Means and methods for displaying FC-containing proteins on cells and selection thereof

Abstract

The invention relates to the field of cell surface display and selection of Fc-containing molecules such as antibodies or alike, and / or selection of Fc-containing molecule-expressing clones. In particular, the invention relates to a method for displaying Fc-containing proteins on a cell, in particular wherein said method further relates to selection of yeast clones recombinantly expressing Fc-containing proteins. The invention further relates to a capturing or binding agent comprising a Fc-binding protein and an oligomannose binding protein, wherein the oligomannose binding protein specifically binds oligomannose or mannan on the outside of a cell, in particular a yeast cell. In particular, the invention discloses a binding agent comprising a bivalent ZZ domain or an antigen-binding domain specifically binding to Fc-tails, and comprising a lectin domain, wherein the lectin preferably binds yeast mannans. The invention further relates to a method for cell surface display of Fc-containing proteins wherein the capturing agent is applied to a population of cells to connect the cell surface via oligomannose-binding to the secreted Fc-containing proteins recombinantly expressed by said cell. In particular, the invention relates to said method further used to isolate the cells with the highest number of secreted Fc- containing proteins by cell sorting of the displayed cells.

Description

[0001] NiCa / PIZZAH / 841PCTMEANS AND METHODS FOR DISPLAYING FC-CONTAINING PROTEINS ON CELLSAND SELECTION THEREOF FIELD OF THE INVENTION The invention relates to the field of cell surface display and selection of Fc-containing molecules such as antibodies or alike, and / or selection of Fc-containing molecule-expressing clones. In particular, the invention relates to a method for displaying Fc-containing proteins on a cell, in particular wherein said method further relates to selection of yeast clones recombinantly expressing Fc-containing proteins. The invention further relates to a capturing or binding agent comprising a Fc-binding protein and an oligomannose binding protein, wherein the oligomannose binding protein specifically binds oligomannose or mannan on the outside of a cell, in particular a yeast cell. In particular, the inventiondiscloses a binding agent comprising a bivalent ZZ domain or an antigen-binding domain specificallybinding to Fc-tails, and comprising a lectin domain, wherein the lectin binds yeast mannans. Theinvention further relates to a method for cell surface display of Fc-containing proteins wherein thecapturing agent is applied to a population of cells to connect the cell surface via oligomannose-bindingto the secreted Fc-containing proteins recombinantly expressed by said cell. In particular, the invention relates to said method further used to isolate the cells with the highest number of secreted Fc- containing proteins by cell sorting of the displayed cells. INTRODUCTIONYeast Surface Display combined with fluorescence-activated cell sorting is an established methodologythat allows to screen up to 108 cells / hour 1. The protein of interest produced by the yeast cell iscovalently linked to the yeast cell wall by fusion to a protein anchor. Yeast display has been widely used for protein engineering purposes, for example, to screen for improved antibody affinity, protein stability, or enzymatic activity1–3. Nevertheless, the detection of higher levels of the antibody-yeast cell wall protein may not unequivocally reflect a better capacity of the cell for antibody secretion, but also better capacity for cell wall protein incorporation. In fact, it has been shown that the effect ofengineered cell lines with enhanced secretion was masked by the dominant effect of the fusion to theanchor protein4. Hence, the number of molecules that can be displayed on the cell wall is limited to about 104-105molecules per cell, thus, probing the improvement of protein secretion is limited to the saturation of this display capacity1. Another important limitation of current yeast display technologies is that the selected clone cannot directly be used for development and antibody production, as anchoring tags or proteins need to be engineered out prior to cell line development. NiCa / PIZZAH / 841PCTA number of methods have been applied to keep the secreted protein, such as the antibodies, in theproximity of the cell that produced it. In the antibody biologicals area, the conventional production hostremained mammalian cells, thus with a focus on cell line generation of mAbs-producing CHO cells. Inyeast as host for antibodies or antibody-like molecules, such as single domain antibody-based formats,such as VHH-Fcs, so far 2 technologies were used. The first is Secretion-and-Capture Technology(SECANT), which relies on in vivo biotinylation of the protein of interest that is captured on theavidinated cell surface5. The advantage of the SECANT technology is that it can be adapted to a wide variety of proteins of interest, including protein complexes such as full-length IgGa5. However, it still requires the presence of biotin-acceptor peptide and the co-expression of the biotin ligase that has to keep up with the antibody expression. In addition, the selected clone cannot immediately be used as host cell line for production and one has to hope that the selected phenotype can be regenerated in aseparate targeted engineering prior to cell line generation.The second technology relating to antibody-producing yeast clones selection is the ReversibleExpression of Antibody Libraries for Selection (REAL-Select), which displays non-modified IgG moleculesusing an antibody-catching reagent. The secreted IgG antibodies are captured by a streptavidin-Fc- binding domain, made by two repeats of the Z domain of protein A (ZZ), which is immobilized on the biotinylated cell surface6. Both of these methods have been validated for screening of antibody libraries with enhanced affinity in S. cerevisiae5,6. A similar technique has been devised by the same authors in which a construct encoding ZZ fused to the anchorin Aga2P (a protein native in S. cerevisiaebut not P. pastoris) is co-expressed with the antibody of interest7. The capturing reagent howeverrequires covalent linkage via strep / biotin to the cells to screen for high affinity binders, and thusrequires modification of the selected clones if desired to use them for direct evolution or cell line generation for antibody manufacturing. For yeast production hosts, the conventional manual picking of clones is still frequently used as a selection method, but very time-consuming. Besides, the currently available microfluidic technologies are lacking a real presence in industry cell line development, and are often too artificial for yeast cells. So there is still room for improvement in the field of surface display of antibody-type molecules andeven more in the technologies to screen and select for clones with the highest number of secretedantibodies or alike, since straightforward identification of these clones directly impacts thedevelopment and manufacturing of antibody-based biologicals at a reduced cost of goods. NiCa / PIZZAH / 841PCT SUMMARY OF THE INVENTION The present invention is based on the search to improve cell-surface-display methods for cloneselection, in particular to identify those clones that recombinantly produce the desired antibody ordesired antibody levels. Furthermore, the application of a method wherein clones or cell lines (or ‘cells’as used interchangeably herein) can be selected without requiring further modification in view of cellline generation for manufacturing has led to the design of a novel capturing agent to be applied as anexogenous addition to the clone population thereby connecting the antibodies to the antibody-expressing cells. Said capturing agent initially tested was composed of a ZZ domain, specifically bindingto Fc-tails, and a multiple-mannose-binding domain, specifically binding to oligomannoses, with the aim to non-covalently attach to oligomannose units present on the cell surface, in particular on the cellsurface of yeast cells. It is known in the art that when the bivalent ZZ domain, specifically binding theFc-part of antibodies, and the bound antibody are dimers, avidity effects drive a very tight non-covalentligand-antibody interaction and strong phenotype-genotype coupling, thereby allowing for thescreening of large combinatorial libraries. So by fusing these bivalent ZZ domain Fc-binders topolypeptides such as lectins that may non-covalently but strong enough interact with the cell surfaceof the antibody-expressing cells, a novel proximity-based detection tool was invented that mayrevolutionize the display and screening of antibody-producing clones in their native state. The finding that the fusion of the ZZdomain with a mini-protein lectin domain, such as the actinohivin(AH) protein, which binds α-mannan glycans on the yeasts’ cell wall, could accomplish such a fine-tunedscreening environment, was surprising in the sense that one could not predict that a clone with a non-covalently bound capturing agent could be filtered for its antibody (or Fc-containing protein) expressionprofile on a high throughput scale. This finding brought about several advantages over the drawbacksof current technologies state here above. Moreover, the invention as presented herein relates to saidcapturing agent as well as the use of said agent in a method for screening and selecting or ranking ofthe clones based on their secretion level. Moreover, as shown herein, the selection methoddetermining the amount of secreted Fc-containing proteins per cell, i.e. the Fc capturing enrichment factor was shown to be in correlation to the difference in expression level. As to further optimize the capturing reagent, in view of large-scale production and purification, several fusion protein combinations have been explored as exemplified herein. Surprisingly, when the ZZ- domain was replaced with an antigen-binding domain, specifically a VHH, which binds to Fc-tails, still we could functionally apply this capture reagent to select Fc-expressing clones, even if said VHH is not present in a dimeric form, we observed that a monovalent binding domain for Fc-containing protein retention to the cell wall sufficiently allowed to monitor the clones selection process. NiCa / PIZZAH / 841PCTAs known from the state of the art, the current display methods also cope with the limitation that themaximum numbers are reached in the screening by the limitation of the display on the cell wall being limited to about 104-105molecules per cell. Surprisingly, the use of our lectin-based capturing agentsas presented herein significantly increased the display capacity in yeast cells expressing VHH-Fcs, asshown in the examples wherein the saturation of display was not yet reached.In particular, in the host Pichia pastoris, which is one of the preferred hosts for recombinant proteinproduction, strain productivity is one of the key elements to achieve a sustainable production process. A number of tools are available to increase recombinant protein secretion, however cell line development is highly unpredictable and still relies on a trial and error process. Screening of multiple clones enhances the chances of hitting the rare high-producing strains, but common practices of clone screening are slow and laborious. In the present invention, we provide a new method for the high-throughput screening of yeast clones with enhanced protein secretion. By generating a non-covalentcatching reagent for Fc-containing proteins that, when added to the secreting cells, retain the secreted protein on the yeast cell surface, in amounts reflecting the Fc-containing protein expression level, these cells could be run on a flow cytometer where the signal correlates with protein secretion observed inbulk, allowing identification of for instance high secretors. This capturing reagent (CapRe) technologycould drastically increase the speed and the number of clones that can be screened for protein secretion, therefore facilitating the future of strain development.In summary, the invention is beneficial to the development of a novel screening platform for antibodysecretion that relies on using an antibody-capturing agent which is a fusion protein of an Fc-bindingdomain, such as two synthetic Z-domains from protein A (ZZ) or an antigen-binding domain such as anFc-specific VHH, and a high mannose glycan-binding lectin, in particular as exemplified herein forinstance using Actinohivin (AH), Avaren, Banlec, FimH, Tectonin, or variants thereof. These catchingreagents bind the Fc region of antibodies or VHH-Fc molecules, while the mannose-binder attaches toα-mannan glycans on the cell wall, thereby non-covalently anchoring the antibody-Fcs to the producingcell. By adding a fluorescent label, e.g. indirectly via the antigen of said Fc-containing antibody producedby the cell, rapid screening by FACS at very high throughput is performed to screen and sort for specificclones, such as high-expressing clones. Advantages of this screening approach include that cells can becultured in bulk and screened immediately in same / desired conditions, and that the selected yeast clone is in its native state, ready for manufacturing / direct evolution. NiCa / PIZZAH / 841PCT DESCRIPTION OF THE FIGURES The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.Figure 1. Cartoon representation of Actinohivin (rainbow cartoon) binding to three α-1,2 mannoseglycans (red sticks) (PDB 4p6a). (A) front and (B) side view. The three carbohydrate binding sites areon the same side of the protein, reducing the chances of mediating cell agglutination. Figure 2. Cartoon model of the fusion between two Z domains (2spz) and Actinohivin (4p6a).Figure 3. SDS-PAGE analysis of ZZAH expressed in E. coli. Protein expression was induced with threeconcentrations of IPTG (0.5, 0.8 and 1mM) at 37 °C or 28 °C. The soluble (S) and insoluble (I) fractionsof induced E. coli culture after sonication was loaded on SDS-PAGE in reducing conditions.Figure 4. Purification and characterization of the ZZAH fusion protein. (A) Elution profile of the ZZAHprotein from the IgG affinity column and SDS-PAGE analysis of the purification fractions. (B) Elution profile of ZZAH protein on Superdex 20010-300 GL and SDS-PAGE analysis of the purification fractions. (C) Detection by western blotting of purified ZZAH in reducing (DTT) and non-reducing conditions. The ZZAH protein was detected with anti-protein A FITC conjugated antibody (goat) primary antibody, followed by anti-goat HRP conjugated secondary antibody.Figure 5. Characterization of Pichia cells treated with ZZAH by flow cytometry. Indirect fluorescencesignal was measured by flow cytometry. Wild-type NRRL Y11-430 cells were labelled with 50 ng / µl of ZZAH. Surface ZZ domain was detected using a FITC-conjugated goat anti-protein A antibody. (A and C) cell polydispersity of untreated (blue) and treated (red) cells. (B) Cells were treated with propidium iodide for cell viability: untreated (orange), treated (red) and ethanol killed (blue) cells. (D) Inhibitionof ZZAH binding to Pichia cell wall by S. cerevisiae mannans. Cells were incubated with 50 ng / µl of ZZAHand serial dilutions of S. cerevisiae mannans. Surface ZZ domain was detected using a FITC-conjugatedgoat-anti-protein A antibody.Figure 6. Titration of ZZAH concentration on yeast cells. Indirect fluorescence signal was measured byflow cytometry. (Top) wild-type NRRLY11-430 and (Bottom) OCH1 knock-out cells were labelled with serial dilutions of ZZAH (0-100 ng / µl). Surface ZZ domain was detected using a FITC-conjugated -anti- protein A antibody (goat). The Kd was estimated using the specific binding with hill slope in GraphPad Prism 9.0, by plotting the mean and the median of the fluorescence signal at different ZZAH concentrations.Figure 7. ZZAH functionalized cells binds to M2eVHH-Fc and GBP-Fc. Display of M2eVHH-Fc wasdetected using (A) anti-hIgG-AF647 detection antibody or (B) anti-hIgG Fab-AF647. Surface ZZ domain NiCa / PIZZAH / 841PCT was detected using a FITC-conjugated -anti-protein A antibody (goat). Cells were first functionalizedwith ZZAH and then incubated with M2eVHH-Fc. (Green) unstained cells; anti-protein A-stained cells(orange); (cyan) anti hIgG stained cells corresponding to (A) anti-hIgG-AF647 detection antibody or (B) anti-hIgG Fab-AF647; (red) double stained cells; (blue) cells functionalized with ZZAH but not incubated with M2eVHHFc, which were stained with (A) anti-hIgG-AF647 detection antibody or (B) anti-hIgG Fab-AF647. (C) Display of GBP-Fc was detected using GFP. Cells were first functionalized with ZZAH and thenincubated with serial dilutions of GBP-Fc. The green population is cells functionalized with ZZAH to which GBP-Fc was not added.Figure 8. Flow cytometric analysis of capturing of secreted GBP-Fc by PiZZAH functionalized cells.Positive control (blue) is non-secreting cells in which GBP-Fc was externally added. Negative control (red) is non-secreting cells. The percentage of positive population (orange) increased over time (0, 4 and 20 hours). The percentage of positive cells differs for two strains with different VHH-Fc productivity in bulk (high secreting cells, left and low secreting cells, right). Capture of secreted GBP-Fc was detected using GFP. The % of positive cells is calculated with FlowJo by selecting the sub-population with fluorescence higher than the negative control. HS is high secreting cells, and LS is low secreting cells.Figure 9. SDS-PAGE of selected (left) PichiaGen3 and (right) PichiaGen4 clones. 