CN117836308A - Means and methods for selecting specific binding agents - Google Patents

Abstract

The present disclosure relates to a novel method for selecting and identifying specific polypeptide binding agents for a target of interest. More specifically, the selection method involves capturing a target of interest on a surface using a first binding agent to form an immobilized antigen complex, selecting a specific antigen binding polypeptide present in the sample, preferably in the form of a display library, and eluting the target protein and the selected polypeptide binding agent using a second binding agent that competes with the first binding agent for the target binding site. More specifically, the selection methods presented herein provide an efficient and highly selective medium-to-high throughput technique suitable for recombinant antibody libraries, including immune and unbiased or whole proteome display libraries, where selection can be performed under physiological conditions without the need for purified target proteins.

Description

Means and methods for selecting specific binding agents

Technical Field

The present disclosure relates to novel methods for selecting and identifying specific polypeptide binding agents for a target of interest. More specifically, the selection method involves capturing a target of interest on a surface using a first binding agent to form an immobilized antigen complex, selecting a specific antigen binding polypeptide present in the sample, preferably as a display library, and eluting the target protein and the selective polypeptide binding agent using a second binding agent that competes for the target binding site of the first binding agent. More specifically, the selection methods presented herein provide efficient and highly selective medium-to-high throughput techniques suitable for recombinant antibody libraries, including immune and unbiased or whole proteomic display libraries, wherein selection can be performed under physiological conditions without the need for purified target proteins.

Introduction to the invention

Various display and selection methods have been reported for many years and used extensively as antibody discovery techniques, providing means and methods for identifying and isolating novel polypeptide binders, mainly monoclonal antibodies (mabs), antibody fragments, etc., starting from a large collection. Monoclonal antibody production requires hybridoma cell lines, i.e., hybrid cell lines produced by fusion of antibody-producing lymphocytes with tumor cells. From the following componentsAnd Milstein was still the most advanced technique for antibody hit discovery at the beginning of 70 s of the 20 th century. However, cloning antibody genes as single domains or fragments in combinatorial libraries has found its way to more and higher throughput methods of antibody discovery. The principle of genotype-phenotype coupling results from this development, as this "barcoding" is critical for up to billions of different protein variants, whose binding agents can be selected by high throughput identification in an iterative process. Several display technologies reveal faster and more efficient selection processes that in combination with clonal identification lay the foundation for antibody screening platforms, but in particular expression of functional antibody fragments on the surface of filamentous phage called phage display for in vitro selection of antibody fragments by McCafferty and Chiswell of cambridge, barbeas of La Jolla, and Breitling and dobel of sea deburg. In addition to phage, selection methods using non-phage display systems have been established, such as ribosome and mRNA display, which are well suited for purified antigens, and yeast display libraries, which are generally applied to cell sorting-based selection methods, even mammalian displays, which are beneficial for therapeutic development using similar hosts, despite slow growth rates. For a recent review of the different methods see, for example, valdorf et al (2021).

Recombinant antibody libraries, universal libraries, naive (non-immune) or immune-based libraries are applied for high throughput selection of target-specific binding agents. Although immune libraries are intended for specific target selection, so-called "universal libraries" can be subdivided into natural, synthetic and semisynthetic methods, allowing unbiased selection of new binders (see also Almagro et al, 2019). After obtaining individual clones from the iterative selection rounds, the polypeptide binders are screened for target binding and biophysical properties using typical antibody binding assays (ranging from ELISA to immunoprecipitation). Alternative selection methods for selecting recombinant display libraries that evolve from "gold standard" are typically based on custom requirements, as reported by Lakzaei et al (2018), which disclose biopanning methods for diphtheria toxin binders, using soluble antibody capture, which allows capture of binders that specifically recognize native epitopes, although acidic elution conditions are still required, and no control of target specific conformation is selected. Another example relates to a female-male biopanning method that provides affinity selection for crude extracts, thus solving the problem of requiring antigen purification, and uses negative selection (blocking agent) to obtain specificity prior to positive phage selection (Lim et al, 2019).

The use of display technologies capable of selecting abs with therapeutic relevance has laid the foundation for a large number of market approved therapeutic agents by the U.S., european or chinese medical authorities. This observation supports the precious impact those platform technologies have on drug discovery. Furthermore, combining display technology with microfluidic systems and/or next generation sequencing in antibody discovery has enabled functional screening, e.g., to further enrich the kits and tubing for high throughput selection methods in the therapeutic field. Current conventional methods or alternatives to phage display and further evolutionary platforms provide high-tech and commonly used display technologies to select polypeptide binders, particularly from recombinant antibody libraries, which have great potential in drug discovery. However, there is still room for improvement to address certain technical bottlenecks, such as for antigens that are difficult to purify under natural conditions, for binders to select conformational epitopes to target antigens, and to increase efficiency and specificity against background when applied in high-throughput mode.

Summary of The Invention

The present disclosure is based on the following findings: the NANEX technique (as further described herein) is suitable for selection and screening of recombinant antibody libraries under physiological conditions without the need for purified antigen.

Nanobody-based exchange chromatography (also known as NANEX) provides a nanobody (Nb) -based immune displacement (displacement) purification method in which a pair of Nb (traps) competing for binding to the same or highly overlapping epitopes on a target is used as a capture agent, and a stripper (stripper) is used as an eluent) to analytically purify the target protein, resulting in a small amount of high purity protein bound to high affinity Nb (stripper) as previously described in PCT/EP 2020/087291. This highly selective one-step purification method has the advantage that no concentration step, dialysis or proteolysis is required and physiological conditions can be maintained during protein capture and elution. Furthermore, where only one specific Nb is available, the use of Nb as an antigen binding portion in the NANEX purification method also allows starting from a single binding agent to obtain a pair via the design of lower affinity or faster dissociating variants (e.g. mutants).

The present application describes proof of concept demonstrating that the integration of the NANEX purification method in display library selection provides a powerful approach that improves the selection procedure to screen binders under mild conditions, even for refractory protein targets. Even when unbiased Nb libraries are used for selection, specific high affinity target binders are identified in a very efficient manner. NANEX-based target protein capture (trapping) allows maintenance of the natural physiological condition of the target, releasing the need for purified targets or antigens, as the targets can be provided in complex samples (e.g. biological samples) and results in new innovative antibody selection methods that are capable of fishing effective binders from polypeptide binders generated against the complete target proteome. It is not possible in such an efficient way using conventional selection procedures and thus positioning the NANEX-based panning or selection method as a breakthrough in challenging target or tool-generated drug discovery. More specifically, using this selection method, a significantly robust enrichment of different antigens was demonstrated after only 2-3 rounds of selection, and furthermore, success was independent of their cell abundance when selected from whole proteomic immune libraries. In addition, several families of natural protein binders can be identified, even with few Nb family members (which typically disappear after 3 rounds of conventional selection). In addition to selecting binders for soluble protein targets, we further demonstrate proof of concept of membrane protein binder screening and demonstrate robust isolation of new binders specifically shown herein for the Nb family, which may even be omitted in conventional selection methods.

Although immune libraries against a variety of proteins have been generated in the past (e.g., against trypanosoma evansi (t. Evansi) secretion proteomes in Li et al 2020), we have surprisingly found that using a whole proteome Nb library, NANEX purified phage display selection generated target specific binding agents against most test targets, demonstrating the selective capacity and reliability of this new selection method. Another benefit is that when using e.g. endogenous GFP-tagged yeast clones in combination with GFP-specific NANEX, or using a collector/stripper pair against endogenous protein epitopes, the need for purification of the antigen is avoided in the whole process, which is very attractive for implementation in the selection of difficult to purify antigens.

Thus, this selection method has the following advantages: target purification and subsequent selection of specific target binders are possible under physiological and natural conditions, while obtaining highly specific and thus selective target protein-polypeptide binder interactions and/or conformation specific target protein binders, all of which are possible under high throughput conditions. Furthermore, when applied to labeled target proteins, such as the GFP-specific capture/release agents shown herein, provide a versatile selection platform using a single capture/release agent (or capture/displacement agent) pair.

Thus, a first aspect of the present disclosure relates to a method of selecting a protein binding agent that specifically binds to a target protein from a plurality of binding agents (e.g. display libraries), wherein NANEX is used to form an immobilized complex between a capture agent (or first protein binding agent, also referred to as a capture agent) and a target, which is presented to the plurality of potential binding agents, thereby allowing selection of protein binding agents associated with the target at different binding sites than the capture agent. The complex is then eluted using an eluent (or a second protein binding agent, also known as a stripper) that outperforms the capture agent in binding to the target and elutes the target associated with the selected binding agent in the manner of a tight target-stripper complex. The advantage of this NANEX-based selection procedure is that targets can be captured from complex natural environments while retaining a specific/natural conformation without the need for purified target proteins, and that the selection allows the identification of more/alternative binders than, or complementary to, conventional selection procedures.

The selection method comprises the following steps:

a) Providing an immobilized first protein binding agent that specifically binds to a target protein and a sample comprising said target protein for obtaining an immobilized complex of said target bound to said first protein binding agent,

b) Adding a solution comprising a plurality of polypeptide binding agents to the immobilized complex of step a) for allowing the polypeptide binding agents to specifically bind to the immobilized target proteins,

c) Providing said solution of step b) with a solution comprising a second protein binding agent that binds to the target protein and competes for said binding with the first binding agent, thereby displacing (display) the target protein for release of the target protein into the solution, and

d) Collecting an eluate of a second protein binding agent that binds to a target protein for separation of a polypeptide binding agent that binds to the target protein.

Further embodiments relate to the method, wherein the second protein binding agent has a higher affinity, or its dissociation rate constant (k _off Value) compared to k of the first binding agent _off The values are lower or equal. Another embodiment relates to the method, wherein the second and/or first protein binding agent comprises an antigen binding domain that specifically binds to a target protein. More specifically, the first and/or second agent antigen binding domain comprises an immunoglobulin foldA stack, which is an antibody or active antibody fragment, a single domain antibody, an Immunoglobulin Single Variable Domain (ISVD), a VHH, a nanobody or an antigen binding chimeric protein (defined as ISVD fused to a scaffold protein by at least two sites, and preferably comprising HopQ, ygjK or derivatives thereof, also known as MegaBody).

Further embodiments of any of the methods described herein provide for the use of the plurality of polypeptide binders of step b) provided in the form of a display library of binders, which may more particularly comprise a recombinant antibody library, including an immune library or a non-immune library, such as a naiveLibraries, including (semi) synthetic libraries, that express and display binders that are (monoclonal) antibodies, single domain antibodies, fab, ISVD, VHH or nanobodies, or any active fragment thereof.

In another specific embodiment, the methods as described herein are performed using phage display, as known to those of skill in the art. Alternatively, specific settings or libraries are applied to perform the method using yeast, ribosome, bacterial or mammalian display.

Other specific embodiments aim at enriching the amount of specific polypeptide binding agent of the target protein, providing a method wherein a iterative process of selecting a polypeptide binding agent is applied in a similar way as known in the art for panning procedures, wherein steps a) to d) are repeated at least once, preferably two or more times, wherein the iterative process is denoted "x-round" selection. Specifically, when using phage display libraries, the phage-containing eluate collected in step d) is reused for infection with E.coli and returned to step a).

In another embodiment, the method as described herein specifically comprises a surface with a first protein binding agent for immobilizing a target protein, wherein the surface comprises a matrix comprising beads, such as beads exemplified herein, such as magnetic beads, or alternatively the surface is provided by a resin, chromatography column or polymeric column, plate arrangement or microchip.

Another embodiment relates to the method, wherein the target protein is present in the sample in step a), which is a complex sample, comprising a mixture of components, such as biological material, cell lysate, extract, soluble proteome, proteome of a certain cell or tissue type, or recombinant target protein as part of a protein mixture. In a particular embodiment, the selection method is provided which uses an immunogen for obtaining a plurality of polypeptide binding agents, wherein the immunogen comprises the same properties and composition as the sample comprising the target protein in step a). For example, the immunogen comprises a cell lysate for use in immunization to produce a display library, such as a recombinant antibody library, wherein such cell lysate is also used in step a) as a sample comprising the target protein.

Alternative methods as described herein provide a universal method wherein the first and second protein binding agents specifically bind to a tag, wherein the tag is present on the target protein, preferably as an N-or C-terminal heterologous tag. In a preferred embodiment, the heterologous tag is specifically recognized by a GFP-specific binding agent, which embodiment relates to a selection method as described herein, wherein the collector comprises at least the sequence of the CDR or VHH of SEQ ID NO:71 and the stripper comprises at least the sequence of the CDR or VHH of SEQ ID NO: 70.

Another embodiment of the method described herein allows selection of polypeptide binding agents which bind specifically to the protein complex to be eluted in step d) in a direct or indirect manner for the target protein. More specifically, for polypeptide binding agents that recognize the target protein itself at a binding site different from the capture/release agent binding site, direct binding to the target is obtained, and indirect binding may be obtained by binding to a tag fused to the target protein or to a polypeptide binding agent that co-captures and binds (via the target protein) to another protein or component of the immobilized surface in step a). Thus, the indirect polypeptide binding agent may comprise a binding agent that specifically binds and recognizes a protein or component that is an interactive agent for a target protein or as a heterologous fusion partner/tag for a target protein.

Thus, in a further aspect, the invention also relates to a protein complex comprising a second protein binding agent which binds to the target protein eluted in step d), wherein the target protein is further bound to at least one further protein, and wherein the method as described herein results in the presence of a polypeptide binding agent in the complex which binds to the further protein.

Another embodiment provides a selection method as described herein, wherein at least two different target proteins are selected in parallel, with the aim of providing in step a) a sample comprising said at least two different target proteins alone or as fusion or cross-linking molecules, and providing in steps a) and c) a first and a second protein binding agent for each of these different target proteins, respectively.

Another method for selecting a polypeptide binding agent specific for a target protein or an antigen protein involves an alternative method of providing a mixture in step c) of the above method, and comprises the steps of:

a) Mixing a target protein sample with a sample (preferably a display library) comprising a plurality of polypeptide binding agents, and

b) Obtaining a complex immobilized on a surface by adding a first protein binder to the mixture of a), which is preferably immobilized on a surface or subsequently immobilized, and

c) Providing said solution of step b) with a solution comprising a second protein binding agent that binds to the target protein and competes for said binding with the first binding agent, thereby displacing the target protein for release of the target protein into solution, and

d) Collecting an eluate of a second protein binding agent that binds to a target protein for separation of a polypeptide binding agent that binds to the target protein.

In particular, the alternative method may preferably use a purified target protein sample. In a preferred embodiment of such a method, the first and/or second protein binding agent comprises nanobodies.

A final aspect of the invention relates to the use of a method as described in any of the embodiments provided herein for selecting a binding agent from an immune library. Alternatively, the use of a method as described in any of the embodiments provided herein for epitope binning on a target protein or for isolating a novel epitope binding agent. Furthermore, the use of the methods as described herein is intended for high throughput purposes in the selection of protein binders, in particular in antibody and drug discovery.

Description of the drawings

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.

FIG. 1 schematic representation of selection of target specific antibodies from an antibody display library using nanobody exchange chromatography (NANEX).

The principle of nanobody exchange chromatography can be applied to select target-specific binding agents, e.g. antibodies, from a display library. A first nanobody (collector) covalently attached to a solid support (herein a bead) is used to immobilize the target. The immobilized targets are then incubated with different libraries of different binders (e.g., antibodies) expressed and displayed in a manner that provides a physical link between phenotype (binding behavior) and encoding genotype, in this case a phage display library. The washing step may optionally be used to remove irrelevant binding agents or antibodies. Similar to NANEX and specific for the present invention, a second nanobody (soluble stripper) competing with the collector is then used to selectively elute the immobilized target associated with the stripper and target-specific binding domain or antibody and its encoding genotype.

FIG. 2 enrichment after first and second rounds of selection on GFP using NANEX.

GFP-specific antibodies were selected from nanobody display libraries using nanobody exchange chromatography (NANEX) according to example 1. GFP-capturing agents (CA 15816 SEQ ID NO: 2) were coupled to the magnetic beads and different NANEX beads were prepared by incubating them with different concentrations of GFP to capture the antigen. Beads coated with collector not incubated with GFP served as negative controls. After incubation with the library, the phage were eluted with GFP-specific stripper (CA 12760 SEQ ID NO: 1) or with trypsin. Two rounds of selection were performed. The output phage from each elution was recovered by infection with E.coli and enrichment was assessed by comparing serial dilutions of these cells according to Pardon et al (2014).

FIG. 3 competitive binding assay of GFP-specific nanobodies by biolayer interferometry.

The binding properties of the newly discovered GFP-specific nanobodies were analyzed by Biological Layer Interferometry (BLI) on OctetRed (Molecular Devices). Streptavidin-coatedThe biosensor was used to capture biotinylated GFP (100 nM). Next, picomoles of GFP stripper (CA 12760, SEQ ID NO: 1) were allowed to bind to GFP until the plateau was reached. These CA12760 saturated biosensors were then incubated in solutions containing each newly discovered Nb (as shown in the legend of the figure), supplemented with CA12760 Nb, respectively. At room temperature, in the case of BSA 0.1% and Tween supplementation ₂₀ The measurement was performed in 0.005% HEPES25mM pH 7.5 and NaCl 150 mM. All nanobodies (CA 17517, CA17676, CA17518 in a; CA17674, CA17673, CA17519, CA17520, CA17675 in B) except for the stripping agent (CA 12760 in a) or the unrelated Nb (CA 8780 in a) caused an increase in apparent mass on the sensor, indicating that they bound an epitope that did not overlap with the epitope of the stripping agent.

FIG. 4 characterization of FBA1 specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP specific collector/stripper pairs.

Four FBAl-specific nanobodies were characterized by co-immunoprecipitation assays (40=nb clone CA17440 corresponding to SEQ ID No. 3, 41=ca 17441-SEQ ID No. 4, 42=ca 17442-SEQ ID No. 5, 43=ca 17443-SEQ ID No. 6). Each FBAl-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these FBA 1-specific nanobodies were incubated with EBY100 lysate or lysate of engineered yeast strain expressing FBA1 as GFP-tagged protein (yeast GFP fusion pool reference: GFP (+) 22, G1) on a spin device for 1 hour at 4 ℃. After washing, these individual beads were resuspended in SDS-PAGE loading dye, boiled and analyzed on SDS-PAGE (A). The isolated proteins were transferred onto PVDF membranes and western blots (B) were developed with GFP-specific antibodies to confirm the presence of GFP-tagged FBA 1.

FIG. 5 characterization of PDC 1-specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP-specific collector/stripper pairs.

Two PDC 1-specific nanobodies were characterized by co-immunoprecipitation assay (51=nb clone CA17451 corresponding to SEQ ID No. 7, 52=ca 17452 corresponding to SEQ ID No. 8). Each PDC 1-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these PDC 1-specific nanobodies were incubated with EBY100 lysates or lysates of engineered yeast strains expressing PDC1 (yeast GFP fusion accession number: GFP (+) 12, F8) as GFP-tagged protein on a rotating device at 4 ℃ for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (A). The isolated proteins were transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies (B) to confirm the presence of GFP-tagged PDC 1.

FIG. 6 characterization of SSA1 specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP specific collector/stripper pairs.

Six SSA 1-specific nanobodies were characterized by co-immunoprecipitation assays (60=nb clone CA17560 corresponding to SEQ ID No. 19, 61=ca 17561-SEQ ID No. 20, 62=ca 17562-SEQ ID No. 21, 63=ca 17563-SEQ ID No. 22, 64=ca 17564-SEQ ID No. 23, 65=ca 17565-SEQ ID No. 24). Each SSA 1-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these SSA 1-specific nanobodies were incubated with EBY100 lysates or lysates of engineered yeast strains expressing SSA1 (yeast GFP fusion pool reference: GFP (+) 10, E4) as GFP-tagged protein on a rotating device at 4 ℃ for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (A). The isolated proteins were transferred to PVDF membranes and western blots were developed with GFP-specific antibodies (B) to confirm the presence of GFP-tagged SSA 1.

FIG. 7 characterization of PGI1 specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP specific collector/stripper pairs.

