High-throughput methods for the rapid generation of affinity reagents and uses thereof

Abstract

Molecules that bind proteins of interest — known as "binders" or "affinity reagents" are useful for basic research, diagnostic, and therapeutic purposes. Provided herein are methods and compositions for generating such molecules in a high-throughput manner without burdensome optimization of selection conditions, as well as uses thereof.

Description

HIGH-THROUGHPUT METHODS FOR THE RAPID GENERATION OF AFFINITY REAGENTS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 712,316, filed October 25, 2024, which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under GM119840 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The instant application contains an Electronic Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing XML was created on October 27, 2025, is named “24-0950-WO_SequenceListing.xml”, and is 880,183 bytes in size.FIELD OF DISCLOSURE

[0004] This disclosure relates generally to molecules that bind proteins of interest, and methods of generating them. In particular, these binders can be used for basic research, as well as diagnostic and therapeutic purposes.BACKGROUND

[0005] Affinity reagents, molecules that bind to a target protein of interest, are critical as basic research tools for measuring or tracking biomolecules1, as probes for studying biological regulation through induced proximity2, as core elements of diagnostics3, and as therapeutics, such as neutralizing antibodies and antibody-drug conjugates4. Affinity reagents are also known as “binders.” However, even for very well studied organisms, including Homo sapiens, antibody -based binders do not exist for many proteins of interest and, when available, are notorious for heterogeneous quality control, function, and specificity5.

[0006] Binders can be developed by generating antibodies to a target of interest through animal immunization, molecular display methods, or by computational design6.Immunization-mediated binder generation typically costs thousands of dollars, takes many months, and is limited to creating antibody-based reagents6. In vitro selection approaches, such as display-based methods (e.g. phage display, mRNA display), cell sorting methods (e.g. FACS, MACS), and growth-based methods (e.g. bacterial-2-hybrid selections) can be used tomine high diversity libraries of protein variants to identify binders to a target of interest7'11. However, these selection-based methods take several months to complete, primarily due to high false positive rates necessitating time-consuming secondary screening12'14. Finally, while rapidly improving, computational approaches require significant computing capacities, expertise, and subsequent display-based selections, affinity maturation, and / or screening15,16.

[0007] Creating a novel binder to a protein generally requires months of highly specialized work, thousands of dollars, and often results in failure. Collectively, the costs and time associated with protein binder generation restrict production to labs with a significant focus and expertise in these techniques, prohibiting exploratory work. In comparison, creating selective binders to DNA or RNA is as simple as designing complementary oligonucleotides (oligos), which can be synthesized and delivered in a matter of days for ~$10. The programmable nature of nucleic acid binders has led to the rapid explosion of diverse CRISPR technologies and other genetic tools.

[0008] Thus, there is an urgent need to address the critical bottleneck of protein affinity reagent creation.SUMMARY OF THE DISCLOSURE

[0009] This disclosure describes methods and composition for selecting and / or identifying novel peptide and protein binders.

[0010] In a first aspect, the present disclosure provides a method of identifying a binder of a target peptide or protein. The method includes the steps of:(a) engineering a host cell to express a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase (RNAP);(b) exposing the host cell to a high diversity library of phage-encoded protein variants each tagged with a second half of the proximity-dependent split RNA polymerase, wherein the phage library is replication deficient, and wherein the phage-encoded protein variants are expressed within the host cell;(c) reconstituting the proximity-dependent split RNA polymerase upon binding of a phage-encoded protein variant to the target peptide or protein, whereby the reconstituted RNAP initiates expression of a phage gene in the phage encoding the binding protein variant to initiate replication of the phage encoding the binding protein variant; and(d) isolating the replicated phage encoding the binding protein variant to identify the binding protein variant as the binder of the target peptide or protein.Optionally, the method can include a method of detecting the replicated phage in order to identify the binding protein variant.

[0011] In some embodiments of the first aspect, the high diversity library comprises 105-1012phage-encoded protein variants.

[0012] In some embodiments of the first aspect, step (d) of the method further comprises serially passaging the library to de-enrich phage that do not encode the binding protein variant and enrich phage encoding the binding protein variant before isolating the replicated phage encoding the binding protein variant.

[0013] In some embodiments of the first aspect, the host cell is E. coli. In some embodiments of the first aspect, the phage is an Ml 3 phage. In some embodiments of the first aspect, the phage gene is gill. In some embodiments of the first aspect, the method is high-fidelity. In some embodiments of the first aspect, the method is high-throughput. In some embodiments of the first aspect, the method is generalizable. In some embodiments of the first aspect, the method can be completed in about 48 hours.

[0014] In some embodiments of the first aspect, the binder is a tool used in research. In some embodiments of the first aspect, the binder is a diagnostic agent. In some embodiments of the first aspect, the binder is a therapeutic agent. In some embodiments of the first aspect, the binder is part of an antibody-drug conjugate.

[0015] In some embodiments of the first aspect, the binder is an inhibitor of peptide or protein function. In some embodiments of the first aspect, the binder is a peptide inhibitor or a protein inhibitor. In some embodiments of the first aspect, the binder promotes degradation of the protein to which it binds, or of some other biomolecule. In some embodiments of the first aspect, the binder regulates enzymatic activity. In some embodiments of the first aspect, the binder affects cellular signaling.

[0016] In some embodiments of the first aspect, the binder alters interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the first aspect, the binder inhibits interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the first aspect, the binder promotes interactions (gluing) between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the first aspect, the target peptide or protein is disordered or lacks secondary structure.

[0017] In some embodiments of the first aspect, the binder is part of a targeted delivery technology. In some embodiments of the first aspect, the binder is part of an engineered cellsurface receptor. In some embodiments of the first aspect, the binder is part of an engineered virus.

[0018] In a second aspect, the present disclosure provides a composition for identifying a binder of a target peptide or protein. The composition can include a host cell containing an expression vector encoding a target peptide or protein tagged with a first half of a proximitydependent split RNA polymerase, as well as a replication-deficient, high diversity phage library. Each phage of the library encodes a different protein variant tagged with a second half of the proximity-dependent split RNA polymerase. The phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, and the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to initiate replication of the phage-encoded protein variant.

[0019] In some embodiments of the second aspect, the high diversity library comprises 105- 1012phage-encoded protein variants. In some embodiments of the second aspect, the host cell is E. coll. In some embodiments, the phage is an Ml 3 phage. In some embodiments of the second aspect, the phage gene is gill. In some embodiments of the second aspect, the binder is a diagnostic agent. In some embodiments of the second aspect, the binder is a therapeutic agent. In some embodiments of the second aspect, the binder is a part of an antibody-drug conjugate.

[0020] In some embodiments of the second aspect, the binder is an inhibitor of peptide or protein function. In some embodiments of the second aspect, the binder is a peptide inhibitor or a protein inhibitor. In some embodiments of the second aspect, the binder promotes degradation of the protein to which it binds, or of some other biomolecule. In some embodiments of the second aspect, the binder regulates enzymatic activity. In some embodiments of the second aspect, the binder affects cellular signaling.

[0021] In some embodiments of the second aspect, the binder alters interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the second aspect, the binder inhibits interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the second aspect, the binder promotes interactions (gluing) between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the second aspect, the target peptide or protein is disordered or lacks secondary structure.

[0022] In some embodiments of the second aspect, the binder is part of a targeted delivery technology. In some embodiments of the second aspect, the binder is part of an engineered cell surface receptor. In some embodiments of the second aspect, the binder is part of an engineered virus.

[0023] In a third aspect, the present disclosure provides a system for identifying a binder of a target peptide or protein. The system can include a host cell containing an expression vector encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase, as well as a replication-deficient, high diversity phage library. Each phage of the library encodes a different protein variant tagged with a second half of the proximity-dependent split RNA polymerase. The phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, and the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to induce replication of the phage-encoded protein variant.

[0024] In some embodiments of the third aspect, the high diversity library comprises 105- 1012phage-encoded protein variants. In some embodiments of the third aspect, the host cell is E. coll. In some embodiments, the phage is an Ml 3 phage. In some embodiments of the third aspect, the phage gene is gill. In some embodiments of the third aspect, the binder is a diagnostic agent. In some embodiments of the third aspect, the binder is a therapeutic agent. In some embodiments of the third aspect, the binder is a part of an antibody-drug conjugate.

[0025] In some embodiments of the third aspect, the binder is an inhibitor of peptide or protein function. In some embodiments of the third aspect, the binder is a peptide inhibitor or a protein inhibitor. In some embodiments of the third aspect, the binder promotes degradation of the protein to which it binds, or of some other biomolecule. In some embodiments of the third aspect, the binder regulates enzymatic activity. In some embodiments of the third aspect, the binder affects cellular signaling.

[0026] In some embodiments of the third aspect, the binder alters interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the third aspect, the binder inhibits interactions between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the third aspect, the binder promotes interactions (gluing) between the target peptide or protein and its endogenous (natural) binding partners. In some embodiments of the third aspect, the target peptide or protein is disordered or lacks secondary structure.

[0027] In some embodiments of the third aspect, the binder is part of a targeted delivery technology. In some embodiments of the third aspect, the binder is part of an engineered cell surface receptor. In some embodiments of the third aspect, the binder is part of an engineered virus.

[0028] In a fourth aspect, the present disclosure provides an expression vector for use in a method of identifying a binder of a target peptide or protein. The vector encodes a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase.

[0029] In a fifth aspect, the present disclosure provides a kit for use in a method of identifying a binder of a target peptide or protein. The kit includes a host cell, an expression vector, and a library of phage-encoded protein variants.BRIEF DESCRIPTION OF DRAWINGS

[0030] FIGS. 1A-1C show a schematic of the PANCS-Binders process. As defined in detail below, PANCS-Binders is a rapid, reproducible, and reliable method for discovering protein binders. FIG. 1A shows a schematic of serial passaging of in vitro, high diversity libraries of protein binders encoded in phage for the rapid discovery of binding variants via conditional phage replication on an E. coli selection strain. Selections include the extinction of inactive variants and enrichment of the active variants. FIG. IB shows a schematic of PANCS-Binders molecular biology. A split RNAP biosensor is used for in vitro selection of binder variants, gill is removed from the Ml 3 phage and placed on the positive selection plasmid (+AP). Phage encode the N-terminal portion of RNAP (RNAPN) fused to a scaffold variant (potential binder). The +AP encodes the target fused to the C-terminal portion of RNAP (RNAPc). If the binder variant interacts with the target, then RNAPN and RNAPc recombine and transcribe gill. A simultaneous counter-selection is performed using a negative selection plasmid (-AP). The -AP encodes an off-target fused to an orthogonal RNAPc. If the binder variant interacts with the off-target, then the RNAPN and RNAPc recombine and transcribe glllns - a dominant negative variant of ZZZ that poisons phage amplification by preventing release of phage from the E. coli host. FIG. 1C shows a representative timeline for 2-week binder discovery using PANCS-Binders: clone the desired target(s) into a +AP(s) and construct the selection strain, 4-6 passages over 2-3 day of PANCS, a plaque assay or qPCR to assess endpoint titer, and then subcloning and sequencing to validate specific enriched variants.

[0031] FIGS. 2A-2E show the development of PANCS-Binders for selection from in vitro-\ke mock libraries. FIG. 2A shows the selection system for mock libraries, whichcomprises an / ;. coli selection strain with KRAS4b (WT)-RNAPC.CGG as the positive selection target (+AP) and ZBneg-RNAPc,T7 as the counter selection target (-AP). The mock library comprises a mixture of two selection plasmids (SP): an active phage with RNAPN-RAF(RBD) and an inactive phage with RNAPN-Affitin (SasA). FIG. 2B shows a phage amplification assay. 1000 PFU of each phage are incubated with 1 mL of a selection strain for 12 hours and then the titer is determined: amplification is output titer / input titer. FIG. 2C shows the amplification rate for RAF variants and the non-binding affitin (SasA) phage on the KRAS selection strain. Published affinities (Kd) are listed above each RAF variant amplification rate30. Each amplification rate was obtained in duplicate, and error bars indicate SD. The upper dashed horizontal line indicates an amplification rate of 20 (no enrichment if passaging at 5%), and the lower dashed horizontal line indicates an amplification rate of 1.FIG. 2D shows four passage PANCS starting from a mock library (10 PFU active phage with 1010PFU inactive phage (affitin (SasA)) in 5 mL KRAS4b (WT) selection strain (as seen in FIG. 2A) with a 12 hr passage outgrowth and 5% transfer of supernatant phage into fresh cells to seed each passage. FIG. 2E shows titers at the end of each passage (passage 0 indicates the initial titer to start PANCS). The limit of detection (LOD) is 5*102PFU / mL (1 PFU). Each phage was passaged in triplicate, and error bars indicate SD.

[0032] FIGS. 3A-3C show the use of PANCS-Binders to discover novel binders from in vitro libraries. FIG. 3A shows the platform: cloning a target +AP panel, transformation of selection strain, and parallel 4-passage PANCS of each selection strain with a 108variant Affibody library. Titers assessed after the fourth passage indicate which selections enriched binders (shown as AlphaFold predictions of the binder-target pair (if titer was high) or target alone (if titer was low), see Table 2 for titers). The affibody sequence from passage 4 phage was then PCR amplified for NGS and subcloned into a luciferase assay system (shown at bottom right) and pET vectors for protein purification for SPR. Binding validation luciferase assay (bottom right): the target is fused to the RNAPc on an expression plasmid, the binder is fused to the RNAPN on a separate expression plasmid, and a reporter plasmid where LuxAB expression is determined by PPI dependent recombination of the spRNAP. FIG. 3B shows the top variant amino acid sequences identified from the NGS (top) and the percentage of reads for each unique variant in the NGS of passage 4 (bottom left) for each successful selection. Variants shown with their associated sequences were examined further in the luciferase and SPR assays. The second S-WYS variant for Mdm2 isolated for testing in lux did not have a single read in the NGS but is indicated as having a single read for plotting on a log scale. Fold-change in luciferase signal (bottom right) for select variants from the in vitroscreens over the template affibody used in cloning the library (a non-binder), and when available, previously published binders to these targets (NS1 Monobody (70 nM Kd31), Z(RAF322) Affibody (190 nM Kd32), C12-based binder (Kd not determined33). Each condition has four independent data points, and error bars indicate SD. The in vitro binding affinity, Kd, is reported as text for select variants (as measured by SPR, see FIG. 20). FIG. 3C shows the percentage of reads for each unique variant in the original PANCS compared to the percentage of reads for each unique variant in a biological replicate of the PANCS at passage 4 (comparison of parallel replicates in FIG. 22). If a variant was present in one NGS sample but not in another, it was coded as 0.01% of reads.

