Libraries and methods

Abstract

The disclosure provides methods for analyzing protein-library interactions, by providing a protein display library and complexing it with partner query elements. Unique protein variants that bind to these partner elements are separated and their nucleic acid identifiers are subsequently detected.

Description

Attorney Docket No.: 123828-02-5139-PCLIBRARIES AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 642,455, filed on May 3, 2024, the entire contents of which are hereby incorporated herein by reference in their entireties.BACKGROUND

[0002] Potential molecule space, including protein-based molecules, is massive. Molecules, including proteins, can cover a vast array of industrial applications, including therapeutics, such as human therapeutics or veterinary applications, as well as agricultural, sustainability, and industrial applications. Wet lab techniques for screening candidate molecules face challenges in cost and scaling, including for more complicated library-on-library or hashing approaches. Traditional antibody discovery and engineering via yeast surface display is limited in its ability to predict variant affinities at library scale. Methodologies, such as Tite-Seq, have been developed to overcome some of these limitations, however, these approaches are still limited in throughput.

[0003] Accordingly, a need exists for improved library techniques — and associated systems for performing them and methods using them— that overcome these and other challenges, preferably with high throughput semi -automated workflow that is able to rank-order variants by affinity, such as within yeast surface display libraries.SUMMARY

[0004] The disclosure provides, inter alia, improved library techniques — and systems for performing them and associated methods using them— that overcome these and other challenges, e.g., with parallel -processing, high throughput, semi-automated workflow able to rank-order large numbers of variants by affinity, such as within yeast surface display libraries.

[0005] The disclosure provides methods for analyzing protein-library interactions, by providing a protein display library and complexing it with partner query elements. Unique protein variants that bind to these partner elements are separated and their nucleic acid identifiers are subsequently detected. This approach is applicable at multiple partner query element concentrations and enables the evaluation of, for example, binding affinity. The method can be automated and can be1DBl / 157546510.2Attorney Docket No.: 123828-02-5139-PC performed efficiently, even with large number of display complexes. The application also includes embodiments involving the use of nucleic acid barcodes, and consideration of different types of library units such as yeast, phage, mammalian, ribosome, and virus-like particles. Associated systems equipped to perform these methods are also provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The figures below serve to help illustrate the principles and utilities of the methods and systems provided by the disclosure.

[0007] FIG. 1 is a diagram illustrating Fluorescence-Activated Cell Sorting (FACS) Tite-Seq methodology, which can achieve library-scale affinity rank-ordering, but is limited in throughput.

[0008] FIG. 2 is a diagram illustrating one aspect of the disclosure: a semi-automated protocol for Magnetic- Activated Cell Sorting (MACS) selection of a yeast surface display library, one implementation of which is a KingFisher protocol.

[0009] FIG. 3 is a line graph illustrating the relative efficiencies of cell recovery under unoptimized and optimized KingFisher MACS protocols.

[0010] FIG. 4 is a line graph and table demonstrating how automated MACS concentration series sorting can predict EC so in a mock library setting.

[0011] FIG. 5 is a line graph and table illustrating that MACS Tite-Seq can distinguish between variants with different affinities.

[0012] FIG. 6 is a series of plots that demonstrate MACS Tite-Seq can accurately rank-order variants within a small-scale library with similar robustness to FACS Tite-Seq.

[0013] FIG. 7 is a series of plots that demonstrate MACS Tite-Seq can accurately rank-order variants within a library comprised of thousands of variants.

[0014] FIG. 8 is a non-limiting diagram illustrating certain embodiments of the disclosure.DETAILED DESCRIPTION

[0015] The disclosure provides, inter alia, methods for analyzing protein-library interactions, by providing a protein display library and complexing it with partner query elements. In embodiments, unique protein variants that bind to these partner elements are separated and their nucleic acid identifiers are subsequently detected. In embodiments, this approach is applicable atDBl / 157546510.2 2Attorney Docket No.: 123828-02-5139-PC multiple partner query element concentrations and enables the evaluation of, for example, binding affinity. In embodiments, the present method and / or system can be automated and can be performed efficiently, even with large number of display complexes. In embodiments, there is also provided a method and / or system involving the use of nucleic acid barcodes, and consideration of different types of library units such as yeast, mammalian, phage, ribosome, and virus-like particles.