Three PGI 1-specific nanobodies were characterized by co-immunoprecipitation assay (55=nb clone CA17455 corresponding to SEQ ID NO:15, 56=ca 17456-SEQ ID NO:16, 57=ca 17457-SEQ ID NO: 17). Each PGI 1-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these PGI 1-specific nanobodies were incubated with EBY100 lysates or lysates of engineered yeast strains expressing PGI1 (yeast GFP fusion pool reference: GFP (+) 12, H11) as GFP-tagged protein on a rotating device at 4 ℃ for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (A). The isolated proteins were transferred to PVDF membranes and western blots stained with GFP-specific antibodies (B) to confirm the presence of GFP-tagged PGI 1.

FIG. 8 characterization of SIS 1-specific, ALD 6-specific, BMH 1-specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP-specific collector/stripper pairs.

One SIS1 specific nanobody (44=nb clone CA17444 corresponding to SEQ ID NO: 9), three ALD6 specific nanobodies (53=ca 17453-SEQ ID NO:10, 54=ca 17454-SEQ ID NO:11, 60=ca 17460-SEQ ID NO: 12), two BMH1 specific nanobodies (58=ca 17458-SEQ ID NO:13, 59=ca 17459-SEQ ID NO: 14) were characterized by co-immunoprecipitation assays. Each target-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these target-specific nanobodies were incubated with EBY100 lysates or lysates of engineered yeast strains expressing the target as GFP-tagged protein (yeast GFP fusion pool reference, for SIS1: GFP (+) 22, E5, for ALD6: GFP (+) 15, F1, for BMH1: GFP (+) 27, D5) on a rotating device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (A). The isolated proteins were transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies (B) to confirm the presence of GFP-labeled targets.

FIG. 9 characterization of SXM1 specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP specific collector/stripper pairs.

One SXM1 specific nanobody (30 = Nb clone CA17530 corresponding to SEQ ID No. 18) was characterized by co-immunoprecipitation assay. SXM 1-specific nanobodies were covalently linked to NHS-sepharose beads. Beads functionalized with the SXM1 specific nanobodies were incubated with EBY100 lysate or lysate of engineered yeast strain expressing SXM1 GFP (+) 04, H9) as GFP-tagged protein on a spin device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (A). The isolated proteins were transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies (B) to confirm the presence of GFP-tagged SXM 1.

FIG. 10 NANEX captures and immobilizes PGI1 binding agent from the soluble fraction of yeast lysate.

The beads were functionalized with PGI1 specific nanobody CA17455 clones (SEQ ID NO: 15) for use as collectors. The CA17455 functionalized beads were then incubated with EBY100 lysate on a spin device at 4 ℃ for 1 hour and washed. Next, these beads were incubated with the same PGI 1-specific nanobody for 1 hour to elute the target (PGI 1) associated with several interacting proteins (LYS 20 Uniprot P48570, TDH3 Uniprot P00359, PNC1 Uniprot P53184) as shown by mass spectrometry analysis.

FIG. 11 characterization of GR-LBD specific nanobodies selected from GR-LBD antibody library by NANEX using GFP-specific collector/stripper pair.

Five GR-LBD specific nanobodies were characterized by co-immunoprecipitation assay (97 = Nb clone CA17797 corresponding to SEQ ID NO:35, 98 = CA17798-SEQ ID NO:36, 99 = CA17799-SEQ ID NO:37, 00 = CA17800-SEQ ID NO:38, 01 = CA17801-SEQ ID NO: 39). Each GR-LBD specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these GR-LBD specific nanobodies were incubated on a rotating device for 1 hour at 4 ℃ with HEK293T lysates (supplemented with DEX or not) transfected with pcdna3.1 plasmid containing GFP-GR (full length) gene expressing full length GR as GFP-tagged protein. After washing, the individual beads were resuspended in SDS-PAGE loading dye, boiled and analyzed on SDS-PAGE (not shown). The isolated proteins were transferred to PVDF membranes and western blots were developed with GR specific antibodies to confirm the presence of GFP-tagged GR. NC represents the negative control Nb. GFP = 27kDa; GFP-GR fusion = 117kDa; GR (cleaved) =90 kDa.

FIG. 12 enrichment after the first and second rounds of selection using NANEX for 94 different GFP-POIs.

According to example 3, the NANEX was used to select POI-specific antibodies from whole proteomic antibody display libraries. GFP traps (CA 15816, corresponding to SEQ ID NO: 2) were coupled to the magnetic beads and distributed in 96 different wells. NANEX beads were incubated with different lysates of engineered yeasts expressing GFP-POI (table 3). After several washing steps, phage were added to the wells. After incubation with the library, the phage was eluted with GFP-specific stripper (CA 12760, corresponding to SEQ ID NO: 1). Two rounds of selection were performed (R1 in A and R2 in B). The output phage from each elution was recovered by infection with E.coli and enrichment was assessed by comparing serial dilutions of these cells according to Pardon et al (2014). Data are presented for each well listed chronologically as in table 3 (A1-F12, G12 and H12 from left to right on the X-axis, as last sample on the X-axis for control) for each of the 94 GFP-targets).

FIG. 13 characterization of POI specific nanobodies selected from whole proteomic antibody libraries by NANEX using GFP specific collector/stripper pairs.

One HSP 104-specific nanobody (04=ca 18504 SEQ ID NO: 40), one MET 6-specific nanobody (05=ca 18505 SEQ ID NO: 41), two SBA 1-specific nanobodies (08=nb clone CA18508 corresponding to SEQ ID NO:42, 09=ca 18509-SEQ ID NO: 43), one SOD 1-specific nanobody (10=ca 18510-SEQ ID NO: 44) and one ENO 1-specific nanobody (38=ca 17938-SEQ ID NO: 45) were characterized by co-immunoprecipitation assays. Each POI-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these POI-specific nanobodies were incubated on a rotating device for one hour at 4℃with lysates of engineered yeast strains expressing POIs as GFP-tagged proteins (yeast GFP fusion accession numbers: GFP (+) 05, A2 (HSP 104), GFP (+) 07, D4 (MET 6), GFP (+) 30, A6 (SBA 1), GFP (+) 33, F8 (SOD 1) and GFP (+) 17, D12 (ENO 1)). After washing, the beads were analyzed on SDS-PAGE (not shown) and Western blots were developed with GFP-specific antibodies to confirm the presence of GFP-labeled POIs.

FIG. 14 characterization of PGI1 specific nanobodies selected from whole proteomic antibody libraries by NANEX using PGI1 specific collector/stripper pairs.

Six PGI 1-specific nanobodies were characterized by co-immunoprecipitation assay (91 = Nb clone CA17791, 92 = CA17792-SEQ ID No. 47, 93 = CA17793-SEQ ID No. 48, 94 = CA17794-SEQ ID No. 49, 95 = CA17795-SEQ ID No. 50, 96 = CA17796-SEQ ID No. 51). Each PGI 1-specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these PGI 1-specific nanobodies were incubated with EBY100 lysates or lysates of engineered yeast strains expressing PGI1 (yeast GFP fusion pool reference: GFP (+) 12, H11) as GFP-tagged protein on a rotating device at 4 ℃ for 1 hour. After washing, the beads (A) were analyzed on SDS-PAGE. The isolated proteins were transferred to PVDF membranes and western blots were developed with GFP-specific antibodies (B) to confirm the presence of GFP-tagged PGI 1. 55 Nb clone CA17455, corresponding to SEQ ID No. 15, was used as a collector/stripper for PGI1 as a positive control.

FIG. 15 characterization of rVGLUT1 specific nanobodies selected from rVGLUT1 antibody library by NANEX using rVGLUT1 specific collector/stripper pairs.

rVGLUT 1-specific nanobodies (25=nb clone CA18425, corresponding to SEQ ID NO: 53) were characterized by co-immunoprecipitation assay. rVGLUT1 specific nanobodies were covalently linked to NHS-sepharose beads. Beads functionalized with the rVGLUT1 specific nanobody were incubated with HEK293T lysate transfected with plasmid expressing full length rVGLUT1 as cMyc-YFP labeled protein on a spin device at 4℃for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye, not boiled, and analyzed on SDS-PAGE (not shown). The isolated proteins were transferred to PVDF membrane and Western blots were developed with c-Myc specific antibodies to confirm the presence of c-Myc labelled rVGLUT 1. NC represents the negative control Nb.

FIG. 16 characterization of rVGLUT1 specific nanobodies selected from a synapse proteome antibody library by NANEX using GFP specific collector/stripper pairs.

Six rVGLUT1-YFP specific nanobodies were characterized by co-immunoprecipitation assay (24=Nb clone CA18024 corresponding to SEQ ID NO:54, 37=CA 18463-SEQ ID NO:55, 38=CA 18438-SEQ ID NO:56, 39=CA 18439-SEQ ID NO:57, 40=CA 1840-SEQ ID NO:58, 41=CA 18441-SEQ ID NO: 59). Each rvguelut 1-YFP specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these rVGLUT1 specific nanobodies were incubated for 1 hour at 4℃on a spin device with HEK293T lysates transfected with plasmids containing the full length rVGLUT1 gene expressing proteins as c-myc-YFP markers. After washing, the beads were resuspended in SDS-PAGE loading dye, not boiled, and analyzed on SDS-PAGE (not shown). The isolated proteins were transferred to PVDF membrane and Western blots were developed with c-Myc specific antibodies to confirm the presence of c-Myc labelled rVGLUT 1. NC represents the negative control Nb.

FIG. 17 characterization of GR specific nanobodies selected from GFP-GR antibody library by NANEX using mCherry specific collector/stripper pair.

Seven GR specific nanobodies were characterized by co-immunoprecipitation assay (98=nb clone CA18498, corresponding to SEQ ID No. 62, 99=ca 18499-SEQ ID No. 63, 01=ca 18501-SEQ ID No. 64, 02=ca 18502-SEQ ID No. 65, 03=ca 18503-SEQ ID No. 66, 85=ca 18585-SEQ ID No. 67, 86=ca 18586-SEQ ID No. 68). Each GR specific nanobody was covalently linked to NHS-sepharose beads. Beads functionalized with these GR specific nanobodies were incubated for one hour at 4 ℃ on a rotating device with HEK293T lysates transfected with pcdna3.1 plasmid containing the mCherry-GR (full length) gene expressing full length GR as mCherry tagged protein. After washing, the individual beads were resuspended in SDS-PAGE loading dye, boiled and analyzed on SDS-PAGE (not shown). The isolated proteins were transferred to PVDF membranes and western blots were developed with GR specific antibodies to confirm the presence of GR. NC represents the negative control Nb.

Detailed description of the preferred embodiments

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. It should be understood, of course, 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, together with its features and advantages, may best be understood by reference to the following detailed description when read in connection with the accompanying drawings. Aspects and advantages of the invention will become 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, the appearances of the phrases "in one (one) embodiment" or "in one (an) embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim.

Definition of the definition

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, other elements or steps are not excluded. 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 under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided only to aid in understanding the present invention. Unless defined otherwise herein, all terms used herein have the same meaning as those skilled in the art to which the present invention pertains. For definitions and terms in the art, practitioners refer specifically to Sambrook et al, molecular Cloning: a Laboratory Manual, 4 th edition, cold Spring Harbor Press, plainsview, new York (2012); and Ausubel et al Current Protocols in Molecular Biology (journal 114), john Wiley & Sons, new York (2016). 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., molecular biology, biochemistry, structural biology, and/or computational biology).

The terms "protein," "polypeptide," and "peptide" are further used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. "peptide" may also refer to a partial amino acid sequence derived from its original protein, e.g., after trypsin digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation, and acetylation. Based on the amino acid sequence and modifications, the atomic or molecular weight or weight of a polypeptide is expressed in (kilodaltons (kDa). A "protein domain" is a defined functional and/or structural unit in a protein. In general, protein domains are responsible for specific functions or interactions that contribute to the overall function of the protein. Domains can exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.

By "isolated" or "purified" is meant that the material is substantially or essentially free of components normally accompanying in its natural state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been purified from a molecule that is alongside it in a naturally occurring state (e.g., a polypeptide binding agent), or a target protein identified and disclosed herein that has been removed from a molecule that is present in a sample or mixture (e.g., a production host) that is adjacent to the polypeptide. The isolated protein or peptide may be produced by amino acid synthesis, or may be produced recombinantly or by purification from complex samples.

As used herein, the term "fused to" and is used interchangeably herein as "linked to," "conjugated to," "linked to," particularly "genetic fusion," e.g., by recombinant DNA techniques, as well as "chemical and/or enzymatic conjugation" that results in stable covalent attachment, such as a heterologous tag that is covalently attached to the target protein.

"a homolog" of a protein "encompasses peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the degree to which sequences are identical on an amino acid-by-amino acid basis over a comparison window. Thus, the "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical amino acid residues (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 herein by a single letter code) occur in the two sequences to produce a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity. "substitutions" or "mutations" or "variants" as used herein are due to the substitution of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid sequence or nucleotide sequence of the parent protein or fragment thereof. It will be appreciated that the protein or fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the protein.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the genes most commonly observed in a population and are therefore arbitrarily designed as "normal" or "wild-type" forms of the genes. In contrast, the terms "modified," "mutation," "engineered" or "variant" refer to a modified gene or gene product that exhibits in-sequence, post-translational modification and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; these are determined by the fact that they have altered characteristics compared to the wild-type gene or gene product.

The term "binding site" refers to a region of a molecule or molecular complex that, due to its shape and charge, advantageously associates with another chemical entity, compound, protein, peptide, antibody or Nb. For antibody-related molecules, the terms "epitope" or "conformational epitope" are also used interchangeably herein. The term "pocket" includes, but is not limited to, a slit, channel, or site. The term "a portion of a binding pocket/site" or "a partially overlapping epitope" refers to less than all amino acid residues defining a binding pocket, binding site or epitope. For example, the atomic coordinates of the residues that form part of the binding pocket may be specific to the chemical environment defining the binding pocket, or may be used to design fragments of inhibitors that can interact with those residues. For example, the residue moiety may be a critical residue for the binding of the target protein, or may be a residue that is spatially related and defines the three-dimensional compartment of the binding pocket, or a residue that confers conformational function. As used herein, "adjacent" or "minimal overlap" binding sites refer to "non-overlapping amino acids (but binding to nearby sites)" in the binding amino acid residues, respectively, or up to about 30% overlap. As used herein, "epitope" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target molecule. The epitope on the target protein may comprise at least one amino acid necessary for binding to the binding agent, but preferably comprises at least 3 amino acids in the spatial conformation, which is unique to the epitope. Typically, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more typically, at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography, multidimensional nuclear magnetic resonance, cryo-EM, deuterium exchange (HDX) -MS, and cross-linked mass spectrometry (XL-MS), epitope binning, or to a lesser extent, neutron scattering, X-ray free electron laser (xfl) or Small Angle Neutron Scattering (SANS) and small angle X-ray scattering (SAXS) techniques. As used herein, a "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation that is characteristic of a folded three-dimensional conformation of a polypeptide. Typically, conformational epitopes are composed of amino acids that are discontinuous in the linear sequence but which are clustered together in the folding structure of the protein. However, conformational epitopes may also consist of linear sequences of amino acids, which adopt a conformation that is characteristic of the folded 3-dimensional conformation of the polypeptide (and which does not exist in a denatured state). In protein complexes, conformational epitopes are composed of amino acids that are discontinuous in the linear sequence of one or more polypeptides that aggregate together when folding different folded polypeptides and they bind in a unique quaternary structure. Similarly, conformational epitopes may also be comprised of linear amino acid sequences of one or more polypeptides that are clustered together and adopt a conformation that is unique in quaternary structure. The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any time. Those skilled in the art will recognize that determinants of conformation or conformational state include the primary structure of the protein, as reflected in the amino acid sequence of the protein (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as the secondary structure of the protein (e.g., alpha-helix, beta-sheet, etc.), tertiary structure (e.g., three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interaction of the polypeptide chain with other protein subunits). Post-translational and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, etc., can affect the conformation of the protein. In addition, environmental factors such as pH, salt concentration, ionic strength and osmolality of the surrounding solution, and interactions with other proteins and cofactors, etc., can affect protein conformation. The conformational state of a protein may be determined by functional assays for activity or binding to another molecule or by physical methods such as X-ray crystallography, NMR or spin labeling, among others. For a general discussion of protein conformation and conformational state, reference may be made to Cantor and Schimmel, biophysical Chemistry, part I: the Conformation of Biological.Macromolecules, W.H.Freeman and Company,1980 and Cright on, proteins: structures and Molecular Properties, W.H. Freeman and Company,1993.

"binding" means any direct or indirect interaction. Direct interaction means contact between the binding partners. Indirect interaction refers to any interaction in which the interaction partner interacts in a complex of more than two molecules. The interaction may be entirely indirect, by means of one or more bridging molecules, or partly indirect, wherein there is still a direct contact between the partners, which is stabilized by further interactions of one or more molecules. As used herein, the term "specific binding" refers to a binding domain that recognizes a particular target but does not substantially recognize or bind other molecules in the sample.Specific binding does not mean exclusive binding. However, specific binding does mean that the protein has some increased affinity or preference for one or several of its binding agents. As used herein, the term "affinity" generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide, so as to shift the equilibrium of the individual protein monomers toward the presence of a complex formed by their binding. Affinity is generally determined by equilibrium dissociation constants (K _D ) To measure and report, equilibrium dissociation constants are used to assess and rank the strength of bimolecular interactions. Binding of antibodies to their antigens is a reversible process and the rate of the binding reaction is proportional to the concentration of the reactants. At equilibrium, [ antibody ]][ antigen ]]The rate of complex formation is equal to dissociation into its components [ antibodies ]]Antigen + [ antigen ]]Is a rate of (a). Measurement of the reaction Rate constant can be used to define the equilibrium or affinity constant (1/K _D ). Briefly, K _D The smaller the antibody, the greater the affinity for its target. The rate constants of the two directions of reaction are referred to as: rate constant of association reaction (k) _on ) Which is used to calculate the "binding rate" (k) _on ) The association reaction rate constant is a constant used to characterize how fast an antibody binds to its target. Vice versa, dissociation reaction rate constant (k _off ) Is used to calculate the "dissociation rate" (k) _off ) Is a constant used to characterize how fast an antibody dissociates from its target. In the measurements as shown herein, the flatter the slope, the slower the dissociation rate, or the stronger the antibody binding. Vice versa, a steeper underside indicates a faster dissociation rate and weaker antibody binding. The dissociation and association rate (k) _off /k _on ) Is used to calculate K _D Values. Several assays are known to those skilled in the art to measure association and dissociation rates and calculate K _D Therefore, the standard error is considered to be a value independent of the assay used.

A "binding agent" or "agent" as used interchangeably herein relates to a molecule capable of binding to another molecule, wherein the 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 source. The binding agents may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as engineered and synthetically produced. Thus, the binding agent may be a small molecule, a chemical, a peptide, a polypeptide, an antibody or any derivative thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, or the like. Binding may be achieved by covalent or non-covalent attachment.

The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain that specifically binds to an antigen. An "antibody" may also be an intact immunoglobulin derived from a natural source or a recombinant source, and may be an immunoreactive portion of an intact immunoglobulin. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that itself has a high affinity for an epitope or epitope and contains one or more CDRs responsible for such specificity. Non-limiting examples include immunoglobulin domains, fab, F (ab)' 2, scFv, heavy-light chain dimers, immunoglobulin Single Variable Domains (ISVD), nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as whole light chains or whole heavy chains.