[0033] FIGS. 4A-4C show high-throughput PANCS-Binders for 95 targets. FIG. 4A shows cloning of a 96-panel set of target selection strains, 96-deep well plate-based parallel PANCS-binder selection of two 108libraries (affibody (FIG. 6) and affitin (FIG. 24)), and using binding assays and qPCR to measure the endpoint titer in a high-throughput manner. FIG. 4B shows an E. coli spRNAP complementation luciferase assay heatmap (FIG.3A) on all preliminary hit variants (titer > 107PFU / mL) when top variant is full-length protein (see Note in Example 4 and FIG. 28 for individual plots). Fold / change in binding >20 is set equal to 20; selections which were not preliminary hits are indicated with an asterisk (*). FIG. 4C shows fold-change in luciferase signal for selection of multiple variants from the in vitro screens assessed across multiple targets and additional controls to assess selectivity. Within each target (y-axis) each binder is normalized to the signal for two non-binders (set to 1): an affibody that was evolved to bind PD-L134and an affitin that was evolved to bind SasA27. Only one off-diagonal had a fold-change >2.

[0034] FIGS. 5A-5C show larger libraries, affinity maturation, and mammalian applications of PANCS-binder hits. FIG. 5A: large, 1010libraries were prepared using parallel transformations (Table 5) that were pooled to initiate large library PANCS-Binders (Table 6), which required starting at an initial volume of 500 mL and decreasing the volume of each passage slowly as the selection progressed (FIG. 33 for NGS results). E. coli spRNAP complementation luciferase assay is heatmap shown for successful selections, with slashes indicating selections that also gave hits in the 95 panel selections (FIG. 4). FIG. 5B shows luciferase assay results for large KRas G12D selection (FIG. 34 for others) with in vitro affinity listed above each bar, comparing literature reference binder (NS1 Monobody), original binder from smaller scale selection (Ab-1 (N-VCD)), and new binder (Ab-N-LHY) (see FIG. 35 for surface plasmon resonance (SPR)). FIG. 5C shows affinity maturation of Mdm2 Affibody hit by PACE. Passage 4 from the Mdm2 selection with the 108affibodylibrary (FIG. 3B) was used to seed PACE. PACE was initiated on a +AP selection strain with lower Mdm2 expression than the original PANCS. After 24 hours, there was a 12-hour mixing step with an even lower expression level strain for an additional 36 hours (total of 60 hours). Titers were monitored by activity independent plaque assay, and variants were subcloned at 60 hours (isolated variants shown here). FIG. 5D: variants were tested in the luciferase binding assay alongside T-WDN and the non-binding Affibody (PDL1) as in FIG.3. Affinities measured by SPR (FIG. 33) are shown above each luciferase bar graph. FIG. 5E: initial KRAS G12D (FIG. 3B) and high affinity KRAS G12D (FIG. 5B) binders were tested for binding in mammalian cells using a split nano-luciferase assay in HEK293T cells. Transfections were performed in triplicate; error bars indicate SD. FIG. 5F is a schematic showing LC3B recruiting element (LIR motif) fused to binders for targeted degradation by autophagy.36FIG. 5G shows a representative Western blot (WB) of endogenous KRAS degradation by Ab N-LHY - LIR (FIG. 38 for full blots and replicates). FIG. 5H shows quantification of replicate degradation results as shown in FIG. 5G (FIG. 38). FIG. 51 shows Mdm2 / Mdm2 binder co-localization: mCherry-tagged Mdm2 and GFP -tagged Mdm2 binder co-localize in the nucleus, while a control GFP does not (replicates in FIG. 39). FIG. 5 J shows a representative WB of Mdm2-p53 PPI inhibition; inhibition of this PPI activates p53 transcription resulting in increased expression of p21 and Mdm2 (FIG. 40 for full blots and replicates). Quantification of replicate WBs for Mdm2-p53 PPI inhibition in U2OS cells: FIG. 5K, Mdm2 expression and FIG. 5L, p21 expression (FIG. 40). For Western blot quantification, statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparison test binder vs. control. *P < 0.05; **P < 0.01.

[0035] FIGS. 6A-6C show construction of the affibody library. FIG. 6A shows the affibody structure with constant residues and randomized residues shaded differently. FIG.6B show the template phage (SEQ ID NO: 188) used when cloning the library (SEQ ID NO: 189) and the position and possible codons in the library variants. FIG. 6C shows monitoring of total phage after transformation. The early timepoints (highlighted) are a lower-bound on the number of variants in the library (5*107) and the 2 h timepoint is the upper-bound (2*109) as the phage were separated from the transformed cells at this timepoint. We refer to this as a 108variant library.

[0036] FIG. 7 shows PACE of the affibody library from FIG. 6 on two targets - RAF and IFNG- with two different selection strength +APs (ori, glll / RNAPc RBS strength). From pl 5 SD8 / SD8 to pSClOl SD8 / SD8 for RAF; from pSClOl SD8 / SD8 to pl 5 SD8 / sd5 for IFNG. In addition to the +AP, each strain had a ZBneg-AP (20-01) and MP6 (see Table 8.3).The first strain proceeded for 24 h (at which point a titer was collected) and then mixed 1 : 1 with the second strain for 12 h, then finally the second strain was used alone for 24 h at which point the titer was again collected. A single 20 mL lagoon was seeded with IO10PFU library phage and filled with strain 1 and with arabinose to induce mutagenesis, after 1 h, the lagoon was flowed out to waste at a rate of 1 vol / h. During the mixing experiment, each selection strain chemostat flowed at half speed to maintain 1 vol / h.

[0037] FIG. 8 shows PACS with a mock library. The KRAS WT (+AP; 3 l-69) / ZBneg (-AP; 20-06) selection strain and active (RAF WT) and inactive (Affitin (SasA)) phage used in FIG. 2 were used here in PACS (duplicate 20 mL lagoons were seeded with 1010PFU library phage, and after 1 h, the lagoons were flowed out to waste at a rate of 1 vol / h. The mock library consisted of 1010inactive phage mixed with varying amounts of active phage (legend). By 36 h, all lagoons had gone extinct except for the mock library spiked with 105active phage. Titers measured by plaque assay in duplicate (LOD of 103PFU / mL).

[0038] FIGS. 9A-9B show the rate of phage infection. FIG. 9A. Phage (RNAPN-RAF WT) were added to 1 mL LB or 1030 at two different concentrations (~ equal cell-to-phage ratio for 1030 and roughly lOx cell-phage ratio for 1030) and their titers were monitored over time. 1030 cells were grown to an OD600 of 0.3 prior to infection. 1030 cells are phage infectible, but do not support phage replication as they cannot supply giii. Duplicate tubes were performed for each combination and the titers were measured overtime. Phage and cells for the 0 hour timepoint were separated by centrifugation ~ 1 minute after combining and mixing. FIG. 9B. Percent of phage infected were determined by the difference between initial number of phage (determined as the average titer of phage in LB) and the remaining number of phage (as determined by the titer of the 1030 culture). Phage infections plateau at -95% at 6 hours. These data indicate that the rate at which phage infect cells decreases logarithmically as the concentration of non-infected (and therefore infectable) cells decreases.

[0039] FIG. 10 shows amplification rates of Raf phage using optimized conditions.Using the amplification rates in FIG. 2C, we predicted the expected enrichment of RAF mutant phage and de-enrichment of non-binder (Affitin (SasA)) phage based on an initial 5 mL volume, 10 or 1010PFU respectively, and a 5% transfer between passages. We constrained the bottom to 0.01 and the top to 1012PFU / mL respectively. The Inactive phage goes to extinction by passage 4 and the active variant enriches to saturation by passage 2. We expect that both rates are over-estimated by the amplification rate: RAF WT should amplify more slowly than in the amplification assay as most cells have been infected by an inactive phage and are therefore non-infectible; similarly, the non-binder should de-enrich moreslowly as some fraction of phage will not infect a cell in the initial passage(s). Therefore, we settled on testing 4 passages in FIG. 2E.

[0040] FIG. 11 shows PANCS with mock library and varied +APs. The active (RAF WT, Q66A K84A) and inactive (Affitin (Sas A)) phage was used in mock library PANCS using different +AP strengths (31-69 (top) and 31-70 (bottom), see Table 8.2). Each PANCS had a 1 mL passage volume with 5% transferred between passages (4 total) that was seeded with 109PFU inactive phage and 10 PFU active phage (duplicate PANCS). PCR was used to estimate the endpoint titer of each PANCS. The PCR cycle number, 25, was optimized so that 106PFU / mL produced a faint band (similar to that observed in the lower gel). The pl 5a, SD8 / SD8 +AP strength was chosen for +AP designs moving forward.

[0041] FIG. 12 shows PANCS with mock library and varied -APs. The active (RAF WT, Q66A K84A) and inactive (Affitin (Sas A)) phage was used in mock library PANCS using different -AP strengths (top-bottom: none, 20-6, 20-5, and 20-1, see Table 8.3). Each PANCS had a 1 mL passage volume with 5% transferred between passages (4 total) that was seeded with 109PFU inactive phage and 10 PFU active phage (duplicate PANCS). PCR was used to estimate the endpoint titer of each PANCS. The PCR cycle number, 25, was optimized so that 106PFU / mL produced a faint band (similar to that observed in the lowest gel on the right). The sd8 / sd8 -AP strength was chosen for PANCS moving forward.

[0042] FIG. 13 shows PANCS with mock library and varied transfer rates. The active (RAF WT, Q66A K84A) and inactive (Affitin (SasA)) phage was used in mock library PANCS using different transfer rates. Each PANCS had a 1 mL passage volume with varying amounts transferred between passages (4 total) that was seeded with 109PFU inactive phage and 10 PFU active phage (duplicate PANCS). The same selection strain was used in each of these PANCS. PCR was used to estimate the endpoint titer of each PANCS. The PCR cycle number, 25, was optimized so that 106PFU / mL produced a faint band.

[0043] FIGS. 14A-14B show PANCS with mock library and varied initial cell-to-phage ratio. FIG. 14A. Amplification rate of 5*105active (RAF WT) phage in 1 mL of KRAS +AP selection strain grown for 12 h with either no inactive phage, simultaneous addition of 1010inactive phage (>10x the number of cells), or prior infection (1 h of 1010inactive phage (>10x the number of cells). Active phage were monitored using activity dependent plaque assays (active phage shows robust plaques, inactive do not produce plaques). Both simultaneous and prior infection of excess phage prevented replication of the active phage when the phage-to-cell ratio was >10:1. FIG. 14B. The active (RAF WT) and inactive (Affitin (SasA)) phage was used in mock library PANCS on the KRAS (+AP) / ZBneg(-AP) using a 5% transfer rate.PCR band size was used to estimate the relative population of active and inactive phage after 4 passages. Each PANCS had a different passage volume and was seeded with 10 PFU active phage and an amount of inactive phage either lOx, lx, or O.lx the number of cells in the passage (assuming 109cells / mL). Shading indicates failure to enrich the active phage during PANCS. These data demonstrate the necessity for having at most a 1 : 1 phage-to-cell ratio in the initial passage.

[0044] FIG. 15 shows mock library selections with additional binder-target pairs.Previously published binders to RAF (Affibody (RAF)2), hSUMOl (Monobody (hSUMOl)3), and KRAS WT (Monobody (KRAS), NS14) were used in mock library (10 PFU active and IO10PFU inactive (Affitin (SasA)5) using 4 passages, 5% transfer, 5 mL starting volume, with pl5a / SD8 / SD8 +APs and sd8 / sd5 (20-01) ZBneg-AP. PANCS was performed in triplicate and endpoint (passage 4) titers were measured with an activity independent plaque assay. LOD was 5*104PFU / mL (0 PFU, data points not shown). Error bars indicate standard deviation.

[0045] FIGS. 16A-16C show tracking enrichment of top variants. For the first 6-target panel PANCS, we collected NGS of the initial affibody library, passage 2, passage 3, and passage 4. This allowed us to monitoring enrichment of the active phage across passages as shown for the top three variants at passage 4 for KRAS G12D (FIG. 16A), RAF RBD (FIG.16B), and IFNG (FIG. 16C). Generally, the relative ratio of variants stabilizes by passage 2 or 3.

[0046] FIG. 17 shows AlphaFold3 predictions of binder-target interactions for binders from 6-target panel PANCS (FIG. 3B). KRAS G12D / N-VCD, RAF / T-VPN, RAF / N-LFN, RAF / Y-LCS, IFNG / S-VVD, IFNG / S-VCD, Mdm2 / S-WFY, Mdm2 / S-WYS, and Mdm2 / T-WDN (top to bottom, left to right).

[0047] FIG. 18 shows specificity of PANCS derived binders. Shown is the fold-change in luciferase signal for select variants from FIG. 3B over the template affibody (PDL1) used in cloning the library (a non-binder). Individual values in the heatmap are the average of triplicates. Only one off-target has a fold-change significantly (p = 0.05) greater than 1.0 (S-VVD on Mdm2).

[0048] FIG. 19 shows purification of GST-tagged binders. Each final purified binder was run on a 12% acrylamide SDS PAGE gel and run next to a gradient of BSA. Stained with Coomassie Blue and imaged using a LICOR. All in vitro binding affinities reported here (Table 3) are from these purified proteins.