191 clones forPichiaGen3 and 191 clones for PichiaGen4 were manually screened for VHH-Fc expression in 24-deep well blocks. Five clones for PichiaGen3 and five clones for PichiaGen4 were selected based on higherband intensities on SDS-PAGE and levels of expression were validated with two independent expressiontests with 4 biological replicates. We selected PichiaGen3 clone 51 (left) and PichiaGen4 clone 20 (left) as the clones with the highest and most robust expression. PichiaGen3 clone 51 (PichiaGen3-HS) expressed twice and PichiaGen4 clone 20 (PichiaGen4-HS) 3.5-fold the amount of VHH-Fc, as comparedto the control strain PichiaGen2(G). Lane 1 Precision Plus Protein™ All Blue Prestained Protein Standard;Lane 2-13 cleared supernatant of four biological replicates of the indicated clones; Lane 14-15 dilutions of a protein standard. Figure 10. The percentage of positive cells on flow cytometry correlates with protein expression inbulk. (Orange) PichiaGen3-HS, (green) PichiaGen4-HS express respectively 2- and 3.5-fold higheramount of protein than the reference strain (blue) PichiaGen2(G), in bulk. Cells were functionalized with ZZAH and secreted VHH72-Fc was detected with biotinylated SARS-CoV-2-RBD-SD1-AviTag followed by Streptavidin-Phycoerythrin (PE). Median fluorescence intensity, Overtone % positive and Chi-Squared T(X) were calculated with FlowJo 10.7.1, referred to the negative control fully stained NCYC2543. NiCa / PIZZAH / 841PCTFigure 11. Fluorescence activated cell sorting of two VHH72-Fc expressing pools. (A-B) Sorting ofPichiaGen3-pool. (Red) negative control is a non-expressing strain, (blue) PichiaGen3-HS, (orange) PichiaGen3 pool. (C-D) Sorting of PichiaGen4-pool. (Red) negative control is a non-expressing strain, (blue) PichiaGen4-HS, (orange) PichiaGen4-pool. (B-D) Gate settings for the sorting of the upmost positive population.Figure 12. FACS sorted cells express VHH72-Fc. (Top) Protein expression of 11 single colonies from thesorted pools was analysed by loading Pichia supernatant on SDS-PAGE and Coomassie staining.(bottom) colony PCR of the tested colonies on the VHH72Fc expression cassette. (A) lane 1 ladder, lane2-12 VHH72Fc_S56A_A(s) expressing colonies, lane 13 wild type strain, lane 14 VHH72_S56A_G expressing clone, lane 15 VHH72_S56A_A(s) expressing clone. (B) lane 1 ladder, lane 2-12 VHH72Fc_5m_A(s) expressing colonies, lane 13 VHH72_S56A_G expressing clone, lane 14 VHH72_5m_A(s) expressing clone. The non-expressing colonies do no longer have the expression cassette, as demonstrated by the absence of the PCR amplicon in the colony PCR (bottom).Figure 13. FACS sorted cells have on average higher expression than unsorted cells. The ‘unsorted’plots show the amount of secreted PichiaGen3 and PichiaGen4 from single clones, estimated by densitometry analysis on SDS-PAGE protein loads. The FACs-sorted clones from the FACS-sorted poolare overall showing a higher intensity. However, in the ‘unsorted’ pool, the red dots are the clonesselected from the manual screening that showed the highest and consistent expression. These clones were selected after three independent expression tests on a sub-population of the unsorted pool. Red dots represent the calculated band intensity during the first screening, and are higher than the sorted pool intensities. Figure 14. A. Crystal structure of Actinohivin (AH; PDB: 4G1R, left) superimposed with a homologymodel of stabilized Actinohivin (As or Avaren, right). Homology modelling was performed with SWISS-MODEL, using AH as a template. Arrows indicate the new disulphide bridges in the first and second modules. Shaded circles approximately indicate the mannose binding pockets in AH. B. Amino acidsequences of ZZAH (fusion of ZZ and AH; SEQ ID NO: 12), stabilized actinohivin (SEQ ID NO:2), and ZZAs(fusion of ZZ and stabilized actinohivin with ΔA46; SEQ ID NO: 14). ZZ domain is indicated in yellow shading, stabilizing mutations in actinohivin are indicated in red and Ala46 is indicated in pink shading.C. Addition of the OmpA signal sequence for periplasmic expression in E. coli (SEQ ID NO:28). OmpAindicated in cyan shading.Figure 15. Production of ZZAH and ZZAs in E. coli BL21-DE3. A. Upon expression in E.coli, most of theZZAH protein is found in the insoluble fraction, while ZZAs is found in the periplasmic fraction. B. NiCa / PIZZAH / 841PCTPurified ZZAs in reducing (+DTT) and non-reducing (-DTT) conditions on SDS-PAGE. C. Elution profile ofZZAs on an IgG sepharose column.Figure 16. Fluorescent signal of VHH72-Fc Pichia expressing strains stained with either PiZZAH orPiZZAs. PichiaGen3-HS secretes 2-fold higher protein in bulk. Positive control is non-expressing strainto which external VHH-Fc was added. HS:LS 1:1 mix is a cell culture deriving by mixing PichiaGen3-HS and PichiaGen2-LS before cell staining in 1:1 to ratioFigure 17. Scatter plots of VHH72-Fc Pichia expressing strains stained with either PiZZAH or PiZZAs.The topmost fluorescent population was sorted using a FACS Melody sorter.Figure 18. Pichia sorting from PiZZAH and PiZZAs cells. A. Analysis of protein expression in sortedclones. Samples indicated with the arrows are respectively PichiaGen2-LS (orange) and PichiaGen3-HS(grey). B. PCR on Pichia sorted colonies. 11 sorted clones from the PiZZAH and PiZZAs stainedpopulations were analysed by PCR. On top, primers specific for PichiaGen2 construct were used. Bottom gel, amplification was performed using PichiGen3 specific primers. Last two lanes are PichiaGen3 and PichiaGen2 positive controls. Remarkably, all the sorted clones are PichiaGen3-HS. Figure 19. Scatter plots of VHH72-Fc Pichia expressing strains stained with PiZZAs. Figure 20. Protein expression of PiZZAs sorted clones compared to 1-copy and 2-copy strains.Figure 21. PiZZAH variants produced in mammalian cells. Top: representative example of SECchromatogram (ZZAs IgG peak 2). Elution peaks are indicated as D1, D2 and D3. Middle: SDS-PAGE of SEC fractions. Bottom: yield overview.Figure 22. Analysis of alternative capture reagents produced in E. coli. Top panel, Coomassie stainedSDS PAGE gel; bottom panel, western blot. The alternative Capture reagents (as listed in Table 1) were detected with a Dylight800 conjugated IgG. A) Expression in supernatant. B) Expression in periplasm. C) Expression in intracellular soluble fraction. Figure 23. Analysis of alternative capture reagents produced in HEK293S cells. Top panel, Coomassiestained SDS PAGE gel ; bottom panel, western blot. Capture reagents were detected with a Dylight800conjugated IgG. Figure 24. Analysis of purified alternative capture reagents. Top panel, Coomassie stained SDS PAGE gel ; bottom panel, western blot. Capture reagents were detected with a Dylight800 conjugated IgG.Figure 25. BLI sensorgrams of alternative capture reagents (CapRe) binding to the Fc tail of a VHH-Fc,bound to an immobilized antigen. NiCa / PIZZAH / 841PCTFigure 26. Binding of alternative capture reagents on Pichia cell walls was detected by flowcytometry. Capture reagents (CapRe) were detected with a FITC conjugated anti-human CD3 antibody or an AF594 conjugated anti-VHH antibody.Figure 27. Captured and displayed secreted VHH-Fcs were detected on Pichia cell surface viaalternative capture reagents. Secreted VHH-Fcs were detected based on their binding to biotinylatedantigen target followed by PE conjugated streptavidin. The profiles from top to bottom: Top linerepresents the high VHH-Fc expressing Pichia strain named HS VHH-Fc, second line represents the medium VHH-Fc expressing strain named MS VHH-Fc, bottom 3 panels represent the negative controls of the assay.Figure 28. The binding and displaying capacity of alternative capture reagents shown on differentPichia strains expressing various VHH-Fc. CapRe5 was used for analysis and secreted VHH-Fcs weredetected based on their binding to biotinylated target followed by PE conjugated streptavidin. HS : High secreter, MS : Medium secreter. DETAILED DESCRIPTION The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification are not necessarily all referring to the same embodiment but may. NiCa / PIZZAH / 841PCT Definitions Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable underappropriate circumstances and that the embodiments, of the invention described herein are capableof operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, and structural biology). “Nucleotide sequence”, “DNA sequence” or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This termrefers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, “caps”substitution of one or more of the naturally occurring nucleotides with an analog. “Coding sequence”is a nucleotide sequence, which is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to messenger RNA (mRNA), cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certaincircumstances. “Gene” as used here includes both the promoter region of the gene as well as the codingsequence. It refers both to the genomic sequence (including possible introns) as well as to the cDNA derived from the spliced messenger, operably linked to a promoter sequence.The terms “protein”, “peptide”, and “polypeptide” are interchangeably used further herein to refer toa polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non- NiCa / PIZZAH / 841PCT naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation, ubiquitination, sumoylation, and acetylation, among others known in the art. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton ((k)Da). By "isolated" or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolatedprotein", "isolated molecule", refers to a protein, or nucleic acid molecule, which has been purifiedfrom the molecules which flank it in a naturally-occurring state, or in its production host, e.g., othersecretion or ER / Golgi residing proteins or cellularly present nucleic acid molecules, as identified anddisclosed herein which have been removed from the molecules present in the sample or mixture, oryeast or cellular environment, such as a production host, that are adjacent to said material, by usingthe detergents, or other agents, and / or purification means as disclosed herein, and as known in the art.An isolated protein or nucleic acid molecule or composition can be generated by chemical synthesisfollowed by further treatments or can be generated by recombinant production or by purification from a complex sample. A “protein domain” is a distinct functional and / or structural unit in a protein. Usually a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. The term “linked to”, or “fused to”, as used herein, and interchangeably used herein as “connected to”, “conjugated to”, “ligated to” refers, in particular, to “genetic fusion”, e.g., by recombinant DNA technology, as well as to “chemical and / or enzymatic conjugation” resulting in a stable covalent link. “Homologue”, “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteinsand enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodifiedprotein in question and having similar biological and functional activity, or in case of a mutanthomologue, a similar deficiency in a certain activity, as the unmodified protein from which they arederived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A “corresponding” NiCa / PIZZAH / 841PCTamino acid, as referred to herein, is meant to provide for the amino acid of a homologous protein thatcorresponds to the same position (and function) of the amino acid of a wild type or reference protein as determined by aligning one or more protein sequences using a pairwise or multiple alignment tool, as known to the skilled person. Theoretically, when for instance two protein sequences are aligned, theamino acids which are structurally and functionally similar will be corresponding in their position in thealignment. A "substitution", or “mutation”, or “variant” as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental or reference protein or a fragment thereof. Similarly, a deletion mutant as used herein refers to the resulting protein or nucleic acid molecule wherein one or more amino acids or nucleotides, resp., have been removed as comparedto an amino acid or nucleotide sequence of a parental or reference sequence, or fragment thereof. Itis understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. The term “wild-type” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene. In contrast, the term “modified”, “mutant” or “variant”refers to a gene or gene product that displays modifications in sequence (e.g. codon-optimized), post-translational modifications and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-typegene or gene product. A ‘functional variant’ thus also refers to ‘variants’ as compared to the wild type,though with the limitation that such a functional variant has retained function and properties relevant for its function.“Binding” means any interaction, be it direct or indirect. A direct interaction implies a contact betweenthe binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term“specifically binds,” as used herein is meant a binding domain which recognizes a specific target, butdoes not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. In the present application, with specific binding of Fc-parts as present in antibodies or VHH-Fcs, this means that the Fc-tail is bound by said ZZ domain withhigh affinity. The term "affinity", as used herein, generally refers to the degree to which a ligand, NiCa / PIZZAH / 841PCT chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding. A “binding agent”, or “agent” as used interchangeably herein, relates to a molecule that is capable of binding to another molecule, via a binding region or binding domain located on the binding agent, wherein said binding is preferably a specific binding, recognizing a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Said binding agent may hence be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivatives thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. A “protein binding agent” is a binding agent of protein nature.The term “capturing agent” , “capture reagent (CapRe)”, or “catching agent” as used herein furtherrefers to said binding agent that is capable of binding an Fc-containing molecule through specific binding to its Fc-tail, and thereby captures or catches said Fc-containing molecule. In the particular casewherein the capturing agent is also bound to the oligomannose present on a cell surface through afurther domain specifically binding oligomannoses, preferably of the cell expressing and secreting said Fc-containing molecule, the capturing or catching agent or reagent functions in attaching or displaying the Fc-containing molecule bound to said catching agent on the cell surface. The term “antibody” refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. “Antibodies” can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, defined by 6 CDRs. Non-limiting examples of active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. The term “antibody fragment” and “active antibody fragment” or “functional variant” as used herein refers to a protein comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding an antigen. Antibodies are typically tetramers of immunoglobulin molecules. The term “immunoglobulin (Ig) domain”, or more specifically “immunoglobulin variable domain” (abbreviated as “IVD”) means an immunoglobulin domain essentially consisting of four NiCa / PIZZAH / 841PCT “framework regions” which are referred to in the art and herein below as “framework region 1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or “CDR1”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2- FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to anantibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, binds to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. An “immunoglobulin single variable domain (ISVD)” as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An “immunoglobulin domain” of this invention refers to “immunoglobulin single variable domains” (abbreviated as "ISVD"), equivalent to the term “single variable domains”, and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR’s. