
  • compositions, methods, and respective component(s) thereof are 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.
  • 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.
  • eukaryotic refers to a nucleated cell or organism, and includes insect cells, plant cells, mammalian cells, animal cells and lower eukaryotic cells.
  • 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 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.
  • 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.
  • 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.
  • Glycosylphosphatidylinositol 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.
  • 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.
  • 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.
  • 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.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.
  • (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
  • 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.
  • an antigen-binding domain of an antibody or fragment thereof refers 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.
  • 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”).
  • 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 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.
  • 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.
  • 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.
  • mutagenesis 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.
  • Plasmid vectors 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.
  • ACS ARS consensus sequence
  • ARS activity may be determined by the methods described e.g. in WO2017055436A1 or other assays known in the art.
  • 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).
  • operably linked refers to the association of nucleotide sequences on a single nucleic acid molecule, e.g.
  • 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
  • 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.
  • 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.
  • 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.
  • a drug a chemical or antimicrobial/ antibio tic drug
  • a drug a chemical or antimicrobial/ antibio tic drug
  • 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.
  • auxotrophy such as arginine, methionine or histidine auxotrophy
  • 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).
  • ARS autonomously replicating sequence
  • 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.
  • 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.
  • BMD1 medium 200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base
  • BMGy 200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base
  • 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.
  • BMM medium 200 mM potassium phosphate buffer pH 6, 13.4 g/1 yeast nitrogen base, 4 x 10’ 4 g/1 biotin
  • 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.
  • approximately 1 x 10 6 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 10 6 cells were washed with ice-cold PBS-F buffer (8 g/1 NaCl, 0.2 g/1 KC1, 1.44 g/1 Na 2 HPO 4 , 0.24 g/1 KH 2 PO 4 , 1 g/1 BSA, pH 7.4) and after centrifugation (5 min, 1500 x g) resuspended in 100 pl PBS-F.
  • PBS-F buffer 8 g/1 NaCl, 0.2 g/1 KC1, 1.44 g/1 Na 2 HPO 4 , 0.24 g/1 KH 2 PO 4 , 1 g/1 BSA, pH 7.4
  • Example 4 Flow cytometric detection of scFv expression and successful Fab fragment surface capture
  • 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
  • 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.
  • Binding efficiencies of different scFv capture matrices were evaluated by Fab titration and subsequent flow cytometry analysis.
  • BD QuantibriteTM Beads (BD Biosciences) for PE fluorescence quantification were used. Approximately 1 x 10 6 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.
  • Example 7 Monitoring and determination of episomal plasmid loss and loss of surface expressed scFv capture matrix
  • MCS magnetic-activated cell sorting
  • 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.
  • staining buffer phosphate - buffered saline, pH 7.2, 5 g/1 BSA and 2 mM EDTA
  • 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.
  • 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.
  • 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 10 7 cells/ml.
  • 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 10 7 cells/ml.
  • 1 x 10 6 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
  • Nunc-Immuno TM 96-well plates 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.
  • assay buffer (1 % BSA in PBS buffer
  • an antibody specific for human Ig light chains of K type HRP conjugated, Miltenyi Biotec
  • 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
  • Example 12 FACS Sorting of high producing antibody clones
  • MACSQuant® Tyto® 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.
  • a staining solution specific for apoptotic Pichia cells were used (propidium iodide staining solution, Miltenyi Biotec) in order to exclude dead cells.
  • Example 14 Determination of residual scFv capture matrix expression of sorted cells after FACS and removal of antibiotic selection pressure
  • scFv capture matrix 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.
  • 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.
  • FOG.10 linearized anti-CD20 Fab expression vector
  • BMD1 Glucose

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Abstract

La présente invention concerne un procédé permettant d'exprimer et d'afficher des protéines d'intérêt (POI) souhaitées à la surface d'un eucaryote inférieur sous une forme accessible pour la détection et l'isolement de clones cellulaires souhaités en introduisant les deux types d'acides nucléiques suivants dans la cellule hôte eucaryote inférieure : I) une première séquence d'acide nucléique comprenant i) un gène codant pour un polypeptide comprenant une protéine d'ancrage à la surface cellulaire fusionnée à un domaine de première fraction de liaison, et ii) un marqueur de résistance antimicrobienne codant pour une protéine conférant une résistance à un produit chimique, ladite séquence d'acide nucléique étant un plasmide comprenant un élément de séquence à réplication autonome (ARS) ; et II) une seconde séquence d'acide nucléique comprenant i) un gène ou des gènes codant pour ladite POI souhaitée, ladite POI souhaitée comprenant un second fragment de liaison capable d'interagir spécifiquement avec ledit premier fragment de liaison et ii) un second marqueur de résistance antimicrobienne codant pour une protéine conférant une résistance à un produit chimique ou à un médicament antimicrobien.
PCT/EP2022/070483 2021-09-29 2022-07-21 Procédé de génération et de sélection d'une cellule productrice WO2023051972A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN117487841A (zh) * 2023-12-29 2024-02-02 南京瑞源生物技术有限公司 一种应用2a肽策略构建双功能酵母展示与分泌系统的方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117487841A (zh) * 2023-12-29 2024-02-02 南京瑞源生物技术有限公司 一种应用2a肽策略构建双功能酵母展示与分泌系统的方法
CN117487841B (zh) * 2023-12-29 2024-03-22 南京瑞源生物技术有限公司 一种应用2a肽策略构建双功能酵母展示与分泌系统的方法

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