[0049] FIG. 20 shows Surface Plasmon Resonance (SPR) dose-response and kinetic model fits for Kas shown in FIG. 3B. See Table 3 for details of model fits. Each target-binder pair required a different concentration range: KRAS G12D with N-VCD (2000, 1000, 500, 250, 62.5, 15.3, 4, 0 nM); RAF with T-VPN (4000, 2000, 1000, 500, 250, 125, 62.3, and 0 nM), N-LFN (24000, 12000, 6000, 3000, 1500, 750, 375, 0 nM), and Y-LCS (4000, 2000, 1000, 500, 250, 125, 62.3, and 0 nM); IFNG with S-VVD (15000, 10000, 5000, 2500, 1250, 625, 313, 0 nM) and S-VCD (24000, 12000, 6000, 3000, 1500, 750, 375, 0 nM); and Mdm2 with S-WFY (1000, 500, 250, 125, 62.5, 31.3, 15.6, 0 nM), S-WYS (2000, 1000, 500, 250, 62.5, 15.3, 4, 0 nM), and T-WDN (2000, 1000, 500, 250, 62.5, 15.3, 4, 0 nM). All interactions were measured in duplicate and all data was fit to a 1 : 1 binding model.

[0050] FIG. 21 shows replicate 6-target panel PANCS. In addition to the initial replicate, technical quadruplicates were performed several months later (FIG. 3A, Table 2). Each of the 5 total PANCS are shown as individual datapoints, error bar indicates SD. For Myc, all four of the parallel replicates had a titer below the limit of detection (LOD) and therefore are shown as being equal to the LOD (500 PFU / mL).

[0051] FIG. 22 shows a comparison of parallel replicates by NGS. Percentage of reads for each unique variant in one of the parallel replicate PANCS compared to the percentage of reads for each unique variant in each of the other parallel replicates. If a variant was present in one NGS sample but not in another, it was coded as 0.001%. of reads. For variants >0.1% of NGS reads, there was a high correlation between parallel replicates (r = 0.95; average of each pairwise Pearson’s Correlation).

[0052] FIGS. 23A-23F show the effect of extending the linker length connecting the target and RNAPc. FIG. 23A.The structure of T7 RNAP (1CEZ) with the split site shown as spheres and the N- and C-termini shaded differently. FIG. 23B. The structure (3OGO) of a nanobody (left) binding to GFP (right) with each terminus that would be fused to spRNAP in this system shown as spheres - the distance between them is >50 A. On GFP, all atoms within 20 A are shaded lighter, indicating the likely radius reachable for a 6 AA linker. FIG.23C. AlphaFold3 prediction of the interaction of HRAS (1-169) with the RAF (RBD) compared to the predicted folding of HRAS-6aa-8aa-RAF and HRAS-60aa-8aa-RAF aligned to HRAS where 6aa, 8aa, and 60aa refer to linker lengths used in our binder / target fusions with spRNAP. The 60 AA linker is predicted to allow HRAS and RAF to bind in the actual PPI conformation, but the 6 AA linker is not. FIG. 23D. Extended linker lengths, sequence, and predicted maximum extended length (assuming 3.5 A / residue) cloned into several target-RNAPc fusion +AP and Lux-C plasmids that function well with a short linker. FIG. 23E.Phage amplification rate with varied linker lengths for several binder-target pairs (each measured as a single replicate). FIG. 23F. Change in luciferase signal when the linker length is extended for several binder-target pairs (same legend as in FIG. 23E). Each pair has three biological replicates of technical quadruplicates (12 total measurements). Each luminescence signal is normalized to the 6 AA linker for each target. Overall, E and F suggest that extending the linker length decreases replication and signal, but to a minimal extent (<10x lower amplification rate and <2x lower luciferase signal).

[0053] FIGS. 24A-24C show construction of the affitin library. FIG. 24A shows the affitin structure with constant residues and randomized residues shaded differently. FIG. 24B shows the template phage (Affitin (SasA) 3X STOP, SEQ ID NO: 190) used when cloning the library (SEQ ID NO: 191) and the position and possible codons in the library variants (all are NNY at the DNA level). FIG. 24C shows monitoring of total phage after transformation. Because this transformation utilized the 10P-1059 (non-infectible E. colt), the titer does not change much between the timepoints (12% increase). We estimate that the likely total number of variants is similar to total titer measured at 40 minutes: 1.5*108.

[0054] FIG. 25 shows background replication rates of all targets. Proximity independent, NWT phage amplification rates on 96-target panel +AP only (left) and +AP with -AP (right). Amplification rates obtained using 40,000 PFU RNAP-NWT phage (70-63; Table 8.1) as the input and the output was measured using activity independent plaque assays. (Left) Left dashed line indicates where a transfer rate of 10% would prevent enrichment during PANCS; right dashed line indicates rate for RAF WT phage on KRAS used in optimizing PANCS (FIG. 2C)

[0055] FIG. 26 shows a 95-target panel PANCS endpoint titer heatmap. Endpoint titer heatmap for each well of the 192 selections (FIG. 4A): legend shows titer (Log PFU / mL) with darker shading indicating a higher titer, white indicating 107, and lighter shading indicating lower titers to extinction. Titers determined by single replicate qPCR and compiled in Table 4.

[0056] FIG. 27 shows analysis of NGS for hits from the 96-target panel PANCS with affibody and affitin. We performed NGS on each hit (see Methods) and show in the figures below each variant that had >1% of reads for a hit (organized as each affibody hit and then each affitin hit below). We then used AlphaFold2 multimer collab (AlphaFold2.ipynb - Colab (google.com)) for predicting the interaction between binder and target for the top 4 variants (above 1% of reads). Alphafold metrics: pLDDT (Average Predicted Local Distance Difference Test): Tests for local structural confidence - Very Low (<50), Low (60), OK (70),Confident (80), Very High (>90); pTM (Predicted Template Modeling Score): Measures structural congruency - pTM > 0.5 is generally recognized as strong enough to accept as inference; and ipTM (Interface Predicted Template Modeling Score): Similar to pTM but looking at multimer interactions - >0.8 indicates high confidence and less than <0.2 indicates no confidence in the predicted interaction interface. Predicted structure is omitted for predictions in which the pLDDT was <50 or the pTM was <0.5. Targets are shown in light grey, binders are other colors corresponding to the pie chart. “Library” sequence in each panel is either SEQ ID NO: 189 or SEQ ID NO: 191.

[0057] FIG. 28 shows E. coli luciferase validation assay for variants isolated from 95-panel target PANCS. Scaffolds from each hit were subcloned into Lux-N expression plasmids and three clones were sequenced prior to testing. If the top variant was not obtained, a second attempt was made; after which, the most dominant variant obtained was tested. Affibody hits are indicated by “Ab-1”, and affitin hits are indicated by “Af-1”. Fold-change in luciferase signal for select variants from the de novo screens over two non-binding protein variants (Non-Binder 1 is an Affibody evolved to bind PD-L16and Non-binder 2 is an Affitin evolved to bind SasA5; for SasA, a third Non-Binder variant was used ((Monobody (hSUMOl)3). Each condition has four independent data points and error bars indicate standard deviation.

[0058] FIGS. 29A-29C summarize the 100 preliminary hitNGS results. FIG. 29A shows the number of variants with >1% of reads within a hit, grouped according to whether top variant was a full-length scaffold or a truncation. FIG. 29B shows the percentage of reads of the most dominant variant within a hit, grouped according to whether the top variant was a full-length scaffold or a truncation. FIG. 29C shows the AlphaFol d2 Multimers prediction iPTM for the top variant in all hits (excluding top hits that were truncated) as assessed by binder luciferase assay (FIG. 28).

[0059] FIG. 30 shows NGS data for select PANCS wells just below the hit criteria threshold. For KRAS WT, FOXP3, and alpha amylase, “library” sequence is SEQ ID NO: 189. For no fusion, GFP, and G3BP, “library” sequence is SEQ ID NO: 191.

[0060] FIG. 31 shows that passaging increases signal -to-noise of hits and decreases false positives. Shown is additional passaging of passage 4 (P4) using a 5% transfer rate for 2 passages (P6). Each data point indicates the titer (measured by qPCR) of a single well in the deep 96-well PANCS plate.

[0061] FIGS. 32A-32H show AlphaFold 3 predictions for every target-binder pairing in the specificity of binding luciferase dataset (FIG. 4C). FIG. 32A, FIG. 32B, and FIG. 32C show pTM, iPTM, and iPTM / PTM heatmaps, respectively. The diagonal indicating positionof binder-target pairing is shown as a black line. FIG. 32D shows the correlation of iPTM and binding. FIG. 32E and FIG. 32F show iPTM separated by target and by binder, respectively. Binders exhibited a >3 -fold change in luciferase signal). FIG. 32G shows statistical categorization of iPTM of predictions for variant-target pairings for binder and non-binder pairs. FIG. 32H shows the percentage of false negatives and false positives within this data set if an iPTM threshold is set as a cut-off for filtering binders from nonbinders.

[0062] FIG. 33 shows sequence prevalence plots for hits from the large (IO10) library PANCS. The percentage of NGS reads is plotted for each unique variant, and variants tested in the luminescence assay are shown. The sequences of all variants with >5% of reads are listed next to their percentage of NGS reads (sequences below 5% included if luminescence assay data were collected for that variant).

[0063] FIG. 34 shows luciferase binding assays of variants from 1010library PANCS. Original best variant from 108library PANCS indicated. New affibody and affitin hits are also shown. Fold-change in luciferase signal for select variants from the de novo screens over two non-binding protein variants (Non-Binder 1 is an Affibody evolved to bind PD-L16 and Non-binder 2 is an Affitin evolved to bind SasA5). Each condition has four independent data points and error bars indicate standard deviation.

[0064] FIG. 35 shows Surface Plasmon Resonance (SPR) dose-response and kinetic model fits for Kas shown in FIG. 5B. See Table 3 for details of model fits. Each target-binder pair required a different concentration range: KRAS G12D with Y-LHD (400, 200, 100, 50, 25, 12.5 0 nM), Y-LHY (400, 200, 100, 50, 25, 12.5 0 nM), N-LHY (100, 50, 25, 5, 1, 0.2, 0 nM), S-LCS (200, 100, 50, 25, 12.5, 6.3, 0 nM), and N-WSS (100, 50, 25, 5, 1, 0.2, 0 nM); and RAF with N-WCN (60, 30, 15, 7.5, 3.8, 1.9, 0 nM) and Y-WID (60, 30, 15, 7.5, 3.8, 1.9, 0 nM). All interactions were measured in duplicate and all data was fit to a 1 : 1 binding model.

[0065] FIG. 36 shows Surface Plasmon Resonance (SPR) dose-response and kinetic model fits for Kas shown in Fig. 5D. See Table 3 for details of model fits. Each target-binder pair required a different concentration range: Mdm2 with Y-WTT (1000, 500, 250, 125, 62.5, 31.3, 15.6, 0 nM), Y-WCT A46T (200, 100, 50, 25, 6.3, 1.5, 0.4, 0 nM), and Y-WCT A46T L51F (1000, 500, 250, 125, 62.5, 31.3, 15.6, 0 nM). All interactions were measured in duplicate and all data was fit to a 1:1 binding model.

[0066] FIGS. 37A-37B show that AlphaFold cannot predict whether proteins bind. FIG.37A shows the correlation between the iPTM (AlphaFold 3 predictions) and the Kd for everybinder for which the in vitro binding affinity was determined. Pearson Correlation of -0.50 (two tailed p = 0.028; 95% CI = -0.78 to -0.06). FIG. 37B shows grouping of iPTM values by affinity. No grouping is statistically different from any other group (all pairwise t-test are not significant at p = 0.05).

[0067] FIG. 38 shows endogenous KRAS degradation. The figure shows KRAS binder (Ab-N-LHY)-LIR fusion targeted degradation of endogenous KRAS in U2OS cells (FIGS.5G-5H). An initial replicate, which included a positive control of NS1 Monobody (previously demonstrated to interact with KRAS in cells) was followed up by two additional replicate transfections. Ab-N-GIY binds to PIK3CB and not KRAS. Other bands in the full blots are from other attempts to screen effectors for degradation and are not related to LIR driven degradation.

[0068] FIG. 39 shows Mdm2 co-localization. Mdm2-mCherry and either GFP or Mdm2 binder (FIG. 5D)-GFP fusions were co-transfected in HEK293T cells.

[0069] FIG. 40 shows Mdm2 inhibition. Mdm2 Binders (FIG. 5D) expressed in U2OS cells inhibit the Mdm2-p53 interaction resulting in p53 driven transcription of Mdm2 and p21 (FIGS. 5J-5L).

[0070] FIG. 41 shows nanobody hits using the methods disclosed herein.DETAILED DESCRIPTION OF THE INVENTION

[0071] It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0072] All publications, including but not limited to journal articles, patents, and patent applications, cited in this specification are herein incorporated by reference as though set forth in their entirety in the present application.Definitions

[0073] Before describing the methods and compositions of the disclosure in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, referenceto “a peptide” means one or more peptide molecules, and reference to “a binding protein variant” means one or more binding protein variants.

[0074] As used herein, a peptide is defined as a molecule that contains two or more amino acids. Peptides that contain many amino acids are called polypeptides or proteins. Thus, as used herein, the terms “peptide” and “protein” are interchangeable. The choice of term makes no assertion regarding any particular properties of the molecule, including but not limited to length, molecular weight, or secondary structure.

[0075] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0076] In some embodiments, percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated disclosure.

[0077] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0078] As used herein, ranges and amounts can be expressed as “about” a particular value or range. “About” also includes the exact amount. For example, “about 5%” means “about 5%” and also “5% .” The term “about” can also refer to ± 10% of a given value or range of values, or to a value on a similar order of magnitude. Therefore, about 5% also means 4.5% -5.5%, for example, or 1-10%.

[0079] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0080] As used herein, the terms “protein tag” is utilized to describe a short sequence of amino acids, joined to each other by peptide bonds, which is appended to another molecule(whether another peptide, a protein, a nucleic acid, and / or a small molecule, for example) in order to impart it with a particular property or to direct its localization in space. The linkage between the protein tag and the other molecule can be a peptide bond, as in a fusion protein, and / or some other chemical or biochemical linkage.