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL- sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH- sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the NiCa / PIZZAH / 841PCT immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH- sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in WO2008 / 020079. “VHH domains”, also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al (1993) Nature 363: 446-448). The term “VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For a further description of VHHs and Nanobody , reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 of Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more The term ‘antibody’ or ‘Fc-fusion’ or ‘ISVD-Fc fusion’ or ‘VHH-Fc fusion’ as used herein further refers to the genetic linking or fusion of antigen-binding fragments or antigen-binding domains with an Fcconstant domain as to obtain dimers forming an antibody structure when expressed in a recombinant NiCa / PIZZAH / 841PCT host. In particular, antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N-terminus of an Fc domain, preferably via a linker or hinge region. Alternatively, antibody fragments, or single domain antibodies such as ISVDs, may be fused at the N-terminus to the C-terminal end of an Fc domain, preferably via a linker or hinge region. Said single domain antibody or ISVD fused to said Fc may comprise one or more VHHs or Nbs, as described herein. “Fc domains” or “Fc-regions” or “Fc-tails”, as interchangeably used herein, and refer to the single Fc chain and / or the dimeric Fc domain of an Fc-containing proteins. Specifically in antibodies, said Fcdomain is thus responsible for antibody function, and ‘antibody Fc engineering’ stands for engineeringfunctions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life. Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post- translational modifications, dimerization behavior, effector function, serum half life, among others. To indicate the variations present in Fc domains based on the sequence of naturally occurring IgGs, conventional antibody numbering annotations are known in the art, such as for instance IMGT numbering (LeFranc, 2014; Frontiers in Immunology.5 (22): 1-22), Kabat numbering (Kabat, E.A. et al.,Sequences of proteins of immunological interest. 5th Edition - US Department of Health and HumanServices, NIH publication n° 91-3242, pp 662,680,689 (1991)), or preferably used herein EU numbering(Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. ProcNatl Acad Sci USA.;63:78–85). As used herein, the terms "determining," "measuring," "assessing,", “identifying”, “screening”, “addressing”, “testing”, and "assaying" are used interchangeably and include both quantitative andqualitative determinations. “Similar” as used herein, is interchangeable for alike, analogous,comparable, corresponding, and -like or alike, and is meant to have the same or commoncharacteristics, and / or in a quantifiable manner to show comparable results i.e. with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less. Detailed description Protein secretion is the favored way to produce recombinant proteins and the screening of the best ormost optimal clones for recombinant protein production is still one of the bottlenecks of cell linegeneration. Particularly for secreted proteins, such as antibodies or alike, methods for linking the host cell genotype to the secreted protein still rely on techniques with limited throughput, which are based on physical separation of cells in microwells. The present invention provides for new means and NiCa / PIZZAH / 841PCTmethods to link the secreted protein to the producing cell, which is based on the catching and non-covalently displaying of Fc-containing protein molecules on the surface of yeast cells, in particular Pichiacells, using the non-covalent binding of a catch reagent.The very first capture reagent, called ZZAH (also referred to herein as PiZZAH), is a fusion proteinbetween a lectin, actinohivin (AH) that binds to the cell wall of Pichia, and two repeats of the Z domainof protein A (ZZ), which binds the Fc region of Fc-containing molecules. We show that the ZZAH fusionprotein can be produced in E. coli in a soluble form and can be purified from the cleared cell lysate. Wefurther showed that the ZZAH fusion protein can bind both the Pichia cell wall glycans and the Fc regionof secreted molecules enabling high-throughput screening of the expression level of a pool of yeaststrains and sorting of the desired population.The recombinantly produced ZZAH fusion protein although soluble appeared (at least partially) inoligomeric state, even though the ZZ domain is reported as a remarkably soluble and stable domain.AH is usually found in inclusion bodies when produced in E. coli, and multimers of AH are observed on SDS-PAGE when it is produced in Nicotiana benthamiana18,23. In fact, around half or more of the ZZAHfusion protein was in the insoluble fraction of the E. coli lysate. Nevertheless, purified soluble proteinwas obtained from the cleared cell lysate and used for the display and selection method describedherein. To provide alternatives to a potential suboptimal production capacity for the PiZZAH reagent,engineered versions of AH, called avaren ‘Av’ (resulting in ‘ZZAv’ ; or with Ala46 deletion ‘As’ resultingin ‘ZZAs’), or Avaren-mut were used as capture reagent mannose binding unit, to improve the stabilityand homogeneity of the capture reagent / lectin. Alternatively, several additional lectin proteins known to have mannose-binding capacity were also tested in the form of a capture reagent fusion protein (with ZZ domain and / or anti-Fc VHH) to apply as recombinant capture reagent. So in a first aspect the invention relates to a binding agent, also named herein ‘capturing ‘ , ‘capture’or ‘catching’ or ‘catch’ agent or reagent, also abbreviated herein as ‘CapRe’, which contains a t least 2protein domains of which one in particular specifically binds the Fc-tail of Fc-containing proteins, such as VHH-Fcs or antibodies, and the second domain specifically binds oligomannose units as present on the cell wall of the Fc-containing protein production host, such as yeast cells, and in particular Pichia cells. Said two domains may be fused or coupled directly or by a linker, preferably a short linker,preferably a linker of maximum 20 amino acids, or maximally 10 amino acids, or most preferablymaximally 5 amino acids. Linkers suitable for the domain-fusions are typically short flexible peptidelinkers as known in the art. Commonly used linkers are stretches of small polar or non-polar aminoacids, such as Glycin (Gly / G) and Serine (Ser / S). One of the most used linker is (GGGGS)n, where n canbe adjusted to create longer linkers. NiCa / PIZZAH / 841PCT Said catching agent was shown herein to efficiently display the secreted Fc-containing molecules on the Fc-containing molecule-producing cells without a cross-contaminating effect, so allowing to screen for high secreting cells or clones in a population of cells or a library. As described in the examples, several types of lectins were used herein to test the efficiency of binding to the yeast mannans and cell surface display capacity of the catching reagents. Moreover, despite theoligomerization observed for the initial E.coli produced ZZAH protein, the binding to the yeast cell wallwas still determined to be in of nanomolar affinity and did not trigger cell agglutination nor showedtoxicity for the cells. Yeast cells functionalized with ZZAH (or ‘PiZZAH’, as used interchangeably herein)catching reagent bound and display VHH-Fc proteins in an efficient manner. By indirect fluorescentdetection of the displayed VHH-Fc, the percentage of positive cells was correlated with the amount ofVHH-Fc bound, and to the level of expression of a yeast strain in bulk. Moreover, the Fc-containingmolecule cell surface display method using said ‘capture reagent’ or in a particular embodiment usingsaid PiZZAH reagent is also generally referred to as the “PiZZAH method” and applied herein todemonstrate that one may enrich for those yeast cells that have higher / desired VHH-Fc secretion levels by screening pools of transformants. Where it is desired to screen for true enrichment of high expressing clones, the average expression should increase over the sorting rounds. It is further envisaged that this method may be used to screen for very rare events within a pool of yeast mutants, given multiple selection rounds. Yeast libraries of mutants can be generated for example by chemical mutagenesis, UV, transposon-mediated mutagenesis and more recently by using whole genome targeting CRISPR guide RNAs libraries30–33. The chances of finding a higher secreting clone are usually quite low, and millions of clones need to be screened to cover the library diversity. In a further aspect of the present invention, a method is described to produce the binding reagent orcatching reagent, comprising the steps of: cultivating a host cell expressing the catching reagentencoding sequence, ideally provided as a genetic fusion of said Fc-binding and oligomannose bindingdomains, and isolating the binding agent from said host cell, preferably through affinity chromatography and / or size exclusion or desalting to obtain monomeric and / or multimeric forms of said catching reagent for applying in the PiZZAH method as described herein. In a specific embodiment, said binding agent or catching agent is produced in a host cell, wherein the ‘host cells’ can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected with the nucleic acid molecule or protein encoding or providing said binding agent. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate NiCa / PIZZAH / 841PCT co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction. A DNA construct capable of enabling the expression of the chimeric protein of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillusspp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells,Pseudomonas spp. cells, and Salmonella spp. cells. Animal host cells suitable for use with the inventioninclude insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO)),and human cell lines, such as HeLa, HEK293 cells. Yeast host cells suitable for use with the inventioninclude species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichiapastoris), Hansenula (e.g. Hansenula polymorpha), Yarrowia, Schwaniomyces, Schizosaccharomyces,Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the mostcommonly used yeast hosts, and are convenient fungal hosts. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals. The recombinant production of said binding agent in said host may be obtained using a constitutive or continuous production, depending on the promoter or system of production, or may be based on an inducible system, such as in E.coli, several option including induction using IPTG, as exemplified herein,or alternatively in E. coli BL21-AI which is induced with 0.2% arabinose. Furthermore, alternativeproduction cells such as HEK293 were tested herein, already providing indications that these may be better positioned to obtain monomeric forms of the catching reagents tested herein. NiCa / PIZZAH / 841PCT Beyond the expression and purification, the catching agent also requires a proper protein stability for its application as additive in screening assays. In view of this requirement, adaptation of the moreproblematic characteristics of lectin domains may be necessary and is already partly investigatedthroughout the experimental settings disclosed herein.For instance, the ZZAs (variants) used herein contain an Ala46 deletion in the stabilized actinohivindomain that is not present in Avaren as presented by Hamorsky et al.26. While ZZAs was proven to befunctional in our PiZZAH strategy, further analysis of the Avaren as previously reported is anticipatedto determine whether this is preferable in view of protein stability. Furthermore, some mannose-binding lectins might interact with sepharose resin in purification columns, which is a feature to analyze in more detail and take into account in the purification steps. Further embodiments disclosed herein relate to capture reagents comprising a Fc-containing protein- binding domain fused to a lectin, wherein said lectin binds oligomannose units, more specifically mannan, even more specifically yeast mannan. Mannans are linear polymers of linked mannose units, typically classified into four different types, linear mannan (β-1,4-mannan), galactomannan, glucomannan, and galactoglucomannan. Yeastmannans include a linear α-1, 6-mannoside backbone branched with α-1, 2-mannoside and α-1, 3-mannoside bonds in the form of mono-, di-, tri-, and tetramers. Specifically for more details on the appearance and characteristics defining yeast mannan, we refer herein to Jones and Ballou (1969.Studies on the structure of yeast mannan. J. Biol Chem. 244(4) p.1043-1051), or to Baek et al. (2024.Yeast cell wall mannan structural features, biological activities, and production strategies. Heliyon.;10(6):e27896).With “lectin specifically binding oligomannose units” is referred herein to those lectins that bind saidmannose polymeric units or mannans present on the cell wall of several organisms, including yeast.These types of lectins are also known in the art s Mannose-binding-lectins or Mannan-binding-lectins (MBL) and the analysis as to known whether a lectin protein is a mannan-binding lectin protein may be performed by Surface plasmon resonance type of assays, wherein the lectin-sugar interaction is characterized. Yeasts are eukaryotic single-cell microorganisms that are easy to grow and therefore one of the most important model organisms for molecular biology and genetics. The genome of the baker’s yeast S.cerevisiae was the first eukaryotic genome to be sequenced in 1996. In the meantime, many otheryeasts started to be investigated. Different to human cells, which synthetize complex glycans by addition of different monosaccharides and sialic acid, yeast glycosylates its proteins with high mannose carbohydrates, and P. pastoris in NiCa / PIZZAH / 841PCTparticular, expresses mainly glucosyltransferases involved in the synthesis of oligomannose typeglycans. Pichia pastoris belongs to the methylotrophic yeasts, providing for further advantages.Methyloptrophic yeast (belonging to Hansenula, Candida, Pichia, Torulopsis genera) indeed are capableto metabolise monocarbonic compounds like methanol and formaldehyde (Pawel, et al.,Biodegradation 12, pp: 169–177, 2001; Negruta et al., Romanian Biotechnological Letters Vol.15, No.4, p5369, 2010), and appear in nature in particular in moulds, fruit and other vegetable products, exudates of trees and their barks (Craveri et al. Taxonomical examination and characterization of a methanol-utilizing yeast, Antonie van Leeuwenhoek 42, pp: 533- 540, 1976). Methylotrophic yeast are Crabtreenegative organisms that do not produce ethanol in anaerobic conditions, hence can grow to extremely high cell densities, which often translates into high product yield. In addition, they can efficiently secrete high molecular weight proteins, which in S. cerevisiae, would be retained in the periplasmic space. Methylotrophic yeast strains which can be modified using the present methods include but arenot limited to yeast capable of growth on methanol such as yeasts of the genera Candida, Hansenula,Torulopsis, and Pichia. A list of species which are exemplary of this class of yeasts can be found in C.Anthony (1982). The Biochemistry of Methylotrophs, 269. Pichia pastoris, Pichia methanolica, Pichiaanomola, Hansenula polymorpha and Candida boidinii are examples of methylotrophic yeasts useful inthe practice of the present invention. Preferred methylotrophic yeasts are of the genus Pichia.Especially preferred are Pichia pastoris strains GS115 (NRRL Y-_15851); GS190 (NRRL Y-_18014) disclosed in U.S. Patent No.4,818,700; PPF1 (NRRL Y-_18017) disclosed in U.S. Patent No.4,812,405;PPY120H and yGC4; as well as strains derived therefrom.In particular, Pichia pastoris, which was recently reclassified as Komagataella phaffii, but hereinafterstill referred colloquially to as Pichia, is now one of the preferred hosts of the biotechnological sector for recombinant protein production. Pichia pastoris triggered the interest to be used as a cheap single cell protein source for animal feed, because of its capacity of growing to extremely high cell densities (130-150 dry cell weight / l) using methanol as its sole carbon source and metabolic energy, and itspotential of becoming