[0016] In embodiments, associated systems equipped to perform all of the disclosed methods are provided.

[0017] In aspects, there is provided a method comprising: a) providing a protein display library, the library comprising a plurality of display complexes, each display complex comprising a unique protein library variant and a nucleic acid identifier of the unique protein library variant; b)complexing the protein display library with a first partner query element under conditions suitable for the partner query element to bind with a portion of the unique protein library variants, wherein the complexing is performed at multiple concentrations of the first partner query element; c) at each of the multiple concentrations of the first partner query element, separating a portion of the unique protein variants bound to the first partner query element to form a bound fraction, from the portion of the unique protein variants not bound to the first partner query element to form an unbound fraction; and d) detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction.

[0018] In embodiments, the detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction is by sequencing the nucleic acid identifiers. In embodiments, the sequencing is via a technique known in the art.

[0019] In embodiments, the sequencing is NGS.

[0020] In embodiments, any high-throughput technique for sequencing nucleic acids can be used in the methods of the disclosure. In embodiments the sequencing is whole genome sequencing. In embodiments, the sequencing is amplicon sequencing ultra deep sequencing to identify genetic variations. In embodiments the sequencing is classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary; sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing; 454 sequencing; allele specific hybridization to a library of labeled oligonucleotide probes; sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation; realDBl / 157546510.2 3Attorney Docket No.: 123828-02-5139-PC time monitoring of the incorporation of labeled nucleotides during a polymerization step; polony sequencing; and SOLiD sequencing.

[0021] In embodiments, the sequencing uses methods that comprise a step of spatially isolating individual molecules on a solid surface where they are sequenced in parallel. In embodiments, such solid surfaces may include nonporous surfaces (such as in Solexa sequencing, e.g., Bentley et al, Nature, 456: 53-59 (2008) or Complete Genomics sequencing, e.g., Drmanac et al, Science, 327: 78-81 (2010)), arrays of wells, which may include bead- or particle-bound templates (such as with 454, e.g., Margulies et al, Nature, 437: 376-380 (2005) or Ion Torrent sequencing, U.S. patent publication 2010 / 0137143 or 2010 / 0304982), micromachined membranes (such as with SMRT sequencing, e.g., Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as with SOLiD sequencing or polony sequencing, e.g., Kim et al, Science, 316: 1481-1414 (2007)).

[0022] In embodiments, the separation of the bound fraction and unbound fraction is by magnetic separation, wherein the partner query element is associated with a magnetic element.

[0023] In embodiments, the magnetic element is a magnetic particle, e.g., a magnetic bead.

[0024] In embodiments, the magnetic element is a paramagnetic particle.

[0025] In embodiments, the magnetic or paramagnetic particle is a nanoparticle, which can be, e.g., a nanobead. In embodiments, the magnetic or paramagnetic particle is a microparticle. In embodiments, the microparticle is a microbead. The magnetic or paramagnetic particle is, in embodiments, a magnetic nano- or microbead, which allows the particle to be held and / or manipulated by magnets. In embodiments, the paramagnetic particle is a metallic nanoparticle coated with a thin (e.g., about or least about 2 nm in diameter) graphene-like carbon layer. In embodiments the paramagnetic particle is coated, e.g., streptavidin- or PEG-coated. Examples of magnetic particles that can be used are DYNABEADs (THERMOFISHER), MACS beads (MILTENYI BIOTEC), TURBOBEADS (TURBOBEADS), ABSOLUTE MAG STREPTAVIDIN MAGNETIC PARTICLES (CREATIVE DIAGNOSTICS), and GOLD NANOPARTICLES (SIGMAALDRICH).