The terms "antibody fragment" and "active antibody fragment" or "functional variant" as used herein refer to a protein comprising an immunoglobulin domain or antigen binding domain comprising the CDRs and/or structural features required for specific binding to a target protein. Antibodies are typically tetramers of immunoglobulin molecules. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") refers to an immunoglobulin domain consisting essentially of four "framework regions", which are referred to in the art and hereinafter as "framework region 1" or "FR1", respectively; "frame region 2" or "FR2"; "frame region 3" or "FR3"; and "frame region 4" or "FR4"; the framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1'; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3". Thus, the general structure or sequence of an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to an antigen by carrying an antigen binding site to an antibody. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form antigen binding sites. 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 definitions, conventional 4-chain antibodies (e.g., igG, igM, igA, igD or IgE molecules; known in the art) or Fab fragments, F (ab') 2 fragments, fv fragments (e.g., disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) derived from such conventional 4-chain antibodies bind to an epitope of the antigen via a pair of (associated) immunoglobulin domains (e.g., light and heavy chain variable domains), i.e., via a VH-VL pair of an immunoglobulin domain that co-binds to the epitope of the corresponding antigen. Immunoglobulin Single Variable Domain (ISVD), as used herein, refers to a protein having an amino acid sequence comprising 4 Framework Regions (FR) and 3 Complementarity Determining Regions (CDRs) according to the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The term "immunoglobulin domain" according to the invention refers to an "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is equivalent to the term "single variable domain", and defines a molecule in which an antigen binding site is present on and formed from a single immunoglobulin domain. This distinguishes an immunoglobulin single variable domain from a "conventional" immunoglobulin or fragment thereof, the two immunoglobulin domains, in particular the two variable domains, interacting 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. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. Thus, a 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 a VHH sequence) or a combination thereof A suitable fragment; so long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit consisting essentially of a single variable domain such that a single antigen binding domain need not interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, an immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or nanobody (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of any of these. In particular, the immunoglobulin single variable domain may be a nanobody (as defined herein) or a suitable fragment thereof. And (3) injection:and->Is a registered trademark of Ablynx n.v. (san ofi Company). For a general description of nanobodies, reference is made to the following further description, as well as to the prior art cited herein, as described, for example, in WO 2008/020079. "VHH domains", also known as VHH, VHH domains, VHH antibody fragments and VHH antibodies, have been described initially as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e. "antibodies lacking light chains"; hamers-Casterman et al (1993) Nature 363:446-448). The term "VHH domain" has been selected to distinguish these variable domains from heavy chain variable domains (which are referred to herein as "VH domains") present in conventional 4-chain antibodies and light chain variable domains (which are referred to herein as "VL domains") present in conventional 4-chain antibodies. For further description of VHH and nanobodies, reference is made to the heddle of Muyldermans The article (Reviews in Molecular Biotechnology 74:277-302, 2001) refers to the following patent applications as a general background: WO 94/04678, WO 95/04079 and WO 96/34103 of 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 97/49505, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 to Alganomics N.V. and Ablynx N.V.; WO 01/90190 of the national research Committee of Canada; WO 03/025020 (=ep 1433793) of the antibodies institute; and Ablynx N.V. WO 04/041687, WO 04/041682, WO 04/041685, WO 04/041683, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, and more published patent applications of Ablynx N.V. Nanobodies (particularly VHH sequences and partially humanized nanobodies) can be characterized, in particular, by the presence of one or more "tag residues" in one or more framework sequences, as described in these references. For numbering of amino acid residues of IVD, different numbering schemes can be applied. For example, numbering can be according to the AHo numbering scheme given by honeygger, a. And pluckthun, a. (j.mol. Biol.309, 2001) for all heavy (VH) and light chain variable domains (VL), as applied to VHH domains from camelids. Alternative methods for amino acid residue numbering of VH domains (which may also be applied in a similar manner to VHH domains) are known in the art. For example, the profiling of FR and CDR sequences can be accomplished by using the Kabat numbering system as applied to VHH domains from camelids in the paper of Riechmann, L. And Muyldermans, S.231 (1-2), J Immunol methods.1999. It should be noted that as in the art for V _H The total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied by the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows), as is well known for VHH domains. This meansIt means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and VHH domain is typically in the range 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein. Determination of CDR regions can also be performed according to different methods, such as based on contact analysis and assignment of binding site morphology, as described by MacCallum et al (j.mol. Biol. (1996) 262, 732-745). Or alternatively, the annotation of the CDRs may be according to the antibody modeling package of AbM (AbM is Oxford Molecular ltd. As described in http:// www.bioinf.o.g.uk/abs/index. Html), chothia (Chothia and Lesk,1987;Mol Biol.196:901-17), kabat (Kabat et al, 1991; 5 th edition, NIH Publication 91-3242), IMGT (LeFranc, 2014;Frontiers in Immunology.5 (22): 1-22), and/or optional annotations including aHo, gelfand and honeygger; for an overview, see, e.g., dondelinger et al, 2018,Front Immunol9:2278). The annotations also include depictions of CDRs and Framework Regions (FR) in proteins containing immunoglobulin domains and are methods and systems known to those skilled in the art, so those skilled in the art can apply these annotations to any immunoglobulin sequence without undue burden. These annotations are slightly different, but each is intended to encompass a region of the loop that participates in binding to the target. When referring to CDRs herein, at least one of the above notes is applicable, preferably IMGT notes.

VHH or Nb are typically classified in different sequence families or even superfamilies, such as clustering of clone-related sequences derived from the same progenitor cells during B cell maturation (descago et al 2017.Front Immunol.10;8:420). This classification is typically based on the CDR sequences of Nb, and wherein, for example, each Nb family is defined as a cluster of (clone) related sequences with a sequence identity threshold for the CDR3 region. Within a single VHH family as defined herein, CDR3 sequences are thus identical or very similar in amino acid composition, preferably having the same length and at least 80% identity, or at least 85% identity, or at least 90% identity in CDR3 sequences, such that Nb of the same family bind to the same binding site with the same effect or functional impact.

As used herein, the terms "determining," "measuring," "evaluating," "identifying," "screening," and "assaying" are used interchangeably and include quantitative and qualitative determinations.

Detailed description of the preferred embodiments

The present invention relates to a novel selection method for identifying specific polypeptide binders of a protein of interest from a plurality of binders, in particular from a library of protein binders, wherein a NANEX-based affinity displacement method is integrated first to isolate and immobilize a target protein by binding to a first protein binder coupled to a surface and second to elute a polypeptide binder complexed to the target protein by using the concept of the affinity displacement principle of NANEX. This integrated selection method provides a medium to high throughput approach in recombinant antibody library screening and provides high selectivity complementary to the capabilities of display technology. This new method is the first method in the field of recombinant antibody library selection and panning and has several advantages inherent to the NANEX-based system, as explained below by elucidating the originally established nanobody exchange chromatography (NANEX). In addition, this new method provides several benefits and improvements over (conventional) in vitro selection methods using phage display, where selection involves exposure to immobilized antigen to allow antigen-specific phage antibodies to bind to their targets during biopanning, then recovering the antigen-bound phage and subsequently infecting in bacteria. Most in vitro selection methods rely on purified antigens and require harsh conditions (high salt, extreme pH, proteolysis) to elute phage from the immobilized target. In contrast, NANEX-based selection allowed us to capture and immobilize complex antigens from natural samples, subsequently apply phage libraries, and then strip antigen-bound phage in a target-specific manner, all under entirely natural conditions. NANEX also allows us to uniquely select Nb that binds to epitopes that do not overlap with the collector-stripper pair.

NANEX affinity displacement

Nanobody exchange chromatography(referred to herein as "NANEX", based on the method previously described in PCT/EP 2020/087291) involves purification of proteins by affinity displacement chromatography, using small antigen binding entities, thereby establishing a favorable kinetic environment. In particular, a pair of target-specific protein binders that specifically bind to an epitope on a target in a competitive manner are used in the context of complementary kinetics. A pair of binding agents directed against the same target may involve competing or non-overlapping binding sites, or different binding sites at non-overlapping or different epitopes. Typically, affinity displacement is effected via the transient sandwich complex by the binding agent within defined dose and kinetic relationships. However, where pairs with identical or overlapping epitopes are used, the binding kinetics and nature of the binding agent affect the balance between cross-blocking or displacement. In NANEX, it was observed that when using an antigen binding domain built on an Immunoglobulin Single Variable Domain (ISVD) or more specifically Nb as a displacement agent, if the dissociation rate constant (k _off ) Lower than the first protein binding agent, most effectively creates a target for the same or largely overlapping and thus competing epitope as a collector on the target protein. Furthermore, purely on the basis of their competitive properties, the same binding agents, such as nanobodies for binding (or trapping) and elution (or stripping), can be used to purify the targets to obtain (suboptimal) satisfactory purified protein yields in the eluted fractions. When looking for binders that are capable of fully outperforming the capture agents that bind to the target as desired in high throughput applications (selection methods as described herein), it will be found that the use of conventional antibody binders (e.g., monoclonal antibodies) directed against the same epitope will largely block any displacement reaction, and rather protein binders, such as antibodies, that bind to epitopes that are adjacent or minimally overlapping compared to the capture agents will be used to avoid such competitive blocking epitopes. However, NANEX provides at least the second protein binder ISVD for the debonding agent, which binds to the same, substantially the same or largely overlapping epitope as the first protein binder, and wherein the second protein binder comprising ISVD has a lower dissociation rate constant (k _off ) By usingNANEX achieves a very efficient displacement of the first protein binding agent, allowing elution of the target protein in high yield and specificity. The compact but highly specific nature of the ISVD-type antigen binding agent therefore provides a kinetic relationship that effectively displaces another antigen binding protein in a competitive binding mode, which is superior to larger conventional antibodies with larger antigen binding sites/paratopes (consisting of residues from 6 CDRs instead of 3 CDRs from ISVD). Furthermore, the reaction can occur under mild physiological conditions and still provide efficient elution, so it is well suited for integration in the selection methods described herein, as polypeptide binding agents from the plurality of binding agents that bind to the target protein can remain in a complex with the target protein and co-elute with the stripper-target complex. Thus, by integrating NANEX into this new selection method, the combined NANEX-purification and selection-conditions established herein lead to the next generation of improved selections for polypeptide binding agents, especially in antibody drug discovery. Indeed, the results as exemplified herein have clearly shown that the integrated NANEX selection procedure provides a robust and reliable selection of displayed polypeptide binding agents in a more efficient and deeper manner than conventional selection panning methods.

"ISVD-based displacement", or more specifically "nanobody exchange" or "nanobody exchange chromatography" or "NANEX", as used interchangeably herein, which was previously focused on analytical purification of direct high purity protein complex elution, now finds its application in antibody drug discovery and target binding agent identification methods. Thus, the advantage of this novel selection method is the use of NANEX to present targets, as Nb-based collectors/strippers compete for the same or highly overlapping epitopes and provide high affinity binders that do not interfere with other binders at different epitopes. This not only makes it possible to find binding agents to new or different epitopes, but also allows to find binding agents of certain conformations of the target (when using a collector/stripper that locks the target in a specific conformation).

When purifying using NANEX, the eluting complex thus contains a stripping or displacement agent, which has the following advantages: allowing the application of an additional functionalized second protein-binding agent comprising ISVD (referred to as a stripper, or nanostripper in the case of nanobodies), i.e. they provide specific functions to the eluted protein complex. Such functionalization may involve visualization of the protein complex (via fluorescence or labeling of the reagent) or involve function as a chaperone or adapter protein (including, for example and without limitation, megaBody), as well as other examples, to elute the target in the functionalized complex (see also below). Furthermore, after the elution step and regeneration procedure, the affinity matrix (which may be any type of surface, such as (magnetic) beads, columns, wells in a plate or a resin) is easy to use for the next affinity purification and/or selection cycle and may be used in a high throughput platform, such as a screening platform, a chip or a microfluidic setup or device.

By using the selection method of NANEX or Nanobody exchange chromatography integration as described herein, a leap in the high throughput selection of binders for several target classes (e.g. difficult to handle proteins or difficult to purify proteins) can be foreseen. In addition, the selection of protein binders having conformational selective recognition of an antigen or target may be selected, which are immobilized by a capture agent in a certain conformational state, such as a stable conformation, an active/inactive conformation, more specifically an agonist, partial agonist or biased agonist conformation. Alternatively, where a heterogeneous tag (e.g., GFP) NANEX-trap/stripper is proposed in the selection method described herein, a versatile high throughput selection platform can be developed, providing further benefits for automated and efficient antibody screening techniques. With the rapid development of this technology in biotechnology, it is expected that the present invention will affect the efficiency and potential of novel therapeutic drug screening, as well as increase the throughput and potential of proteomics, MS-based, structural and other assays.

Selection of NANEX-based polypeptide binding agents

Thus, a method for selecting a polypeptide binding agent specific for a target protein involves in a first aspect a method comprising the steps of:

a) Mixing a first protein binding agent immobilized on a surface and specifically binding to a target protein with a sample comprising the target protein for obtaining a complex on the surface,

b) Providing a sample comprising a plurality of polypeptide binding agents to the complex of step a),

c) Adding a sample comprising a second protein binding agent to the mixture of step b), the second protein binding agent competing with the first binding agent for binding to the target protein and displacing the first binding agent from the target protein by specific binding to the target protein, and

d) Eluting the second protein binding agent that binds to the target protein for isolating the polypeptide binding agent that binds to the target protein.

The feature "competing for binding to the target protein" is required for efficient elution of the target-stripper complex associated with the polypeptide binding agent, which may be interpreted as that the stripper competes for the same epitope, or may also mean competing in a different way (such as kinetic or allosteric), since in particular in the Nb field, allosteric interactions with the target are known to represent binding patterns. Thus, in one embodiment, the debonding agent may compete for binding to the target by binding to a minimally overlapping or adjacent epitope, or alternatively, the debonding agent may disrupt interactions between the capture agent and the target even by binding to an allosteric site on the target, by inducing conformational changes in the target. Competitive binders may be established using several methods known in the art, such as, but not limited to, competitive ELISA, alphalisa, octet measurement or Biological Layer Interferometry (BLI), SPR Biacore, micro thermophoresis (MST), and the like.

Thus, the competitive elution mode serves to elute the target from the surface to which the target is immobilized, and thus does not involve any competition for the selected polypeptide binding agent (which ideally also binds to the target during the elution phase). However, the use of at least one ISVD as an antigen binding second protein binding or stripping agent is desirable in such competitive elution as used in NANEX, as the use of ISVD or Nb provides more favourable kinetics than, for example, the use of large conventional antibodies as a displacement agent.

The method of the invention comprises a second protein binding agent which is in solution and is soluble under elution conditions. The elution conditions preferably relate to physiological conditions, as known to the person skilled in the art. The term "soluble" as used herein refers to the fact that a protein binding agent is in a functional form, meaning that it is capable of specifically binding its target within its intended range of affinities for the epitope. For the method of the invention, the first protein binding agent is immobilized in step a), wherein it may be immobilized on the surface via covalent or other coupling means. The reagents may be coupled to beads, which may be agarose beads or magnetic beads, or may be present on a surface or substrate, more particularly packed on an affinity column, which may be suitable for the scale of preparation and analysis, more particularly, which may be a microcolumn of the order of less than 1mL of column volume, or even a sub-micromolar volume, or even provided on a chip using microfluidic technology. Most preferably, the first protein binder is immobilized on a solid support or resin. "resin" or "affinity resin" as used interchangeably herein is an activated affinity chromatography carrier for immobilization of biomolecules such as ISVD or other protein binders. In a specific embodiment, the first protein binder comprises ISVD and is coupled to the resin using coupling methods known in the art (see examples). The method as presented herein has the following advantages: in the elution in step d), physiological conditions are used. Optionally, depending on the type of sample used in a) and the duration of selection, a gentle regeneration step of the immobilized surface may allow for the reuse of the immobilized complex in a second round, although preferably more selection rounds include repeating method steps a) to d), thereby ensuring the integrity and stability of the target protein.

Depending on the nature of the sample, after step a) or step b) of the method of the invention it may be necessary to optionally wash the immobilized surface under neutral, or very mild, or rather harsh conditions, or it may be necessary to repeat the washing step to remove any unbound abundant components present in the sample comprising the target protein, or to remove unbound remainder of the various polypeptide binding agents.

In a further embodiment, the method as described herein comprises k of a second protein binding agent, with a first protein binding agent _off In contrast, k of the second protein binding agent _off Lower. Furthermore, the method delivers optimal results when the first binding agent or capture agent has a higher dissociation rate or lower or equal affinity than the second binding agent, and vice versa, when the second binding agent has a lower dissociation rate and/or the same or higher epitope affinity than the first binding agent. According to current state of the art knowledge, the dissociation rate constant (or dissociation rate or k _off ) And association rate constant (or binding rate or k) _on ) Is related as K _D ＝k _off /k _on Wherein K is _D Defined as the dissociation constant, which is inversely related to the affinity of the binding agent for its target, as also described in detail in the definition above. Thus, if the dissociation constant K _D The value (lower) is higher (higher) if k _on Are identical). Alternatively, if k _on Higher, then K _D Lower and higher affinity (if k _off Are identical). Thus, for the selection methods of the invention, the protein binders described herein are directed to k of the same, substantially the same or a majority of overlapping epitopes _off And/or affinity (or K) _D ) Relatively different. The methods as described herein more particularly refer to k of the second binding agent for the same, substantially the same or a majority of overlapping epitopes of the target protein _off K lower than the first binding agent _off Wherein "lower" herein means at least 2-fold lower, 5-fold lower, or 10-fold lower, or at least 30-fold lower, or at least 100-fold lower, or at least 200-fold lower, at least 300-fold lower, at least 400-fold lower, or at least 500-fold lower. More preferably, k to the first protein binding agent _off In contrast, the second binding agent k _off Values are in the range of at least 2-fold lower to at least 10-fold lower, or at least 5-fold lower to at least 20-fold lower, or at least 10-fold lower to at least 30-fold lower, or at least 100-fold lower.

Similarly, affinity of the second binding agent for an epitope of the target proteinThe force may be equal to or higher than the affinity of the first binding agent for the epitope of the target protein, wherein "higher affinity" refers to K to the first protein binding agent _D Value of "K" of the second protein binding agent _D The value "K is at least 2-fold lower, or at least 5-fold lower, 10-fold lower, 20-fold lower, or 100-fold lower, or in the range of at least 2-fold lower to at least 2000-fold lower _D Values. In a preferred embodiment, the purification method as described herein discloses having from 1mM to about 1 nanomolar K for an epitope of a target protein _D The first binder having a value and disclosed a K having a value of 1 nanomolar or less, optionally as low as 1 picomolar _D A second protein binding agent of value, optionally having substantially the same or a substantial portion of overlapping epitopes to the target. More preferably, the first binding agent has a K in the nanomolar to millimolar range (i.e., 10E-9 to 10E-3) _D Value, and the second binding agent has a K in the femtomolar to micromolar range (i.e., 10E-12 to 10E-6) _D The value, most preferably the relative difference between the first binding agent and the second binding agent is at least 2-fold. In one embodiment, the first protein binder is K _D Value ratio of K of displacement agent _D The value is at least 2 times higher, this is due to k _off The difference in values drives the difference, especially when the displacement agent binds to the same or a large part of the overlapping epitopes.

Multiple polypeptide binding agents

The methods described herein are selected from samples comprising "multiple polypeptides or protein binders" which may be virtually any type of protein or peptide or polypeptide, such as in the broadest sense a collection of proteins that are candidates for specific binding to a target protein, wherein the "multiple polypeptide binders" in the broadest sense may thus be derived, for example, from a cell extract, a specific tissue or signal transduction cascade, or an unbiased proteomic sample. In a more specific aspect, a "plurality of protein binders" are provided as a sample comprising a repertoire of binders as fragments expressed and/or displayed from a library. More specifically, the display library is predominantly a molecule of known antibody type. Thus, recombinant antibody libraries are within the scope of such samples having multiple protein binders, as such antibody libraries will typically provide protein binders selected for specific binding to a target protein.

Thus, a "plurality of polypeptide binders" may be provided structurally by binders comprising a binding domain (potentially binding a target protein), and by proteins, peptides or peptidomimetics, more particularly "antigen binding" domains, as present in a number of different antibodies and antibody-like molecules, as described herein, for example but not limited to antibodies or active antibody fragments such as Fab, fab 'and F (ab') 2, fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (dsFv) and fragments comprising VL or VH domains, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (minibodies), variable domains derived from camelid heavy chain antibodies (VHH or nanobodies), variable domains derived from neoantigen receptors of shark antibodies (VNARs), protein scaffolds including Alphabody, engineered ankyrin repeat domains (pin), fibronectin type III repeats, anti-cargo proteins (antt), proteins (knoodin), engineered CH2 domains, and the like.

In a preferred embodiment, the "plurality of polypeptide binders" is thus provided by a sample comprising a display library that allows selection of phenotype-genotype couplings, as described herein, which are critical to facilitate selection of antigen-specific antibodies or binders (antigen binding domain containing polypeptides), are key features of antibody display technology for identification of therapeutic hits. Thus, the term "display library" as used herein provides those recombinant libraries that allow for the physical linking of phenotypes (antigen binding behavior) to genotypes of polypeptide binding agent libraries.