an excellent organism for heterologous protein expression, became also clearfrom further developments, such as a commercial Pichia expression system (Invitrogen Corporation (USA)), which is currently used worldwide. The original Pichia strain was deposited as a patent deposition, in two culture collection entries, NRRL Y-11430 and CBS7435. The commonly used GS115 strain is an auxotrophic his4 strain derived from CBS7435 by random mutagenesis with N-methyl-Nʹ- nitro-N-nitrosoguanidine (NTG). In the early 2000s, Pichia obtained the GRAS status (Generally Recognized as Safe) for pharmaceutical production, after which the first Pichia-produced recombinant biopharmaceutical product ecallantide (Kalbitor®), a plasma kallikrein inhibitor, was approved by the FDA in 2009 for the treatment of hereditary angioedema and in the prevention of blood loss in NiCa / PIZZAH / 841PCT cardiothoracic surgery. Today, there are more than 70 Pichia-produced biopharmaceutical products on the market or in late stage development. Furthermore, it was shown herein that engineered yeast strains, such as engineered Pichia strains, even glyco-engineered yeast strains, are still applicable as production host for the method of the present invention for applying the capture reagent. Indeed, as a non-limiting example, an OCH1 ko strain was analyzed for its binding retention to the capture reagent (Figure 6), which lacks a functional Och1 gene. Och1 encodes an α1,6-mannosyltransferase protein, required for high mannose glycoproteins. Non-functional OCH1 thus prevents hypermannosylation of said yeast cells, though we have shown hereinthat this has no significant impact on the Capture reagent binding to the cell wall of OCH1 KO Pichia cells. An examples of such an OCH1 defective strain is for instance reported in Callewaert et al. (2024, WO2024 / 032891).A further aspect of the invention relates to the method for cell surface display of Fc-containing proteinmolecules, using the catching reagent as described herein. As exemplified in the present application, the detection of the displayed VHH-Fc by using fluorescently tagged antigens has the advantage that the sorting experiment directly screens for antigen binding capable VHH-Fc productivity while the integrity of the Fc is already screened for by the use of the protein A derived ZZ domains. However, it requires a different reagent for every VHH-Fc target. A more generic approach of detection may involve the use of a synthetic Fab fragment binding to the scaffold of nanobodies with picomolar affinity35,allowing the detection of VHH-Fcs in general.In view of the embodiments wherein said method is applied to the screening for clones with enhanced(Fc-containing) protein secretion, the technology described herein was optimized for maximum capturing capacity and quantitative detection of displayed VHH-Fc. Alternatively, it is envisaged hereinthat this PiZZAH technology may possibly be used to sort libraries of affinity enhanced VHH-Fc mutants,similarly to what was shown for the REAL-SELECT method6. A further embodiment thus relates to said method for cell surface display of Fc-containing proteins,comprising the steps of: providing a host cell comprising the recombinant construct or expressing theFc-containing protein, or alternatively provide a population of cells expressing Fc-containing proteins, or a library of Fc-containing protein-expressing clones; and mixing said cell culture or population or library with the catching reagent of the present invention, wherein said mixing or addition of the purified catching reagent is only performed at the moment where the presence of secreted Fc-containing proteins is minimal or nihil (i.e. the cells should not yet be induced, or the expressing cellsshould be pretreated to wash away the secreted Fc-containing proteins present at that point); and NiCa / PIZZAH / 841PCT provide incubation time for binding of the catching reagent to the oligomannoses on the cells, and to obtain cells displaying their secreted Fc-containing proteins.In a further embodiment, said culture of host cells, or said population of cells or library of clonesdisplaying said Fc-containing proteins is further used for sorting the cells by using a detection reagentfor said Fc-containing proteins by for instance FACS, as to identify those clones with a desired number or preferred range of secreting molecules, which may be a low amount a mid-range or high amount of secreting molecules, depending on the goal, preferably the clones with the highest amount of secreting protein. A final aspect of the invention relates to a kit for cell surface display of Fc-containing protein-secreting cells, comprising the binding agent as described herein, and optionally a solution or buffer for incubation of the binding agent with a population of cells. It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods and products according to the disclosure,various changes or modifications in form and detail may be made without departing from the scope ofthis invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims. EXAMPLES Introduction We envisaged to develop a method for the screening of enhanced antibody secretion in P. pastoris, butwithout the need for chemical functionalization of the cells or co-expression of a capture reagent, suchthat we could use the tool in native clones, simply by spiking in the reagent at any stage of cultivationof a library of clones (e.g., in productive phase of a fed batch mini-fermentation). We generated acatching reagent that directly binds to the yeast cell surface. The yeast outermost layer of the cell wallis a dense mat of heavily mannosylated N-and O-glycosylated proteins, which can be bound by carbohydrate-binding proteins (lectins). For example, the lectin of the seeds of jack bean (Canavalia ensiformis), named Concanavalin A (ConA), binds specifically to the non-reducing termini of α-D-mannosyl and α-D-glucosyl containing sugars 8. Already in the 70s, fluorescein-conjugated ConA (FITC-ConA) had been used to stain the highly branched α-mannans on the cell wall of S. cerevisiae9, however, because of the multimeric nature of ConA, it also triggers cell agglutination, which can hamper cell viability and dramatically complicates single-cell analysis10. In the last two decades, several newmannose-specific lectin mini-proteins have been reported, particularly in the field of HIV research. The NiCa / PIZZAH / 841PCT HIV-1 gp120 protein is highly glycosylated with high mannose carbohydrates, therefore specific oligomannose binding agents can prevent viral entry into the cell. Among them, cyanovirin-N (from Nostoc ellipsosporum) and actinohivin (from Longispora albida)11,12are interesting in this context because of their specificity for high-mannose type glycans. Cyanovirin-N (CV-N) shows the highest affinity for oligomannose oligosaccharides such as Man8 and Man913. A CV-N monomer has two mannose-binding pockets, however, it predominantly occurs as a multimer (dimers, trimer, andtetramers) and structural studies revealed that nonamannoside (Man9) mediated CV-N cross-linking,therefore similarly to ConA, it may mediate cell agglutination14,15. Contrary, Actinhovin (AH) is naturally monomeric, and the three closely spaced carbohydrate-binding sites are situated on the same side ofthe protein (Figure 1), therefore by using this lectin a reduced agglutination of yeast cells was expectedas compared to other lectins such as ConA and CV-N. Moreover, AH shows a higher binding affinity (Kd=0.034 µM) 16 than ConA (KD= 0.1-1.1 µM) 17 for yeast mannan and its recombinant production in E. colihas previously been reported18. Staphylococcal protein A (SpA) is one of the most characterized IgG-binding proteins and it is used in different types of applications19. Structural and functional analysis have shown that primarily the B domain interacts with the CH2 and CH3 domains within the Fc region20. An engineered and more stable variant of domain B, called domain Z, contains an Ala1Val and a Gly29Val substitution and it no longer binds the Fab domain, making it truly specific for the Fc region. The 6.7 kDa Z domain is easy to producerecombinantly, stable, and has the capacity to refold21. The bivalent ZZ domain exhibits an 8-foldreduced dissociation rate and 17-fold increased affinity (3 nM vs.50 nM) to human Fc as compared to monomer Z domain22. In addition, since both the Fc and the ZZ domain are dimers, the binding could be enhanced by avidity effects. Rhiel et al. used a Streptavidin-ZZ fusion protein for the non-covalent display of full-length antibodies on the chemically biotinylated yeast cell wall, which allowed strong phenotype-genotype coupling even after 20 hours of incubation6. As shown herein, we succeeded to generate a new ‘capture’ reagent to catch and display Fc-containingmolecules on the yeast cell surface composed of a translational fusion of a lectin, in particular and firstlythe lectin Actinohivin, to the bivalent ZZ domain, also called ‘ ZZAH’ herein (Figure 2). When added tothe yeast cells, on one side the AH domain of the fusion protein binds to the high-mannosecarbohydrates of the cell wall, while on the other side it can bind soluble Fc- containing molecules. Theso-caught IgGs or VHH-Fcs or other Fc-containing molecules can be fluorescently detected for example with IgG or VHH or Fc-targeting antibodies or fluorescently labeled antigens and sorted by FACS. In thefollowing examples, we describe the development of the Pichia ZZAH (PiZZAH) catch and displayreagent and methods using the same, to retain secreted VHH-Fc on the Pichia cell wall and moreoverallowing selection of the highest secreting cells. Besides the advantage that the capturing and / or NiCa / PIZZAH / 841PCTselection of the cells with the highest producing Fc-containing molecules, such as VHH-Fc antibodies, isperformed in a non-covalent manner, which allows easy recycling of the clones / Fcs, this type ofcapturing agent is superior in that it facilitates screening of cells cultured in bulk and screenedimmediately in the same / desired conditions, wherein the selected yeast clone is in its native state, ready for manufacturing and / or direct evolution. Based on the present invention, we introduced a general nomenclature used herein referring to “PiZZAH” for the display and capture / sorting technology, and “ZZAH” or “ZZAs” for the capture reagent proteins.Example 1. Production and purification of the ZZAH catching reagentThe ZZAH fusion protein was produced in E. coli BL21-DE3. To test which are the best conditions forZZAH expression, we induced protein expression with 0.5, 0.8, or 1 mM IPTG. Protein expression was performed overnight at 37 °C or 28 °C. The cells were lysed with lysozyme followed by sonication, and the soluble fraction was separated from the insoluble fraction by centrifugation. The soluble (S) andthe insoluble (I) fractions of the induced E. coli cultures were analyzed on SDS-PAGE, which showedthat more than half of the protein was in the insoluble fraction, however there is still a reasonable amount of soluble protein in the cleared cell lysate that could be purified (Figure 3). For protein purification, we expressed the ZZAH protein overnight at 28 °C, by inducing expression with 0.8 mM IPTG. After cell lysis, the soluble fraction was loaded on an IgG affinity chromatography column. The protein (25.7 kDa) was eluted in a single slightly asymmetric peak, which contained proteins that run at different molecular weights on SDS-PAGE (Figure 4A). The fractions were pooled, and gel filtrated on a Superdex 20010-300 GL column. Unexpectedly, all the proteins eluted with the column void, which means that they form a protein complex with a hydrodynamic volume bigger than ~200 kDa (Figure 4B). To better understand the nature of these interactions, the fraction 3 of the gel filtration elution (Figure 4B) was analyzed by western blot in reducing and non-reducing conditions (Figure 4C). The high- MW contaminants were recognized by the anti-protein A antibody, which means that these are probably multimers of the ZZAH monomer (Figure 4C). Surprisingly, these oligomers were the majority of the protein in non-reducing conditions (Figure 4C). Even though Actinhovin is reported to function as a monomer, dimers and bigger oligomers have been reported for recombinant expression in E. coli and Nicotiana benthamiana23. After several attempts to reduce the presence of these oligomers bybuffer formulation, we decided to investigate the activity of the ZZAH oligomers on the Pichia cells.Example 2. The ZZAH reagent binds and displays VHH-Fc to the Pichia cell wall.To investigate whether the ZZAH fusion protein can bind to the yeast cell surface, we incubated serialdilutions (0-100 ng / µl) of the ZZAH reagent with Pichia wild type cells (NRRL Y-11430) and Pichia OCH1 NiCa / PIZZAH / 841PCT knock-out cells. The OCH1 knock-out strain (Callewaert et al., WO2024032891A1) has reduced hypermannosylation as compared to the wild type strain, and it is often used for recombinant protein production. The ZZAH catching reagent was detected with anti-protein A FITC conjugated antibody (goat) and the stained yeast cells were analyzed on flow cytometry. No differences in cell size, agglomeration nor viability were observed between treated and untreated cells (Figure 5A-C). The binding specificity of ZZAH for the yeast’s high mannose glycans was investigated by incubating the cellswith 50 ng / µl of ZZAH in the presence of mannans from S. cerevisiae. Although not complete at thetested concentration, the yeast mannans inhibit the binding of the catching reagent to the yeast cell wall (Figure 5D). To investigate the capacity of the immobilized ZZAH to bind a Fc tail from a human Immunoglobulin G(hIgG-Fc) and display it on the yeast cell surface, we incubated the cells pre-treated with ZZAH withpurified M2eVHH-Fc, wherein the Fc part was generated based on the sequence from hIgG.Homogenous loading with ZZAH was detected with anti-protein A-FITC antibody, and bound M2eVHH- Fc was detected with antihIgG-AF647. The tested double-stained cell samples showed a positive signalfor ZZAH display and VHH-Fc binding, indicated by a double positive fluorescent signal, as compared tothe respective negative controls labeled with only anti-protein A or only anti-Fc (Figure 7A). However, incubation of the goat anti-hIgG-AF647 detection antibody without VHH-Fc also gave some positivesignal (Figure 7A dark blue), likely because it was also captured by the catching reagent. Protein A issupposed to have a very low affinity for goat IgGs24, however, it is possible that because of avidity, it still binds to the catching reagent. This would mask the signal of the VHH-Fc binding, especially for the detection of low VHH-Fc amounts. To reduce this undesired binding, we replaced the full-length antibody with an anti-hIgG Fab fragment20. Nevertheless, we still saw an increase in the AF647 positive population when ZZAH stained cells were incubated with the anti-hIgG Fab (Figure 7B), but not when cells were not pre-stained with ZZAH (not shown). To obviate this problem, fluorescently labeled antigen may be used, which is common practice in yeastsurface display. The GFP binding protein (GBP) is a VHH that binds GFP 25. We produced a GBP-Fcresembling the M2eVHH-Fc fusion and we used this VHH-Fc for the further optimization of the methodsince it simplifies the detection of the VHH-Fc displayed on the Pichia cell wall. Three GBP-Fc dilutionswere incubated with cells pre-stained with ZZAH and after removal of unbound GBP-Fc, the displayed VHH-Fcs were detected with GFP. Detection of the immobilized ZZAH was omitted because the FITC signal of the available anti-protein A conjugate, would be in the same channel as the GFP. By using GFP for detection we obtained an even higher signal, with a shift of four orders of magnitude at the lowest VHH-Fc concentration tested (Figure 7C). As desired, increasing concentrations of GBP-Fc resulted in NiCa / PIZZAH / 841PCT an increase of fluorescent signal and therefore it can be used for quantitative determination of VHH-Fc displayed on the yeast surface.Example 4. The ZZAH reagent can capture and display VHH-Fcs that are secreted from Pichia cells.To investigate the capacity of the PiZZAH method to capture secreted VHH-Fc and display them on thePichia cell wall, we stained GBP-Fc Pichia expressing strains with the ZZAH catching reagent andincubated the cells while shaking at 28 °C for protein expression. The occupation of the immobilized ZZAH with GBP-Fc was monitored with GFP at different time points. As controls, we used a non- expressing strain, which was incubated with external GBP-Fc for the positive control. For the positive control, the excess of ZZAH was removed by centrifugation before incubation