[0026] In embodiments, the magnetic beads are nanoparticles with a superparamagnetic Fe2O3 core and a biocompatible outer coating. The surface of the beads can be activated, e.g., with carboxyl groups. In embodiments, the reporter particles described herein may include a biocompatible coating that may be activated with amine groups or carboxyl groups to facilitateDBl / 157546510.2 4Attorney Docket No.: 123828-02-5139-PC amid coupling. In embodiments, the particles described herein may be activated with amine groups or carboxyl groups to facilitate amid coupling.

[0027] In embodiments, the magnetic or paramagnetic particles described herein are nanoparticles (e. , nanobeads), which are smaller than about 1 micrometer in diameter (e.g., about 5 to about 500 nanometers, e.g., about 5 nanometers, or about 10 nanometers, or about 50 nanometers, or about 100 nanometers, or about 250 nanometers, or about 500 nanometers). In embodiments, the nanoparticles (e.g. , nanobeads) have a mean particle diameter of 25-500 nm+ / -5 nm, 25-500 nm+ / - 10 nm, 25-500 nm+ / -15 nm, 25-500 nm+ / -20 nm, 25-500 nm+ / -25 nm, 25-500 nm+ / -30 nm, 25- 500 nm+ / -35 nm, 25-500 nm+ / -40 nm, 25-500 nm+ / -45 nm, or 25-500 nm+ / -50 nm.

[0028] In embodiments, the nanoparticles (e.g., nanobeads) are smaller than about 1 micrometer in diameter (e.g., about 5 to about 500 nanometers, e.g., about 5 nanometers, or about 10 nanometers, or about 50 nanometers, or about 100 nanometers, or about 250 nanometers, or about 500 nanometers). In embodiments, the nanoparticles (e.g., nanobeads) have a mean particle diameter of 25-500 nm+ / -5 nm, 25-500 nm+ / -10 nm, 25-500 nm+ / -15 nm, 25-500 nm+ / - 20 nm, 25-500 nm+ / -25 nm, 25-500 nm+ / -30 nm, 25-500 nm+ / -35 nm, 25-500 nm+ / -40 nm, 25- 500 nm+ / -45 nm, or 25-500 nm+ / -50 nm. In embodiments, the nanoparticles (e.g., nanobeads) have a mean particle diameter of about 20 to about 200 nm.

[0029] In embodiments, the magnetic or paramagnetic particle is composed of oxides, such as ferrites, maghemite, magnetite, or iron oxide, optionally modified by surfactants, silica, silicones or phosphoric acid derivatives. In embodiments, the magnetic or paramagnetic particle is composed of ferrites with a shell (e.g., a silica shell, optionally modified). In embodiments, the magnetic nanoparticle is metallic (e.g., iron, cobalt, etc.). In embodiments, the magnetic or paramagnetic particle is a metallic or paramagnetic nanoparticle comprising a shell (e.g., of gentle oxidation, surfactants, polymers and metals (e.g., of gold, graphene, palladium, platinum, etc.)).

[0030] In embodiments, the method comprises, at each of the multiple concentrations of the first partner query element, separating the bound fraction and the unbound fraction without further binning of the bound fraction. In embodiments, the separation uses techniques known in the art.

[0031] In embodiments, the separation is effected magnetically, e.g. by applying a magnetic field to the magnetic or paramagnetic element. In embodiments, the separation is effected via centrifugation, filtration, dialysis, and the like. In embodiments, the separation step in c) isDBl / 157546510.2 5Attorney Docket No.: 123828-02-5139-PC performed using an automated liquid handling system, automated magnetic bead handling system, or both.

[0032] In embodiments, the nucleic acid identifiers of the display complexes are nucleic acids encoding the unique protein library variant.

[0033] In embodiments, the method comprises estimating the binding affinity of the unique protein library variants to the first partner query element.