In addition, the types of recombinant antibody libraries contemplated herein include mAb libraries based on immune fragments (i.e., biased toward certain specificities found in immunized animals or naturally immunized or infected humans) or priming fragments (not biased toward specificities found in the immune system). The latter type of fragment may be derived from non-immune natural or semisynthetic sources. The non-immune (or priming) library was derived from a natural, non-immune, rearranged V gene (e.g., from IgM B cell pool) to reduce antigen-induced bias in all components, and was the first library to isolate anti-autoantibodies-otherwise difficult to obtain by immunization. Synthetic antibody libraries are constructed entirely in vitro, using oligonucleotides that introduce regions of complete or custom degeneracy into the CDRs of one or more V genes.

As is known in the art, several display techniques allow the use of different methods in the selection of recombinant antibody libraries, with phage display being the primary technique, alternatives including yeast, ribosome, bacterial and mammalian displays are also contemplated in the methods as described herein.

In a specific embodiment, the selection method as described herein is used for multiple rounds of selection, enriching for polypeptide binding agents present in a library (phage display library as used in the methods shown herein) during the repeat process. Specifically, when the collected eluate contains phage displaying target-specific Nb, which binds to the target-stripper complex, phage solution for re-infecting escherichia coli is provided by the collected eluate in order to provide phage for the next round.

Target protein sample and collector/stripper pairs in NANEX-based selection

As previously mentioned, the target proteins of the methods described herein provide epitopes for the first and second protein binders (capture and release agents) applied in steps a) and d) of the methods, wherein the epitopes may be naturally occurring, naturally occurring and/or endogenously available epitopes on the target proteins. When the capture and stripping agents recognize native or endogenous proteins present in the sample of step a), this will allow capturing the target from the sample to obtain an immobilized antigen-capture agent complex. Another option is provided by presenting epitopes on recombinantly produced target proteins that are provided as purified or partially purified samples, which do not themselves require tags. For protein-binding agent pairs (collector/stripper) that specifically bind to unlabeled target proteins, one can screen for competition for the same targetAnd selecting to provide a pair of competing binders, or a higher affinity and lower affinity protein binder pair may be designed. Indeed, the use of a 3D structure of the debonder or second protein binder bound to the target allows for the design of mutations in the binding site of the protein binder, which will result in reduced affinity or higher k _off And thereby provide a compatible collector or first protein binder. Furthermore, a more direct method that does not require structural information once the sequence is known also allows the pairing to be determined based on a single binding agent. As shown in the examples, in a non-limiting manner, for GFP, when used as a "target protein", new pairs with lower dissociation rate constants can be identified for use as collectors in the NANEX method by analyzing their competitive properties using BLI to map the positions or, alternatively, based on the sequence of nanomolar binders or strippers, also performing alanine mutation scans in the CDR3 region known to be most critical in defining binding kinetics. In this way, at different k _off Or pairs of protein binding agents that bind affinity to the same epitope can be obtained simply by introducing single or multiple mutations.

In alternative embodiments involving methods in which the trapping agent and stripping agent comprise ISVD, a "monovalent" form may be used as the trapping agent, and a "multivalent" form may be used as the stripping agent, since the multivalent form is at a higher k than the monovalent form _off With higher affinity, which also results in better elution yields and target protein purity. The term "monovalent form" as used herein refers to an ISVD as used herein that recognizes only one epitope, while the term "multivalent" form refers to an ISVD as used herein that recognizes more than one epitope, such as, but not limited to, a divalent, trivalent, or tetravalent form. Furthermore, instead of multivalent stripping agents, multi-paratope or multi-specific stripping agents are also contemplated, wherein the stripping agent may comprise the same building block binding to the same epitope, and at least one or more building block binding, which may be different and may bind to the same or another table on the target protein A site, or an epitope on another target protein that is complexed with the first target protein.

In a further embodiment, the method as described herein utilizes a first and a second protein binding agent (collector and stripper), wherein at least one of those binding agents comprises an antigen binding domain as defined herein, or more specifically at least one antibody, ISVD, VHH, nanobody or antigen binding chimeric protein (defined herein as ISVD fused to a scaffold protein by at least two sites, and the scaffold protein domain preferably comprises HopQ, ygjK or derivatives or variants thereof). The latter definition of the antigen binding chimeric protein is also known in practice as a macroantibody, which can thus be applied as a first and/or second protein binding agent in the methods of the invention. The term macroantibody as used herein refers to a novel fusion protein disclosed in steyart et al (WO 2019/086548 A1), to a fusion protein comprising an antigen binding domain linked to a scaffold protein, wherein the scaffold protein is coupled to the antigen binding domain at one or more amino acid sites accessible or exposed at the surface of the domain, resulting in disruption of the topology of the antigen binding domain. The antigen binding chimeric protein is further characterized in that it retains its antigen binding function as compared to an antigen binding domain not fused to the scaffold protein. The macroantibodies described herein relate to specific macroantibodies or antigen binding chimeric proteins, the antigen binding domain of which comprises an Immunoglobulin Single Variable Domain (ISVD) or nanobody, which is fused or linked to a scaffold protein at the accessible surface (β -turn or loop, excluding CDRs) of the ISVD domain, resulting in disruption of the topology of the antigen binding domain and retaining its antigen binding function, i.e. specific epitope recognition. In a specific embodiment, the second protein binding agent involves a macroantibody or antigen binding chimeric protein comprising an ISVD linked to a scaffold protein by inserting the scaffold protein in the first β -turn linking β -chains a and B of the ISVD (as defined according to IMGT nomenclature and as defined in WO2019/086548 A1). In even more specific embodiments, the scaffold protein used herein is a HopQ or YgjK scaffold protein, wherein fusion of the scaffold protein interrupts the topology of the ISVD, but does not interrupt its overall 3D structure, nor its epitope binding specificity. As used herein, a "HopQ" or "HopQ-derived" scaffold refers to a protein scaffold (protein database: PDB 5LP 2) of the adhesin domain of HopQ type 1 of H.pylori strain G27, or a circularly permutated protein thereof, also known as chopQ or c7HopQ (see also WO2019/086548A 1). "YgjkK" or "YgjK derived" scaffolds as used herein relate to the protein scaffold of E.coli K12 YgjK (PDB 3W 7S), or the circularly permutated gene encoding said protein thereof, also known as cYgjK (see also WO2019/086548A 1).

Another embodiment relates to a method for selecting, wherein the sample comprising the target protein in step a) as described herein provides a target protein having epitopes recognized by the first and second protein binding agents present on a scaffold protein (target protein) in a diabody. The macroantibodies may preferably be constructed using scaffold proteins derived from HopQ or YgjK proteins, so that the HopQ or YgjK protein scaffold contains an epitope that specifically binds to the protein binding agent of the method. The protein binding agent pair that specifically binds to a scaffold protein epitope present on a macroantibody as disclosed herein or as disclosed in steyart et al (WO 2019/086548A) or as otherwise may be described has the further advantage that the method can be applied to capture or remove target proteins bound to a macroantibody from a complex mixture or can be applied as a general selection tool, similar to other labeled target proteins.

Another embodiment relates to a method as described herein, wherein the target protein comprises a tag or a heterologous tag or label or a detectable label. The term "detectable label" or "labeling" or "tagging" refers to a detectable label or tag that allows for the detection, visualization and/or isolation, further purification and/or immobilization of a target protein of interest, or, when present on a stripper, allows for the detection, visualization and/or isolation, further purification and/or immobilization of an eluted complex or isolated or purified (poly) peptide or complex as described herein, and is intended to include any label/tag known in the art for such purposes. In a further implementation In one embodiment, the protein binding agent specifically binds to an epitope on a tag present on a fusion protein comprising the target protein. Particularly preferred are fluorescent labels or tags (i.e., fluorochromes/fluorophores), such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.) and fluorochromes (e.g., FITC, TRITC, coumarin, and cyanine); luminescent labels or tags, such as luciferase; and (other) enzyme labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease, or glucose oxidase). Also included are affinity tags, such as Chitin Binding Protein (CBP), maltose Binding Protein (MBP), glutathione-S-transferase (GST), poly (His) (e.g., 6 XHis or His 6),And->A solubilising tag, thioredoxin (TRX), poly (NANP) and ubiquitin, or a small ubiquitin-like modifier (SUMO) or SMT3; chromatographic tags, such as FLAG tags; epitope tags, milk V5-tags, myc-tags and HA-tags, or EPEA (CaptureSelect C-tag, US9518084B 2), or furthermore an intein-chitin binding domain (intein-CBD), streptavidin/biotin based tag, his-Patch ThioFusion (thioredoxin based), or HaloTag, or even reporter tags, such as HRP, or alkaline phosphatase. Many non-limiting examples are provided, for example, in Kimple et al (2015 Table 9.9.1). Combinations of any of the foregoing markers or tags are also included.

In a further embodiment, the method as described herein may comprise a sample comprising the target protein to be applied in step a), which is a "complex sample" or a complex mixture of proteins and/or other components. The sample may be an in vitro mixture of components or a biological sample, wherein the term "biological sample" refers to serum, urine, cells and tissues, and other body fluids. The "complex mixture" may also be provided as a lysate or cell extract or any mixture of components containing target proteins, which may also be recombinantly produced target proteins, optionally incorporated into the complex mixture (in order to capture certain conformations under different conditions or in specific circumstances). The "composite sample" may also be provided as a whole proteome or soluble proteome solution containing the entire protein pool of organisms, cellular environments or tissues, optionally corresponding to a certain signaling cascade, disease stage or disorder, etc.

In a specific embodiment, a "complex sample" comprising the target protein as provided in step a) of the method has also been applied as an immunogen or antigen to an immunized animal to obtain a "plurality of polypeptide binders" for use in step b) of the method. In such methods, a display library comprising a plurality of polypeptide binders, which is less or partially or unbiased, is applied to selection, rather than a biased library that is typically generated only against a specific (single) antigen (see examples).

As described above, the method as described herein may result in elution of a complex comprising not only the stripper and the target protein bound to the selected polypeptide binding agent, but also the complex may comprise other proteins bound to the target, especially at the beginning of the complex sample in step a), wherein the binding of the target protein to the surface via the first protein binding agent thus already results in the binding of the target+ interactant to the surface. In such embodiments, selecting a protein binding agent upon addition of a plurality of protein binding agents may thus result in the identification of a protein binding agent that directly or indirectly binds to the target protein. Direct binding means that there is a direct interaction and specificity for the target protein, whereas indirect binding means herein that the polypeptide binding agent binds to another component of the eluted protein complex, such as a protein that has itself bound and is present in the complex that binds the target protein (from the complex sample used in step a). Thus, in a specific embodiment, the method allows not only the identification of polypeptide binding agents of target proteins, but also the identification of polypeptide binding agents of co-immobilized interactions of target proteins in method step a).

Finally, it should be clear to a person skilled in the art how and when the method for selecting polypeptide binders is useful, as sufficient utility can be identified, as non-limiting examples, for selecting specific binders from recombinant antibody libraries in drug discovery, and for epitope binning or identifying neoepitopes on targets, i.e. epitopes other than known binders (e.g. capture and stripping agents). As contemplated herein, the selection method is suitable for medium-throughput and even high-throughput purposes.

It is to be understood that although specific embodiments, specific configurations, and materials and/or molecules have been discussed herein with respect to methods, samples, and products according to the present disclosure, various changes or modifications in form and detail may be made without departing from the scope of the present invention. The following examples are provided to better illustrate specific embodiments and should not be construed as limiting the application. The application is limited only by the claims.

Examples

Introduction to the invention

The present invention is based on the principle of nanobody exchange chromatography methods (herein referred to as "NANEX", based on the methods previously described in PCT/EP 2020/087291), which are herein applied to the selection of target-specific binding agents, e.g. antibodies, from a display library. In particular, a first binding agent (referred to herein as a "collector"), in particular a nanobody, is attached (preferably covalently) to a solid support for immobilization of an antigen or target or protein of interest (as used interchangeably herein). The immobilized antigen is then incubated with different libraries of different binding agents (e.g., antibodies) expressed and displayed in a manner that provides a physical link between the phenotype (binding behavior) and the encoding genotype of each binding domain, e.g., by phage display, yeast display, ribosome display, or any other method. The washing step may optionally be used to remove irrelevant binding agents or antibodies. Similar to the NANEX purification method, and specific to the present method, a second binding agent (referred to herein as a "stripper" or "displacement agent"), particularly a nanobody, competing with the capture agent for binding to the antigen is then used to selectively elute the immobilized antigen associated with the stripper and antigen-specific binding domain or antibody and its encoding genotype. The binding agent can then be amplified to create a new pool of antigen-specific binding agents or antibodies, allowing the cycle to be repeated until the binding agent dominates the population and can be characterized as a monoclonal binding domain or antibody (fig. 1). Accordingly, provided herein is a novel selection method for specific target protein binding agents that integrates the principles of NANEX-based immunodisplacement purification for the first time to increase the efficiency and selectivity of the selection process.

Example 1: NANEX selects new GFP-specific nanobodies from antibody libraries using GFP-specific collector/stripper pairs and GFP protein as targets.

Production of GFP antibody libraries. Llama (llama) was immunized once a week, six times in total, with 850 μg GFP (SEQ ID NO: 25) and phage display libraries were generated as described (materials and methods).

Discovery of novel GFP-specific nanobodies. To find new GFP-specific nanobodies that bind non-overlapping epitopes (compared to the epitope of the collector/stripper pair), we immobilized a GFP-specific collector (SEQ ID NO: 2) with the reference CA15816 on magnetic beads according to the manufacturer's instructions, with an affinity of 2.7nM for GFP. These collector coated beads were blocked and washed 5 times with PBS prior to use. Various concentrations of GFP (SEQ ID NO: 25) ranging from 0.1nM to 100nM were mixed with these collector-coated beads to trap GFP on NANEX beads. Collector coated beads not incubated with GFP served as negative controls. After incubation with GFP (antigen), all beads were routinely washed 3 times and incubated with phage-displayed GFP antibody libraries in 96-well plates. After incubation for 2h at 4℃the beads were routinely washed 12 times with PBS-Tween. To select new GFP-specific antibodies that bind to epitopes that do not overlap with those of the collector/stripper pair, the non-covalent interactions between the collector and GFP are selectively disrupted by adding a high affinity stripper CA12760 (SEQ ID NO: 1) that competitively binds to epitopes that overlap with the collector epitope. Thus, the beads were incubated with high affinity CA12760 (GFP-specific stripper) that bound the same epitope on GFP for 30min. GFP with bound phage was selectively eluted with this GFP-specific stripper. Phage are then amplified to produce enriched A novel repertoire of antibodies. For comparison, we eluted bound phage with trypsin from collector-coated beads prepared with 100nM GFP as described (Pardon et al, 2014).

For round 2 selection using NANEX, we followed the same strategy, using output phage obtained from collector coated beads (prepared with 100nM GFP on all beads). The enrichment observed after 1 or 2 rounds of selection is shown in figure 2. A significantly higher background was observed after 2 rounds of selection if elution was performed by trypsin compared to NANEX. Eight different families of GFP-specific nanobodies were selected based on their CDR3 sequences (Nb clone CA17517 corresponds to SEQ ID NO:26, CA17518-SEQ ID NO:27, CA17519-SEQ ID NO:28, CA17520-SEQ ID NO:29, CA17673-SEQ ID NO:30, CA17674-SEQ ID NO:31, CA17675-SEQ ID NO:32, CA17676-SEQ ID NO: 33).

Epitope analysis.To demonstrate that the newly selected nanobody family binds to a different epitope on the target than the epitope of the collector/stripper, we propose a Biological Layer Interferometry (BLI) binding experiment. To this end, we loaded the streptavidin biosensor with biotinylated GFP, and next we bound GFP with picomolar affinity to GFP-stripper CA12760 (SEQ ID NO: 1). After washing, the biosensor loaded with GFP-CA12760 complex was incubated with newly discovered Nb. In each case we observed a further increase in mass, indicating that all these nanobodies bound to another epitope and did not displace CA12760 (fig. 3).

Example 2 nanex new target specific nanobodies were selected from antibody libraries using GFP-specific collector/stripper pairs and GFP-tagged target proteins.

The collector/stripper pair, such as the GFP-specific pair introduced in example 1, can also be used first to capture GFP-tagged proteins of interest from complex mixtures (e.g., cell lysates) and to selectively elute GFP-tagged proteins of interest from the matrix (beads, plates, etc.). Here, we show by phage immunization against the human glucocorticoid receptor ligand binding domain (GR-LBD) generated by immunization of llamasLibrary of nanobodies of the library. GFP-specific capture reagents covalently attached to a solid support were used to immobilize GFP-tagged forms of GR. Then use KingFisher ^TM The immobilized antigen GFP-GR was incubated with a nanobody display library derived from immunized animals by Flex purification System (thermo scientific). Similar to NANEX and in particular the present invention, GFP-specific stripping agents competing with the capture agents were then used to selectively elute immobilized GFP-GR associated with LBD-specific nanobodies displayed on phage. The phage selectively recovered with this GFP-specific stripper is then amplified to produce a new pool enriched in antibody-specific stripper, and then amplified to produce a new pool enriched in LBD-specific antibodies (particularly Nb herein), allowing the cycle to be repeated until the LBD-specific nanobodies predominate in the population to be characterized as monoclonal LBD binders.

Production of GR-LBD antibody library. Constructs encoding the ligand binding domain (LBD, 369-777) of the human Glucocorticoid Receptor (GR) are used to express recombinant LBD in the cytoplasm of E.coli in the presence of dexamethasone (Dex). The GR-LBD domain was purified to homogeneity by affinity purification (Ni-NTA) as a soluble protein, followed by buffer (20 mM NaH ₂ PO ₄ pH 8.0, 150mM NaCl, 10% glycerol, 1mM DTT, and 10. Mu.M Dex). Llamas were immunized once a week with a total of 110 μg GR-LBD over a period of 6 weeks and phage display libraries were generated as described (materials and methods).

Discovery of novel GR-LBD specific nanobodies. The full length gene encoding the human glucocorticoid receptor (NR 3C 1) was introduced into a pCDNA3 expression vector to produce GFP-tagged target fusion. This vector was transfected into HEK293T cells using Polyethylenimine (PEI) as a transfection agent. 48h after transfection, 5000 ten thousand cells were harvested, washed with ice-cold PBS buffer and washed with Dounce homogenizer in 5ml lysis buffer (10mM Hepes pH 7.4, 10. Mu.M Dex,10% glycerol, 10. Mu.M ZnCl) supplemented with 50. Mu.g/ml DNAse I and protease inhibitor ₂ ，2.5mM MgCl ₂ 2.5mM DTT,0.5%NP40 substitute). The lysate was clarified by centrifugation at 20,000 g. Collecting Supernatant containing GFP-labeled GR (target) was incubated at 4 ℃ in 96-well deep-well blocks for 1 hour with 20 μl of magnetic beads that were covalently functionalized with GFP-specific capture agents to immobilize GFP-labeled GR on these beads. Using a KingFisher flex apparatus, the beads were collected and washed with 0.5mL of wash buffer (10mM HEPES pH 7.4, 10. Mu.M Dex, 10% glycerol, 10. Mu.M ZnCl) ₂ 、2.5mM MgCl ₂ 2.5mM DTT) and then they were washed at 4℃with phage displaying GR-LBD nanobody library (1,4.10) ¹⁴ Individual phages) were incubated for 1h. Next, the beads were washed 9 times with 0.5mL of wash buffer. Next, immobilized GR associated with the GR specific nanobody displayed on the phage is then selectively eluted using GFP-specific stripper nanobody. The eluted phages were used to infect exponentially growing E.coli TG1 cells and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/mL ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g, and the cell pellet was resuspended in LB (supplemented with 100. Mu.g/ml ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Lysates of untransfected HEK293T (which did not contain any GFP-tagged proteins) were spiked with 20 μg of wild-type GFP to serve as (negative) controls.