[0081] It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the methods and compositions as described herein or to imply that certain features are critical, essential, or even important to the structure or function of the subject matter recited in the claims.

[0082] As utilized in accordance with the present disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the meaning commonly understood by one of ordinary skill in the art.Overview

[0083] PANCS-Binders is a rapid, reproducible, and reliable method for discovering protein binders. The entire process of cloning a target into the +AP / RNAPc expression plasmids, selection, assessment, and secondary validation assays can routinely be performed in 2 weeks without the requirement for highly specialized expertise or equipment. The speed of PANCS-Binders comes from its strong de-enrichment of weak and non-binders and high enrichment of binders, which abrogates the need for secondary screening campaigns common in display-based selection techniques. We propose that two fundamental aspects of such techniques limit the relative enrichment of binding variants over non-binding variants commonly achieved in display methods: threshold selection (bound or not bound) and activity-independent amplification. PANCS-Binders utilizes a split RNAP -based biosensor with a large dynamic range that links the degree of variant function to the degree of phage replication for that variant, both creating a gradient rather than threshold selection and removing the activity-independent amplification step. Notably, neither Alphafold2 Multimer (FIG. 29) nor AlphaFold3 (FIG. 32 and FIG. 37) could predict binding (false negatives) or non-binding (false positives) from our screen, illustrating the enduring importance and value of real experimentation and limitations still inherent in computational modeling.

[0084] The ability to utilize high diversity libraries in phage-assisted selections and evolutions is a powerful tool in the directed evolution arsenal and should expand the range of evolutions possible. PACE and PANCE have been applied to alter or tune the specificity of a variety of protein functions8,17'19’21'23,25’29’38; however, because mutations accumulate incrementally, these campaigns require a nearly continuous evolutionary pathway startingfrom low or non-functional initial variants. In a recent tour de force, the evolution of a protein that binds a small molecule-protein complex, an elaborate pathway was needed to access the 5 mutations needed for minimal function and the 8 mutations eventually reached for high function. This included repeated high mutagenesis drift periods, a 3-position randomized library, steppingstone states (a panel of 16 small molecules), and testing of a wide range of selection stringencies (PACE and PANCE across 26 different stringency selection plasmids). High diversity libraries, like those used here in PANCS-Binders, are prepared using in vitro diversification techniques capable of making tens of targeted mutations in the initial variant. PANCS is a powerful approach to jumpstart more difficult evolutionary campaigns by increasing the navigable distance between functional states.

[0085] PANCS-Binders can screen multiplexed libraries of IO10phage-encoded variants across dozens of targets in 2-3 days, yielding high affinity, selective binders with sufficient fidelity such that hits can be directly used in secondary assays, such as mammalian cell experiments. In our 96-well based high-throughput PANCS-Binders with 108variant libraries, we achieved a 55% hit rate across a range of targets with a high correlation between endpoint titer and validated binding (79 / 92). Repeating a subset of failed selections with a 100-fold larger library produced hits for 38% of those initial failures, showcasing how simply scaling up library size can yield hits for otherwise challenging targets. Additionally, either with large 1010+libraries or through rapid affinity maturation via PACE, high affinity binders (<10 nM) can be obtained quickly.

[0086] PANCS-Binders generated hits for disordered protein targets and proteins that are challenging or impossible to purify, showcasing the potential of this screening and selection platform to discover binders for proteins that lack structural data or are incompatible with in vitro selection strategies. PANCS-Binders did not require significant optimization of selection conditions, as is commonly the case for 2-hybrid selections; while target solubility does impact the target-RNAPc expression level, the differences were small enough to abrogate the need for target dependent tuning of expression. In other words, with PANCS-Binders, one can “plug-and-play” targets into the system, creating a generalizable platform for the identification of novel binders.

[0087] The present disclosure generally describes methods of identifying or selecting affinity reagents (e.g., peptides and / or proteins) that bind proteins or peptides of interest, as well as methods of identifying said reagents. This disclosure also describes compositions, systems, expression vectors, and kits for use in performing such methods.

[0088] The present disclosure contemplates, inter alia, a method of selecting or identifying an affinity reagent, also known as a “binder,” to a target peptide or protein. The method includes the steps of:(a) engineering a host cell to express a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase;(b) exposing the host cell to a library of phage-encoded protein variants each tagged with a second half of the proximity-dependent split RNA polymerase, wherein the phage library is replication deficient and wherein the phage-encoded protein variants are expressed within the host cell;(c) reconstituting the proximity-dependent split RNA polymerase upon binding of a phage-encoded protein variant to the target protein, whereby the reconstituted RNAP initiates expression of a phage gene in the phage encoding the binding protein variant to initiate replication of the phage encoding the binding protein variant; and(d) isolating the replicated phage encoding the binding protein variant and thereby identifying the binding protein variant as the binder of the target peptide or protein.Optionally, the method can include a method of detecting the replicated phage in order to identify the binding protein variant.

[0089] In some embodiments, a method of detecting the binding protein variant is included. Modalities for detecting the binding protein variant can include luminescence or another readout. In some embodiments, these features of the method enable automation.

[0090] In some embodiments, the binder is a genetically encoded molecule. In some embodiments, the binder is a bifunctional molecule. In some embodiments, bifunctional molecules are engineered by connecting two separate binders, each binding a distinct and unique target, by a linker. In some embodiments, bifunctional molecules include PROTACs (proteolysis targeting chimeras) and bsAbs (bispecific antibodies).

[0091] In some embodiments, engineering a host cell to express a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase, in some cases using a linker, can include genetic manipulation and protein design. Methods of engineering cells in this way are well known to those of ordinary skill in the art. Usually, such engineering includes linking a target peptide or protein to a tag. Methods of linking usually comprise synthesizing or cloning a DNA sequence encoding a target peptide or protein and a tag into a suitable expression vector, followed by transformation or transfection of a suitable host cell with the expression vector under conditions amenable to expression of the target peptide or protein. In some embodiments, the expression vector is a pET vector. Insome embodiments, the host cell is a bacterium. In particular embodiments, the bacterium is E. coll. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an animal cell.

[0092] As used herein, the term “tagged” refers to a protein, polypeptide, or peptide that is covalently or operably linked to an additional sequence or moiety (a tag) that facilitates one or more functions such as detection, purification, immobilization, localization, quantification, or modulation of activity. The tag may be peptidic (e.g., an epitope tag, affinity tag, fluorescent protein, or degradation tag) or non-peptidic (e.g., a chemical label, fluorophore, or biotin group), and may be positioned at the N-terminus, C-terminus, or an internal site of the protein. Unless otherwise indicated, the term encompasses both direct fusions and indirect linkages through a spacer, linker, or adaptor moiety.

[0093] In some embodiments, the method incorporates a library of potential binders. In some embodiments, the library of potential binders includes peptide or protein variants. In some embodiments, the library of peptide or protein variants is a library encoded by bacteriophages (also known as phages), where each phage encodes a different peptide or protein variant. In some embodiments, contemplated libraries of the present disclosure may encode about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, or more variants. Libraries containing this high number of variants, wherein each variant is unique, are termed “high diversity” libraries. In some embodiments, the library is diversified in vitro using methods such as error-prone PCR or parallel single-site saturation mutagenesis (SSM). In preferred embodiments, the phage is an Ml 3 phage. In preferred embodiments, the phage is replication-deficient. In some embodiments, the peptide or protein variants carried by the phage library are tagged with a second half of a proximity-dependent split RNA polymerase. In some embodiments, exposing the host cell to the phage library leads to uptake of the phages into the cell and expression of the peptide or protein variants encoded by the phages.

[0094] In some embodiments, the first half of the proximity-dependent split RNA polymerase is the C-terminal half (RNAPc), and the second half of the proximity-dependent split RNA polymerase is the N-terminal half (RNAPN). In other embodiments, the first half of the proximity-dependent split RNA polymerase is the N-terminal half (RNAPN), and the second half of the proximity-dependent split RNA polymerase is the C-terminal half (RNAPc). Examples of split RNA polymerases are taught by U.S. Patent No. 11,913,081, incorporated by reference in its entirety herein.

[0095] As used herein, a “first half’ can be a first portion of two portions that need not be equal in size. Similarly, a “second half’ can be a second portion of two portions that need not be equal in size. Taken together, the first half and second half constitute the whole of the proximity-dependent split RNA polymerase.

[0096] In some embodiments, the method is high-fidelity - meaning it repeatedly and thoroughly assesses a library. In some embodiments, the method is high-throughput, amenable to running multiple selections in parallel in multi -well (e.g., 96-well) plates. In some embodiments, the method is generalizable. Generalizability means that one practicing the invention can choose any target peptide or protein of interest and successfully perform the method to identify a binder.

[0097] In some embodiments, the method can be completed more quickly than preexisting methods in the field. In some embodiments, the method can be completed in about 24 hours. In some embodiments, the method can be completed in about 48 hours (about 2 days). In some embodiments, the method can be completed in about 72, 96, 120, 144, 168 hours, or an interval of time encompassed therein. In some embodiments, the method can be completed in about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or an interval of time encompassed therein.

[0098] In some embodiments, the binder can be an experimental tool used in research. Such research tools can be used to investigate protein-protein interactions within a biological system of interest. Such research tools can also be used to measure or track biomolecules (PMID: 36370112) or find use as probes for studying biological regulation through induced proximity (PMID: 29590011).

[0099] In some embodiments, the binder can be a diagnostic agent. Diagnostic agents can be used to determine the presence, location, concentration, and / or amount of a target peptide or protein that can be indicative of a disease, condition, or disorder. Examples of diagnostic agents can be employed for use in detection assays for target peptides. For example, a contemplated detection assay can include an ELISA (enzyme-linked immunosorbent assay), including those that detect viral or bacterial antigens from pathogens such as S. pyogenes, influenza viruses, and coronaviruses. Additional applications of the diagnostic agents are contemplated herein.

[0100] In some embodiments, the binder can be a therapeutic agent. Therapeutic agents can be used to prevent or treat a disease, condition, or disorder that is mediated through the presence or activity of a specific peptide or protein. Examples of therapeutic agents caninclude antibody-drug conjugates, antibody-based drugs, cell-penetrating peptides, and therapeutic peptides, such as insulin and GLP-1 agonists.

[0101] There are a variety of ways in which binders and affinity reagents affect the proteins or peptides to which they bind. In some embodiments of the method described herein, the binder is an inhibitor of peptide or protein function. In some embodiments, the binder is an activator or inducer of peptide or protein function. Binders with these inhibitory or activating properties can be known as antagonists or agonists, respectively.

[0102] In some embodiments, the binder promotes degradation of the peptide or protein to which it binds, or of some other biomolecule. Examples of processes that promote degradation of the target peptide or protein include proteolysis-dependent mechanisms (including E3 ubiquitin ligase-dependent proteolysis via the proteasome), autophagy, binding-induced self-degradation, binding-induced misfolding or destabilization, or allosteric or orthosteric inhibition of a stabilizing interaction. Examples of processes that promote degradation of some other biomolecule include activation of a protease or nuclease, alteration of reactive species, or blocking transcription or translation of another biomolecule.

[0103] In some embodiments, the binder regulates the activity of a peptide or protein, which may be an enzyme. Such regulation can occur through modulation of catalytic activity, for example by directly occupying or altering the active site of the enzyme, or by binding to an allosteric site of the target peptide or protein to modulate its conformation or functional state. The binder can also regulate activity by modulating one or more post-translational modifications (e.g., phosphorylation, ubiquitination, or acetylation) or by recruiting enzymes that catalyze such modifications. In some embodiments, regulation of activity occurs through interference with protein-protein or protein-nucleic acid interactions, control of subcellular localization, or alteration of protein stability or degradation. In other embodiments, activity is regulated by recruitment of other proteins, activation or inhibition of a regulatory enzyme, or modulation of transcriptional or translational activity.

[0104] In some embodiments, the binder alters interactions between the target peptide or protein and its endogenous binding partners. Endogenous binding partners are also known as natural binding partners. In some embodiments, altering interactions includes inhibiting interactions, and in some embodiments, altering interactions includes promoting interactions. The promotion of these interactions is also known as molecular gluing. In some embodiments, the target peptide or protein has a well-defined structure. In other embodiments, the target peptide or protein is disordered or lacks secondary structure.

[0105] In some embodiments, the binder affects cellular signaling. The binder can modulate one or more signal transduction pathways by altering the activity, localization, or stability of signaling proteins such as kinases, phosphatases, transcription factors, receptors, or adaptor proteins. In some embodiments, the binder activates or inhibits upstream or downstream components of a signaling cascade, thereby modulating the production of second messengers, the phosphorylation state of pathway intermediates, or the expression of target genes. In other embodiments, the binder affects signaling by promoting or disrupting proteinprotein or protein-nucleic acid interactions that mediate complex formation or transcriptional responses. The binder can also alter the assembly or disassembly of multiprotein complexes involved in receptor activation, intracellular trafficking, or feedback regulation, thereby enhancing or attenuating a cellular response to an internal or external stimulus.

[0106] In some embodiments, the binder is part of a larger system for delivery of payloads to a cell. In some embodiments, the binder is part of a targeted delivery technology. The binder can be conjugated or otherwise linked to a delivery moiety, carrier, or scaffold that directs a therapeutic, diagnostic, or functional agent to a specific cell type, tissue, organ, or subcellular compartment. In certain embodiments, the binder recognizes and selectively binds a cell-surface receptor, transporter, or extracellular matrix component, thereby facilitating uptake, internalization, or retention of the conjugated agent. Examples of targeted delivery technologies include engineered viruses and lipid nanoparticles decorated with protein or peptide binders. In other embodiments, the binder can be incorporated into nanoparticles, liposomes, viral vectors, or polymer-based delivery systems, where it guides the complex to the intended target site. The binder can also be part of bifunctional or multifunctional constructs that simultaneously achieve targeting and modulation of cellular activity. In some embodiments, binder-mediated targeting enhances the specificity, efficacy, or safety profile of the delivered agent by limiting off-target effects and improving pharmacokinetics or biodistribution.