[0034] In embodiments, the method is a high-throughput method comprising parallel processing of about or least about 100, about or least about 200, about or least about 300, about or least about 400, about or least about 500, about or least about 600, about or least about 700, about or least about 800, about or least about 900, about or least about 1000, about or least about 3000, about or least about 5000, about or least about 10000, or more display complexes; optionally wherein the method is about or least about 2, about or least about 3, about or least about 4, about or least about 5, about or least about 6, about or least about 7, about or least about 8, about or least about 9, about or least about 10, about or least about 11, about or least about 12, about or least about 13, about or least about 14, about or least about 15, about or least about 16, about or least about 17, about or least about 18, about or least about 19, or about or least about 20-fold faster than a FACS-based screening, such as Tite-Seq method (e.g., the method provided in / / doi.org / 10.7554 / eLife.23156).

[0035] In embodiments, the method is performed in multi-well plates and the multiple concentrations of the first partner query element are evaluated in parallel.

[0036] In embodiments, the display complex, first partner query element, or both, is complexed with a nucleic acid barcode. In embodiments, the nucleic acid barcode is a distinct sequence from the nucleic acid identifiers of the display complexes. In embodiments, the nucleic acid barcode shares less than about 99%, or less than about 98%, or less than about 97%, or less than about 96%, or less than about 95%, or less than about 90%, or less than about 80%, or less than about 70%, or less than about 60%, or less than about 50%, or less than about 30% sequence identity with the nucleic acid identifiers of the display complexes

[0037] In embodiments, the method further comprises evaluating a plurality of partner query elements, comprising at least one (e.g., about or least about 2, about or least about 3, about or least about 4, about or least about 5, about or least about 10, about or least about 50, about or least about 100, about or least about 500, about or least about 1000, about or least about 5000, about or least about 10000, or more) additional partner query elements in addition to the first partnerDBl / 157546510.2 6Attorney Docket No.: 123828-02-5139-PC query element. In embodiments, the evaluating is or comprises a library-on-library method (hashing). In embodiments, the additional partner query elements comprises a different molecular structures from each other and the first partner query element.

[0038] In embodiments, the library comprises at about or least about 10, about or least about 30, about or least about 50, about or least about 100, about or least about 200, about or least about 500, about or least about 1000, about or least about 5000, about or least about 10A5, about or least about 5xl0A5, about or least about 10A6, about or least about 5xl0A6, about or least about 10A7, about or least about 5x10A7, or more, unique protein library variants.

[0039] In embodiments, the display library is a mammalian display library. In embodiments, the display library is a yeast library. In embodiments, the display library is a phage library. In embodiments, the display library is a ribosome library. In embodiments, the display library is a VLP library.

[0040] In embodiments, the unique protein library variants are immunoglobulins, or an antigen binding fragment thereof. In embodiments, the immunoglobulin is a monoclonal antibody or a polyclonal antibody, or a binding fragment thereof. In embodiments, the immunoglobulin is a single-domain antibody, single-chain antibody (scFv), Fv, Fab, Fab', and F(ab')2. In embodiments, the immunoglobulin is an antibody format selected from binding fragment-His (e.g., VHH-His or scFv-His), binding fragment-Fc (e.g., VHH-Fc or scFv-Fc), a biparatopic molecule (e.g., biparatopic VHH-Fc), tetravalent molecule (e.g., a tetravalent VHH-Fc), bidisulfide-bond stabilized scFv (ds-scFv), bi-specific antibody, Tri-TAC, single chain Fab (scFab), Fc- heterodimeric molecules, di- and multimeric antibody formats like dia-, tria- and tetra-bodies, Fab- IgG, IgG-Fab, and IgG-VHH.

[0041] In embodiments, the unique protein library variants are receptors or a functional fragment thereof.

[0042] In embodiments, the first partner query element, or optionally additional partner query element(s), is an antigen, such as a polypeptide antigen.

[0043] In embodiments, the first partner query element, or optionally additional partner query element(s), is a polypeptide ligand. In embodiments, the polypeptide ligand is a cytokine or signaling molecule.DBl / 157546510.2 7Attorney Docket No.: 123828-02-5139-PC

[0044] In embodiments, the first partner query element, or optionally additional partner query element(s), is a polypeptide associated with, or expressed by, a cell, a tissue a VLP, an exosome, a vesicle, a lipid nanoparticle (LNP), and / or a virus.