with the external GBP- Fc. For the expressing strains, the excess of ZZAH was not washed, as we hypothesized it could work as a decoy for “escaped” GBP-Fc. To check whether we could relatively quantify the amount of the secreted protein we used two GBP-Fc expressing strains that have a 3-fold difference in expression (measured in 24 deep-well blocks). Protein expression was constitutive and controlled by the GAPpromoter. For this experiment, we used the Pichia OCH1 knock-out strain. The GBP-Fc secreted fromboth strains was detectable already at time zero, and the positive signal increased over time (Figure 8). After 20 hours of incubation, the signal of the positive population was still increasing, which implies that the catching reagent was still intact. We assumed that the positive control has all the ZZ sites occupied by GBP-Fc, which means that after 20 hours, the ZZ sites of the expressing strains are most likely not fully occupied, and longer incubation time may be used if needed (Figure 8). Example 5. Screening of VHH-Fc expressing strains: looking for enhanced secretion.The PiZZAH method was validated for its application to screen Pichia libraries expressing two VHH-Fcmolecules of biopharmaceutical relevance. We generated ‘PichiaGen3’ and ‘PichiaGen4’, two SARS-CoV-2 targeting VHH-Fcs effective in preventing SARS-CoV-2 infections in a hamster model. One of the most common strategies to enhance protein expression is to increase the gene copy number of the recombinant protein. Therefore, we generated two pools of transformants (PichiaGen3-pool and PichiaGen4-pool), aiming at multiple-copy integration by using high amounts of DNA during transformation and selection at high antibiotic concentrations. These two pools of transformants have a diversity of VHH-Fc expression levels, due to the number of copies of integrated plasmids and their genomic locations. We first screened 192 clones per strain by picking single colonies and performing an expression test in 24-deep well plates. After manual picking, all the colonies were scraped from the transformation plates, and each pool was resuspended in YPD and stored in 20% glycerol at -80 °C. From the manual screening, we selected two high secreting clones (Figure 9), PichiaGen3-HS (clone 51) NiCa / PIZZAH / 841PCT and PichiaGen4-HS (clone 20), for which we observed consistently 2 and 3.5-fold higher expression than PichiaGen2(G), which is a previously characterized clone selected in initial past experiments. The copiesof integrated plasmids were determined by quantitative RT-PCR, which resulted in 2-copies forPichiaGen3-HS and 8-10-copies for PichiaGen4-HS.To verify whether we could translate the PiZZAH method to the SARS-CoV-2 targeting VHH-Fc, we firstvalidated it on the three clones of known productivity: PichiaGen2(G), PichiaGen3-HS and PichiaGen4- HS (Figure 10). Captured VHH-Fc was detected with biotinylated SARS-CoV-2-RBD-SD1-AviTag followed by Streptavidin-Phycoerythrin (PE). Contrary to what was observed for GBP-Fc, we found that toefficiently catch the secreted VHH-Fc, the excess of ZZAH had to be removed. After 4 hours ofexpression, all the expressing strains had a positive signal. The percentage of the positive populationwas also correlated with the level of expression that we observed in bulk, where the selectedPichiaGen3-HS and PichiaGen4-HS showed respectively 2-fold ad 3.5-fold increased expression as compared to the PichiaGen2(G) control strain (Figure 10). Encouraged by these observations, we testedthe method for the screening of two pools of Pichia transformants. We performed the PiZZAH protocolon the PichiaGen3 and PichiaGen4 Pichia pools and we sorted the upmost population, after 3 hours ofincubation (Figure 11). The sorted pools were plated on selective YPD plates, and 11 colonies were tested for VHH-Fc expression in deep-well plates. 4 / 11 clones for PichiaGen3 and 7 / 11 clones for PichiaGen4, were expressing (Figure 12). To understand the nature of the non-expressing clones, we amplified the VHH-Fc expression cassette by PCR (Figure 12). Interestingly, the non-expressing clonesdid not have the VHH-Fc expression vector, which explains why we do not see protein expression forthese clones. The occurrence of non-expressing clones observed during the manual screening was very low, therefore we believe that these colonies were the result of a contamination occurring after sorting. We performed densitometric quantification of the protein loads stained with Coomassie following SDS-PAGE to evaluate the expression level of the screened Pichia clones. The signals were normalized andreferenced to the PichiaGen2(G) control. On average, FACS-sorted clones (Figure 13, orange dots) had2.7 and 1.4-fold higher expression than those analyzed by manual screening (Figure 13, green dots).However, the analyzed sorted clones had on average 24.0 % and 43.6 % lower expression than the twoselected clones from the manual sorting (Figure 13 red dots). Of course, densitometry analysis of SDS-PAGE gels has a rather limited accuracy.Example 6. Generation of a stabilized PiZZAH reagentWe observed that the purified ZZAH formed oligomers larger than 200 kDa, providing for a batch ofheterogenous oligomers, which may hamper experimental reproducibility and robustness. In addition, protein stability will be impacted thereby, so we aimed to verify that the capture reagent can be NiCa / PIZZAH / 841PCTprovided by alternative forms of Actinohivin or even further lectins specific for binding multiplemannoses (see Example 10). One option is to produce a stabilized mutant of Actinohivin named Avaren(actinohivin variant expressed in Nicotiana), which was designed by structure-guided modificationswith the aim of improving overall surface charge properties26. Actinohivin is composed of three binding domains that are nearly identical, except for few amino acid differences. To reduce the aggregation propensity of Actinohivin, the authors designed multiple variants by replacing the amino acids in oneor two of the three domains, so that they all had a similar sequence and surface properties (particularlyin surface-exposed loops)26. Avaren was identified as the variant with the highest soluble expression inN. benthamiana and retained gp-120 binding capacity. It carries 15 substitutions in the 114 AH aminoacid sequence (Figure 14)26. We introduced these stabilizing mutations in our ZZAH fusion protein to improve the stability and homogeneity of the catch reagent, with in addition an Ala46 deletion, yielding the variant ‘ZZAs’ as used herein. Because the stabilized variant contains two additional disulfide bonds,the recombinant protein expression was targeted to the periplasmic space of E. coli. To this aim, theOmpA signal peptide was fused at the N-term of the ZZAs sequence (Figure 14C) to promote periplasmiclocalization and disulphide bond formation27. The sequence was ordered synthetically (sequence optimized IDT proprietary algorithm) and cloned into a pDEST17 plasmid by golden gate assembly. The insert sequence was verified by Sangersequencing and the final construct was transformed into E. coli BL21-DE3. Protein expression wasperformed in similar conditions as for the expression of the PiZZAH (Example 1). The periplasmicfraction was isolated by osmotic shock and the different cells fractions were analysed by SDS-PAGE. Asexpected, the majority of ZZAs protein was found in the periplasmic fraction, while the ZZAH proteinwas found in the insoluble fraction (Figure 15A), showing that the ZZAs, when targeted to the periplasm, is more soluble than ZZAH. Considered these results, we decided to name this proteinPiZZAs. For protein purification, protein expression was carried overnight at 28 °C after induction with0.8 mM IPTG. The periplasmic fraction was separated by osmotic shock and loaded on an IgG affinitychromatography column for protein purification. The protein (25.7 kDa) was eluted as two overlappingpeaks (Figure 15C). Protein analysis on SDS-PAGE showed that both fractions contain protein of different molecular weights, in a similar way (Figure 15B). In addition, when the protein is not reduced with DTT prior loading, a certain amount of protein runs at high MW (Figure 15B). As the two peaks looked identical on SDS-PAGE, these were pooled and buffer exchanged in PBS. NiCa / PIZZAH / 841PCTExample 7. The ZZAs reagent can capture and display VHH-Fcs that are secreted from Pichia cellssimilarly to ZZAH.To investigate whether the stabilized mutant is able to capture and display VHH-Fcs as good as theoriginal reagent, we stained the VHH72-Fc Pichia expressing strains described in Example 5, with thetwo reagents. The occupation of the displayed VHH-Fc was monitored with biotinylated SARS-CoV-2- RBD-SD1-AviTag followed by Streptavidin-Phycoerythrin (PE). Similar to what we obtained with the ZZAH reagent, the percentage of the positive population after 3 hours of expression was correlated with the level of expression observed in bulk, where the PichiaGen3-HS showed 2-fold increasedexpression as compared to the PichiaGen2(G) (Figure 10 and Figure 16). Interestingly, fluorescent signalfor Pichia cells stained with PiZZAs strategy looks more homogenous than for those stained with PiZZAH(Figure 16). To validate that the method works for enriching a population with higher protein secretion we mixed the above described strains in 1:1 ratio and we sorted the population with the highest signal (Figure17). Sorted cells were recovered in YPD supplemented with 1 % BSA and plated on selective YPD platesimmediately after sorting. Since in previous experiments, an important subpopulation of the sorted cells had lost the expression cassettes, we tested eleven clones sorted from the ZZAH or ZZAs-stained populations for protein expression in BMGY. Interestingly, we saw that all the sorted clones were still expressing the protein (Figure 18A). Two-fold differences in expression levels (as expected between PichiaGen2-LS and PichiaGen3-HS) would not be visible in the tested conditions, as cultures were not started at the same OD and were not grown in glucose limiting conditions. To understand whether these clones were high or low-secretors, specific primers were designed to amplify either the PichiGen3-HS or PichiaGen2-LS expression cassette (Figure 18B). Remarkably, all the sorted clones carried the expression construct for PichiaGen3-HS, which we know to be expressed 2-fold higher than in PichiaGen2 strain, confirming that the method is suitable for enriching cells with higher secretion in a mixed population.Example 8. Sorting of a Pichia library results in enrichment of clones with higher expression.Next, we applied the PiZZAH strategy with ZZAs reagent to sort a library of Pichia cells, obtained froma transformation aimed at multi-copy plasmid integration (similar to the experiment described in Example 5). We recently found that Kar2 secretion signal leads a two-fold increased protein secretion compared to the previously used Ost1 secretion signal. The effort to generate a strain with high VHH- Fc secretion, lead to the development of the following strains that express PichiaGen(4) protein. First, we found that overexpression of HAC1 in a single-copy strain leads to a 2-fold increase (Figure 19, 1- copy strain+HAC1). Then, we generated a multi-copy strain by transformation with 30 µg of plasmid NiCa / PIZZAH / 841PCT DNA and selection in high antibiotic concentration. By manually screening 191 individual clones, we selected a clone with 1.3-fold increased expression compared to the single-copy strain. We eventuallydetermined by qPCR that the selected strain has two copies of the expression plasmid integrated in thegenome (Figure 19, 2-copy strain). The above described transformation pool was used to validatewhether PiZZAs is suitable to replace and even improve the manual screening. The PiZZAs protocol wasperformed on the following Pichia strains: 1-copy strain, 2-copy strain, 1-copy strain + HAC1 and the transformation pool. Samples were stained with RBD-Avi and Strep-PE after 3 hours of incubation at 28 C. Interestingly, the fluorescent signal nicely correlates with protein secretion observed in bulk. Thetransformation pool has on average a higher signal than 1-copy strain, which is used as reference strain.From the transformation pool sample, we screened about 6 million cells, of which about 10,000 weresorted (Figure 19, transformation pool). Sorted cells were grown to single colonies in YPD + Zeocin. Therest of the sorted pool was grown for 60 hours in YPD+Zeo and stored in 20 % glycerol for futureanalysis. 22 single colonies from the sorted population were analysed for protein expression in YPC- EnPump. Interestingly, most of the sorted clone seems to express on average as much VHH-Fc as the 2-copy strain (Figure 20). Example 9. Optimization of ZZAs expression and purification in mammalian host cells.Considering previous complications to obtain monomeric stable ZZAH or variants, expression of severalnew ZZAs variants was tested at small-scale in mammalian HEK293S cells. In addition to ZZAs(containing three disulphide bonds), three new variants ‘ZZ_As’, ‘Z_Zas’ and ‘Z_Z_As’ were designedthat contain additional Gly4Ser linkers between the fused protein domains (indicated above as ‘_’ )to potentially allow for interdomain flexibility, which might affect their tendency for aggregation.As observed previously upon E. coli production, IgG affinity chromatography yielded two peaks. After adesalting step, the protein in both fractions appeared similar on SDS-PAGE and a total 42 to 53 mg ofprotein per liter of HEK293S culture was obtained for each of the ZZAs variants. In all variants, higherorder multimers were observed that are not visible upon addition of DTT to the SDS-PAGE samples,indicating multimerization is likely cysteine-mediated. After IgG affinity chromatography, a fraction ofZZAS and ZZ_As were further purified via size exclusion chromatography (SEC, Error! Reference source not found.). This revealed that a majority (~75%) of the protein eluted in a main peak (D2), which mostly seemed to contain monomeric 25 kDa ZZAs protein on SDS-PAGE. A shoulder to the left (D1) contained mostly cysteine-mediated multimers, whereas a late peak co-eluting with the salt peak (D3) contained monomer too. NiCa / PIZZAH / 841PCT Example 10. Design of alternative mannose-binding (lectin)-Fc-binding domain capture reagents.We have described a novel method herein which is based on the use of a protein agent that links thesecreted Fc-containing protein to the host cell recombinantly producing said Fc-containing protein,through specific non-covalent binding of said protein agent to both, the Fc region of the secretedmolecules and the mannose sugars present on the surface of the Pichia cells.We showed that the ZZAH fusion protein can be produced in E.coli in a soluble form and can be purifiedfrom the cleared cell lysate. Further, we showed that ZZAH fusion protein can bind both Pichia cell wallglycans and Fc region of secreted molecules that enables us to screen the level of expression of a yeast strain in bulk. Next, we investigated the engineered version of AH, called Avaren (with Ala46 deletion), to improve stability and homogeneity of the capture reagent. We showed that the fusion protein of ZZ and Avaren,called ZZAs, can be also produced in E.coli expression system, with the help of signal peptide (ompA)targeting protein to periplasmic space of E.coli, ZZAs can be purified from the periplasmic extraction(PE). Next to that, ZZAs can capture and display secreted molecules on Pichia cell surface and ZZAs issuitable for screening of yeast cells with different expression levels, similar to ZZAH. Despite to initial promising results on protein expression and solubility, exploring further option to provide capturereagent based on the fusion between the ZZ domain and (multiple) mannose-binding proteins is still inscope with a first strategy provided in this example to perform the PiZZAH methodology usingalternative capture reagents based on their lectin properties, which are preferably monomeric uponpurification from recombinant expression (in E.coli, or mammalian cells, and / or using a host wherein its recombinant expression has been demonstrated). We propose herein the use of various lectin folding types, including but not limited to CVN trefoil-fold, beta-prism, beta-sandwich, beta-propellor,alpha-beta, which were identified from Barre et al.36.Specifically, we applied protein engineering approaches on existing capture reagents, ZZAH and ZZAs,to improve addressed concerns, and more specifically by using the ompA signal peptide fused to N-term of the ZZAH and ZZAs to promote periplasmic location and ease the purification process; as shown,insert a short glycine-serine linker (G4S) at different locations, in between ZZ and AH or Avaren, toprovide structural flexibility that can effect the biophysical characterization of capture reagent; and testthe original Avaren sequence (with Ala46 as presented Hamorsky et al. doi:10.1016 / j.ymthe.2019.07.021). Furthermore, we apply a version of avaren, called avaren-mut oravaren-FT, designed by substituting one back-mutation at position 75, G75F, and by inserting a threonine residue at position 113. NiCa / PIZZAH / 841PCTAdditionally, an Fc-binding VHH (SEQ ID NO: 42) was considered herein as an alternative Fc-bindingdomains to the ZZ domain of the capture reagents.Moreover, different lectins are envisaged as instead of the actinohivin or avaren to apply as a mannose-binding-protein in the fusion of the capture reagent. A selection of lectins with a different mannosebinding capacity and various lectin folding types was made to further test within this context of thecapturing reagent (so in fusion to the ZZ domain or the anti-Fc VHH domain) for the PiZZAHmethodology: ^Type 1 fimbrin D-mannose specific adhesin from E.coli – FimHFimH scaffold is β sandwich, it is a monomer and contains one mannose binding site. It’s N-termsequence is involved in recognition of mannoses. Thus, ZZ is fused at the C-term of FimH.