[0107] In some embodiments, the binder is part of an engineered cell surface receptor. The binder can be incorporated into the extracellular domain of the receptor to confer selective binding to a target ligand, peptide, protein, small molecule, or other biomolecule. In certain embodiments, binding of the target to the receptor induces a conformational change that modulates intracellular signaling, enzymatic activity, or gene expression. The engineered receptor can be designed to recruit or activate downstream signaling proteins, adapter molecules, or transcriptional effectors upon ligand binding. In other embodiments, the binder enables the receptor to mediate internalization, trafficking, or retention of the ligand orreceptor-ligand complex. Engineered receptors can be incorporated into cells, including immune cells, stem cells, or other somatic or engineered cell types, to provide controlled or programmable cellular responses. In some embodiments, the binder-mediated receptor enhances the specificity, potency, or safety of cellular therapies by restricting activation or signaling to cells presenting the intended ligand.

[0108] In some embodiments, the binder is part of an engineered virus. The binder can be incorporated into viral surface proteins or other structural components to confer selective binding to a target cell, tissue, organ, or biomolecule. In certain embodiments, binding of the virus to the target facilitates targeted delivery of viral genetic material, protein payloads, or other cargo into the desired cells. The binder can also influence viral tropism, internalization, or retention, thereby enhancing specificity and reducing off-target effects. In other embodiments, the binder is part of a multifunctional viral construct that simultaneously enables targeting and modulation of cellular activity, such as activation, repression, or recruitment of endogenous signaling pathways. Engineered viruses can be designed for therapeutic, diagnostic, or research applications, and binder-mediated targeting can improve safety, efficacy, and controlled delivery of viral or non-viral payloads.

[0109] The present disclosure also contemplates a composition for identifying a binder of a target peptide or protein, the composition includes:(a) a host cell comprising an expression vector encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase; and(b) a replication deficient phage library, wherein each phage of the library encodes a different protein variant tagged with a second half of the proximity-dependent split RNA polymerase,wherein the phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, andwherein the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to induce replication of the phage-encoded protein.

[0110] The present disclosure also contemplates a system for identifying a binder of a target peptide or protein, the system includes:(a) a host cell comprising an expression vector encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase; and(b) a replication deficient phage library, wherein each phage of the library encodes a different protein variant tagged with a second half of the proximity-dependent split RNA polymerase,wherein the phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, andwherein the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to induce replication of the phage-encoded protein.[OHl] The present disclosure also contemplates an expression vector for use in a method of identifying a binder of a target peptide or protein. The vector encodes a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase.

[0112] The present disclosure also contemplates a kit for use in a method of identifying a binder of a target peptide or protein. The kit can contain any one or more of the elements disclosed in the above methods and compositions. The kit can include a host cell, expression vectors, and a high diversity library of phage-encoded protein variants. The kit can also include packaging and instructions for use.

[0113] In some embodiments, engineering a host cell for use in the kit can include genetic manipulation and protein design. Methods of engineering cells in this way are well known to those of ordinary skill in the art. Usually, such engineering includes linking a target peptide or protein to a tag. Methods of linking can comprise synthesizing or cloning a DNA sequence encoding a target peptide or protein and a tag into a suitable expression vector, followed by transformation or transfection of a suitable host cell with the expression vector under conditions amenable to expression of the target peptide or protein. In some embodiments, the expression vector is a pET vector. In some embodiments, the host cell is a bacterium. In particular embodiments, the bacterium is E. coli. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an animal cell. In some embodiments, the host cell is, for example, a HEK293, HEK293T, or Chinese hamster ovary (CHO) cell, or another cell amenable to large-scale cell culture.

[0114] In some embodiments, the method incorporates a library of (bacteriophage-encoded protein variants, each tagged with a half of a proximity-dependent split RNA polymerase. In some embodiments, each phage encodes a different protein variant. In some embodiments, contemplated libraries of the present disclosure may encode about 10, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about IO10,about 1011, about 1012or more variants. Libraries containing a high number of variants, wherein each variant is unique, are termed “high diversity” libraries. In some embodiments, the library is diversified in vitro using methods such as error-prone PCR or parallel single-site saturation mutagenesis (SSM). In preferred embodiments, the phage is an M13 phage. In preferred embodiments, the phage is replication deficient. In some embodiments, the replication deficiency can be due to the lack of a gene required for replication. In some embodiments, the gene required for replication can be gill.

[0115] In some embodiments, exposing a host cell to a phage library leads to uptake of the phages into the cell (infection) and expression of the protein variants encoded by the phages within the host cell.

[0116] In some embodiments, the first half of the proximity-dependent split RNA polymerase is the C-terminal half (RNAPc), and the second half of the proximity-dependent split RNA polymerase is the N-terminal half (RNAPN). In other embodiments, the first half of the proximity-dependent split RNA polymerase is the N-terminal half (RNAPN), and the second half of the proximity-dependent split RNA polymerase is the C-terminal half (RNAPc). Examples of split RNA polymerases are taught by U.S. Patent No. 11,913,081, incorporated by reference in its entirety herein.

[0117] As used herein, a “first half’ can be a first portion of two portions that need not be equal in size. Similarly, a “second half’ can be a second portion of two portions that need not be equal in size. Taken together, the first half and second half constitute the whole of the proximity-dependent split RNA polymerase.

[0118] In some embodiments, the kit includes one or more reagents for use in the methods contemplated herein. The reagents may be supplied in any suitable container and may include, for example, one or more reaction or storage buffers. The reagents can be furnished in a form ready for immediate use in an assay, or in a form that requires addition of other components prior to use, such as a concentrated or lyophilized formulation. The buffer may be any suitable buffer, including, without limitation, sodium carbonate, sodium bicarbonate, borate, Tris, MOPS, HEPES, or combinations thereof.EXAMPLES

[0119] The examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.EXAMPLE 1:Phage-Assisted Non-Continuous Selection of Protein Binders (PANCS-Binders)

[0120] In the work described herein, we established Phage- Assisted Non-Continuous Selection of protein Binders (PANCS-Binders; FIG. 1 A), a viral life cycle-based selection platform that can comprehensively screen high diversity (1010+) libraries of M13 phage-encoded protein variants and identify binders to panels of dozens or more proteins of interest in a matter of days. PANCS-Binders uses replication-deficient phage that encode protein variant libraries tagged with one half of a proximity-dependent split RNA polymerase (RNAPN) biosensor (FIG. IB)17.

[0121] E. coli host cells are engineered to express a target protein of interest tagged with the other half of the split RNA polymerase (RNAPc). Protein-protein interaction (PPI) between a phage encoded variant and the target reconstitutes the RNA polymerase (RNAP) and triggers expression of a required phage gene, allowing phage encoding that variant to replicate, in line with the basic principles of PACE18,19. After optimization and trial selections, we demonstrated the versatility of PANCS-Binders by performing selections on 95 different protein targets with two in vitro phage-encoded protein variant libraries, each encoding ~108unique protein variants, thereby completing 190 independent selections in 2 days. The hit rate of this screen was 55%, resulting in new binders for 52 diverse targets. We scaled up our library size 100-fold (~1010), which expanded the hit rate to 72% and dramatically improved the affinity of hits from PANCS - a 40-2000x improvement with affinities as low as 206 pM. Additionally, we showcased how hits can be quickly affinity matured though PACE, resulting in >20x improvement in affinity (to 8.4 nM). Finally, we demonstrated that the binders for two targets, Mdm2 and KRAS, engage their targets in mammalian cells: our Mdm2 binders inhibit the Mdm2-p53 interaction and fusion of our KRAS binder with an LIR motif leads to LC3B mediated degradation of endogenous KRAS20. The ease-of-use, speed, and reliability of PANCS-Binders will facilitate a transition of binder generation from an expensive specialty requiring months of work with high failure to a laboratory tool requiring less than 2 weeks (FIG. 1C) and available to any researcher.EXAMPLE 2: Optimizing PANCS-Binders

[0122] Recently, we established a split RNAP -based PPI-PACE platform for reprogramming the binding specificity of proteins21(FIG. IB), which we demonstrated could swap the binding specificity of BCL2 and MCL1 using continuous evolution. In general, PACE has been shown to be powerful for altering or tuning existing functions of molecules22'25, primarily from initial variants with minimal or closely related function, rather than in vitro discovery of function. We aimed to adapt the components of our PPI-PACE platform for the use of mining high diversity libraries for in vitro discovery of binders.

[0123] To accomplish this, we cloned a phage-encoded, RNAPN-tagged 108unique variant affibody library (FIG. 6)26. We then performed PACE with this library on two targets, the RAS binding domain of RAF (RAF) and IFNG (see Table 1 for target details). Both evolutions went extinct (FIG. 7). Prior efforts have established that PACE can enrich active phage from pools of inactive phage (1:1000 active-to-inactive ratio)19,25; however, in vitro libraries are likely to have an active-to-inactive ratio closer to 1 : 107+. To assess if the PACE evolution process itself led to phage extinction (as opposed to no binders being present in our library), we constructed a mock library selection system that included known, active variants. We performed PACS (Phage- Assisted Continuous Selection19), PACE without the mutagenesis plasmid, using KRAS as a protein target (+AP) and a mock library of containing a mixture of active phage encoding RAF that binds KRAS and inactive phage encoding an affitin, evolved to bind SasA, that does not bind KRAS (FIG. 2A)27. From these mock selections, we found that PPI-PACS could successfully enrich active phage from mock libraries of 1 : 105(active inactive phage; FIG. 8), but failed to do so at any lower ratio (1 : 106'9). This indicates that continuous selection does not sample every variant in the mock library in the initial infection step. PANCE, non-continuous passaging, has frequently been used as a less stringent version of PACE, and we suspected that part of this lower stringency could be due to a higher percentage of phage that infect cells prior to being washed away in the continuous flow versus in passaging28,29. We hypothesized that by extending the incubation time of phage with selection cells, we could more completely sample every variant in our in vitro library, and therefore succeed in in vitro selections.

[0124] To test this hypothesis, we optimized a non-continuous selection procedure. First, we established 6 hours as a minimum time for incubating phage and cells to obtain nearly complete infection of our phage sample by monitoring the rate at which phage infect our cells (FIG. 9); 12 hour incubations were chosen for convenience. To determine how quickly active phage would enrich and how quickly inactive phage would de-enrich, we measured the rate of amplification for active (RAF variants with known affinities) and inactive phage (Affitin (SasA)) in a KRAS selection strain (FIGS. 2A-2B). The amplification rates spanned 8 orders of magnitude: 106for high affinity WT RAF, 101'2for low affinity RAF mutants, and 10'1'2for non-binders (FIG. 2C).

[0125] Based on the replication rates, we predicted that serial passaging with 5% of phage transferred between passages would result in selective enrichment of the high affinity WT RAF from 10 phage to >109phage in just 2 passages and the complete de-enrichment of the inactive phage from 109to 0 in just 4 passages (FIG. 10). We tested this prediction by passaging mock libraries of 10 phage of each RAF variant spiked into IO10inactive Affitin (SasA) phage (FIG. 7D). As expected, over 2 days, the high affinity WT RAF variant enriched (a >1015-fold relative enrichment) during the four-passage selection; all weaker binders and inactive phage went extinct (FIG. 2E). We performed additional mock PANCS to understand the effects of several variables on this relative enrichment rate: +AP selection stringency (FIG. 11), -AP selection stringency (FIG. 12), transfer rate (FIG. 13), and initial cell-to-phage ratio (FIG. 14). Finally, we tested mock selections using several published binder-target pairs using our optimized AP strengths, transfer rate, and initial cell-to-phage ratios (FIG. 15). Collectively, these mock selections indicate that this new system, which we named Phage- Assisted Non-Continuous Selection of Protein Binders (PANCS-Binders), can perform in vitro selections of up to 1010+variant libraries (above the typical IO9'10E. coli transformation limit) in 2 days, using simple serial phage out-growths in culture tubes or even 96-well plates. Therefore, we next performed pilot selections with an in vitro phage-encoded binder library to demonstrate that PANCS-Binders can be used to discover novel binders.