[0045] In embodiments, the unique protein library variants, first partner query element, or optional additional partner query element(s), is a polypeptide, optionally wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is labelled. In embodiments, the label is biotin, fluorescein isothiocyanate (FITC), dinitrophenol (DNP), digoxigenin (DIG), Etag, FLAG, Myc, HA, SNAP, and / or CLIP.

[0046] In embodiments, the unique protein library variants, first partner query element, or optional additional partner query element(s), is a polypeptide, optionally wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is biotinylated.

[0047] In embodiments, the method is as described in FIG. 8.

[0048] In aspects, there is provided a system for performing the methods described herein, wherein the system comprises an automated liquid handling system, automated magnetic bead handling system, or both, optionally further comprising computer-readable instructions for performing any step of any of the foregoing methods, including performing any of the foregoing methods, further optionally wherein the system comprises a purification instrument, e.g, a KingFisher system (THERMO, see JALA 2007;12:195-201).

[0049] Features provided by the disclosure can include one or more of the following enumerated embodiments.Embodiment 1 A method comprising: a) providing a protein display library, the library comprising a plurality of display complexes, each display complex comprising a unique protein library variant and a nucleic acid identifier of the unique protein library variant; b)complexing the protein display library with a first partner query element under conditions suitable for the partner query element to bind with a portion of the unique protein library variants, wherein the complexing is performed at multiple concentrations of the first partner query element; c) at each of the multiple concentrations of the first partner query element, separating a portion ofDBl / 157546510.2 8Attorney Docket No.: 123828-02-5139-PC the unique protein variants bound to the first partner query element to form a bound fraction, from the portion of the unique protein variants not bound to the first partner query element to form an unbound fraction; and d) detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction.Embodiment 2 The method of embodiment 1, wherein the detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction is by sequencing the nucleic acid identifiers, such as by NGS.Embodiment 3 The method of any one of the preceding embodiments, wherein the NGS provides a mean bin (weighted average) readout.Embodiment 4 The method of any one of the preceding embodiments, wherein the separation of the bound fraction and unbound fraction is by magnetic separation, wherein the partner query element is associated with a magnetic element, such as a magnetic bead.Embodiment 5 The method of any one of the preceding embodiments, wherein the method comprises, at each of the multiple concentrations of the first partner query element, separating the bound fraction and the unbound fraction without further binning of the bound fraction.Embodiment 6 The method of any one of the preceding embodiments, wherein the nucleic acid identifiers of the display complexes are nucleic acids encoding the unique protein library variant.Embodiment 7 The method of any one of the preceding embodiments, wherein the method comprises estimating the binding affinity of the unique protein library variants to the first partner query element.Embodiment 8 The method of any one of the preceding embodiments, wherein the separation step in c) is performed using an automated liquid handling system, automated magnetic bead handling system, or both.Embodiment 9 The method of any one of the preceding embodiments, wherein the method is a high-throughput method comprising parallel processing of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 3000, 5000, 10000, or more display complexes; optionally wherein the method is at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-foldDBl / 157546510.2 9Attorney Docket No.: 123828-02-5139-PC faster than a FACS-based screening, such as Tite-Seq method (e.g., the method provided in / / doi.org / 10.7554 / eLife.23156 and / or the method provided in Cell 2020 Sep 3; 182(5): 1295— 1310 doi: 10.1016 / j .cell.2020.08.012).Embodiment 10 The method of any one of the preceding embodiments, wherein the method is performed in multi-well plates and the multiple concentrations of the first partner query element are evaluated in parallel.Embodiment 11 The method of any one of the preceding embodiments, wherein the display complex, first partner query element, or both, is complexed with a nucleic acid barcode, wherein the nucleic acid barcode is a distinct sequence from the nucleic acid identifiers of the display complexes.Embodiment 12 The method of any one of the preceding embodiments, wherein the method further comprises evaluating a plurality of partner query elements, comprising at least one (e.g., 2, 3, 4, 5, 10, 50, 100, 500, 1000, 5000, 10000, or more) additional partner query elements in addition to the first partner query element, mutatis mutandis, for example, as a library-on-library method (hashing), wherein the additional partner query elements comprises a different molecular structures from each other and the first partner query element.Embodiment 13 The method of any one of the preceding embodiments, wherein the library comprises at least about: 10, 30, 50, 100, 200, 500, 1000, 5000, 10A5, 5x10A5, 10A6, 5x10A6, 10A7, 5xlOA7, or more, unique protein library variants.Embodiment 14 The method of any one of the preceding embodiments, wherein the display library is a mammalian display library.Embodiment 15 The method of any one of the preceding embodiments, wherein the display library is a yeast library.Embodiment 16 The method of any one of the preceding embodiments, wherein the display library is a phage library.Embodiment 17 The method of any one of the preceding embodiments, wherein the display library is a ribosome library.Embodiment 18 The method of any one of the preceding embodiments, wherein the display library is a VLP library.DBl / 157546510.2 10Attorney Docket No.: 123828-02-5139-PCEmbodiment 19 The method of any one of the preceding embodiments, wherein the unique protein library variants are immunoglobulins, or an antigen binding fragment thereof.Embodiment 20 The method of any one of the preceding embodiments, wherein the unique protein library variants are receptors or a functional fragment thereof.Embodiment 21 The method of any one of the preceding embodiments, wherein the first partner query element, or optionally additional partner query element(s), is an antigen, such as a polypeptide antigen.Embodiment 22 The method of any one of the preceding embodiments, wherein the first partner query element, or optionally additional partner query element(s), is a polypeptide ligand, such as a cytokine or signaling molecule.Embodiment 23 The method of any one of the preceding embodiments, wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is a polypeptide, optionally wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is biotinylated.Embodiment 24 The method of any one of the preceding embodiments, wherein the method is as described in FIG. 8.Embodiment 25 A method as described in FIG. 8.Embodiment 26 A system for performing the method of any of the preceding embodiments, wherein the system comprises an automated liquid handling system, automated magnetic bead handling system, or both, optionally further comprising computer-readable instructions for performing any step of any of the foregoing methods, including performing any of the foregoing methods, further optionally wherein the system comprises a Kingfisher system.EXEMPLIFICATION