After 3 rounds of selection by panning using the NANEX technique, individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family were selected and the encoding plasmids were transformed into E.coli WK6 expressing strains to express and purify each nanobody of interest (Nb clone CA17797 corresponds to SEQ ID NO:35, CA17798-SEQ ID NO:36, CA17799-SEQ ID NO:37, CA17800-SEQ ID NO:38, CA17801-SEQ ID NO: 39). The specificity of GR specific nanobodies was verified by attaching individual nanobodies to NHS-sepharose beads for co-immunoprecipitation assays. Beads functionalized with these GR specific nanobodies were incubated with lysates of HEK293T cells transfected with GFP-GR vectors on a spin device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE. The isolated proteins were transferred to PVDF membranes and western blots were developed with GR specific antibodies to confirm the presence of GFP-tagged GR (fig. 11).

We conclude from this example that the NANEX-based selection method allows easy selection of target-specific antibodies from an antibody library using GFP-specific collector/stripper pairs and GFP-tagged target proteins.

Example 3 nanex uses GFP-specific capture/stripper pairs and GFP-tagged targets to select new target-specific binders from whole proteomic antibody libraries.

GFP-tagged proteins of interest can also be first captured from complex mixtures (e.g., cell lysates) on a substrate (beads, plates, etc.) using a capture/stripper pair, such as the GFP-specific pair introduced in example 1. Similar to NANEX and in particular the present method, a second nanobody (stripper) competing with the collector is then used to selectively elute the immobilized antigen associated with the antigen-specific binding domain or antibody and its encoding genotype.

For example 3, we immunized llamas with all soluble yeast proteins (soluble yeast proteomes) to generate an immune response against all soluble yeast proteins. The nanobody library of the immunized animal is displayed on phage to produce a whole proteome antibody library. In parallel, GFP-labeled forms of FBA1 were immobilized using GFP-specific capture reagents covalently attached to a solid support (Huh et al, 2003). The immobilized antigen (GFP-FBA 1) was then incubated with a nanobody display library derived from immunized animals using a KingFisher Flex apparatus. Similar to NANEX and in particular the present invention, GFP-specific stripping agents competing with the capture agents were then used to selectively elute immobilized GFP-FBA1 associated with FBA 1-specific nanobodies displayed on phage. The phage selectively recovered with this GFP-specific stripper was then amplified to generate a new pool enriched in FBA 1-specific antibodies, allowing this cycle to be repeated until the master population of FBA 1-specific nanobodies was characterized as monoclonal FBA1 binders.

Generation of whole proteome antibody libraries. Within 6 weeks, saccharomyces cerevisiae strain EBY100 #, was usedMYA-4941 ^TM ) To generate a whole proteomic antibody library. To prepare a whole proteome mixture of such soluble antigens, 1 liter EBY100 cultures were grown in YPD medium and harvested in mid-log phase (od600=0.6). Cells were collected by centrifugation and resuspended in 50. Mu.g/ml DNase I and EDTA-free protease inhibitor (cOmplet) ^TM Roche) in PBS. The cells were then lysed using a French press and centrifuged at 20,000g for 30min. The supernatant containing all soluble proteins was collected, filtered through a syringe using a 0.22 μm filter, aliquoted and stored at-80 ℃. Quantification by BCA protein (Pierce ^TM BCA protein assay kit 23225, thermo fisher) assay, total protein concentration in lysate was 1.5mg/mL. After immunization, phage display libraries were generated as described (materials and methods).

Discovery of novel FBA1 specific nanobodies. We used NANEX to selectively capture GFP-FBA1 onto collector coated magnetic beads from lysates of engineered yeast strains (yeast GFP fusion accession number: GFP (+) 22, G1) expressing FBA1 (System name YKL 060C) as GFP-tagged protein (Huh et al, 2003) (see example 1). Washing the beads, and phage displaying whole proteome nanobody library (1,4.10) ¹⁴ Individual phages) were incubated together and washed again. Next, GFP-specific stripper nanobodies were then used to selectively elute immobilized GFP-FBA1 associated with the FBA 1-specific nanobodies displayed on phage using a KingFisher Flex apparatus. Eluted phage for infection indexThe E.coli TG1 cells were grown and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/ml ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The following day, the cultures were centrifuged at 3000g and the cell pellet resuspended in LB (supplemented with 100. Mu.g/ml ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Yeast lysates from reference strain EBY100 (which did not contain any GFP-tagged protein) were spiked with 20 μg of wild type GFP to serve as (negative) controls.

Characterization of FBA1 specific nanobodies. After 3 rounds of selection by panning using the NANEX technique, individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected e.coli TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family are selected. Four different FBAl-specific nanobodies derived from four different families of sequences were selected based on their CDR3 sequences (Nb clone CA17440 corresponds to SEQ ID NO:3, CA17441-SEQ ID NO:4, CA17442-SEQ ID NO:5 and CA17443-SEQ ID NO: 6). Their encoding plasmids were transformed into E.coli WK 6-expressing strains to express and purify each nanobody of interest. The specificity of the FBA1 specific nanobodies was confirmed by attaching individual nanobodies to NHS-sepharose beads for co-immunoprecipitation assay. Beads functionalized with these FBA 1-specific nanobodies were incubated with lysates of EBY100 expressing native FBA1 or engineered yeast strains expressing GFP-tagged protein forms of FBA1 (GFP (+) 22, G1) on a spin device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (FIG. 4). The isolated proteins were also transferred onto PVDF membranes and western blots stained with GFP-specific antibodies to confirm the presence of GFP-tagged FBA1 (fig. 4). For each of the FBA 1-specific nanobodies analyzed in this way, an EBY100 lysate was used Giving a major band observed at 39kDa, corresponding to the expected molecular weight of FBA 1. In addition, a major band was observed at 66kDa when incubated with lysates of yeast strains expressing GFP-tagged protein forms of FBA1, which is consistent with the molecular weight of GFP-FBA1 (27kda+39kda=66 kDa). Western blot analysis with anti-GFP antibody confirmed that the 66kDa band contained a GFP tag. The identity of FBA1 was further confirmed by mass spectrometry of the relevant bands (39 kDa and 66 kDa).

We conclude from this example that the NANEX-based selection method allows easy selection of target specific antibodies from whole proteome antibody libraries.

Example 4 nanex was used to select target-specific binders in parallel from whole proteomic antibody libraries using GFP-specific collector/stripper pairs and GFP-tagged targets alone.

To demonstrate that the NANEX-based selection method as described herein is widely applicable for the discovery of antibodies against different targets, we selected 12 different specific binders of soluble yeast proteins from a whole proteomic antibody library in parallel (table 1). Thus, 12 engineered yeast strains were selected, each expressing another GFP-tagged protein of interest (collection of yeast GFP fusions, thermoFisher). These proteins were selected based on their abundance in Saccharomyces cerevisiae, from one of the most abundant proteins in yeast, FBA1 (YKL 060C), spanning to RIF2 (YLR 453C), a protein that controls telomere length and has less than one thousand molecules per cell (SGD Project http:// www.yeastgenome.org).

Table 1: properties of 12 GFP-tagged proteins used as representative targets of the soluble yeast proteome.

Standard nameWeighing scale	System name	Median abundance (molecules/cells)	Yeast GFP fusion Consortium reference
				FBA1	YKL060C	737009	GFP(+)22,G1
PDC1	YLR044C	581219	GFP(+)12,F8
				SSA1	YAL005C	255901	GFP(+)10,E4
PGI1	YBR196C	158738	GFP(+)12,H11
				ALD6	YPL061W	135000	GFP(+)15,F1
BMH1	YER177W	65471	GFP(+)27,D5
				ACC1	YNR016C	29049	GFP(+)01,B2
SIS1	YNL007C	28050	GFP(+)22,E5
				SXM1	YDR395W	7202	GFP(+)04,H9
top2	YNL088W	3960	GFP(+)01,H11
				TAM41	YGR046W	1666	GFP(+)20,A8
RIF2	YLR453C	976	GFP(+)20,E1

The GFP-specific capture agents introduced in example 1 were used to capture each GFP-tagged target separately on beads from cell lysates of the corresponding engineered yeast strain. These different beads are then incubated separately with a whole proteomic antibody library and the different GFP-tagged proteins of interest associated with the antigen-specific binding domain or antibody and its encoding genotype are selectively eluted using a GFP-specific stripper. Phage recovered with this GFP-specific stripper are then amplified to generate a new pool enriched in antibodies specific for each target protein or protein of interest (POI), allowing the cycle to be repeated until the POI-specific nanobodies predominate in the population to be characterized as monoclonal binders.

Generation of whole proteome antibody libraries. The same library described in example 3 was also used to perform the experiment described in example 4.

Discovery of novel POI-specific nanobodies. Similarly to examples 1, 2 and 3, we used NANEX to selectively capture 12 different GFP-POIs from lysates of engineered yeast strains (Huh et al, 2003) expressing different GFP-tagged protein forms of POIs (table 1) on 12 separate collector coated magnetic beads. After washing, these individual magnetic beads were combined with phage displaying whole proteome nanobody libraries (1,4.10 ¹⁴ Individual phages) were incubated together and washed again. In particular for this example, immobilized GFP-POIs associated with POI-specific nanobodies displayed on phage were then selectively eluted using GFP-specific stripper nanobodies. The selectively recovered phage were used to infect exponentially growing E.coli TG1 cells and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/mL ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g, and the cell pellet was resuspended in LB (supplemented with 100. Mu.g/mL ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Yeast lysates from reference strain EBY100 (which did not contain any GFP-tagged protein) were spiked with 20 μg of wild type GFP to serve as (negative) controls.

For each target selection of binders in this example, individual clones were isolated from the enriched sub-library by plating in LB 10-fold serial dilutions of phage-infected e.coli TG1 cells grown overnight after 3 rounds of selection by panning using the NANEX technique, as detailed in table 2. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family are selected. Their encoding plasmids were transformed into E.coli WK 6-expressing strains to express and purify each nanobody of interest. The specificity of the target-specific nanobodies was confirmed by attaching individual nanobodies to NHS-sepharose beads for co-immunoprecipitation assays. Beads functionalized with these target-specific nanobodies were incubated with lysates of either EBY100 lysates expressing the native target protein or engineered Yeast strains expressing the target protein in GFP-tagged protein form (Yeast GFP fusion accession numbers, as described in Table 2) on a rotating device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (as in the graph designated for each target in table 2). The isolated proteins were transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies to confirm the presence of GFP-labeled targets. For each target-specific nanobody analyzed in this way, co-immunoprecipitation with EBY100 lysate gave a major band corresponding to the expected molecular weight of the target protein. In addition, major bands were observed at MW consistent with the molecular weight of GFP-target when incubated with lysates of yeast strains expressing targets for GFP-tagged proteins. Western blot anti-GFP confirmed that the latter band contained a GFP tag.

Table 2. Yeast protein targets for selection of Nb binders (as described in example 4).

From example 4 we conclude that NANEX allows easy selection of target specific antibodies against several different targets from a whole proteomic antibody library.

Example 5. High throughput NANEX uses GFP-specific capture/stripper pairs and GFP-tagged targets alone for parallel selection of target-specific binders from whole proteomic antibody libraries.

Example 4 describes parallel selection of antigen-specific antibodies against different targets from a whole proteomic antibody library using NANEX with GFP-specific collector/stripper pairs. In example 5, we have scaled up this method in a parallel (high throughput) method for the discovery of antigen specific binders for 94 different GFP-tagged antigens from a whole proteomic antibody library. 96 different selections were performed in parallel using a KingFisher instrument (ThermoFisher Scientific), allowing most steps to be automated.

Thus, 94 representative yeast strains were selected based on MW and abundance of proteins, each expressing another GFP-tagged protein of interest (Yeast GFP fusion pool, thermoFisher) (Table 3). 2 negative controls (yeast without GFP fusion protein and lysis buffer without yeast lysate) were added to 96-well plates.

For examples 3 and 4, we immunized llamas with all soluble yeast proteins (soluble yeast proteomes) to generate an immune response against all soluble yeast proteins. The nanobody repertoire of immunized animals is displayed on phage to produce a whole proteomic antibody library.

GFP-specific collector CA15816 (SEQ ID NO: 2) introduced in example 1 was covalently attached to a solid support (magnetic beads) and distributed in 96 different wells. Each condition was used to capture each GFP-tagged target on beads separately from cell lysates of the corresponding engineered yeast strain. The captured GFP-labeled targets were incubated with the whole proteome antibody libraries from examples 3 and 4, respectively, and the different GFP-labeled proteins of interest associated with the antigen-specific binding domains or antibodies and their encoding genotypes were selectively eluted using the GFP-specific stripper CA12760 (SEQ ID NO: 1). Phage selectively recovered with the GFP-specific stripper are then amplified to generate a new pool enriched in antibodies specific for each POI, allowing the cycle to be repeated until the POI-specific nanobodies predominate, to be characterized as a population of monoclonal binders.

Table 3.94 representative yeast strains, each expressing GFP-tagged proteins of interest.

Information about Molecular Weight (MW) and estimated molecules/cells comes from SGD databasehttps:// www.yeastgenome.org/)。

Generation of whole proteome antibody libraries. The experiment described in example 5 was performed using the same library described in example 3.

Discovery of novel POI-specific nanobodies. Similarly to examples 2 to 4, we used NANEX to selectively capture 94 different GFP-POIs from lysates of engineered yeast strains (Huh et al, 2003) expressing different GFP-tagged protein forms of POI (table 3) on 94 separate collector coated magnetic beads. After washing, these individual magnetic beads were combined with phage displaying whole proteome nanobody libraries (1,4.10 ¹⁴ Individual phages) were incubated together and washed again. In particular for the present invention, immobilized GFP-POIs associated with the POI-specific nanobodies displayed on phage are then selectively eluted using GFP-specific stripper nanobodies. The eluted phages were used to infect exponentially growing E.coli TG1 cells and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/mL ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The following day, the culture was centrifuged at 3000g The cell pellet was resuspended in LB (supplemented with 100. Mu.g/ml ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Yeast lysates or lysis buffers (without yeast) from reference strain EBY100 (which did not contain any GFP-tagged proteins) were spiked with 20 μg of wild-type GFP to serve as (negative) controls. The 2 rounds of panning were sufficient to observe significant enrichment of almost all 94 different POI-specific phages (fig. 12).

For each target selection of binders in this example, individual clones were isolated from the enriched sub-library by plating in LB 10-fold serial dilutions of phage-infected e.coli TG1 cells grown overnight after 2 rounds of selection by panning using the NANEX technique, as detailed in table 3. 10 different POIs showing different enrichment in R2 were selected, for each POI 12 individual clones were selected and grown in 96-well plates containing LB supplemented with 100 μg/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into sequence families (materials and methods). Representative members of each sequence family are selected. Their encoding plasmids were transformed into E.coli WK 6-expressing strains to express and purify each nanobody of interest. The specificity of the target-nanobody was confirmed by attaching individual nanobodies to NHS-sepharose beads for co-immunoprecipitation assay. Beads functionalized with these target-specific nanobodies were incubated with lysates of engineered yeast strains (Yeast GFP Fusion Collection Reference, as shown in table 3) expressing GFP-tagged protein versions of the target proteins on a rotating device at 4 ℃ for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE. The isolated proteins were transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies (GFP mouse mAb (GF 28R), MA5-15256,ThermoFisher Scientific) to confirm the presence of GFP-labeled targets (fig. 13).

From example 5 we conclude that NANEX allows easy selection of target specific antibodies against several different targets in parallel on a high throughput scale from a whole proteomic antibody library.

Table 4. Yeast protein targets for selection and characterization of Nb binders (as described in example 5).

Example 6 NANEX novel yeast PGI 1-specific binding agents were selected from whole proteomic antibody libraries using PGI 1-specific collector/stripper pairs and endogenous PGI1 from crude yeast cell lysates.

Examples 1 to 5 use a collector/stripper pair to immobilize GFP or specific GFP-tagged targets as targets onto a substrate, followed by elution of this target associated with an antigen specific binding domain or antibody and its encoding genotype. In example 6 we show that the collector/stripper pair can bind directly to unlabeled target proteins other than GFP for immobilization onto a substrate and subsequent elution.

Generation of whole proteome antibody libraries. The experiment described in example 6 was performed using the same library described in example 3.

Discovery of novel PGI 1-specific nanobodies. Similar to examples 3, 4 and 5, we used NANEX to selectively capture POI from yeast lysates. Specifically, as described previously, PGI 1-specific nanobody CA17455 (SEQ ID NO: 15) was immobilized on magnetic beads, and used as a trapping agent. These CA17455 coated beads were incubated on a rotating device for 1 hour at 4 ℃ with either EBY100 lysates containing endogenous PGI1 target protein or lysates of engineered yeast strains expressing GFP-tagged protein forms of PGI1 (systematic name YBR196C, table 1) (yeast GFP fusion pool reference GFP (+) 12, H11) (Huh et al, 2003). After washing, these beads were combined with phage displaying whole proteome nanobody libraries (1,4.10 ¹⁴ Individual phages) were incubated together and washed again. Next, the same PGI 1-specific nanobody (herein acting as a stripper) was then used to selectively elute immobilized PGI1 associated with the PGI 1-specific nanobody displayed on phage. Selectively recovered phage for infection with exponentially growing E.coliTG1 cells were incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/ml ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g and the cell pellet resuspended in LB (supplemented with 100. Mu.g/ml ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Yeast lysis buffer (which does not contain any yeast protein) was used as a (negative) control. Two rounds of panning were sufficient to observe significant enrichment of PGI 1-specific phage.

Individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected e.coli TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family are selected. Six different PGI 1-specific nanobodies (Nb clones CA17791, CA17792-SEQ ID NO:47, CA17793-SEQ ID NO:48, CA17794-SEQ ID NO:49, CA17795-SEQ ID NO:50 and CA17796-SEQ ID NO: 51) derived from four different sequence families were selected based on their CDR3 sequences. Their encoding plasmids were transformed into E.coli WK 6-expressing strains to express and purify each nanobody of interest. The specificity of PGI 1-specific nanobodies was confirmed by ligating individual nanobodies onto NHS-sepharose beads for a co-immunoprecipitation assay. Beads functionalized with these PGI 1-specific nanobodies were incubated with EBY100 lysates expressing native (unlabeled) PGI1 or lysates of engineered yeast strains expressing GFP-tagged protein forms of PGI1 (GFP (+) 12, H11) on a rotating device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and analyzed on SDS-PAGE (FIG. 14A). The isolated proteins were also transferred onto PVDF membranes and western blots were developed with GFP-specific antibodies to confirm the presence of GFP-tagged PGI1 (fig. 14B). For each PGI 1-specific nanobody analyzed in this way, co-immunoprecipitation with EBY100 lysate gave a major band observed at 61kDa, corresponding to the expected molecular weight of PGI1. In addition, a major band was observed at 88kDa when incubated with lysates of yeast strains expressing GFP-tagged protein forms of PGI1, consistent with the molecular weight of GFP-PGI1 (27kda+61kda=88 kDa). Western blot analysis with anti-GFP antibody confirmed that the 88kDa band contained a GFP tag.

We conclude from this example that the NANEX-based selection method allows easy selection of target-specific antibodies from whole proteomic antibody libraries using target-specific collector/stripper pairs and natural (unlabeled) targets, wherein the natural targets are endogenously expressed and captured directly from crude cell lysates without any additional purification steps.

Furthermore, we have found that PGI1 is captured from yeast lysate and then eluted with a stripper, not only is PGI1 captured from yeast lysate, but a number of interacting proteins are co-eluted and can be identified as binding agents for PGI1, as captured herein from its cellular background. Specifically, as described above, PGI 1-specific nanobody CA17455 (SEQ ID NO: 15) was immobilized on magnetic beads as a trapping agent. These CA17455 coated beads were incubated with EBY100 lysate comprising endogenous PGI1 target protein on a rotating device at 4 ℃ for 1 hour and washed. Next, these coated beads were incubated with the same PGI 1-specific nanobody (used herein as a stripper) for 1 hour to elute the target protein (PGI 1), which showed several protein bands on SDS-PAGE (fig. 10), the identity of which was confirmed by mass spectrometry to be involved in several PGI1 interacting proteins (LYS 20 UniProt P48570, TDH3 UniProt P00359, PNC1 UniProt P53184) (fig. 10). This discovery supports that the method allows for the provision of target proteins in a physiological environment (including potential interaction partners) for selection of new binders from libraries.