^ Banana lectin from M.paradisiaca - BanLecBanLec scaffold is β prism, it has dimerization potential which is known to be very stable and containstwo mannose binding sites. Since it’s N-term sequence is free, ZZ is fused at the N-term of BanLec.^ Tectonin2 from L.bicolor – TectoninTectonin scaffold is β propeller, it has tetramerization potential which is known to be very stable andcontains six mannose binding sites. Since it’s N-term sequence is free both in monomer and tetramer,ZZ is fused at the N-term of Tectonin.Similar to ZZAH / ZZAv / ZZAs, the OmpA signal peptide was fused to the N-terminal end of these capturereagents sequence to promote periplasmic location and ease the purification process and a G4S linkerwas introduced in between ZZ / anti-Fc VHH and the lectin portion of the amino acid sequence to providestructural flexibility which may affect the biophysical characterization of the capture reagent.Alternative the IgK leader sequence is N-terminally added for production in HEK cells. As a proof of concept, 13 different combinations of capture reagents were generated by utilizing ZZdomain of protein A or anti-Fc VHH to provide binding to Fc domain of secreted molecules and sixlectins (actinohivin, avaren, avaren-FT, banana lectin, tectonin2, FimH) to provide Pichia cell wallbinding (Table 1).Table 1. Alternative Capture reagent proof of concept designsName Acronym FcMannose Lectin# MannoseMolecular Binding Scaffold binding sites weight (kDa) CapRe1 ZZAH ZZ Actinohivin (AH) CVN-fold 3 25.8CapRe2 ZZ_AH ZZ Actinohivin (AH) CVN-fold 3 26.1CapRe3 ZZA ZZ Avaren (A) CVN-fold 3 25.3 NiCa / PIZZAH / 841PCT Name Acronym FcMannose Lectin# MannoseMolecular Binding Scaffold binding sites weight (kDa)CapRe4 ZZ_A ZZ Avaren (A) CVN-fold 3 25.7CapRe5 ZZAmut ZZ Avaren (A-FT) – mutated CVN-fold 3 25.5CapRe6 ZZ_Amut ZZ Avaren (A-FT) – mutated CVN-fold 3 25.8CapRe7 ZZBanLec ZZ BanLec β-prism 2 27.9CapRe8 ZZ_BanLec ZZ BanLec β-prism 2 28.2CapRe9 ZZTectonin ZZ Tectonin2 β-propeller 6 37.0CapRe10 ZZ_Tectonin ZZ Tectonin2 β-propeller 6 37.4CapRe11 FimHZZ ZZ FimH Adhesin β-sandwich 1 30.2CapRe12 FimH_ZZ ZZ FimH Adhesin β-sandwich 1 30.5CapRe13 aFcVHH_A VHH Avaren (A) CVN-fold 3 25.4Example 11. Production and purification of the alternative capture reagents.The designed capture reagents shown in Table 1 were codon optimized for expression in E. coli. An N-terminal signal peptide, ompA, was fused to the capture reagents to direct their secretion to the oxidizing environment of the periplasm to allow proper disulfide bond formation. Recombinantexpression of the new capture reagents was investigated in E. coli BL21-AI. Protein expression wasinduced with L-arabinose according to the manufacturer’s protocol. The supernatant (SN), periplasmic extract (PE) and soluble intracellular fraction were analyzed on SDS-PAGE and western blot to detect the capture reagents (Figure 22). The protein bands appeared according to their molecular weights. Sufficient amount of CapRe1-6 and CapRe13 were secreted to supernatant facilitating the downstream processing (Figure 22A). The secreted CapRe7, CapRe8 were detectable both in periplasmic extract and soluble intracellular fraction (Figure 22B,C), but the absolute protein amounts were considerably less than for the others. Production was only achieved for the linker variants of tectonin2 and FimH, CapRe10 and CapRe12, respectively. A small amount of CapRe10 could be observed in the soluble intracellular fraction(Figure 22C). Comparable amounts of CapRe12 were found in both periplasmic extract and soluble intracellular fraction (Figure 22B,C). To investigate whether the expression yield of the capture reagents can be enhanced, the capture reagents were expressed in HEK293 cells. For this, the designed capture reagents were codon optimized for mammalian expression and a signal peptide was included for secretion to the medium. The transient expression was terminated when cell viability had dropped below 75%, harvested culture medium was analyzed on SDS-PAGE and western blot to detect the capture reagents (Figure 23). Expression of the FimH variants, CapRe11 and CapRe12, was not tested in HEK293 cells, as FimH is naturally occurring in NiCa / PIZZAH / 841PCTE. coli. The expression test in HEK293 cells resulted in similar expression levels of CapRe1-6 as can beachieved in E. coli. Expression of CapRe2 could not be tested due to technical issues. CapRe7 andCapRe8 showed slightly better expression while no expression was detected for the tectonin2 variants, CapRe9 and CapRe10.For protein purification, the capture reagents were produced in E. coli, except for the HEK-producedCapRe7 and CapRe8. Soluble protein containing fractions (culture supernatant for CapRe1-6, CapRe13, soluble intracellular fraction for CapRe10 and periplasmic extract for CapRe12) were loaded on an IgG affinity chromatography column and purified according to the manufacturer’s protocol. The elution fractions were pooled for each capture reagent and samples were buffer exchanged into PBS pH 7.4. Elution attempts of CapRe12 with a low pH buffer resulted in insufficient amount of protein. Further unfolded CapRe12 sample was collected in a high pH CIP solution and refolded through overnight dialysis to PBS. Purity of all proteins was assessed via SDS-PAGE analysis (Figure 24). High purity was observed for most of the capture reagents, while purity of CapRe7 and CapRe8 were lower compared to others. Unexpectedly, CapRe10 showed highly degraded profile possibly due to harsh intracellular extraction protocol. Nevertheless, intact size of CapRe10 (37 kDa) was detected via western blot based on ZZ domains, which means contaminant bands were lack of ZZ domain. Example 12. Alternative Capture reagents bind to Fc containing molecules as well as to Pichia cell walls. The interaction between the Fc domain of a Pichia pastoris-produced VHH-Fc and the Fc-bindingdomain of the capture reagents was assessed via biolayer interferometry (BLI). An antigen of theparticular VHH of the VHH-Fc was biotinylated and immobilized on a streptavidin-coated biosensor,followed 500 nM of VHH-Fc. Association of capture reagents at 400 nM was measured for 120 s at 25°C, followed by 60 s dissociation. No aspecific binding was observed between the immobilizedbiotinylated target molecule or biosensor and the capture reagents in the absence of VHH-Fcs. Afterdouble reference subtraction, all capture reagents showed binding to immobilized VHH-Fc (Figure 25) showing that the Fc binding parts of the capture reagents are functional and structurally available independently of their lectin domain. To investigate the binding of the capture reagents to the Pichia cell wall, serial dilutions of the capture reagents were incubated with Pichia cells (NCYC 2543). Capture reagents were detected with a FITC conjugated anti-human CD3 antibody or an AF594 conjugated anti-VHH antibody. Stained cells wereanalyzed on the flow cytometry (Figure 26). Highest 100 μg / mL concentration of capture reagent wastested except CapRe13 (10 μg / mL) due to limited amount of material. The fluorescence signal increasedwith the increasing concentration of the capture reagents. Even though the capture reagents were not NiCa / PIZZAH / 841PCT at saturating concentrations, their binding to Pichia cell wall was distinctly observed compared to unstained and secondary stained controls. Example 13. Capture reagents can capture and display VHH-Fcs secreted from Pichia The VHH-Fc expressing Pichia strains (NCYC 2543) at high and medium yields were tested to evaluate the competence of the capture reagents in both capturing and displaying the secreted molecules and distinguishing the expression levels. VHH-Fc expressing Pichia cells were incubated with the capturereagents in an induction medium while shaking at 28 °C for 2 h for protein expression. Captured andsurface displayed VHH-Fcs were detected through their binding to their biotinylated antigen target molecule with PE conjugated streptavidin. The level of secreted VHH-Fcs from both strains were detectable and separable after 2 h of protein expression for all capture reagents (Figure 27). CapRe10 was not tested as it’s degraded profile might affect the analysis. Next, different Pichia strains expressing various VHH-Fcs were tested to investigate whether capture reagents could perform comparably in distinguishing the expression levels of different VHH-Fcexpressing Pichia backgrounds. OPENPichia and OPENPichia HAC1 strains are derivatives of the NCYC2543 type strain, engineered for higher transformation efficiency and increased protein expression(Claes et al. 2024; Nature Microbiology, Vol.9 p 864–876). Protein expression driven by differentconstitutive promoters was observed to be constitutive for all strains. Same steps as previousexperiment were followed. Pichia cells were incubated with one of the capture reagents, CapRe5, inan induction medium while shaking at 28 °C for 2 h for protein expression. Captured and surfacedisplayed VHH-Fcs were detected through their binding to their biotinylated target molecule with PE conjugated streptavidin. For all strains, increased fluorescence signal was observed compared to their untreated controls, the VHH-Fc expression level of different strains were separable and correlated with the level of expression observed in bulk. VHH-Fc expressing OPENPichia HAC1 strain showed the highest expression signal followed by VHH-Fc expressing OPENPichia strain. The lower fluorescence signal was detected from VHH-Fc expressing NCYC 2543 strains possibly due to their thicker mannoprotein layercompared to engineered strains of OPENPichia platform (Figure 28). Additionally, a higher fluorescencesignal was observed from untreated VHH-Fc expressing OPENPichia cells (Figure 28, third panel from the top) compared to unstained sample. Determining of high-expressing cells based on the amount of transiently trapped proteins on the surface is a frequently used approach for high-throughputmammalian cell screening (Brezinsky et al. 2003; J Immunol Methods 277(1-2):141-55). A similarapproach was also successfully applied to detect retained IgG molecules on a glycoengineered Pichia cells (Lin, et al.2010; J Immunological Methods, Vol 358, 1–2, P.66-74). Therefore, it is possible that the more accessible cell wall of engineered strains of OPENPichia platform could allow detection of NiCa / PIZZAH / 841PCT transiently trapped proteins without a capture reagent. On the other hand, capture reagent treated VHH-Fc expressing OPENPichia cells (Figure 28, second panel from the top) demonstrated at least 10- fold higher fluorescence signal indicating a better separation compared to untreated OPENPichia cells.Based on the analyses described herein above, we can provide a characterized set of functional capturereagents, and in view of specific purposed select preferred capture reagents to produce at large scale.Materials and methods Strains, media, and reagentsEscherichia coli (E. coli) MC1061 was used for standard molecular biology manipulations. For plasmidpropagation, E. coli were cultured in LB broth (0.5% yeast extract, 1% tryptone, and 0.5% NaCl)supplemented with 25 µg / mL chloramphenicol (MP Biomedicals), 50 µg / mL carbenicillin (DuchefaBiochemie) and 50 µg / mL Zeocin® (Life Technologies). E. coli BL21DE3 and E. coli BL21-AI were used forrecombinant protein expression. Protein expression was performed in LB Broth supplemented with100 µg / mL ampicillin (for pDEST17 plasmid) or 100 µg / mL carbenicillin and induced with 1mMIsopropyl β-d-1-thiogalactopyranoside (IPTG) or 0.2% arabinose (final concentrations). P. pastorisNCYC2543 strain was provided by the National Collection of Yeast Culture. Yeast cultures were grown in liquid YPD (1% yeast extract, 2% peptone, 2% D-glucose) or on solid YPD-agar (1% yeast extract, 2% peptone, 2% D-glucose, 2% agar) at pH 7.5 and selected with 100 µg / mL Zeocin. For protein expression, cultures were grown in a shaking incubator (28°C, 225 rpm) in BMGY (same composition but with 1% glycerol replacing the 2% D-glucose) or BMMY (same composition but with 1% methanol replacing the 2% D-glucose). Generation of expression plasmids The pET39b(+)-ZZAH expression vector was cloned using the Gibson Assembly® Cloning Kit. The DNAcoding sequence for ZZAH was codon optimized for expression in E. coli using an IDT proprietaryalgorithm and ordered as a synthetic gBlock. The pET39b(+) was amplified by PCR using Phusion® High-Fidelity DNA Polymerase with primers with 5’ and 3’ ZZAH overlapping regions (SEQ ID NO: 30, 31),using pET39b(+)_FadAORF_Ctag as a template. The PCR reaction was treated with DpnI for 1 hour at 37°C, to digest the template DNA, and it was purified with Wizard® DNA Clean-Up System (Promega). The pET39b(+) backbone and the ZZAH insert were mixed in a 1:2 and 1:5 ratio together with the Gibson Assembly mix and incubated for 1 hours at 50°C. The two reactions were transformed into E. coliMC1061. Plasmid was isolated by single colonies using PureYield™ Plasmid Miniprep System andsequence verified. NiCa / PIZZAH / 841PCTThe OmpA-ZZAs coding sequence was cloned using golden gate designed for modular cloning in apDEST17 plasmid. The pET39b(+)-ZZAs expression vector was cloned using the Gibson Assembly®Cloning Kit. The DNA coding sequence for ZZAs was amplified from pDEST-17_OmpA_ZZAs by PCR usingPhusion® High-Fidelity DNA Polymerase with primers with 5’ and 3’ pET39+ overlapping regions. ThePCR reaction was treated with DpnI for 1 hour at 37°C, to digest the template DNA, and it was purified with Wizard® DNA Clean-Up System (Promega). The pET39b(+) backbone and the ZZAH insert were mixed in a 1:2 and 1:5 ratio together with the Gibson Assembly mix and incubated for 1 hours at 50°C.The two reactions were transformed into E. coli MC1061. Plasmid was isolated by single colonies usingPureYield™ Plasmid Miniprep System and sequence verified.The DNA coding sequences of the designed alternative capture reagents were codon optimized forexpression in E. coli and ordered as synthetic genes. The ompA signal peptide was fused to the N-termof the DNA sequences to target the proteins to the periplasm of E. coli. The genes were cloned usinggolden gate assembly into a modular cloning plasmid called pDEST17. The constructed expressionvectors were sequence verified via Eurofins.HEK293S expression of ZZAH fusion protein variantsThe DNA coding sequences of designed capture reagents were codon optimized for expression in HEK using IDT proprietary algorithm and ordered as synthetic gBlocks. Commonly used signal peptide, murine Igĸ leader sequence, was fused to N-term of DNA sequences to target the proteins tosupernatant. The coding sequences are cloned into pcDNA3.4-TOPO vector using pcDNA™3.4 TOPO™TA Cloning Kit. Final sequence constructed expression vectors are verified via NGS via Eurofins.A HEK293S cell line is used for protein expression, which is performed in combination of FreeStyle™293 Expression Medium and EX-CELL® 293 Serum-Free Medium in 1:1 ratio. Supernatant of growth was used for protein isolation. Purification of ZZAH fusion protein and variants For protein isolation, supernatant, periplasmic fraction and intracellular fractions were analyzed by SDS-PAGE for their protein level. Selected fractions were used for purification.Protein purification was performed using a column packed in-house with 20 ml IgG Sepharose 6 FastFlow resin on an ÄKTA Pure device, according to the manufacturer’s protocol. Briefly, the column was equilibrated for protein binding