[0050] FIGs 1-8 illustrate aspects and embodiments of the disclosure and are more fully described in their figure descriptors and can be more readily understood by reference to the appended claims. Together these evidence that Applicant has developed an automated MACS-based workflow with a significantly improved throughput as compared to traditional Tite-Seq, which enables libraryscale affinity measurement for all variants. These methods are applicable to libraries with increased diversity and down-sampling data can further inform scalability.DBl / 157546510.2 11Attorney Docket No.: 123828-02-5139-PC

[0051] FTG. 1 is a diagram illustrating Fluorescence-Activated Cell Sorting (FACS) Tite-Seq methodology, which can achieve library-scale affinity rank-ordering, but is limited in throughput. Substantially increased throughput is one improvement provided by the instant disclosure, as illustrated in the figures that follow.

[0052] FIG. 2 is a diagram illustrating one aspect of the disclosure: a semi-automated protocol for Magnetic- Activated Cell Sorting (MACS) selection of a yeast surface display library, one implementation of which is a KingFisher protocol. FIG. 8 is a further illustrative embodiment.

[0053] FIG. 3 is a line graph illustrating the relative efficiencies of cell recovery under unoptimized and optimized KingFisher MACS protocols. Protocol optimization resulted in ~4-fold increase in cell recovery.