[0126] Table 1. Sequence and details of target proteins.Target AA Sequence SEQID AlphaFold3 Other Notes NO: StructureNo Fusion MKRAS MTEYKLVWGADGVGKSALTIQLIQNHF 1 Uniprot: P01116. (G12D) VDEYDPTffiDSYRKQWIDGETCLLDILD Human, cytoplasm,TAGQEEYSAMRDQYMRTGEGFLCVFAI GTPase, oncogene, NNTKSFEDIHHYREQIKRVKDSEDVPMV full length isoform 4b LVGNKCDLPSRTVDTKQAQDLARSYGIP G12D mutant FIETSAKTRQGVDDAFYTLVREIRKHKE KMSKDGKKKKKKSKTKCVIM ZB MASEQLEKKLQALEKKLAQLEWKNQAL 2 Synthetic construct;EKKLAQ PMID: 15631464HPV-pE7 MHGDTPTLHEYMLDLQPETTDLYCYEQ 3 Uniprot: P03129. LNDSSEEEDEIDGPAGQAEPDRAHYNIVT Human FCCKCDSTLRLCVQSTHVDIRTLEDLLM Papillomavirus GTLGIVCPICSQKP (HPV), involved in controlling cell cycle and viral genome replication, partially structured, full length. p65 (17- MSGPYVEIIEQPKQRGMRFRYKCEGRSA 4 Uniprot: Q04206. 293) GSIPGERSTDTTKTHPTIKINGYTGPGTVR J&, Human, aka NF-KB,ISLVTKDPPHRPHPHELVGKDCRDGFYE nucleus, transcription AELCPDRCIHSFQNLGIQCVKKRDLEQAI factor, structured SQRIQTNNNPFQVPIEEQRGDYDLNAVR region LCFQVTVRDPSGRPLRLPPVLSHPIFDNR APNTAELKICRVNRNSGSCLGGDEIFLLC DKVQKEDIEVYFTGPGWEARGSFSQAD VHRQVAIVFRTPPYADPSLQAPVRVSMQ LRRPSDRELSEPMEFQYLPDTD FGFRlc MAPYWTNTEI<MEI<RLHAVPAANTVI<F 5 Uniprot: P21802. domain D2 RCPAGGNPMPTMRWLKNGKEFKQEHRI IkO ’ Human, single-pass (153-251) GGYKVRNQHWSLIMESWPSDKGNYTC type 1 membrane WENEYGSINHTYHLDWER cSS protein, extracellular domainTCIM MKAKRSHQAVIMSTSLRVSPSIHGYHFD 6 Uniprot: Q9NR00.TASRKKAVGNIFENTDQESLERLFRNSG ? Human, DKKAEERAKIIFAIDQDVEEKTRALMAL nucleus / speckle / nucle KKRTKDKLFQFLKLRKYSIKVH '4 olus, full length, disordered TRIM21 MGLKKMLRTCAVHITLDPDTANPWLILS 7 Uniprot: P19474. (277-475) EDRRQVRLGDTQQSIPGNEERFDSYPMV Human,LGAQHFHSGKHYWEVDVTGKEAWDLG cytoplasm / nucleus, VCRDSVRRKGHFLLSSKSGFWTIWLWN E3 ligase, structured KQKYEAGTYPQTPLHLQVPPCQVGIFLD domain, Fc binding YEAGMVSFYNITDHGSLIYSFSECAFTGP region LRPFFSPGFNDGGKNTAPLTLCPLNIGSQ GSTDYPDE6D (1- MSAKDERAREILRGFKLNWMNLRDAET 8 Uniprot: 043924. 150) GKILWQGTEDLSVPGVEHEARVPKKILK Human,CKAVSRELNFSSTEQMEKFRLEQKVYFK cytoplasm / membrane, GQCLEEWFFEFGFVIPNSTNTWQSLIEAA binds / shuttles PESQMMPASVLTGNVIIETKFFDDDLLVS prenylated proteins TSRVRLFYV including RAS family proteins; full length RAF (52- MSKTSNTIRVFLPNKQRTWNVRNGMSL 9 Uniprot: P04049. 131) HDCLMKALKVRGLQPECCAVFRLLHEH Human, cytoplasm,KGKKARLDWNTDAASLIGEELQVDFL proto oncogene, RAS binding domain NRAS MTEYKLVWGAGGVGKSALTIQLIQNHF 10 Uniprot: P01111.VDEYDPTIEDSYRKQWIDGETCLLDILD Human, TAGQEEYSAMRDQYMRTGEGFLCVFAI cytoplasm / cell NNSKSFADINLYREQIKRVKDSDDVPMV membrane / Golgi LVGNKCDLPTRTVDTKQAHELAKSYGIP apparatus membrane, FIETSAKTRQGVEDAFYTLVREIRQYRM proto oncogene, full KKLNSSDDGTQGCMGLPCVVM length Beclin (125- MIMSGQTDVDHPLCEECTDTLLDQLDTQ 11 Uniprot: Q14457. 215) LNVTENECQNYKRCLEILEQMNEDDSEQ Human, cytoplasm,LQMELKELALEEERLIQELEDVEKNRKIV central role in AENLEKV autophagy and the PI3K complex, region that interacts with BCL2 family proteins.Mdm2 (1- MCNTNMSVPTDGAVTTSQIPASEQETLV 12 Uniprot: Q00987. 188) RPKPLLLKLLKSVGAQKDTYTMKEVLFY Human,LGQYIMTKRLYDEKQQHIVYCSNDLLGD cytoplasm / nucleus, LFGVPSFSVKEHRKIYTMIYRNLVVVNQ E3 ligase and proto QESSDSGTSVSENRCHLEGGSDQKDLVQ oncogene, region that ELQEEKPS SSHLVSRPSTS SRRRAISETEE binds p53.NSDELSGERQRKRHKSDSCalprotectin MTCKMSQLERNIETIINTFHQYSVKLGHP 13 Uniprot: P05109 and heterodimer DTLNQGEFKELVRKDLQNFLKKENKNE P06702. Human, with 12aa KVIEHIMEDLDTNADKQLSFEEFIMLMA secreted, calcium and linker RLTWASHEKMHEGDEGPGHHHKPGLGE zinc binder involved GTPGGSGGSGGSGGSMLTELEKALNSIID in inflammation and VYHKYSLIKGNFHAVYRDDLKKLLETEC immune response, full length of eachPQYIRKKGADVWFKELDINTDGAVNFQE isoform fused with a FLILVIKMGVAAHKKSHEESHKE 12 amino acid linker. HlgA (30- MGHHHHHHAMENKIEDIGQGAEIIKRTQ 14 Uniprot: P0A074.309) DITSKRLAITQNIQFDFVKDKKYNKDAL Staphylococcus VVKMQGFISSRTTYSDLKKYPYIKRMIW aureus, secreted PFQYNISLKTKDSNVDLINYLPKNKIDSA hemolytic toxin, DVSQKLGYNIGGNFQSAPSIGGSGSFNYS folded domain KTISYNQKNYVTEVESQNSKGVKWGVK (accidentally included ANSFVTPNGQVSAYDQYLFAQDPTGPA His-tag).ARDYFVPDNQLPPLIQSGFNPSFITTLSHE RGKGDKSEFEITYGRNMDATYAYVTRH RLAVDRKHDAFKNRNVTVKYEVNWKT HEVKIKSITPKBcl2 MAHAGRTGYDNREIVMKYIHYKLSQRG 15 Uniprot: P10415.YEWDAGDVGAAPPGAAPAPGIFSSQPGH Human, TPHPAASRDPVARTSPLQTPAAPGAAAG cytoplasm / mitochond PALSPVPPWHLTLRQAGDDFSRRYRRD rion outer FAEMSSQLHLTPFTARGRFATWEELFR membrane / nucleus DGVNWGRIVAFFEFGGVMCVESVNREM ........ . membrane, SPLVDNIALWMTEYLNRHLHTWIQDNG f suppresses apoptosis,' W., GWDAFVELYGPSMRPLFDFSWLSLK I full length.Calpro mon MTCKMSQLERNIETIINTFHQYSVKLGHP 16 Uniprot: P06702. s!00a9 DTLNQGEFKELVRKDLQNFLKKENKNE Human, secreted, KVIEHIMEDLDTNADKQLSFEEFIMLMA calcium and zinc RLTWASHEKMHEGDEGPGHHHKPGLGE binder involved in GTP inflammation and immune response, full length.KIX MGNIGSLSTIPTAAPPSSTGVRKGWHEH 17 Uniprot: Q92793.VTQDLRSHLVHKLVQAIFPTPDPAALKD Human, nucleus, RRMENLVAYAKKVEGDMYESANSRDE transcription factor, YYHLLAEKIYKIQKELEEKRRSRL CREB Binding ♦ Protein (CBP) KIX domain (568-673) HRAS MTEYKLVWGAGGVGKSALTIQLIQNHF 18 Uniprot: P01112.VDEYDPTffiDSYRKQWIDGETCLLDILD Human, cytoplasm, TAGQEEYSAMRDQYMRTGEGFLCVFAI GTPase, oncogene, NNTKSFEDIHQYREQIKRVKDSDDVPMV full length.LVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQHKLRKLNPPDESGPGCMSCKCVLS KRAS wt MTEYKLVWGAGGVGKSALTIQLIQNHF 19 Uniprot: P01116.VDEYDPTffiDSYRKQWIDGETCLLDILD Human, cytoplasm, TAGQEEYSAMRDQYMRTGEGFLCVFAI GTPase, oncogene, NNTKSFEDIHHYREQIKRVKDSEDVPMV full length isoform LVGNKCDLPSRTVDTKQAQDLARSYGIP 4b. FIETSAKTRQGVDDAFYTLVREIRKHKE KMSKDGKKKKKKSKTKCVIMG3BP (11- MVGREFVRQYYTLLNQAPDMLHRFYGK 20 Uniprot: Q13283.139) NSSYVHGGLDSNGKPADAVYGQKEIHR Human, cytoplasm,KVMSQNFTNCHTKIRHVDAHATLNDGV RAS GTPase WQVMGLLSNNNQALRRFMQTFVLAPE activating protein, GSVANKFYVHNDIFRYQDEVFG NIX MSSHLVEPPPPLHNNNNNCEENEQSLPPP 21 Uniprot: 060238.AGLNSSWVELPMNSSNGNDNGNGKNG / Human, nucleus GLEHVPSSSSIHNGDMEKILLDAQHESGQ envelope / ER / mitocho SSSRGSSHCDSPSPQEDGQIMFDVEMHTS ndrial outer RDHSSQSEEEWEGEKEVEALKKSADW membrane, singleVSDWSSRPENIPPKEFHFRHPKRSVSLSM pass membrane RKSGAMKKGGIFSAEFLKVFIPSLFLSHV protein involved in LALGLGIYIGKRLSTPSASTY apoptosis by suppressing BC12, full length.PP2A-B MAGAGGGNDIQWCFSQVKGAVDDDVA 22 Uniprot: P63151.EADIISTVEFNHSGELLATGDKGGRWIF Human, cytoplasm, QQEQENKIQSHSRGEYNVYSTFQSHEPEF regulatory subunit of DYLKSLEIEEKINKIRWLPQKNAAQFLLS a phosphatase, full TNDKTIKLWKISERDKRPEGYNLKEEDG length. RYRDPTTVTTLRVPVFRPMDLMVEASPR RIFANAHTYHINSISINSDYETYLSADDLR INLWHLEITDRSFNIVDIKPANMEELTEVI TAAEFHPNSCNTFVYSSSKGTIRLCDMR ASALCDRHSKLFEEPEDPSNRSFFSEIISSI SDVKFSHSGRYMMTRDYLSVKIWDLNM ENRPVETYQVHEYLRSKLCSLYENDCIF DKFECCWNGSDSWMTGSYNNFFRMFD RNTKRDITLEASRENNKPRTVLKPRKVC ASGKRKKDEISVDSLDFNKKILHTAWHP KENIIAVATTNNLYIFQDKVNCalpro mon MLTELEKALNSIIDVYHKYSLIKGNFHAV 23 Uniprot: P05109. sl00a8 YRDDLKKLLETECPQYIRKKGADVWFK Human, secreted, ELDINTDGAVNFQEFLILVIKMGVAAHK calcium and zinc KSHEESHKE binder involved in inflammation and immune response, full length.Parkin MIVFVRFNSSHGFPVEVDSDTSIFQLKEV 24 Uniprot: 060260.VAKRQGVPADQLRVIFAGKELRNDWTV Human, cytoplasm, QNCDLDQQSIVHIVQRPWRKGQEMNAT E3 ligase, full length. GGDDPRNAAGGCEREPQSLTRVDLSSSV LPGDSVGLAVILHTDSRKDSPPAGSPAGR SIYNSFYVYCKGPCQRVQPGKLRVQCST CRQATLTLTQGPSCWDDVLIPNRMSGEC QSPHCPGTSAEFFFKCGAHPTSDKETSVA LHLIATNSRNITCITCTDVRSPVLVFQCNS RHVICLDCFHLYCVTRLNDRQFVHDPQL GYSLPCVAGCPNSLIKELHHFRILGEEQY NRYQQYGAEECVLQMGGVLCPRPGCGA GLLPEPDQRKVTCEGGNGLGCGFAFCRE CKEAYHEGECSAVFEASGTTTQAYRVDE RAAEQARWEAASKETIKKTTKPCPRCHV PVEKNGGCMHMKCPQPQCRLEWCWNC GCEWNRVCMGDHWFDV KRAS MTEYKLVWGAVGVGKSALTIQLIQNHF 25 Uniprot: P01116. (G12V) VDEYDPTffiDSYRKQWIDGETCLLDILD Human, cytoplasm,TAGQEEYSAMRDQYMRTGEGFLCVFAI GTPase, oncogene, NNTKSFEDIHHYREQIKRVKDSEDVPMV full length isoform 4b LVGNKCDLPSRTVDTKQAQDLARSYGIP G12V mutant. FIETSAKTRQGVDDAFYTLVREIRKHKE KMSKDGKKKKKKSKTKCVIMSosl (1136- MSSISLTKGTDEVPVPPPVPPRRRPESAP 26 Uniprot: Q07889. 