with binding buffer containing 50 mM Tris, 150 mM NaCl and 0.05% Tween20 pH 7.6 (TST buffer). The soluble fraction of the lysate was loaded onto the column and unbound protein was washed with TST buffer followed by a wash with 5 mM NH4Ac pH 5. Protein was eluted with 100% 0.5 M CH3COOH pH 3.4, and eluted fraction were immediately neutralized to pH 6.5 NiCa / PIZZAH / 841PCT with 1 M Tris-HCl pH 9. The fractions containing protein were pooled and concentrate to a volume of 10 ml with Amicon Ultra-1510 kDa cutoff and injected on a HiLoad® 16 / 600 Superdex® 200 pg column (GE-Healthcare) or HiPrep 26-10 desalting column (GE-Healthcare) and eluted in TST. The obtainedfractions were analyzed by SDS-PAGE and the ZZAH (variant) containing fractions were pooled together.Protein concentration was measured by 280 nm absorbance vs. a buffer blank and concentrated with Amicon 10 kDa MWCO spin columns. Purified protein was snapfrozen in liquid nitrogen and stored at - 80 °C. Yeast cell manipulation for IgG display and flow cytometry Yeast cells were stained with the in-house produced ZZAH catch reagent. To achieve this, an overnight preculture was first cooled down for about 1 hour on ice and washed twice in ice-cold BMGY to slow down yeast replication and protein expression, and remove the secreted protein in the medium. The cells were resuspended to a final OD600 of 1 and mixed 1:1 with 650 µg / ml of ZZAH in TST buffer and incubated on a rotating wheel for 1 hour at 4°C. Unbound ZZAH was removed by centrifugation for 5 minutes at 6,500g at 4°, and the cell pellet was washed twice with TST. For detection of ZZAH staining, cells were incubated with anti-protA-FITC diluted 1:2000 (Abcam). For the positive control, cells functionalized with ZZAH were resuspended in BMGY containing 13 µg / ml of VHH-Fc for 1 hour at 4°C, after which the excess of VHH-Fc was removed by centrifugation and washed twice in TST. For expression, cells were resuspended in BMGY and incubated up to 20 hours at 28°C while shaking. To allow oxygen transfer, tubes were let open and sealed with airpore tape (Qiagen). For detection of captured M2eVHH-Fc, cells were incubated with either goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 1:2000 (Abcam) or Alexa Fluor® 647 AffiniPure Fab Fragment Goat Anti-Human IgG (H+L) 1:400 (Jackson Immunoresearch). For detection of GBP-Fc cells were incubated with recombinant His-GFP (in house produced) at 6 µg / ml. For detection of anti-SARS-Cov2 VHH-Fc cells were incubated with 0.2 µg / ml of SARS-CoV-2-RBD-SD1-Avi (in house produced), followed by incubation with 1 / 400 Streptavidin-Phycoerythrin (BD Pharmingen). Detection antibodies were incubated for 30-60 minutes at 4°C in a rotating wheel, after which the cells were washed three times with TST and kept on ice until analysis. Flow cytometric analysis was performed on LSR II Flow Cytometer. Generation of multi-copy strains To promote stable multi-copy integration and increase clone-clone variability, the expression plasmids were linearized in a non-homologous region with PstI (Neb). Linearized DNA was gel purified before transformation to avoid any contamination with bacterial DNA. To promote multi-copy integration 100 ng, 500 ng or 40 μg of linearized vector were used to transform electrocompetent cells prepared as NiCa / PIZZAH / 841PCT described above. Transformants were selected on YPD-agar supplemented with 500 µg / mL or 1000 µg / mL of Zeocin®. After transformation, 192 clones of each construct were manually screened for protein secretion in 24 deep well plates. Analysis of protein secretion in bulk and protein quantification For analysis of protein secretion in bulk, single yeast colonies were inoculated in 2 ml BMGY in a 24 deep well block and incubated in a shaking incubator (28 °C, 250 rpm) 24-30 hours. The preculture was used to inoculate in the controlled glucose release EnPump200 medium (Enpresso GmBH), containingYPC (1% yeast extract, 2% peptone, 100 mM citrate buffer pH 6) with 50 g / l EnPump200 substrate and14 U / l of EnPump ReagentA enzyme solution. Medium was collected by centrifugation (1,500 g, 4 °C for 5 minutes) after 50 hours of expression in a shaking incubator (28 °C, 225 rpm). Protein expression was analyzed by loading 20 μl of medium with 5 μl of 5x laemli on SDS-PAGE. Samples were boiled at 98 °C for 5 minutes and centrifuged before loading. Media from expressing clones was used immediately for analytics purposes or stored at -20 °C. Crude supernatant was loaded on SDS-PAGE to evaluate protein expression levels. Densitometric quantification of the protein loads stained with Coomassie, was performed using imageJ and BandPeakQuantification Macro29. The signals were normalized to the signal of the control strain, which was loaded in every gel. Isolation of genomic DNA and determination of gene copy number by real time quantitative PCRFor preparation of genomic DNA from P. pastoris we used MasterPure™ Yeast DNA Purification Kit(Lucigen), as described by the supplier’s protocol. To remove the excess of RNA, an additional treatment with RNaseA (QIAGEN) was performed overnight at room temperature. RNase A was removed by precipitation with 2 M NH4Ac. Concentration of isolated genomic DNA was measured with a spectrophotometer at 260 nm (Nanodrop). Copy number was determined by RT-qPCR on genomic DNA on a Lightcycler 480 (Roche). Reactions were set up in 10 μl with final concentrations of 300 nM forward primer, 300 nM reverse primer, 1x SensiFast SYBR no rox mastermix (Bioline), 9-18ng gDNA and the following cycling conditions: 3 min denaturation at 95°C, followed by 45 cycles of 95°C for 3s, 60°C for 30s at ramp rate 2.5°C / s, 72°C for 1s, ending with 0.11°C / s from 65°C to 95°C for melting curve determination (5 acquisitions / s). OCH1 and IRE1 were used as reference genes to normalize the data.Copy number of integrated plasmids was calculated relatively to the wild type strain using the ΔΔCtmethod on pAOX1 or pGAP and AOX1tt, assuming that NCYC2543 strain contains only one copy of these sequences and that any extra copy belongs to the integrated plasmids. List of primers used for qPCR below. NiCa / PIZZAH / 841PCT Table 2.Primer sequences Primer Sequence SEQ ID NO:pAOX1_fw CCATCCGACATCCACAGGTC 32pAOX1_rv TCGATGGCAAAAGTGGGTGT 33pGAP_fw CCGGGGTAAAACTTAAATGTG 34pGAP_rv GGGTACACGACCTCCGTTTT 35AOX1tt_fw TGCCATTTGCCTGAGAGATG 36AOX1tt_rv TTCCCAAACCCCTACCACAAG 37ALG9_fw CTTTAGTGGGATGTTACCAG 38ALG9_rv CAACGTAAAAATCACACTCC 39OCH1_fw CCTCTGATAGTTCCTTTCCG 40OCH1_rv AAGACTTCTGGTACACGTTC 41Screening of yeast cell libraries for enhanced secretion For the sorting of the yeast transformant pools, the two libraries were grown overnight in BMDY. The cultures were first cooled down for 30 minutes on ice and washed twice with ice-cold BMGY. The cellswere resuspended to a final OD600 of 1 in fresh BMGY medium.250 µl of cell suspension were mixedone volume of 650 µg / ml ZZAH in TST buffer and incubated four 1 hour at 28 °C. Free ZZAH was removed by centrifugation for 5 minutes at 1,500 g at 4 °C, and the cell pellet was washed twice with BMGY. The cell suspension was incubated for another hour at 28 °C, after which they are stained for sorting. For the positive control, cells functionalized with ZZAH were resuspended in BMGY containing 13 µg / ml of VHHFc for 1 hour at 4°C, after which the excess of VHH-Fc was removed by centrifugation and washed twice in TST. For detection of captured VHH-Fc, cells were incubated with 0.2 µg / ml of SARS-CoV-2-RBD-SD1-AviTag (in house produced) for 1 hour, followed by incubation with 1 / 400 Streptavidin-Phycoerythrin (BDPharmingen) for another hour. After staining, the cells were washed three times with TST and kept onice until analysis. Sorting was performed with a BD FACSMelody™ Cell Sorter.Biolayer interferometryThe binding between the Fc region and the Fc binding domain of the alternative capture reagents wasassessed via BLI on an Octet RED96 system. A target protein of the VHH-Fc was biotinylated and immobilized on a streptavidin-coated biosensor, followed by loading 500 nM of VHH-Fc diluted in 1x NiCa / PIZZAH / 841PCTkinetics buffer (PBS supplemented with 0.05% polysorbate-20, 0.2% bovine serum albumin and 0.02%sodium azide). Capture reagents were prepared at 400 nM in the same 1x kinetics buffer and association to the VHH-Fc ligand was measured for 120 s at 25 °C, followed by 60 s dissociation. Biosensors were regenerated by three times 5 s exposure to 0.1 M glycine pH 3. Double reference subtraction was performed in ForteBio Data Analysis 9.0 software. Functionality AssaysFunctionality of the alternative capture reagents was analyzed via Fluorescence-activated cell sorting(FACS). The binding of the capture reagents on NCYC 2543 Pichia cells was determined by using aconcentration range of the capture reagents. The saturating concentrations were determined based onthe binding curves enabling further analyses. Next, the capture and display capacity of the capturereagents was tested on non-expressing, medium-expressing and high-expressing Pichia clones via flowcytometry. The optimal conditions for sorting experiments was determined based on the signaldifferences of Pichia clones with known expression levels. Finally, FACS experiments were performedwith a transformant pool to sort out the high expressing Pichia clones. Expression levels of sorted clonesand randomly picked unsorted clones were analyzed by SDS-PAGE and flow cytometry.Sequence listing The amino acid sequences of the constructs and fusion proteins, or parts thereof, are provided as one SEQ ID, or can be obtained by combining several amino acid sequences of the SEQ ID NOs listed herein (e.g. a signal peptide OmpA fused to a ZZ domain, followed by a linker GlySer, and a Banlec lectin sequence= OmpA-ZZ_Banlec provided as SEQ ID NOs:9+8+11+5 or 9+22).>SEQ ID NO: 1 : Actinohivin (AH)ASVTIRNAQTGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKW LFYSNGYIQNVETGRVLDSNYNGNVYTLPANGGNYQKWYTG>SEQ ID NO: 2 : Avaren or Av (actinohivin stabilized variant 8; including Ala46 in bold)ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKW TGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG >SEQ ID NO: 3: Avaren -delAla46 or As (actinohivin stabilized variant 8 w / o Ala46) ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWT GPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG >SEQ ID NO: 4: Avaren-mut (G75F and 113+T) ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWT FPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTTG NiCa / PIZZAH / 841PCT >SEQ ID NO: 5: Banlec MNGAIKVGAWGGNGGSAFDMGPAYRIISVKIFSGDVVDAVDVTFTYYGKTETRHFGGSGGTPHEIVLQEGEYLVG MKGEFGNYHGVVVVGKLGFSTNKKSYGPFGNTGGTPFSLPIAAGKISGFFGRGGDFIDAIGVYLEP >SEQ ID NO: 6: FimH FACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKY SGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVV VPT >SEQ ID NO: 7: Tectonin MPWKGISGSLSRISAGSVTNVWGVNAANNIYRYTGDDAKPWVQIPGALTDIGAAADGTVWGVNAAGNIYRYVW DSNHWTQIKGALKRISAGSRTNVWGVNAGGAIYRYTGDDANPWVQIPGVLSDIGAGADGTVWGVNAAGEIYRYT GDQGDPNHWVKIPGALSAISAGIKTNVWGVNSANNIYTSTGDDKNPWLGIGGSLVDIGAGTDGVVWGVNAGGGI YRWIRD >SEQ ID NO: 8: ZZ domain VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK >SEQ ID NO: 9: OmpA signal peptide sequence (for E.coli periplasmic expression) MKKTAIAIAVALAGFATVAQA >SEQ ID NO: 10: IgKleader sequence (for HEK expression) MGWSCIIFFLVATATGVHS >SEQ ID NO: 11: GlySer linker GGGGS>SEQ ID NO:12: ZZAH (ZZ part in italics)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASVTIRNAQTGRLLDSNYNGNVYTLPANGGNYQRWT GPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWLFYSNGYIQNVETGRVLDSNYNGNVYTLPANGGNYQK WYTG>SEQ ID NO:13: ZZAv (A46 in bold)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWT GPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQ KWTG>SEQ ID NO:14: ZZAs (fusion of 2 Z domains (ZZ) and stabilized Avaren (A) with ΔA46)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWT GPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQK WTG>SEQ ID NO:15: ZZ_As (ZZAs_G4S: linker (underlined) between ZZ domain and As) NiCa / PIZZAH / 841PCT VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSASGTIRNAETGRCLDSNYNGNVYTLPCNGGNY QRWTGPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGG NYQKWTG >SEQ ID NO:16: Z_ZAs (ZZAs_(ZZ)G4S: linker between 2 Z domains) VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSVDNKFNKEQQNA FYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASGTIRNAETGRCLDSNYNGNVYTLPCNGGN YQRWTGPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNG GNYQKWTG>SEQ ID NO:17: Z_Z_As (ZZAHs_2xG4S: linker between 2 Z domains and 2nd Z and As)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSVDNKFNKEQQNA FYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSASGTIRNAETGRCLDSNYNGNVYTLP CNGGNYQRWTGPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYT LPCNGGNYQKWTG>SEQ ID NO:18: ZZ-linker-Av (A46 in bold)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSASGTIRNAETGRCLDSNYNGNVYTLPCNGGN YQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCN GGNYQKWTG>SEQ ID NO:19: ZZAv-mut (G75F and 113+T)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWT GPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTFPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQK WTTG>SEQ ID NO:20: ZZ_Av-mut (linker; G75F and 113+T)VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSASGTIRNAETGRCLDSNYNGNVYTLPCNGGN YQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTFPGDGTIQNAETGRCLDSNYNGNVYTLPCN GGNYQKWTTG>SEQ ID NO:21: ZZ-BanLecVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKMNGAIKVGAWGGNGGSAFDMGPAYRIISVKIFSGDV VDAVDVTFTYYGKTETRHFGGSGGTPHEIVLQEGEYLVGMKGEFGNYHGVVVVGKLGFSTNKKSYGPFGNTGGTPF SLPIAAGKISGFFGRGGDFIDAIGVYLEP>SEQ ID NO:22: ZZ-linker - BanLecVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSMNGAIKVGAWGGNGGSAFDMGPAYRIISVKI FSGDVVDAVDVTFTYYGKTETRHFGGSGGTPHEIVLQEGEYLVGMKGEFGNYHGVVVVGKLGFSTNKKSYGPFGNT GGTPFSLPIAAGKISGFFGRGGDFIDAIGVYLEP>SEQ ID NO:23: FimH- ZZFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKY SGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVV NiCa / PIZZAH / 841PCT VPTVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYE ILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK>SEQ ID NO:24: FimH- linker-ZZFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSNFSGTVKY SGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVV VPTGGGGSVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQ QNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK >SEQ ID NO:25: ZZ-Tectonin VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKMPWKGISGSLSRISAGSVTNVWGVNAANNIYRYTGD DAKPWVQIPGALTDIGAAADGTVWGVNAAGNIYRYVWDSNHWTQIKGALKRISAGSRTNVWGVNAGGAIYRYT GDDANPWVQIPGVLSDIGAGADGTVWGVNAAGEIYRYTGDQGDPNHWVKIPGALSAISAGIKTNVWGVNSANN IYTSTGDDKNPWLGIGGSLVDIGAGTDGVVWGVNAGGGIYRWIRD >SEQ ID NO:26: ZZ-linker-Tectonin VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHL PNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSMPWKGISGSLSRISAGSVTNVWGVNAANNIY RYTGDDAKPWVQIPGALTDIGAAADGTVWGVNAAGNIYRYVWDSNHWTQIKGALKRISAGSRTNVWGVNAGG AIYRYTGDDANPWVQIPGVLSDIGAGADGTVWGVNAAGEIYRYTGDQGDPNHWVKIPGALSAISAGIKTNVWGV NSANNIYTSTGDDKNPWLGIGGSLVDIGAGTDGVVWGVNAGGGIYRWIRD>SEQ ID NO:27: OmpA-ZZAH (OmpA for periplasmic expression in bold)MKKTAIAIAVALAGFATVAQAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQ APKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASVTIRNAQTGRLLDS NYNGNVYTLPANGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWLFYSNGYIQNVETGRV LDSNYNGNVYTLPANGGNYQKWYTG >SEQ ID NO:28: OmpA-ZZAs MKKTAIAIAVALAGFATVAQAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQ APKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKASGTIRNAETGRCLDS NYNGNVYTLPCNGGNYQRWTGPGDGTVRNETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCL DSNYNGNVYTLPCNGGNYQKWTG >SEQ ID NO:29: OmpA-ZZ-linker-AH MKKTAIAIAVALAGFATVAQAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQ APKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSASVTIRNAQT GRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWLFYSNGYIQN VETGRVLDSNYNGNVYTLPANGGNYQKWYTG>SEQ ID NO: 30 : primer fwttgaacttgttatcaaccatATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC>SEQ ID NO: 31 : primer revagaaatggtacacgggctaaTTGATTAATACCTAGGCTGCTAAACAAAG>SEQ ID NO:32-41 : Table 2 primer sequences>SEQ ID NO: 42: Fc-binding VHH amino acid sequence NiCa / PIZZAH / 841PCT QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKLREFVAAIRRSGGSTYYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCAAGDDYVDEYDYWGQGTQVTVSS>SEQ ID NO:43: Anti-Fc VHH-Linker-Avaren capture reagent amino acid sequenceQVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKLREFVAAIRRSGGSTYYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCAAGDDYVDEYDYWGQGTQVTVSSGGGGSASGTIRNAETGRCLDSNYNGNVYTLP CNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNV YTLPCNGGNYQKWTG