[0054] FIG. 4 is a line graph and table demonstrating how automated MACS concentration series sorting can predict ECso in a mock library setting. An input library of 2 labeled clones (70% of a non-specific clone, and 30% target-specific clone) were used. Selection was against biotinylated target at a series of concentrations and cell recovery was with streptavidin magnetic beads using our optimized KingFisher MACS protocol. Readout was by flow cytometry by determining the number of cells recovered of the specific clone (in the bead-bound fraction) and plotting cell recovery (% cells selected) vs concentration to determine ECso This result was compared to the ECso determined by a “FACS isogenic yeast titration” experiment (data not shown). In this experiment, the target protein was titrated against yeast that displayed the target-specific clone. The readout was the fluorescence intensity measured by flow cytometry, which was obtained by fluorescent detection of the target protein.

[0055] FIG. 5 is a line graph and table illustrating that MACS Tite-Seq can distinguish between variants with different affinities. The input library was a 4-member library composed of 25% nonspecific variant, 25% target-specific variant A (higher affinity), 25% target-specific variant B (wildtype), and 25% target-specific variant C (lower affinity). Cell counts and subsequently mean bin analysis (i.e., weighted average of number / counts in the bound and unbound bins) were determined either by flow cytometry or a “Computational Readout” from next generation sequencing (NGS). ECso values for each variant were calculated from a plot of mean bin vs. concentration. Values were compared to “FACS isogenic yeast titration” ECso values (as described within FIG. 4).DBl / 157546510.2 12Attorney Docket No.: 123828-02-5139-PC

[0056] FIG. 6 is a series of plots that demonstrate MACS Tite-Seq can accurately rank-order variants within a small-scale library with similar robustness to FACS Tite-Seq. The input library was 25% non-specific clone and 75% of equal distribution of 20 clones and both our semiautomated MACS Tite-Seq protocol and FACS Tite-Seq were performed. Readout was by NGS (next generation sequencing) and mean bin was calculated and plotted vs. concentration for all variants and results were compared to: Experimental ECso from FACS isogenic yeast titrations (as described within FIG. 4) and EC50 calculated by FACS Tite-Seq. These results show that the high throughput MACS Tite-Seq workflow provided by the disclosure accurately rank-orders variants by affinity within a yeast surface display library. MACS concertation series sorting can distinguish between variants of different affinity and computation analysis of NGS data allows for preforming MACS Tite-Seq at scale.

[0057] FIG. 7 is a series of plots that demonstrate MACS Tite-Seq can accurately rank-order variants within a library comprised of thousands of variants. A semi-automated MACS Tite-Seq protocol on a highly diverse input library (1000s of unique variants) was preformed and the readout was by NGS (next generation sequencing) and mean bin was calculated and plotted vs. concentration for all variants. Results of select clones from the library were compared to: EC50 from FACS isogenic yeast titrations (as described in FIG. 4) and Kd calculated by Octet of the soluble protein variant binding to its target. These results show that the high throughput MACS Tite-Seq workflow provided by the disclosure accurately rank-orders variants by affinity within a yeast surface display library and this affinity-rank ordering correlates with affinities of the soluble proteins against their target.

[0058] FIG. 8 is a diagram illustrating certain embodiments of the disclosure.

[0059] It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2- 4, 2-5, 3-4, 3-5, and 4-5, et cetera.

[0060] For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where anyDBl / 157546510.2 13Attorney Docket No.: 123828-02-5139-PC conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.

[0061] Headings used in this application are for convenience only and do not affect the interpretation of this application.

[0062] Preferred features of each of the aspects provided by the disclosure are applicable to all of the other aspects of the disclosure mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the disclosure, including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the disclosure. For example, for materials that are disclosed, while specific reference of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements A-D is disclosed, then, even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C- E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions.DBl / 157546510.2 14Attorney Docket No.: 123828-02-5139-PC

[0063] The forgoing aspects of the disclosure, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art — thus, to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.DBl / 157546510.2 15

Claims

Attorney Docket No.: 123828-02-5139-PCCLAIMSWhat is claimed is:

1. A method compri sing : a) providing a protein display library, the library comprising a plurality of display complexes, each display complex comprising a unique protein library variant and a nucleic acid identifier of the unique protein library variant; b) complexing the protein display library with a first partner query element under conditions suitable for the partner query element to bind with a portion of the unique protein library variants, wherein the complexing is performed at multiple concentrations of the first partner query element; c) at each of the multiple concentrations of the first partner query element, separating a portion of the unique protein variants bound to the first partner query element to form a bound fraction, from the portion of the unique protein variants not bound to the first partner query element to form an unbound fraction; and d) detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction.