1333) AESSPSKIMSKHLDSPPAIPPRQPTSKAYS Human, cytoplasm,PRYSISDRTSISDPPESPPLLPPREPVRTPD promotes RAS VFSSSPLHLQPPPLGKKSDHGNAFFPNSP GTPase nucleotide SPFTPPPPQTPSPHGTRRHLPSPPLTQEVD exchange, disordered LHSIAGPPVPPRQSTSQHIPKLPPKTYKRE domain only.HTHPSMHRDGPPLLENAHS SPP2A-B' MSSSSPPAGAASAAISASEKVDGFTRKSV 27 Uniprot: Q15172. RKAQRQKRSQGS SQFRSQGSQ AELHPLP Human, cytoplasm, QLKDATSNEQQELFCQKLQQCCILFDFM "||| regulatory subunit of DSVSDLKSKEIKRATLNELVEYVSTNRG a phosphatase, full VIVESAYSDIVKMISANIFRTLPPSDNPDF length. DPEEDEPTLEASWPHIQLVYEFFLRFLES PDFQPSIAKRYIDQKFVQQLLELFDSEDP RERDFLKTVLHRIYGKFLGLRAFIRKQIN NIFLRFIYETEHFNGVAELLEILGSIINGFA LPLKAEHKQFLMKVLIPMHTAKGLALFH AQLAYCWQFLEKDTTLTEPVIRGLLKF WPKTCSQKEVMFLGEIEEILDVIEPTQFK KIEEPLFKQISKCVSSSHFQVAERALYFW NNEYILSLIEENIDKILPIMFASLYKISKEH WNPTIVALVYNVLKTLMEMNGKLFDDL TSSYKAERQREKKKELEREELWKKLEEL KLKKALEKQNSAYNMHSILSNTSAEmCherry MVSKGEEDNMAIIKEFMRFKVHMEGSV 28 Synthetic evolved NGHEFEIEGEGEGRPYEGTQTSKLKVTK protein from DsRed GGPLPFAWDILSPQFMYGSKAYVKHPAD of Discosoma sea IPDYLKLSFPEGFKWERVMNFEDGGWT anemones. Red VTQDSSLQDGEFIYKVKLRGTNFPSDGP fluorescent protein VMQI<I<TMGWEASSERMYPEDGAL1<GEI full length.KQRLKLKDGGHYDAEVKTTYKAKKPV QLPGAYNVNIKLDITSHNEDYTIVEQYER AEGRHSTGGMDELYKMyc (DBD) MNVKRRTHNVLERQRRNELKRSFFALR 29 Uniprot: P01106.DQIPELENNEKAPKWILKKATAYILSVQ Human, nucleus, AEEQKLISEEDLLRKRREQLKHKLEQL proto oncogene transcription factor, DNA binding domain (368-451).PP2A-B" MQILQETLTTSSQANLSVCRSPVGDKAK 30 Uniprot: Q06190. (622-1150) DTTSAVLIQQTPEVIKIQNKPEKKPGTPLP Human, cytoplasm,PP ATSPS SPRPLSP VPHVNNWNAPLSINI regulatory subunit of PRFYFPEGLPDTCSNHEQTLSRIETAFMDI a phosphatase, EEQKADIYEMGKIAKVCGCPLYWKAPMFRAAGGEKTGFVTAQSFIAMWRKLLNN primarily the HHDDASKFICLLAKPNCSSLEQEDFIPLL structured domain. QDWDTHPGLTFLKDAPEFHSRYITTVIQ RIFYTVNRSWSGKITSTEIRKSNFLQTLAL LEEEEDINQITDYFSYEHFYVIYCKFWEL DTDHDLYISQADLSRYNDQASSSRIIERIF SGAVTRGKTIQKEGRMSYADFVWFLISE EDKRNPTSIEYWFRCMDVDGDGVLSMY ELEYFYEEQCERMEAMGIEPLPFHDLLC QMLDLVKPAVDGKITLRDLKRCRMAHIF YDTFFNLEKYLDHEQRDPFAVQKDVEN DGPEPSDWDRFAAEEYETLVAEESAQAQ FQEGFEDYETDEPASPSEFGNKSNKILSA SLPEKCGKLQSVDEE MBP (27- MKIEEGKLVIWINGDKGYNGLAEVGKKF 31 Uniprot: P0AEX9.396) EKDTGIKVTVEHPDKLEEKFPQVAATGD ■fit Escherichia coli, GPDIIFWAHDRFGGYAQSGLLAEITPDKA periplasm, maltose FQDKLYPFTWDAVRYNGKLIAYPIAVEA binding protein LSLIYNKDLLPNPPKTWEEIPALDKELKA structured region. KGKSALMFNLQEPYFTWPLIAADGGYAF KYENGKYDIKDVGVDNAGAKAGLTFLV DLIKNKHMNADTDYSIAEAAFNKGETA MTINGPWAWSNIDTSKVNYGVTVLPTFK GQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSY EEELAKDPRIAATMENAQKGEIMPNIPQ MSAFWYAVRTAVINAASGRQTVDEALK DAQTRITKFibronectin MGVFTTLQPGS SIPPYNTE VTETTI VITWT 32 Uniprot: P02751. (1080-1541) PAPRIGFKLGVRPSQGGEAPREVTSDSGS Human, secreted,IWSGLTPGVEYVYTIQVLRDGQERDAPI extracellular matrix,1 VNKWTPLSPPTNLHLEANPDTGVLTVS binds cell surfaces, WERSTTPDITGYRITTTPTNGQQGNSLEE various fibronectin WHADQSSCTFDNLSPGLEYNVSVYTVK type domains. DDKESVPISDTIIPEVPQLTDLSFVDITDSS IGLRWTPLNSSTIIGYRITWAAGEGIPIFE DFVDSSVGYYTVTGLEPGIDYDISVITLIN GGESAPTTLTQQTAVPPPTDLRFTNIGPD TMRVTWAPPPSIDLTNFLVRYSPVKNEE DVAELSISPSDNAWLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFS DITANSFTVHWIAPRATITGYRIRHHPEH FSGRPREDRVPHSRNSITLTNLTPGTEYV VSIVALNGREESPLLIGQQSTVSDV VHL (61- MPVLRSVNSREPSQVIFCNRSPRWLPV 33 Uniprot: P40337. 209) WLNFDGEPQPYPTLPPGTGRRIHSYRGH Human, cytoplasm / LWLFRDAGTHDGLLVNQTELFVPSLNV nucleus, E3 ligase, DGQPIFANITLPVYTLKERCLQWRSLVK structured domain. PENYRRLDIVRSLYEDLEDHPNVQKDLE RLTQERIAHQ BTK (PH) MAAVILESIFLKRSQQKKKTSPLNFKKRL 34 Uniprot: Q06187.FLLTVHKLSYYEYDFERGRRGSKKGSID Human, cytoplasm, VEKITCVETWPEKNPPPERQIPRRGEESS kinase, PH domain EMEQISIIERFPYPFQWYDEGPLYVFSPT (1-133).EELRKRWIHQLKNVIRCalpro het MTCKMSQLERNIETIINTFHQYSVKLGHP 35 Uniprot: P05109 and 6aa DTLNQGEFKELVRKDLQNFLKKENKNE P06702. Human, KVIEHIMEDLDTNADKQLSFEEFIMLMA secreted, calcium and RLTWASHEKMHEGDEGPGHHHKPGLGE zinc binder involved GTPGGSGGSMLTELEKALNSIIDVYHKY in inflammation and SLIKGNFHAVYRDDLKKLLETECPQYIR immune response, KKGADVWFKELDINTDGAVNFQEFLILV full length of each IKMGVAAHKKSHEESHKE isoform fused with a 6 amino acid linker. GAB ARAP MKFVYKEEHPFEKRRSEGEKIRKKYPDR 36 Uniprot: 095166.VPVIVEKAPKARIGDLDKKKYLVPSDLT Human, cytoplasm, VGQFYFLIRKRIHLRAEDALFFFVNNVIP involved in PTSATMGQLYQEHHEEDFFLYIAYSDES autophagy, full VYGL length. GTF2I (1- MAQVAMSTLPVEDEESSESRMWTFLM 37 Uniprot: P78347. 106) SALESMCKELAKSKAEVACIAVYETDVF Human, nucleus, WGTERGRAFVNTRKDFQKDFVKYCVE transcription factor, EEEKAAEMHKMKSTTQANRMSVDA DNA binding domain.OmpF (233- MAEWRVQLDVKFDFDKSKVKENSYAD 38 Uniprot: A6V748.350) IKNLADFMKQYPSTSTTVEGHTDSVGTD Pseudomonas AYNQKLSERRANAVRDVLVNEYGVEGG aeruginosa, outer RVNAVGYGESRPVADNATAEGRAINRR membrane protein, VEAEVEAEAK extracellular domain.14-3-3z MDKNELVQKAKQAEQAEDYDDMAAC 39 Uniprot: P63104. Monomeric MKSVTEQGAELSNEERNLLSVAYKNW Human, cytoplasm,GARRSEWRWSSIEQKTEGAEKKQQMA interacts with REYREKIETELRDICNDVLSLLEKFLIPNA phosphorylated SQAESKVFYLKMKGDYYRYLAEVAAGD proteins, full length. DKKGIVDQSQQAYQEAFEISKKEMQPTH Modified to prevent PIRLGLALNFSVFYYEILNSPEKACSLAK dimerization (L12Q, TAFDEAIAELDTLSEESYKDSTLIMQLLR R18E and S58E). DNLTLWTSDTQGDEAEAGEGGEN PCNA-AF MVRTKADSVPGTYRKWAARAPRKVLG 40 Uniprot: Q15004.SSTSATNSTSVSSRKAENKYAGGNPVCV Human, nuclear, RPTPKWQKGIGEFFRLSPKDSEKENQIPE regulator of DNA EAGSSGLGKAKRKACPLQPDHTNDEKE replication,intrinsically disordered, full length.TEV (2038- MGESLFKGPRDYNPISSTICHLTNESDGH 41 Uniprot: Q88507.2274) TTSLYGIGFGPFIITNKHLFRRNNGTLLVQ Tobacco etch virus,SLHGVFKVKNTTTLQQHLIDGRDMIIIRM protease, proteolytic PKDFPPFPQKLKFREPQREERICLVTTNF domain.QTKSMSSMVSDTSCTFPSSDGIFWKHWI QTKDGQCGSPLVSTRDGFIVGIHSASNFT NTNNYFTSVPKNFMELLTNQEAQQWVS GWRLNADSVLWGGHKVFMVKPEEPFQP VKEATQLMNFnbpA (307- MKYYTNLNGSIETFNKADNKFTHVAYV 42 Uniprot: P14738.461) KPINGNKSESVSITGSLTQGSNVSGDSPIV Staphylococcus KVYEYQGKETDLPKSVSVNLTDNSKFK aureus, cell DVTSDMQNKLTVQENGNYQLNLEKLDK surface / secreted, TYVIHYTGEYSKETDEVNFRTQVSAYPE fibronectin binding NSYRYYSYYNNHYTLTW domain.IBTK MLIDIISSKMIALTTKENNSGMNSMETVL 43 Uniprot: Q9P2D0. (1159-1339) FTPSKVPKPVNAWASSLHSVSSKSFRDFL Human,LEEKKSVTSHSSGDHVKKVSFKGIENSQ cytoplasm / nucleus, APKIVRCSTHGTPGPEGNHISDLPLLDSP J / ..f involved in B-cell NPWLSSSVTAPSMVAPVTFASIVEEELQQ development through EAALIRSREKPLALIQIEEHAIQDLLVFYE BTK and NF-kb, AFGNPEEFVIVERTPQGPLAVPMWNKHG disordered domain. CPAL MLQMQQLQQNNIVYFDLDKYDIRSDFA 44 Uniprot: P0A912. QMLDAHANFLRSNPSYKVTVEGHADER Escherichia coli, GTPEYNISLGERRANAVKMYLQGKGVS outer membrane ADQISIVSYGKEKPAVLGHDEAAYSKNR protein involved in RAVLVY cell division,extracellular domain. PIK3CB (3- MFSFIMPPAMADILDIWAVDSQIASDGSI 45 Uniprot: P42338. 115) PVDFLLPTGIYIQLEVPREATISYIKQML Human,WKQVHNYPMFNLLMDIDSYMFACVNQ cytoplasm / nucleus, TAVYEELEDETRRLCDVRPFLPVLKLVT kinase, adaptor RS binding domain (ABD).SortaseA MAKPHIDNYLHDKDKDEKIEQYDKNVK 46 Uniprot:(25-206) EQASKDKKQQAKPQIPKDKSKVAGYIEI A0A0H2XJ22.PDADIKEPVYPGPATPEQLNRGVSFAEEN Staphylococcus S 4* ESLDDQNISIAGHTFIDRPNYQFTNLKAA aureus, cell KKGSMVYFKVGNETRKYKMTSIRDVKP surface / secreted, TDVGVLDEQKGKDKQLTLITCDDYNEK extracellular domain. TGVWEKRKIFPilQ (468- MGVRWGGAYHKGNWSGYGKDGNIGIK 47 Uniprot: P34750. 549) DEDGMNCGPIAGSCTFPTTGTSKSPSPFV Psudomonas DLGAKDATSGIGIGFITDNIILDLQLSA aeruginosa, outer 3 membrane protein, involved in formation of pili, part of the extracellular domain. DltD (ef) MDISNPETIRRASSSMSVNVLKGDAIKNY 48 Uniprot: Q3XZT4. (41-422) ALSEKQYIPFFGSSELSRISPFHPSVLAEK Enterococcus YQRNYRPFLLGAPGTQSLSQYMMMRSA faecium, outer GDAMKNKKVVFIISPQWFVKNGVKTDY membrane protein FNTYYSELQTYDWLFSMKKVTPADRYL involved in cell wall ARRLLTFSKVKENDTLTAILQTIKKGKLP synthesis, LPESLNQLRSQWNMLKREDEVFSNIGLK extracellular domain. DRQQKIDHESKRLPKQYQETELSILANQI GERETTNNPFGLKNDFYTHRIRAHEPEL KQSQKNWDYRFSPEFSDFQLVLDQLAK NHNEVLFIIPPVNEKWSDYTGLSQEMLQ GFAKKIKFQLNSQGFNRIADFVNQAGTN YFMEDTIHLGWKGWLAADQQIRPFLEENHITASKYHLDDAFFSKSWQHQIPDKLQLK TNFR2 (33- MAPEPGSTCRLREYYDQTAQMCCSKCSP 49 Uniprot: P20333. 