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Claims

NiCa / PIZZAH / 841PCT CLAIMS1. A binding agent for capturing Fc-containing protein-secreting cells wherein said reagentcomprises: a. a polypeptide domain specifically binding to the Fc part of Fc-containing proteins, andb. a polypeptide domain specifically binding oligomannose,wherein said oligomannose-binding polypeptide domain is characterized in that it binds to oligomannose units present on a cell surface.

2. The binding agent of claim 1, wherein said polypeptide domains are fused directly or via a linker.

3. The binding agent of claims 1 or 2, wherein the Fc-containing protein binding polypeptidecomprises a bivalent ZZ domain, for instance as present in SEQ ID NO:8, or an antigen-bindingdomain specifically binding an Fc-tail, such as a single domain antibody, an immunoglobulin singlevariable domain, or a VHH, for instance as present in SEQ ID NO:42.

4. The binding agent of any one of claims 1 to 3, wherein the oligomannose-binding polypeptidedomain comprises a lectin.

5. The binding agent of any one of claims 1 to 4, wherein the oligomannose-binding polypeptidedomain specifically binds yeast mannans.

6. The binding agent of any one of claim 4 or 5, wherein the lectin is selected from any one of thegroup of lectins of Actinohivin, Avaren, Banlec, FimH or Tectonin, or a functional mutant orvariant of any one thereof, or specifically selected from the group of any one of SEQ ID NOs: 1-7.

7. The binding agent of any one of claims 1 to 6, wherein the binding reagent comprises any one ofSEQ ID NOs: 12 to 29 or 43.

8. A nucleic acid molecule encoding the binding agent of any one of claims 1 to 7.

9. A host cell comprising the nucleic acid molecule of claim 8, or the binding agent of anyone ofclaims 1 to 7.

10. A method for cell surface display of Fc-containing proteins, comprising the steps of:a. Cultivating cells recombinantly expressing Fc-containing proteins, andb. Providing the binding reagent from anyone of claims 1 to 7 to said population of cultivated Fc-containing protein-expressing cells,NiCa / PIZZAH / 841PCT wherein the binding agent is added just prior to induction of the recombinant expression, or wherein a pretreatment is performed to the culture for removing the secreted proteins priorto adding the binding agent, for allowing attachment of the binding agent to the cells’ surface oligomannose moieties, andc. Incubating the mixture of step b. during which Fc-containing proteins are secreted and boundto the mannose-bound binding agent displayed on the cell surface.

11. The method of claim 10, further comprising the step of:d. isolating the Fc-containing protein-secreting cells with Fc-containing proteins displayed on thecell through cell sorting using a detection reagent for the Fc-containing protein, and / or e. optionally , determine the intensity of the detection reagent for identifying the cells with thehighest number of displayed Fc-containing proteins.

12. The method of any one of claims 10 or 11, wherein the population of Fc-containing protein-secreting cells are yeast cells.

13. The method of any one of claims 10 to 12, wherein cell sorting in step d. is performed by FACsand the detection reagent is a fluorescent label.

14. The method of any one of claims 10 to 13, wherein the population of Fc-containing protein-secreting cells comprises a library of expression clones.

15. The method of any one of claims 10 to 14, wherein the Fc-containing proteins are VHH-Fcproteins.

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