2. The method of claim 1, wherein the detecting the nucleic acid identifiers of the display complexes in the bound fraction and the unbound fraction is by sequencing the nucleic acid identifiers, such as by NGS.

3. The method of claim 1, wherein the separation of the bound fraction and unbound fraction is by magnetic separation, wherein the partner query element is associated with a magnetic element, such as a magnetic bead.

4. The method of claim 1, wherein the method comprises, at each of the multiple concentrations of the first partner query element, separating the bound fraction and the unbound fraction without further binning of the bound fraction.

5. The method of claim 1, wherein the nucleic acid identifiers of the display complexes are nucleic acids encoding the unique protein library variant.DBl / 157546510.2 16Attorney Docket No.: 123828-02-5139-PC6. The method of claim 1, wherein the method comprises estimating the binding affinity of the unique protein library variants to the first partner query element.

7. The method of claim 1, wherein the separation step in c) is performed using an automated liquid handling system, automated magnetic bead handling system, or both.

8. The method of any one of the preceding claims, wherein the method is a high-throughput method comprising parallel processing of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 3000, 5000, 10000, or more display complexes; optionally wherein the method is at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-fold faster than a FACS-based screening, such as Tite-Seq method (e.g., the method provided in / / doi.org / 10.7554 / eLife.23156).

9. The method of any one of the preceding claims, wherein the method is performed in multiwell plates and the multiple concentrations of the first partner query element are evaluated in parallel.

10. The method of claim 1, wherein the display complex, first partner query element, or both, is complexed with a nucleic acid barcode, wherein the nucleic acid barcode is a distinct sequence from the nucleic acid identifiers of the display complexes.

11. The method of any one of the preceding claims, wherein the method further comprises evaluating a plurality of partner query elements, comprising at least one e.g., 2, 3, 4, 5, 10, 50, 100, 500, 1000, 5000, 10000, or more) additional partner query elements in addition to the first partner query element, mutatis mutandis, for example, as a library-on-library method (hashing), wherein the additional partner query elements comprises a different molecular structures from each other and the first partner query element.

12. The method of any one of the preceding claims, wherein the library comprises at least about: 10, 30, 50, 100, 200, 500, 1000, 5000, 10A5, 5xl0A5, 10A6, 5xlOA6, 10A7, 5xlOA7, or more, unique protein library variants.

13. The method of any one of the preceding claims, wherein the display library is a mammalian display library, a yeast library, a phage library, a ribosome library, and / or a VLP library.DBl / 157546510.2 17Attorney Docket No.: 123828-02-5139-PC14. The method of any one of the preceding claims, wherein the unique protein library variants are immunoglobulins, or an antigen binding fragment thereof; or receptors or a functional fragment thereof.

15. The method of any one of the preceding claims, wherein the first partner query element, or optionally additional partner query element(s), is an antigen, such as a polypeptide antigen.

16. The method of any one of the preceding claims, wherein the first partner query element, or optionally additional partner query element(s), is a polypeptide ligand, such as a cytokine or signaling molecule.

17. The method of any one of the preceding claims, wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is a polypeptide, optionally wherein the unique protein library variants, first partner query element, or optional additional partner query element(s), is biotinylated.

18. A system for performing the method of any of the preceding claims, wherein the system comprises an automated liquid handling system, automated magnetic bead handling system, or both, optionally further comprising computer-readable instructions for performing any step of any of the foregoing methods, including performing any of the foregoing methods, further optionally wherein the system comprises a KingFisher system.DBl / 157546510.2 18

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