Example 7 nanex uses rvgnlut 1 specific collector/stripper pairs and rvgnlut 1 from cell lysates to select membrane proteins as target specific novel nanobodies from antibody libraries.

Examples 1 to 6 used a collector/stripper pair to select binders for soluble proteins of interest. Here, we used our method to select from phage display nanobody libraries against immune libraries generated by membrane proteins, in particular rat vesicle glutamate transporter 1 (rvgclut 1, uniProt entry Q62634). In this example, rVGLUT 1-specific nanobody CA17875 (SEQ ID NO: 52) was immobilized on magnetic beads as previously described herein to act as a collector for the membrane protein rVGLUT1. The immobilized antigen is then incubated with a nanobody display library derived from an immunized animal. In particular for the present invention, the same nanobody is used as a collector and then as a stripper to selectively elute immobilized rVGLUT1 associated with target-specific nanobody displayed on phage. Phage selectively recovered with the rVGLUT1 specific stripper are then amplified to generate a new pool of nanobodies enriched in rVGLUT1 specificity.

Generation of rVGLUT1 antibody libraries. Rat VGLUT1 was purified and llama immunization was performed as described in Schenck et al, 2017. After immunization, phage display libraries were generated as described (materials and methods).

Discovery of novel rVGLUT1 specific nanobodies. Full-length rat VGLUT1 was generated in HEK293T cells as disclosed (Schenck et al, 2017) and cells were lysed in 5mL ice-cold lysis buffer (250mM NaCl,25mM HEPES pH 7.5, 10% glycerol, 2% DDM) supplemented with protease inhibitors by incubation on a rotating device for 1h at 4 ℃. The lysate was clarified by centrifugation at 20,000g for 20 min. The supernatant containing the target was collected and incubated with 5. Mu.L of magnetic beads covalently functionalized with rVGLUT1 specific nanobody CA17875 (SEQ ID NO: 52) on a spin device at 4℃for 1h to immobilize rVGLUT1 on these beads. The beads were collected using a magnet and washed with wash buffer (150 mM NaCl, 20mM HEPES pH 7.5, 10% glycerol and 0.03% DDM) and then they were incubated at 4℃with phage displaying rVGLUT1 nanobody library (1,4.10) ¹⁴ Individual phages) were incubated for 1h. Next, the beads were washed 9 times with 0.5mL of wash buffer. In particular for the present invention, the same rVGLUT1 specific nanobody (herein acting as a stripper) is then used to selectively elute and phage rVGLUT 1-specific nanobody-associated immobilized rVGLUT1 displayed on body. The eluted phage were used to infect exponentially growing E.coli TGI cells and incubated at 37℃for 30 minutes without shaking. Next, LB medium (supplemented with 100. Mu.g/ml ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g and the cell pellet resuspended in LB (supplemented with 100. Mu.g/ml ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. The lysis buffer (which does not contain any target protein) was used as a (negative) control.

After 2 rounds of selection by panning using the NANEX technique, individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). A representative member of the family of sequences of interest was selected and the encoding plasmid was transformed into an E.coli WK6 expressing strain for expression and purification (Nb clone CA18425 corresponds to SEQ ID NO: 53). The specificity of rVGLUT1 specific nanobody was verified by co-immunoprecipitation assay by attaching it to NHS-sepharose beads. Beads functionalized with the rVGLUT1 specific nanobodies were incubated with lysates of HEK293T cells transfected with rVGLUT1 labeled with Venus-YFP at the C-terminus and containing a C-Myc tag on a spin device for 1 hour at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and loaded onto SDS-PAGE. The isolated proteins were transferred onto PVDF membrane and Western blots were developed with c-Myc specific antibodies to confirm the presence of c-Myc labeled rVGLUT1 (88 kDa for rVGLUT1-c-Myc-YFP construct, FIG. 15).

From this example we conclude that the NANEX-based selection method allows selection of target specific antibodies from an antibody library using a specific collector/stripper pair and a membrane target protein.

Example 8 nanex a new rvguet 1 specific nanobody was selected from a synapse proteome antibody library using GFP-specific collector/stripper pair and YFP-labeled target protein.

The GFP-specific capture/stripper pair introduced in example 1 can also be used to capture Yellow Fluorescent Protein (YFP) labeled targets. Since the introduction of mutations into GFP to produce YFP proteins does not alter the epitope important for collector/stripper binding, the GFP-specific collector/stripper pair can be used to efficiently purify YFP-tagged proteins from complex mixtures (e.g., cell lysates) and to selectively elute YFP-tagged proteins of interest from the matrix (beads, plates, etc.). Here we phage display a nanobody repertoire of an immune library generated against the synaptic proteome by immunizing llama with mouse brain extract enriched with synaptic vesicles containing the vesicle glutamate transporter 1 (VGLUT 1) as well as other membrane proteins. In parallel, a GFP-specific capture reagent CA15816 (SEQ ID NO: 2) covalently attached to a solid support was used to immobilize the C-terminal Venus-YFP labeled version of rat VGLUT1. The immobilized antigen (rVGLUT 1-YFP) was then incubated with a nanobody display library derived from the immunized animal. Similar to NANEX and is the invention in particular, immobilized rVGLUT1-YFP associated with target-specific nanobodies displayed on phage is then selectively eluted using GFP-specific stripper CA12760 (SEQ ID NO: 1) competing with the collector.

Generation of synaptic proteome antibody libraries. Mouse brain extracts enriched in synaptic vesicles were prepared as described previously (Takamori et al, 2006). Llamas were immunized weekly over a period of 6 weeks and phage display libraries were generated as described (materials and methods).

Discovery of novel VGLUT1 specific nanobodies. Full-length rat VGLUT1 is produced in HEK293T cells as a C-terminal Venus-YFP tagged protein as disclosed (Schenck et al, 2017). Cells were lysed in 5mL ice-cold lysis buffer (250mM NaCl,25mM HEPES pH 7.5, 10% glycerol, 2% ddm) supplemented with protease inhibitors by incubation on a rotating device for 1h at 4 ℃. The lysate is clarified by centrifugation at 20,000g for 20 min. Collecting the content ofThe supernatant of the target was incubated with 5. Mu.L of magnetic beads covalently functionalized with GFP-specific nanobody collector CA15816 (SEQ ID NO: 2) at 4℃for 1h on a spin device, and YFP-labeled rVGLUT1 was immobilized on these beads. Beads were collected using magnet and washed with wash buffer (150 mM NaCl, 20mM HEPES pH 7.5, 10% glycerol and 0.03% DDM) and then they were incubated at 4℃with phage displaying a library of synaptic proteome nanobodies (1,4.10) ¹⁴ Individual phages) were incubated for 1h. Next, the beads were washed 9 times with 0.5mL of wash buffer. Specifically for this example, immobilized rVGLUT1-YFP associated with rVGLUT 1-specific nanobody displayed on phage was then selectively eluted using GFP-specific stripper nanobody CA12760 (SEQ NO: 1). Even with mouse brain extracts, VGLUT1 from mice and rats differs in its sequence by only 1 amino acid, so cross-reactive binders are expected to be present in the mouse-derived library. The eluted phage were used to infect exponentially growing E.coli TG1 cells and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/ml ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g and the cell pellet resuspended in LB (supplemented with 100. Mu.g/mL ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Lysis buffer (which did not contain any target protein) was spiked with 6 μg of wild-type GFP to serve as (negative) control.

After 2 rounds of selection by panning using the NANEX technique, individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family were selected and encoding plasmids were transformed into E.coli WK6 expressing strains to express and purify five nanobodies of interest (Nb clone CA18024 corresponds to SEQ ID NO:54, CA 184637-SEQ ID NO:55, CA18438-SEQ ID NO:56, CA18439-SEQ ID NO:57, CA 1840-SEQ ID NO:58 and CA18441-SEQ ID NO: 59). Specificity of rVGLUT1 specific nanobody was verified by co-immunoprecipitation assay by attaching individual nanobodies to NHS-sepharose beads. Beads functionalized with these rVGLUT1 specific nanobodies were incubated with lysates of HEK293T cells transfected with rVGLUT1-YFP, which also contains a c-Myc tag, on a spin device at 4℃for 1 hour. After washing, the beads were resuspended in SDS-PAGE loading dye and loaded onto SDS-PAGE. The isolated proteins were transferred to PVDF membrane and Western blots were developed with c-Myc specific antibodies to confirm the presence of c-Myc labeled rVGLUT1-YFP (88 kDa, FIG. 16).

From this example we conclude that the NANEX-based selection method allows selection of a set of new antibodies specific for membrane protein targets from a whole proteome antibody library using a specific GFP-collector/stripper pair and YFP-tagged membrane proteins as targets.

Example 9 nanex new target-specific nanobodies were selected from antibody libraries using mCherry specific collector/stripper pairs and mCherry labeled target proteins.

Examples 1 to 5 and 8 used a GFP-specific collector/stripper pair to capture GFP, GFP-tagged proteins of interest or GFP variants, such as YFP, for NANEX-based selection. In example 9, a capture/stripper pair specific for the mCherry tag (Shaner et al, 2004) was used to capture soluble proteins of interest from complex mixtures and selectively elute the proteins of interest from the matrix (beads, plates, etc.). Here, our phage displayed a nanobody repertoire of an immune library generated by immunizing llamas against the NANEX purified, cross-linked, full-length, GFP-tagged human Glucocorticoid Receptor (GR). In parallel, the mCherry-labeled form of full-length GR was immobilized using mCherry-specific collector Nb clone CA16964 (SEQ ID NO: 60) covalently attached to a solid support. The immobilized antigen (mCherry-GR) was then incubated with a nanobody display library derived from an immunized animal. Similar to NANEX, the mCherry-specific stripper Nb clone CA17302 (SEQ ID NO: 61) competing with the collector Nb was then used to selectively elute and phages Immobilized mCherry-GR associated with GR specific nanobodies displayed on body. Phage selectively eluted with the mCherry specific stripper were then amplified to create a new pool enriched in GR specific nanobodies, allowing the cycle to be repeated until GR specific nanobodies predominated to characterize the population as monoclonal GR binders.Generation of full Length GR antibody library. Constructs encoding human glucocorticoid receptor (NR 3C 1) labeled with GFP were used to express recombinant GFP-GR in HEK293T cells after transient transfection. 48 hours after transfection, cells were collected and homogenized using a Dounce homogenizer at 10mM HEPES pH 7.4, 10% glycerol, 20mM sodium molybdate, 50. Mu.g/ml DNase I, 10. Mu.M ZnCl ₂ 、2.5mM MgCl ₂ Cleavage in 2.5mM DTT, 0.5% NP40 replacement and 1 tablet of Complete EDTA-free protease inhibitor. GFP-GR was purified to homogeneous soluble protein by NANEX purification using a pair involving GFP-specific collector CA15816 (SEQ ID NO: 2) and stripper CA12760 (SEQ ID NO: 1). GFP-GR eluate was used to immunize llamas weekly over a period of 6 weeks. After immunization, phage display libraries were generated as described (materials and methods).

Discovery of novel GR specific nanobodies . The full length gene encoding the human glucocorticoid receptor (NR 3C 1) was introduced into a pcdna3.1 expression vector to produce mCherry-tagged target fusion. The vector was transfected into HEK293T cells using Polyethylenimine (PEI) as a transfection agent. At 48 hours post-transfection, 5000 ten thousand cells were harvested, washed with ice-cold PBS buffer and washed with Dounce homogenizer in 5mL lysis buffer (10mM Hepes pH 7.4, 10% glycerol, 20mM sodium molybdate, 10. Mu.M ZnCl) supplemented with 50. Mu.g/mL DNase I and protease inhibitor ₂ 、2.5mM MgCl ₂ 2.5mM DTT, 0.5% NP40 surrogate). The lysate was clarified by centrifugation at 20,000 g. Supernatant containing mCherry-labeled GR was collected and incubated with 5. Mu.L of magnetic beads covalently functionalized with mCherry-specific collector with the reference name CA16964 (SEQ ID NO: 60) on a spin device at 4℃for 1h to immobilize mCherry-labeled GR on these beads. These beads were collected and used with a KingFisher ^TM Flex purification System (thermo scientific) with 0.5mL wash buffer (10mM HEPES pH 7.4, 10% glycerol, 20mM sodium molybdate, 10. Mu.M ZnCl) ₂ 2.5mM DTT, 0.05% Tween 20) and then washed at 4℃with phage displaying GFP-tagged full-length GR nanobody library (1,4.10) ¹⁴ Individual phages) were incubated for 1h. Next, the beads were washed 9 times with 0.5mL of wash buffer. In particular for the present invention, immobilized mCherry-GR associated with GR-specific nanobody displayed on phage was then selectively eluted using mCherry-specific stripper nanobody CA17302 (SEQ ID NO: 61). The exponentially growing E.coli TG1 cells were infected with the eluted mCherry-GR target protein containing the associated phage and incubated at 37℃for 30min without shaking. Next, LB medium (supplemented with 100. Mu.g/mL ampicillin and 2% wt/vol glucose) was added, and the culture was grown overnight at 37 ℃. The next day, the culture was centrifuged at 3000g and the cell pellet resuspended in LB (supplemented with 100. Mu.g/mL ampicillin and 20% glycerol (vol/vol)) and stored as a glycerol stock at-80℃for later use. Untransfected HEK293T lysates (which contained no mCherry-tagged protein) were spiked with 10 μg of wild-type mCherry (mCherry, escherichia coli recombinant protein, TP790040, origin) for use as (negative) controls.

After three rounds of selection by panning using the NANEX technique, individual clones were isolated from the enriched sub-library by plating ten-fold serial dilutions of phage-infected TG1 cells grown overnight in LB. 96 individual clones were picked and grown in 96-well plates containing LB supplemented with 100. Mu.g/ml ampicillin. Plasmids containing nanobody encoding genes were purified, sequenced and grouped into families of sequences (materials and methods). Representative members of each sequence family were selected and encoding plasmids were transformed into E.coli WK6 expressing strains to express and purify seven nanobodies of interest (Nb clone CA18498 corresponds to SEQ ID NO:62, CA18499-SEQ ID NO:63, CA18501-SEQ ID NO:64, CA18502-SEQ ID NO:65, CA18503-SEQ ID NO:66, CA18585-SEQ ID NO:67, CA18586-SEQ ID NO: 68). The specificity of GR specific nanobodies was verified by attaching individual nanobodies to NHS-sepharose beads for a co-immunoprecipitation assay. Beads functionalized with these GR specific nanobodies were incubated with lysates of HEK293T cells transfected with mCherry-GR expression vectors for 1h on a spin device at 4 ℃. After washing, the beads were resuspended in SDS-PAGE loading dye and loaded onto SDS-PAGE. The isolated proteins were transferred onto PVDF membrane and Western blots were developed with GR specific antibodies (GR (G-5) mouse IgG2b mAb, sc-393232,Santa Cruz Biotechnology) to confirm the presence of mCherry-labeled GR (FIG. 17).

From this example we conclude that the NANEX-based selection method allows easy selection of target-specific antibodies from an antibody library using mCherry specific collector/stripper pairs and mCherry labeled target proteins.

Example 10 NANEX in plate format new GFP-specific nanobodies were selected from antibody libraries using GFP-specific collector/stripper pairs and GFP protein as targets.

In examples 1 to 9, magnetic beads were usedMyone ^TM Tosylactiva, thermo fisher) to make NANEX-based selections. To demonstrate that the NANEX-based selection methods as described herein are widely applicable to different types of matrices, in this example we show that the collector/stripper pair can also be used to immobilize a specific target on the surface of a plastic plate by non-covalent interactions and elute this target associated with the antigen-specific binding domain of an antibody and its encoding genotype.

Production of GFP antibody libraries. The same library described in example 1 was also used to perform the experiment described in example 10.

Discovery of novel GFP-specific nanobodies. To find new GFP-specific nanobodies in plate format, we immobilized biotinylated GFP-specific collector reference CA15816 (SEQ ID NO: 2) on a neutral avidin coated flat bottom 96-well plate (Nunc Immuno Plate F Maxisorp, 43954, thermoFisher) as compared to magnetic beads. The collector coated wells were blocked with 4% milk in PBS and used PBS was washed 5 times, and then 100. Mu.L of 100nM GFP (SEQ ID NO: 25) was added. The collector coated wells not incubated with GFP served as negative controls. After incubation with GFP (antigen), all wells were routinely washed 5 times with PBS and incubated with GFP antibody libraries displayed on phage. After incubation for 1h 30min at room temperature, the plates were incubated with PBS-Tween ₂₀ 0.05% regular wash 15 times. To select for new GFP-specific antibodies, the non-covalent interactions between the collector and GFP are selectively disrupted by the addition of a high affinity stripper CA12760 (SEQ ID NO: 1) that competitively binds to an epitope overlapping the collector epitope. Thus, wells were incubated for 30min with high affinity CA12760 (GFP-specific stripper) that bound the same epitope as the collector on GFP.

For round 2 selection using NANEX, we followed the same strategy, using the output phage obtained from the first round. Four different nanobody families were identified based on their CDR3 sequences. Of these, the three largest families are identical to the three families found and identified for GFP binding by Biological Layer Interferometry (BLI) in example 1 (CA 17518-SEQ ID NO:27, CA17520-SEQ ID NO:29, CA 176774-SEQ ID NO: 31).

From this example we conclude that the NANEX-based selection method allows easy selection of target specific antibodies from an antibody library using different types of matrices coating target specific collector nanobodies.

Materials and methods

And (3) cells.

EBY100(ATCC*MYA-4941")

Genotype: MATA AGA1:: GAL1AGA1:: URA3 URA352 trp1 leu2delta200 his3delta200 pep4:: HIS3prbd1.6R can1 GAL

Yeast GFP fusion pool (Yeast GFP Fusion Collection), thermoFisher, catalog number: 95702, (Huh et al, 2003).

Coli TG1 (electrocompetent cells; lucigen, catalog No. 60502-1) was used to clone and prepare phage libraries.

Coli WK6 non-inhibitory strain (su-) (Zell et al, 1987) for expression of nanobodies.

HEK 293T(ATCC*293T CRL-3216 ^TM )

Monoclonal antibodies for Western blotting。

anti-GFP: mouse mAb (GF 28R), MA5-15256,ThermoFisher Scientific.

anti-GR (G-5) mouse IgG2b mAb, sc-393232,Santa Cruz Biotechnology.

anti-c-Myc: mouse mAb (clone 9E 10), 11667203001, roche.

Nanobody coated magnetic beads for panning. Magnetic beads coated with GFP-specific collector nanobodies (CA 15816) according to the manufacturer's protocolMyOne ^TM Tosylactiva, thermo fisher) to make NANEX-based selections. 2mg of purified CA15816 was coated on 50mg of magnetic beads (. About.40. Mu.g antibody/mg beads) and then the beads were resuspended in 1mL of PBS.

Yeast lysates for NANEX-based selection. Each selected yeast clone was grown in 200mL YPB medium at 30℃for 72h (shaking at 175 rpm). Cells were collected by centrifugation at 4000rpm for 5 minutes. Cell pellet was weighed to normalize all different lysates. Yeast lysis reagent Yper (Y-PER) ^TM PLUS, yeast protein extractant, thermoFisher) cell lysates were prepared, 1g of cell pellet was resuspended in 2.5mL Yper (supplemented with 1mM DTT and protease inhibitor without EDTA) and incubated for 1 hour at 37 ℃. The cell lysate was centrifuged at 20,000g for 10 min, and the soluble fraction was collected and stored at-80 ℃.

Negative control. Yeast EBY100 was grown in 200mL YPB medium at 30℃for 72h (shaking at 175 rpm). Cells were collected by centrifugation at 4000rpm for 5 minutes. Yeast lysis reagent Yper (Y-PER) ^TM PLUS, yeast protein extraction reagent, thermoFisher) prepared cell lysates, 1g of cell pellet was resuspended in 2.5mL Yper (supplemented with 1mM DTT and protease inhibitor without EDTA) and incubated for 1 hour at 37 ℃. The cells are then culturedThe lysate was centrifuged at 20,000g for 10 min, and the soluble fraction was collected and stored at-80 ℃.

Generation of antibody libraries . The antigen, protein or proteome of interest is used to immunize a llama. After immunization, blood samples are collected to clone a diverse collection of affinity matured nanobodies with target or protein specificity of interest. Peripheral Blood Lymphocytes (PBLs) are isolated from non-coagulated blood for purification of total RNA and cDNA synthesis. The cDNA was used as a template to amplify the open reading frame encoding the variable domain (Nb) of the heavy chain antibody and the Nb fragment was cloned into a suitable phage display vector and phage particles were prepared according to Pardon et al, 2014.

Selection procedure for GFP-nanobodies. Panning or selection of new GFP nanobodies was performed primarily as described (Pardon et al, 2014) and the capture and elution steps were adjusted. Briefly, GFP-collector coated beads were used and incubated with GFP at various concentrations ranging from 0.1nM to 100 nM. GFP was trapped on these NANEX beads in a total volume of 100 μl, after which all beads were routinely washed 3 times. Next, these beads were incubated in 96-well plates with GFP antibody libraries displayed on phage. After incubation for 2h at 4℃the beads were routinely washed 12 times with PBS-Tween. To elute GFP-specific phage, the beads were incubated with 20. Mu.M high affinity CA12760 (GFP-stripper) that bound the same epitope on GFP in a total volume of 100. Mu.L for 30 minutes, as the collector or phage was non-specifically eluted from the beads with trypsin (250. Mu.g/mL) for 30 minutes.

^TM Parallel selection procedure using KinaFisher Flex purification System (thermo scientific). For panning experiments, 300 μl of CA15816 coated magnetic beads were resuspended in 2200 μl PBS/4% skim milk and blocked overnight at 4 ℃ on a rotating device. Prior to panning, the beads were washed twice in PBS using a magnet. The beads were then resuspended in 480 μl PBS.

12 different yeast lysates were thawed and 400. Mu.L of each lysate was added to wells of a 96-deep well plate. As a negative control for each panning, 20. Mu.g of purified GFP was used to supplement 400. Mu.L of EBY100 strain lysate. To each well 20 μl of pre-blocked CA15816 coated magnetic beads was added. The beads and lysate were incubated on a shaking platform at 4℃for 1h30min.

Using KingFisher ^TM The Flex purification system was used for the next selection using 96-well plates. The magnetic beads from each well were harvested and washed with 500. Mu.L PBS-Tween for 30s. Next, these beads were incubated with phage-displayed whole proteome antibody library for 1h30, then washed with 500. Mu.L PBS-Tween 9 times for 30s each. To elute GFP-tagged antigen-specific phage, the beads were incubated for 30min in a total volume of 100 μl with 20 μΜ high affinity CA12760 (GFP-stripper) that binds the same epitope on the collector on GFP.

Identification of selected nanobody binders. The plasmid containing the nanobody encoding gene was purified and then sequenced using MP57 as a sequencing primer. Will have similar CDR3 sequences (same length and>nanobodies of 80% sequence identity are grouped into a family of sequences (Pardon et al, 2014). Nanobodies from the same sequence family are known to be derived from the same B cell lineage and bind the same epitope on the target.

Nanobody coated sepharose beads for antigen co-immunoprecipitation. Antigen-specific nanobody beads were prepared using NHS-Activated Sepharose 4Fast Flow (cytova) and purified nanobody. Coupling on the beads was performed according to the manufacturer's protocol. 0.5mg of purified nanobody was coupled to 150. Mu.L of bead, and then resuspended in 0.5mL PBS.

Biotinylation. GFP (SEQ ID NO: 25) and GFP-specific Nb CA15816 (SEQ ID NO: 2) were biotinylated for BLI or NANEX selection, respectively, by using the Thermo Scientific EZ-Link sulfo-NHS-LC-biotinylation kit according to the manufacturer's instructions.

Epitope mapping of GFP nanobodies by Biological Layer Interferometry (BLI). Streptavidin-coatedBiosensor for capturing Biotinylated GFP (100 nM). Unbound biotinylated GFP was washed from the biosensor by two washing steps (60 seconds in buffer). Next, streptavidin coated with GFP ++>The biosensor was first incubated with 20nM Ca12760 (GFP stripper) for 400 seconds, briefly washed, and then incubated in a premix of 20nM Ca12760 with the different Nb to be tested for 400 seconds. Data were analyzed on OctetRed (Molecular Devices). All assays were supplemented with BSA 0.1% and Tween at room temperature ₂₀ 0.005% HEPES25mM pH 7.5, naCl 150 mM.

Sequence listing

The amino acid sequences as set forth in SEQ ID NOS.1-68 provide the binders named therein (except for SEQ ID NO: 25) and are used in the examples, including the C-terminal 6XHis tag (also referred to herein as His tag) and the EPEA tag, as set forth. Further embodiments also include the use of the amino acid binding molecules having the VHH sequences of SEQ ID NO:1 and SEQ ID NO:2 (e.g., SEQ ID NO:70 and SEQ ID NO: 71), respectively, in the absence of a tag, or having an alternative tag, substituted or appended to the 6xHis and EPEA tags, linked at the N-terminus or C-terminus to the remainder of the amino acid sequence.

SEQ ID NO. 1:CA12760 GFP-stripper amino acid sequence (including C-terminal 6XHis+EPEA tag)

>SEQ ID NO. 2-CA 15816T 54A/V55A mutant GFP-collector amino acid sequence (mutant residues areAdding and removing Bold drawn lineThe method comprises the steps of carrying out a first treatment on the surface of the C-terminal 6xHis+EPEA)

SEQ ID NO. 3:17440 FBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 4:CA17441 FBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 5:17442 FBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 6:17443 FBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 7:CA17451 PDC1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 8:17452 PDC1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 9:17444 SIS1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 10:17453 ALD6 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 11:CA17454 ALD6 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 12:CA17460 ALD6 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO. 13:17458 BMH1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 14:17459 BMH1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 15:17455 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 16:17456 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

17:CA17457 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 18:CA17530 SXM1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 19:CA17560 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 20:CA17561 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 21:CA17562 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 22:CA17563 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 23:CA17564 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 24:CA17565 SSA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 25 GFP protein

SEQ ID NO. 26:CA17517 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 27:CA17518 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 28:CA17519 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 29:CA17520 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 30:CA17673GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 31:CA17674 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 32:CA17675 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 33:CA17676 GFP binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 34:CA8780 unrelated binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 35:CA17797 GR-LBD binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 36:CA17798 GR-LBD binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 37:CA17799 GR-LBD binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 38:CA17800 GR-LBD binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 39:CA17801 GR-LBD binding agent amino acid sequence (His-tag and EPEA-tag)

40:CA18504 HSP104 binding agent amino acid sequence (His-tag and EPEA-tag)

SEQ ID NO. 41: CA18505 MET6 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 42:CA18508 SBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 43:CA18509 SBA1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 44:CA18510 SOD1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 45:CA17938 ENO1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 46:CA17791 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 47:CA17792 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 48:CA17793 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 49:CA17794 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 50:CA17795 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 51:CA17796 PGI1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 52:CA17875 VGLUT1 binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO 53:CA18425 VGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 54:CA18024 VGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 55:CA18437 rVGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 56:CA18438 rVGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 57:CA18439 rVGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 58:CA18440 rVGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 59:CA18441 rVGLUT1 binding agent amino acid sequence (with His tag and EPEA tag)

60:CA16964 mCherry-collector binding agent amino acid sequence (His-tag and EPEA-tag)

61:CA17302 mCherry-stripper binding agent amino acid sequence (His-tag and EPEA-tag)

SEQ ID NO. 62:CA18498 GR binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 63:CA18499 GR binding agent amino acid sequence (with His-tag and EPEA-tag)

SEQ ID NO. 64:CA18501 GR binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO. 65:CA18502 GR binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 66: CA18503 GR binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 67: CA18585 GR binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO. 68:CA18586 GR binding agent amino acid sequence (with His tag and EPEA tag)

SEQ ID NO 69:6XHis-EPEA tag

SEQ ID NO. 70:CA12760 GFP-stripper VHH amino acid sequence

SEQ ID NO 71-amino acid sequence of CA15816 GFP-collector VHH

Reference to the literature

Almagro, J.C. et al, (2019) Phage display libraries for antibody therapeutic discovery and development.antibodies 8:44.

Huh WK et al, (2003) Global analysis of protein localization in budding eye. Nature 425 (6959): 686-91.Lakzaei et al (2018) A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin. J. Cell Physiol.234 (6), p.9486-9494.

Li et al, (2020) An unbiased immunization strategy results in the identification of enolase as a potential marker for Nanobody-based detection of Trypanosoma evansi.Vaccines 8,415.

Lim et al, (2019) Development of a Phage Display Panning Strategy Utilizing Crude Antigens: isolation of MERS-CoV Nucleoprotein human anti-diagnostics, scientific Reports 9,6088.

Pardon et al, (2014) A general protocol for the generation of Nanobodies for structural biology. Nature protocols.9:674-693.

Schenck et al, (2017) Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of transport biochemistry 56 (30): 3962-3971

Shaner et al, (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp.red fluorescence protein Nat Biotechnol 22,1567-1572.

Takamori et al, (2006) Molecular anatomy of a trafficking organic. Cell 127 (4): 671-3.

Valdorf et al, (2021) Antibody display technologies: selecting the cream of the crop. Biol. Chem. Https:// doi. Org/10.1515/hsz-2020-0377.

Zell et al, (1987) DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of-methyl-cytosine residues EMBO J.6,1809-1815

Sequence listing

<110> non-profit organization of the national institute of general college of biotechnology

University of Brussell free

<120> means and methods for selecting specific binding agents

<130> JaSt/NANEX-PANNING/728

<150> EP 21181272.2

<151> 2021-06-23

<150> EP 21181405.8

<151> 2021-06-24

<160> 71

<170> PatentIn version 3.5

<210> 1

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA12760 GFP-stripper amino acid sequence

<400> 1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Ala

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val

35 40 45

Ala Gly Ile Tyr Trp Thr Val Gly Ser Thr Tyr Tyr Ala Asp Ser Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Arg Arg Gly Phe Thr Leu Ala Pro Thr Arg Ala Asn Glu

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 2

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA 15816T 54A/V55A mutant GFP-collector amino acid sequence

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Ala

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val

35 40 45

Ala Gly Ile Tyr Trp Ala Ala Gly Ser Thr Tyr Tyr Ala Asp Ser Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Arg Arg Gly Phe Thr Leu Ala Pro Thr Arg Ala Asn Glu

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 3

<211> 129

<212> PRT

<213> artificial sequence

<220>

<223> CA17440 FBA1 binding Agents

<400> 3

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Ala

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ile Thr Trp Ser Gly Ala Gly Val Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gln Ala Leu Ser Gln Gly Asp Phe Gly Ser Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu

115 120 125

Ala

<210> 4

<211> 141

<212> PRT

<213> artificial sequence

<220>

<223> CA17441 FBA1 binding agent amino acid sequence

<400> 4

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Val Thr Glu Gly Val Gly Ala Gly Asn Cys Gly Val Ser Asp

100 105 110

Leu Asp Gly Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr

115 120 125

Val Ser Ser His His His His His His Glu Pro Glu Ala

130 135 140

<210> 5

<211> 129

<212> PRT

<213> artificial sequence

<220>

<223> CA17442 FBA1 binding agent amino acid sequence

<400> 5

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Thr

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Ser Gly Lys Glu Arg Glu Phe Leu

35 40 45

Ala Arg Ile Thr Trp Ser Gly Ala Gly Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gln Gly Leu Asp Gln Arg Asp Phe Ser Ser Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu

115 120 125

Ala

<210> 6

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> CA17443 FBA1 binding agent amino acid sequence

<400> 6

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Leu Arg Ile Tyr

20 25 30

Ser Met Gly Trp Ser Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ser Ile Thr Arg Ala Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Asn Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn

85 90 95

Ala Asn Pro Val Tyr Asp Gly Ser Ala Gly Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 7

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> CA17451 PDC1 binding agent amino acid sequence

<400> 7

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ser Phe Ser Ile Asn

20 25 30

Thr Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Val Thr Thr Gly Gly Ser Thr Tyr Tyr Val Asp Ser Ala Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Leu Glu Arg Tyr Gly Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser

100 105 110

Ser His His His His His His Glu Pro Glu Ala

115 120

<210> 8

<211> 130

<212> PRT

<213> artificial sequence

<220>

<223> CA17452 PDC1 binding agent amino acid sequence

<400> 8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Thr Tyr

20 25 30

Ser Met His Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Thr Asn Trp Gly Gly Asn Ser Lys Phe Tyr Ala Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala His Arg Thr Val Val Gly Lys Thr Pro Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro

115 120 125

Glu Ala

130

<210> 9

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> CA17444 SIS1 binding agent amino acid sequence

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Val Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Gln Ile Thr Ser Thr Gly Cys Pro Asp Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His

115 120 125

Glu Pro Glu Ala

130

<210> 10

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> CA17453 ALD6 binding agent amino acid sequence

<400> 10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ile Arg Ser

20 25 30

Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Ala Leu Val Ala Ser

35 40 45

Ile Phe Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg

50 55 60

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ile Leu Glu Met

65 70 75 80

Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Arg

85 90 95

Val Arg Ser Gly Thr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser His His His His His His Glu Pro Glu Ala

115 120

<210> 11

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> CA17454 ALD6 binding agent amino acid sequence

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Arg Asn

20 25 30

Tyr Ile Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Arg Lys Thr Gln Ser Gly Ala Thr Val Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Thr Asn Asn Ala Lys Asn Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ile Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Gly Leu Ile Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser His His His His His His Glu Pro Glu Ala

115 120

<210> 12

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> CA17460 ALD6 binding agent amino acid sequence

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Thr Asn

20 25 30

Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Lys Tyr Ser Thr Gly Gly Thr Thr Tyr Ala Asn Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Gly

85 90 95

Gly Ala Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser His

100 105 110

His His His His His Glu Pro Glu Ala

115 120

<210> 13

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> CA17458 BMH1 binding agent amino acid sequence

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Leu Ser Leu Arg

20 25 30

Arg Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Thr Leu Thr Ser Gly Gly Glu Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ile Asn Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 14

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA17459 BMH1 binding agent amino acid sequence

<400> 14

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Arg Leu Asn Phe Gly

20 25 30

Ser Trp Asn Trp Tyr Arg Lys Arg Gly Glu Asn Tyr Arg Glu Leu Val

35 40 45

Ala Ala Met Gly Pro Ser Thr Gly His Thr His Tyr Gly Asp Ser Met

50 55 60

Lys Gly Arg Val Thr Ile Ser Leu Asn Ala Ala Lys Asp Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Asp Gly Leu Gly Phe Pro Arg Gly Ser Phe Tyr Gly

100 105 110

Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 15

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> CA17455 PGI1 binding agent amino acid sequence

<400> 15

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Pro Ser Tyr

20 25 30

Arg Leu Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Asp Gly Gly Thr Thr Asn Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Lys Ala Gln Tyr Phe Ser Thr Pro Leu Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 16

<211> 129

<212> PRT

<213> artificial sequence

<220>

<223> CA17456 PGI1 binding agent amino acid sequence

<400> 16

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Val Thr Ile Asn

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ala Thr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Leu Arg Gly Thr Gly Tyr Asn Tyr Gly Glu Asn Tyr Trp Gly Lys Gly

100 105 110

Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu

115 120 125

Ala

<210> 17

<211> 142

<212> PRT

<213> artificial sequence

<220>

<223> CA17457 PGI1 binding agent amino acid sequence

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly His Phe Ser Asn Leu

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Gly Arg Ser Gln Phe Asn Leu Leu Tyr Ser Asn Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Asn Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ser Ser Gly Gly Val Ser Asp Trp Arg Gly Ile Met Thr Thr Gly

100 105 110

Ala Arg Leu Ala Arg Asn Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val

115 120 125

Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

130 135 140

<210> 18

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> CA17530 SXM1 Binder amino acid sequence

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Lys Thr Leu Phe Ser Ile Asn

20 25 30

Ala Met Ala Trp Tyr Arg Gln Ser Pro Gly Lys Pro Arg Glu Leu Val

35 40 45

Ala Gly Trp Thr Ser Gly Gly Arg Thr Ser Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Asp Val Lys Pro Glu Asp Thr Ala Val Tyr His Cys Ala

85 90 95

Ala Asp Tyr Leu Val Val Ala Gly Lys Pro Gly Tyr Asp Tyr Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His

115 120 125

His Glu Pro Glu Ala

130

<210> 19

<211> 137

<212> PRT

<213> artificial sequence

<220>

<223> CA17560 SSA1 binding agent amino acid sequence

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Ala Phe Asn Asp Tyr

20 25 30

Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val

35 40 45

Ala Ala Ile Thr Trp Ser Ser Gly Val Thr Ser Tyr Ser Asp Ser Val

50 55 60

Arg Gly Arg Phe Val Ile Ser Arg Asp Ile Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Asn Pro Glu Glu Thr Ala Val Tyr Val Cys

85 90 95

Ala Ala Arg Leu Gly Val Ser Ser Ser Pro Asn Ser Gln Ile Ser His

100 105 110

Arg Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His

115 120 125

His His His His His Glu Pro Glu Ala

130 135

<210> 20

<211> 137

<212> PRT

<213> artificial sequence

<220>

<223> CA17561 SSA1 binding agent amino acid sequence

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Arg Thr Phe Ser Asn Tyr

20 25 30

Val Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Gly Ala Ile Asn Trp Ser Gly Gly Thr Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asp Leu Asn Pro Glu Asp Thr Ala Asn Tyr Tyr Cys

85 90 95

Ala Ala Arg Arg Gly Thr Ile Ala Arg Thr Ala Leu Val Lys Tyr Leu

100 105 110

Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His

115 120 125

His His His His His Glu Pro Glu Ala

130 135

<210> 21

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> CA17562 SSA1 binding agent amino acid sequence

<400> 21

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Asn

20 25 30

Arg Leu Asn Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Gly Ser Asn Gly Pro Arg Tyr Ala Asp Ser Val Lys Asp

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Glu Lys Asn Thr Val Tyr Leu Glu

65 70 75 80

Met Asn Ser Val Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala

85 90 95

Arg Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His

100 105 110

His His His His Glu Pro Glu Ala

115 120

<210> 22

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA17563 SSA1 binding agent amino acid sequence

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Thr Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Arg Ala Leu Thr Pro Tyr

20 25 30

Thr Met Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Ala Ile Gly Arg Thr Gly Ser Gly Thr Arg Tyr Ala Glu Ala Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Gln Glu Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asn Arg Arg Leu Thr Thr Ser Leu Glu Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 23

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> CA17564 SSA1 binding agent amino acid sequence

<400> 23

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Val Gln His Phe Gly Asn Tyr

20 25 30

Ile Val Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Ala Ile Ser Arg Gly Leu Arg Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Gly Ser Arg Leu Leu Glu Gly Ala Pro Arg Asp Ser Arg Gly Tyr Asn

100 105 110

Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His

115 120 125

His His Glu Pro Glu Ala

130

<210> 24

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> CA17565 SSA1 binding agent amino acid sequence

<400> 24

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Arg Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Gly Met Ser Trp Tyr Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Ser Glu Ser Thr Trp Ala Asp Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Arg Asp Gln Val Pro Trp Pro Thr Trp Gly Pro Gly Thr Gln Val

100 105 110

Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 25

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> GFP protein

<400> 25

Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe

50 55 60

Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 26

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17517 GFP nanobody amino acid sequence

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr

20 25 30

Ile Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Ser Asp Thr Val Tyr Ala Ser Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Lys Asp Ala Val Gly Tyr Trp Ser Ser Asp Val Tyr Ser Glu

100 105 110

Ala Lys Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 27

<211> 140

<212> PRT

<213> artificial sequence

<220>

<223> CA17518 GFP nanobody amino acid sequence

<400> 27

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Ser Ile Ser Ser Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Trp Ala Ala Trp Pro Ser Ile Arg Val Gly Val Gly Val

100 105 110

Pro Ala Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser His His His His His His Glu Pro Glu Ala

130 135 140

<210> 28

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA17519 GFP nanobody amino acid sequence

<400> 28

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Ile Tyr

20 25 30

Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Gln Ile Thr Asn Ser Gly Arg Thr Lys Phe Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Asp Leu Asp Trp Tyr Gly Gly Thr Ala Asn Asn Asp Phe Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 29

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17520 GFP nanobody amino acid sequence

<400> 29

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Val Arg Thr Phe Ser Asn Tyr

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Ile Asn Thr Tyr Tyr Ala Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Lys Ser Ala Val Gly Tyr Tyr His Asp Ser Tyr Ala Ser Arg

100 105 110

Ala Glu Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 30

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17673 GFP binding agent amino acid sequence

<400> 30

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Arg Ala Phe Met Asn Tyr

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Val Thr Thr Asp Tyr Ala Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Lys Ser Ala Val Gly Tyr Tyr Ser Asp Ile Tyr Asn Ser Arg

100 105 110

Ala Lys Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 31

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17674 amino acid sequence of GFP binding agent

<400> 31

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Phe Leu Arg Leu Ser Cys Ala Ala Ser Asp Arg Thr Phe Ser Asn Tyr

20 25 30

Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Ser Asp Thr Val Tyr Ala Ser Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Lys Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ala Lys Tyr Ala Val Gly Tyr Trp Gly Asn Asp Val Ala Ser Glu

100 105 110

Ala Lys Asp Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 32

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA17675 GFP binding agent amino acid sequence

<400> 32

Gln Val Gln Leu Val Glu Ser Gly Gly Lys Leu Val Pro Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr

20 25 30

Ile Ile Gly Trp Phe Arg Gln Val Ser Gly Lys Glu Arg Lys Phe Val

35 40 45

Ala Ala Ile Arg Arg Ser Asp Gly Glu Thr Val Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Tyr Leu Asn Asn Arg Asn Ser Arg Gly Ser Met Asp Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 33

<211> 140

<212> PRT

<213> artificial sequence

<220>

<223> CA17676 GFP binding agent amino acid sequence

<400> 33

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Gly Thr Phe Ser Ala Tyr

20 25 30

Thr Met Gly Trp Phe Arg Gln Gly Leu Gly Glu Glu Arg Glu Leu Val

35 40 45

Ala Cys Ile Ser Arg Ser Gly Ala Ser Thr Phe Tyr Thr Asp Pro Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Trp Thr Val Ile Asp Glu Ile Thr Glu Gln Arg Ser Lys

100 105 110

His Gln Thr Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser His His His His His His Glu Pro Glu Ala

130 135 140

<210> 34

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> CA8780 irrelevant binding agent amino acid sequence

<400> 34

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly Pro Phe Ser Pro Asn Ser

20 25 30

Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

35 40 45

Val Met Thr Ile Asp Gly Arg Thr Asn Tyr Gln Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Tyr Val Lys Asn Thr Ala Tyr Leu Gln

65 70 75 80

Met Asn Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr Ile Cys Asn Ala

85 90 95

Glu Thr Arg Gly Phe Met His Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser His His His His His His Glu Pro Glu Ala

115 120

<210> 35

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17797 GR-LBD binding agent amino acid sequence

<400> 35

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Ser Ser Ser Ser Asp

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ser Ala Ile Arg Trp Asp Thr Ala Leu Lys Tyr Tyr Gly His Ser

50 55 60

Val Lys Gly Arg Phe Ala Val Ser Arg Asp Lys Ala Thr Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Ala Ala Ala Thr Phe Leu Gln Thr Ala Ala Glu Ala Thr Ser

100 105 110

Ala Thr Arg Tyr Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 36

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> CA17798 GR-LBD binding agent amino acid sequence

<400> 36

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Phe Phe Thr Arg

20 25 30

Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Gly Asp Ser Arg Arg Thr His Tyr Ala Asp Pro Val Lys

50 55 60

Gly Arg Tyr Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Ala Thr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Ala Leu Ile Thr Pro Glu Thr Met Val Glu Pro Leu Ser Ser Glu

100 105 110

Ala Trp Thr Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser His His His His His His Glu Pro Glu Ala

130 135

<210> 37

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA17799 GR-LBD binding agent amino acid sequence

<400> 37

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Asn Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ala Phe Val

35 40 45

Ser Ala Ile Ser Ala Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Asn Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Asn Leu Gln Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala His Ala Gln Ser Ile Pro Gln Leu Lys Arg Lys Asn Pro His Asp

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 38

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA17800 GR-LBD binding agent amino acid sequence

<400> 38

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Val Gly Gly

1 5 10 15

Ser Leu Ser Val Ser Cys Ala Ala Ser Gly Arg Pro Phe Asp Asn Tyr

20 25 30

Gly Met Gly Trp Phe Arg Arg Lys Pro Gly Leu Asp Tyr Glu Phe Val

35 40 45

Ser His Ile Thr Trp Asp Gly Glu Tyr Thr Arg Tyr Ser Asp Ala Val

50 55 60

Lys Gly Arg Phe Phe Val Ser Arg Asp Asn Ala Lys Ser Thr Val Thr

65 70 75 80

Leu Lys Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Lys Ala Lys Thr Asn Thr Arg Pro Tyr Tyr Tyr Glu Glu Asn

100 105 110

Tyr Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 39

<211> 130

<212> PRT

<213> artificial sequence

<220>

<223> CA17801 GR-LBD binding agent amino acid sequence

<400> 39

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Pro Gly Ser Val Arg Ser Asn Ile

20 25 30

Asp Met Ala Trp Phe Arg Gln Ala Gln Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ser Ile Thr Gly Asp Gly Thr Thr Asn Tyr Val Asp Ser Val Arg

50 55 60

Gly Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Arg Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asn Arg Met Thr Gly Trp Gly Ala Lys Arg Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro

115 120 125

Glu Ala

130

<210> 40

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> CA18504 HSP binding agent amino acid sequence

<400> 40

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Gly Ser Gly Arg Ala Phe Asp Ser Ile

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Asn Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His

65 70 75 80

Leu Gln Met Asn Ser Leu Gln Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

His Ala Asp Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105 110

His His His His His His Glu Pro Glu Ala

115 120

<210> 41

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> CA18505 MET6 Binder amino acid sequence

<400> 41

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

Ala Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Val Ile Thr Ser Val Gly Gly Arg Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ser Arg Gly Ser Ser Trp Tyr Phe Gly Thr Pro Gln Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His

115 120 125

Glu Pro Glu Ala

130

<210> 42

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> CA18508 SBA1 binding agent amino acid sequence

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Thr

20 25 30

Ala Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Val Ser Thr Ser Ala Gly Gly Gly Thr Asp Tyr Ala Asp Ser Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Tyr Ala Val Gly Arg Tyr Arg Ser Ser Trp Arg Leu Leu Pro Phe Gly

100 105 110

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His

115 120 125

His His Glu Pro Glu Ala

130

<210> 43

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> CA18509 SBA1 Binder amino acid sequence

<400> 43

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ala Gly Phe Thr Phe Arg Met Leu

20 25 30

Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Val Ser Gly Thr Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Arg Met Ala Ala Trp Gly Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 44

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> CA18510 SOD1 binding agent amino acid sequence

<400> 44

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Glu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Pro Thr Ala Tyr His Tyr Ser Thr Thr Arg Asp Glu Tyr Asp

100 105 110

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His

115 120 125

His His Glu Pro Glu Ala

130

<210> 45

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA17938 ENO1 Binder amino acid sequence

<400> 45

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Thr Trp

20 25 30

Ala Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Asp Arg Glu Gly Ala

35 40 45

Ala Cys Ile Ser Ser Glu Pro Ser Ser Thr Tyr Tyr Gly Glu Phe Val

50 55 60

Asn Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ala Asp Arg Leu Ser Ala Tyr Cys Ser Gly Ser Gly Gly Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 46

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> CA17791 PGI1 binding agent amino acid sequence

<400> 46

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Ser Gly Gly Ser Thr Asp Tyr Ala Val Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Leu Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val Tyr Tyr Cys His

85 90 95

Ser Gln Tyr Ser Tyr Gly Pro Leu Glu Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 47

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> CA17792 PGI1 binding agent amino acid sequence

<400> 47

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ser Phe Gly Ser Tyr

20 25 30

Thr Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Met Val

35 40 45

Ala Asp Ile Thr Thr Gly Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Ala Asp Phe Ala Tyr Gly Arg Asp Leu Thr Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 48

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> CA17793 PGI1 binding agent amino acid sequence

<400> 48

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ser Tyr

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Thr Gly Gly Ser Thr Asn Tyr Ala Val Ser Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Arg Val Thr Leu

65 70 75 80

Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Ala Tyr Tyr Cys His

85 90 95

Ser Arg Phe Ala Tyr Gly Pro Leu Glu Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 49

<211> 129

<212> PRT

<213> artificial sequence

<220>

<223> CA17794 PGI1 binding agent amino acid sequence

<400> 49

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Thr Phe Ser Arg Tyr

20 25 30

Ala Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Ala Gly Ser Ser Thr Asn Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Tyr Ala Asp His Tyr Gly Leu Gly Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu

115 120 125

Ala

<210> 50

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> CA17795 PGI1 binding agent amino acid sequence

<400> 50

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Phe Asn Ile Asn

20 25 30

Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Val Ile Ala Ser Ser Gly Arg Thr Asn Tyr Ala Asp Ser Val Ser

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ser Lys Ile Arg Gln Ala Ser Trp Asn Leu Val Leu Ser Asp His Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His

115 120 125

Glu Pro Glu Ala

130

<210> 51

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> CA17796 PGI1 binding agent amino acid sequence

<400> 51

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ser Phe His Ser Tyr

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Thr Asp Tyr Ala Val Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Asn Ser Glu Asp Thr Ala Val Tyr Tyr Cys His

85 90 95

Ser Gln Tyr Ala Tyr Gly Pro Leu Glu Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 52

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> CA17875 VGLUT1 binding agent amino acid sequence

<400> 52

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Val Arg Thr Trp

20 25 30

Ser Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Gly Ile Ser Trp Ser Gly Ser Gly Thr Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Gln Thr Gly Trp Asn Pro Gly Ser Asp Phe His Arg Trp

100 105 110

Gly Lys Gly Thr Gln Val Thr Val Ser Ser His His His His His His

115 120 125

Glu Pro Glu Ala

130

<210> 53

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> CA18425 VGLUT1 binding agent amino acid sequence

<400> 53

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Thr

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Val Ser Trp Arg Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asn Pro Thr Gly Ser Tyr Gly Ala Ala Thr Ser Arg Tyr Asn

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His

115 120 125

His His Glu Pro Glu Ala

130

<210> 54

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA18024 VGLUT1 binding agent amino acid sequence

<400> 54

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Arg Ala Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Arg Gly Gly Phe Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Ser Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Ala Gly Ile Arg Gln Ile Arg Val Ser Tyr Glu Gln Asp Tyr Tyr Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 55

<211> 137

<212> PRT

<213> artificial sequence

<220>

<223> CA 18463 rVGLUT1 binding agent amino acid sequence

<400> 55

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Leu Gly Asn Ser Phe Ser Thr Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Gly Ala Val Asn Trp Arg Gly Asp Ile Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Glu Thr Thr Gln Pro Met Gly Ile Thr Leu Ser Pro Asn

100 105 110

Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His

115 120 125

His His His His His Glu Pro Glu Ala

130 135

<210> 56

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA18438 rVGLUT1 binding agent amino acid sequence

<400> 56

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Ala Ile Asn

20 25 30

Lys Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Asn Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Ile Leu Asp Ser Asp Tyr Gly Glu Asp Glu Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 57

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA18439 rVGLUT1 binding agent amino acid sequence

<400> 57

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

Leu Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Ile Ile

35 40 45

Ala Val Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala His Ile Ala Thr Val Ser Glu Gly Tyr Ser Arg Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 58

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> CA 1840 rVGLUT1 binding agent amino acid sequence

<400> 58

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser His Ile Asn Gly Gly Gly Gly Thr Thr Ala Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Glu Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Glu Ser Ser Ile Gly Asn Val Trp Ala Pro Ser Tyr Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His His His

115 120 125

Glu Pro Glu Ala

130

<210> 59

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> CA18441 rVGLUT1 binding agent amino acid sequence

<400> 59

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Val Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ser Phe Ser Gly Asp

20 25 30

Val Met Gly Trp Tyr Arg Gln Pro Pro Gly Gln Gln Arg Glu Leu Val

35 40 45

Ala Arg Ile Ser Ser Asn Gly Arg Arg Thr Ile Ala Glu Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Gly Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu

65 70 75 80

Gln Met Asp Ser Leu Arg Pro Gln Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Asn Ser Arg Glu Thr Gly Ile Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 60

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> CA16964 mCherry-trapper binding agent amino acid sequence

<400> 60

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Phe Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Gly Leu Arg Ile Gly Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Thr Thr Val His

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gly Ser His Ala Thr Thr Lys Ala Gln Leu Tyr Asp Tyr Glu

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His His

115 120 125

His His Glu Pro Glu Ala

130

<210> 61

<211> 136

<212> PRT

<213> artificial sequence

<220>

<223> CA17302 mCherry-stripper binding agent amino acid sequence

<400> 61

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Leu Pro Phe Ser Arg Tyr

20 25 30

Asn Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Asp Ile Asp Trp Ser Gly Asp Ser Thr Tyr Asp Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Leu Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala His Phe Lys Phe Ile Leu Ala Gln Arg Ile Lys Trp Gly Asp

100 105 110

Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser His His

115 120 125

His His His His Glu Pro Glu Ala

130 135

<210> 62

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> CA18498 GR binding agent amino acid sequence

<400> 62

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Arg Phe Ser Arg Tyr

20 25 30

His Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Thr Ser Val Asp Arg Thr Lys Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Leu Arg Lys Gly Gly Ala Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 63

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> CA18499 GR binding agent amino acid sequence

<400> 63

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Thr Pro Gly Gly Val Tyr Arg Thr Tyr Ala Ala Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr

65 70 75 80

Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Lys Asp Gly Ala Val Arg Pro Arg Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 64

<211> 131

<212> PRT

<213> artificial sequence

<220>

<223> CA18501 GR binding agent amino acid sequence

<400> 64

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Ser Ala Ser Ile Phe Arg

20 25 30

Ser Asn Val Met Gly Trp Tyr Arg Gln Pro Pro Gly Asn Gln Arg Glu

35 40 45

Leu Val Ala Thr Ile Thr Ala Gly Gly Ser Pro Asn Tyr Ala Asp Ser

50 55 60

Val Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Val

65 70 75 80

Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Asn Leu Arg Ser Arg Ser Ser Tyr Pro Tyr Met Asp Tyr Trp Gly

100 105 110

Lys Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu

115 120 125

Pro Glu Ala

130

<210> 65

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> CA18502 GR binding agent amino acid sequence

<400> 65

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Thr Ala Ser Ile Arg

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Leu Val

35 40 45

Ala Ala Phe Thr Ser Ala Gly Ser Ile Asn Tyr Glu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Lys Asp Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Leu Gln Gln Tyr Glu Arg His Leu Lys Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 66

<211> 130

<212> PRT

<213> artificial sequence

<220>

<223> CA18503 GR binding agent amino acid sequence

<400> 66

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Thr Ile Arg Arg Gly Gly Asp Val Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Lys Ala Asp Arg Pro Gly Trp Gly Ser Glu Trp Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro

115 120 125

Glu Ala

130

<210> 67

<211> 135

<212> PRT

<213> artificial sequence

<220>

<223> CA18585 GR binding agent amino acid sequence

<400> 67

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ile Phe

20 25 30

Asn Asn Tyr His Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

35 40 45

Glu Leu Ala Ala Leu Ile Ser Gly Ile Gly Thr Val Thr Asn Tyr Ala

50 55 60

Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Thr Lys Asn

65 70 75 80

Thr Val Ser Leu Gln Met Asn Ser Leu Glu Ala Glu Asp Thr Ala Val

85 90 95

Tyr Phe Cys Gly Gly Gly Arg Pro Ser Ser Arg Ser Pro Tyr Gly Met

100 105 110

Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser His His His

115 120 125

His His His Glu Pro Glu Ala

130 135

<210> 68

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> CA18586 GR binding agent amino acid sequence

<400> 68

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Asn Leu Tyr Met Gly

20 25 30

Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Ala Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Arg Thr Val Tyr Leu

65 70 75 80

Gln Met Asp Thr Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Gly

85 90 95

Ser Asn Ser Gly Thr Tyr Ser Leu Arg Phe Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser His His His His His His Glu Pro Glu Ala

115 120 125

<210> 69

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> 6 xHis+EPEA tag

<400> 69

His His His His His His Glu Pro Glu Ala

1 5 10

<210> 70

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> CA12760 GFP-stripper amino acid sequence

<400> 70

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Ala

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val

35 40 45

Ala Gly Ile Tyr Trp Thr Val Gly Ser Thr Tyr Tyr Ala Asp Ser Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Arg Arg Gly Phe Thr Leu Ala Pro Thr Arg Ala Asn Glu

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 71

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> CA15816 GFP-collector amino acid sequence

<400> 71

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Ala

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val

35 40 45

Ala Gly Ile Tyr Trp Ala Ala Gly Ser Thr Tyr Tyr Ala Asp Ser Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Arg Arg Gly Phe Thr Leu Ala Pro Thr Arg Ala Asn Glu

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Claims (17)

1. A method for selecting a polypeptide binding agent, the method comprising the steps of:

a) Mixing a first protein binding agent immobilized on a surface and specifically binding to a target protein with a sample comprising the target protein for obtaining a complex on the surface,

b) Providing a sample comprising a plurality of polypeptide binding agents to the complex of step a),

c) Adding to the mixture of step b) a sample comprising a second protein binding agent which competes with the first binding agent for binding to the target protein and which displaces (displace) the first binding agent from the target protein by specifically binding to the target protein, and

d) Eluting the second protein binding agent bound to the target protein for isolating the polypeptide binding agent bound to the target protein.

2. The method of claim 1, wherein the dissociation rate constant (k _off Value) and k of the first binding agent _off The values are lower or equal.

3. The method of claim 1 or 2, wherein the second and/or first protein binding agent comprises an antigen binding domain.

4. The method of claim 3, wherein the antigen binding domain comprises an Immunoglobulin Single Variable Domain (ISVD), a VHH, a nanobody, or an antigen binding chimeric protein defined as an ISVD fused to a scaffold protein by at least two sites, and the scaffold protein domain preferably comprises HopQ, ygjK, or a derivative thereof.

5. The method of any one of claims 1 to 4, wherein the sample comprising the plurality of polypeptide binding agents in step b) comprises a display library of binding agents.

6. The method of claim 5, wherein the display library is a recombinant library and/or an immune library or a (semi) synthetic, non-immune or initial library of binding agents, wherein the binding agents comprise antibodies, single domain antibodies, ISVD, VHH or nanobodies.

7. The method of any one of claims 1 to 6, wherein the method is performed using phage, yeast, ribosome, bacterial or mammalian display, and/or wherein after step d), steps a) to d) of the method are repeated at least once, preferably two or more times, to enrich the amount of polypeptide binder eluted in step d.

8. The method of any one of claims 1 to 7, wherein the surface comprises (magnetic) beads, resins, columns, plates or chips.

9. The method of any one of claims 1 to 8, wherein the sample comprising the target protein in a) comprises a complex mixture, such as a biological sample, a cell lysate or a proteomic sample.

10. The method of claim 9, wherein the complex mixture is used as an immunogen for obtaining a plurality of polypeptide binders or in particular the display library of claim 6.

11. The method of any one of claims 1 to 10, wherein the first protein binding agent and/or the second protein binding agent specifically binds to a heterologous tag present on the target protein.

12. The method of claim 10, wherein the tag is GFP or YFP and/or the first protein binder comprises the CDRs of SEQ ID No. 71 and the second protein binder comprises the CDRs of SEQ ID No. 70.

13. The method of any one of claims 1 to 12, wherein the target protein captured on the surface is a protein complex comprising at least one or more other proteins, and/or the polypeptide binding agent binds to the complex via binding to at least one or more proteins comprised in the protein complex.

14. The method of any one of claims 1 to 13, wherein steps a) and b) are replaced by the steps of:

a. mixing a target protein sample with a sample, preferably a display library, comprising a plurality of polypeptide binding agents, and

b. the immobilized complex on the surface is obtained by adding a first protein binder to the mixture of a), preferably immobilized on the surface or subsequently immobilized.

15. Use of the method of any one of claims 1 to 14 for selecting a binding agent from a library of recombinant antibodies.

16. Use of the method of any one of claims 1 to 15 for target protein epitope binning or identifying neoepitopes on a target protein.

17. Use of the method of any one of claims 1 to 15 for high throughput selection of specific binding agents.