205) GQHAKVFCTKTSDTVCDSCEDSTYTQL Human, cell <7 WNWVPECLSCGSRCSSDQVETQACTRE S' membrane, involved QNRICTCRPGWYCALSKQEGCRLCAPLR in regulating KCRPGFGVARPGTETSDWCKPCAPGTF apoptosis, SNTTS STDICRPHQICNWAIPGNASMD A extracellular domain. VCTSTSP CDKN1A MSEPAGDVRQNPCGSKACRRLFGPVDSE 50 Uniprot: P38936.QLSRDCDALMAGCIQEARERWNFDFVT Human, ETPLEGDFAWERVRGLGLPKLYLPTGPR cytoplasm / nucleus, RGRDELGGGRRPGTSPALLQGTAEEDHV kinase inhibitor DLSLSCTLVPRSGEQAEGSPGGPGDSQG involved in cell cycle, RKRRQTSMTDFYHSKRRLIFSKRKP intrinsically disordered, full length.SH3BP5 MLNHATQRVMEAEQTKTRSELVHKETA 51 Uniprot: C9JK30.ARYNAAMGRMRQLEKKLKRAINKSKPY Human, cytoplasm, FELKAKYYVQLEQLKKTVDDLQAKLTL guanine nucleotide AKGEYKMALKNLEMISDEIHERRRSSAM exchange factor GPRGCGVGAEGSSTSVEDL (GEF), full length.0TUB1 (1- MKPSWLSRTEFSKRLLCRTLWCQSGWSS 52 Uniprot: Q96FW1.102) RSYTRSMLKMTTSINRRSRTSTKSTRTSA Human, cytoplasm,RPGLTATVSIGLSDSPTWRHCWMTARSC ' ' de-ubiquitinase, SGGEADLCRRPAGLLQ intrinsically disordered, full length.DltD (strep) MDYYKVKDNSIRYSTSYEKYKSRDILTS 53 Uniprot: X8KHP8. (31-390) NITPNTLVLMGSSELVATINEDYHPNKIF Streptococcus sp.NYNDFNIMQIGTSYSQNIIQATTLGSIEGS CM6, outer MTKRKVAIVESVQWFEKDGTHQDAFLN membrane protein KASQEHIFHMLDNGKISKETKEKLINRIIE involved in cell wall ITKGNKQQNDIYKKYKSYFIDGKGTIVD synthesis, KKLLEFENAMYSFKLKQTFYQKHAKSD extracellular domain. YPSLGDKTPDYDWQKMTNQFVEEVKQK TDNNDYAVDNNYYNTYLKDRYASLKDS NKDLSYLESPEYSDMELFLTVAKELGIEV EVIIFPVNGKWNDYTGVSREMREKTYKKIEDVAKSHGATVLNYGNREYDDYFLFDVMHVGVKGWMEVEKELYKFANQAN PINK (107- MGIPEFEEKQAESRRAVSACQEIQAIFTQ 54 Uniprot: Q9BXM7.353) KSKPGPDPLDTRRLQGFRLEEYLIGQSIG Human,KGCSAAVYEATMPTLPQNLEVTKSTGLL cytoplasm / mitochond PGRGPGTSAPGEGQERAPGAPAFPLAIK non membrane, MMWNISAGSSSEAILNTMSQELVPASRV kinase, kinase ALAGEYGAVTYRKSKRGPKQLAPHPNII domain, partially RVLRAFTSSVPLLPGALVDYPDVLPSRLH disordered.PEGLGHGRTLFLVMKNYPCTLRQYLCV NTPSPRLAAMMLLQLLEGVDHL RBI (1-103) MPPKTPRKTAATAAAAAAEPPAPPPPPPP 55 Uniprot: P06400.EEDPEQDSGPEDLPLVRLEFEETEEPDFT Human, nucleus, ALCQKLKIPDHVRERAWLTWEKVSSVD A regulates cell cycle, GVLVLRSPVFKPWLTMGN disordered domain (isoform 4).SasG (1060- MPI<TGTPETI<TVEIPFETI<REFNPI<LQPG 56 A Uniprot:1140) EERVKQEGQPGSKTITTPITVNPLTGEKV A0A0H2WXE4.GEGQPTEEITKQPVDKIVEFGGE Staphylococcus aureus, cell surface, part of the extracellular domain. SasG (930- MKYGPVKGDSIVEKEEIPFEKERKFNPDL 57 Uniprot:1060) APGTEKVTREGQKGEKTITTPTLKNPLTG A0A0H2WXE4.EIISKGESKEEITKDPVNELTEFGGEKIPQ Staphylococcus GHKDIFDPNLPTDQTEKVPGKPGIKNPDT aureus, cell surface, GKVIEEPVDDVIKHG part of the extracellular domain. FKBP (39- MGVQVETISPGDGRTFPKRGQTCWHYT 58 Uniprot: Q0VDC6.145) GMLEDGKKFDSSRDRNKPFKFMLGKQE Human,VIRGWEEGVAQMSVGQRAKLTISPDYA cytoplasm / sarcoplasm YGATGHPGIIPPHATLVFDVELLKLE ic reticulum membrane, cytoplasmic domain (excluding the membrane anchor).BRD4 (349- MKVSEQLKCCSGILKEMFAKKHAAYAW 59 Uniprot: 060885. 459) PFYKPVDVEALGLHDYCDIIKHPMDMST Human, nucleus, IKSKLEAREYRDAQEFGADVRLMFSNCY chromatin reader, KYNPPDHEWAMARKLQDVFEMRFAK bromo 2 domain.A MPDIsdB (125- MNQELREAIKNPAIKDKDHSAPNSRPIDF 60 Uniprot: Q99UX5.269) EMKKENGEQQFYHYASSVKPARVIFTDS Staphylococcus KPEIELGLQSGQFWRKFEVYEGDKKLPI aureus, cell KLVSYDTVKDYAYIRFSVSNGTKAVKIV surface / secreted, SSTHFNNKEEKYDYTLMEFAQPIYNSAD heme receptor, NEAT KFKT 1 domain.HEWL (19- MKVFGRCELAAAMKRHGLDNYRGYSL 61 Uniprot: P00698. 147) GNWVCAAKFESNFNTQATNRNTDGSTD Gallus gallus YGILQINSRWWCNDGRTPGSRNLCNIPC (chicken), secreted, SALLSSDITASVNCAKKIVSDGNGMNAW lysozyme, structured VAWRNRCKGTDVQAWIRGCRL domain (excluding signal domain). HSPB1 (90- MHTADRWRVSLDVNHFAPDELTVKTKD 62 w. Uniprot: P04792. 171) GWEITGKHEERQDEHGYISRCFTRKYT Human,LPPGVDPTQVSSSLSPEGTLTVEAPMPK cytoplasm / nucleus, molecular chaperone, structured domain only.KRAS MTEYKLVWGACGVGKSALTIQLIQNHF 63 Uniprot: P01116. G12C VDEYDPTffiDSYRKQWIDGETCLLDILD Human, cytoplasm,TAGQEEYSAMRDQYMRTGEGFLCVFAI GTPase, oncogene, NNTKSFEDIHHYREQIKRVKDSEDVPMV full length isoform 4b LVGNKCDLPSRTVDTKQAQDLARSYGIP G12C mutant.FIETSAKTRQGVDDAFYTLVREIRKHKE KMSKDGKKKKKKSKTKCVIMMst2 linker MIEHNSTMLESDLGTMVINSEDEEEEDG 64 Uniprot: Q13188. (350-380) TMK Human,1 „cytoplasm / nucleus, pro-apoptotic kinase, disordered domain that interacts with TRIM21.1LMO2 MSSAIERKSLDPSEEPVDEVLQIPPSLLTC 65 Uniprot: P25791. GGCQQNIGDRYFLKAIDQYWHEDCLSC Human, nucleus, ,7: K / DLCGCRLGEVGRRLYYKLGRKLCRRDY transcription factor, LRLFGQDGLCASCDKRIRAYEMTMRVK mostly disordered, DKVYHLECFKCAACQKHFCVGDRYLLI full length.NSDIVCEQDIYEWTKINGMI HIF-la (1- MEGAGGANDKKKISSERRKEKSRDAAR 66 Uniprot: Q16665.350) SRRSKESEVFYELAHQLPLPHNVSSHLD Human,KASVMRLTISYLRVRKLLDAGDLDIEDD cytoplasm / nucleus, MKAQMNCFYLKALDGFVMVLTDDGDM transcriptional IYISDNVNKYMGLTQFELTGHSVFDFTHP regulator, structured CDHEEMREMLTHRNGLVKKGKEQNTQ domain only.RSFFLRMKCTLTSRGRTMNIKSATWKVL HCTGHIHVYDTNSNQPQCGYKKPPMTC LVLICEPIPHPSNIEIPLDSKTFLSRHSLDM KFSYCDERITELMGYEPEELLGRSIYEYY HALDSDHLTKTHHDMFTKGQVTTGQYR MLAKRGGYVWVETQATVIYNTKNSQPQ CIVCVNYWSGIIQHDL MXD1 MAAAVRMNIQMLLEAADYLERREREAE 67 Uniprot: Q05195.HGYASMLPYNNKDRDALKRRNKSKKN f Human, nucleus, NSSSRSTHNEMEKNRRAHLRLCLEKLKG transcriptional LVPLGPESSRHTTLSLLTKAKLHIKKLED repressor, CDRKAVHQIDQLQREQRHLKRQLEKLGI intrinsically ERIRMDSIGSTVSSERSDSDREEIDVDVES disordered, full TDYLTGDLDWSSSSVSDSDERGSMQSLG length. SDEGYSSTSIKRIKLQDSHKACLGLOmpA (ec, MADKQEAELRRQMEGTGVEVQRQGDDI 68 Uniprot: Q9I5A7.84-224) KLIMPGNITFATDSANIAPSFYAPLNNLA Psudomonas NSFKQYNQNTIEIVGYTDSTGSRQHNMD aeruginosa, outer LSQRRAQSVAGYLTAQGVDGTRLSTRG membrane protein, MGPDQPIASNSTADGRAQNRRVEVNLRP extracellular domain. V CD3z MKWKALFTAAILQAQLPITEAQSFGLLD 69 Uniprot: P20963.PKLCYLLDGILFIYGVILTALFLRVKFSRS Human, cell ADAPAYQQGQNQLYNELNLGRREEYDV membrane, T-cell LDKRRGRDPEMGGKPQRRKNPQEGLYN surface receptor, ELQKDKMAEAYSEIGMKGERRRGKGHD single pass- GLYQGLSTATKDTYDALHMQALPPR transmembrane protein, fullydisordered, full length.PIK3CA (1- MPPRPS SGEL WGIHLMPPRILVECLLPNG 70 Uniprot: P42336. 105) MIVTLECLREATLITIKHELFKEARKYPL Human, cytoplasm,HQLLQDESSYIFVSVTQEAEREEFFDETR kinase, adaptor RLCDLRLFQPFLKVIEPV binding domain (ABD).UEV (1- MAVSESQLKKMVSKYKYRDLTVRETVN 71 Uniprot: Q99816.145) VITLYKDLKPVLDSYVFNDGSSRELMNL Human,TGTIPVPYRGNTYNIPICLWLLDTYPHNP cytoplasm / nucleus, PICFVKPTSSMTIKTGKHVDANGKIYLPY binds ubiquitinated LHEWKHPQSDLLGLIQVMIWFGDEPPV proteins, UEV FSRP domain.FRB (2019- MVAILWHEMWHEGLEEASRLYFGERNV 72 Uniprot: P42345. 2114) KGMFEVLEPLHAMMERGPQTLKETSFN Human, cytoplasm,QAYGRDLMEAQEWCRKYMKSGNVKDL kinase (mTOR), TQAWDLYYHVFRRISKQ FKPB rapamycin associated domain (FRB.PD-1 (25- MLDSPDRPWNPPTFSPALLWTEGDNAT 73 Uniprot: Q15116.160) FTCSFSNTSESFVLNWYRMSPSNQTDKL Human, cell AAFPEDRSQPGQDSRFRVTQLPNGRDFH membrane, inhibitor MSWRARRNDSGTYLCGAISLAPKAQIK of T-cell activation, ESLRAELRVTERRAEVPTAHPSPSP extracellular domain.EIF4A MSASQDSRSRDNGPDGMEPEGVIESNW 74 Uniprot: P60842.NEIVDSFDDMNLSESLLRGIYAYGFEKPS Human, cytoplasm, AIQQRAILPCIKGYDVIAQAQSGTGKTAT translation initiation FAISILQQIELDLKATQALVLAPTRELAQ factor, full length. QIQKWMALGDYMGASCHACIGGTNVR AEVQKLQMEAPHIIVGTPGRVFDMLNRR ^0" YLSPKYIKMFVLDEADEMLSRGFKDQIY DIFQKLNSNTQVVLLSATMPSDVLEVTK KFMRDPIRILVKKEELTLEGIRQFYINVER EEWKLDTLCDLYETLTITQAVIFINTRRK VDWLTEKMHARDFTVSAMHGDMDQKE RDVIMREFRSGSSRVLITTDLLARGIDVQ QVSLVINYDLPTNRENYIHRIGRGGRFGRKGVAINMVTEEDKRTLRDIETFYNTSIEEMPLNVADLISas A (154- MADNNFNKEQQNAFYEILNMPNLNEEQ 75 Uniprot: P02976.211) RNGFIQSLKDDPSQSANLLSEAKKLNESQ Staphylococcus APK aureus, secreted, IgG binding protein, Ig binding domain A. LC3B MPSEKTFKQRRTFEQRVEDVRLIREQHP 76 Uniprot: A6NCE7.TKIPVIIERYKGEKQLPVLDKTKFLVPDH Human, VNMSELIKIIRRRLQLNANQAFFLLVNGH cytoplasm / autophago SMVSVSTPISEVYESEKDEDGFLYMVYA some membrane, SQETFGMKLSV autophagy cargo receptor, full length. ALKBH5 MAAASGYTDLREKLKSMTSRDNYKAGS 77 Uniprot: Q6P6C2.REAAAAAAAAVAAAAAAAAAAEPYPV Human, nucleus, SGAKRKYQEDSDPERSDYEEQQLQKEEE RNA demethylase, ARKVKSGIRQMRLFSQDECAKIEARIDE partially disordered, WSRAEKGLYNEHTVDRAPLRNKYFFG full length.EGYTYGAQLQKRGPGQERLYPPGDVDEI PEWVHQLVIQKLVEHRVIPEGFVNSAVI NDYQPGGCIVSHVDPIHIFERPIVSVSFFS DSALCFGCKFQFKPIRVSEPVLSLPVRRG SVTVLSGYAADEITHCIRPQDIKERRAVII LRKTRLDAPRLETKSLSSSVLPPSYASDR LSGNNRDPALKPKRSHRKADPDAAHRP RILEMDKEENRRSVLLPTHRRRGSFSSEN YWRKSYESSEDCSEAAGSPARKVKMRR HSRGSRA HIFlb (1- MAATTANPEMTSDVPSLGPAIASGNSGP 78 Uniprot: P27540.474) GIQGGGAIVQRAIKRRPGLDFDDDGEGN Human, nucleus, SKFLRCDDDQMSNDKERFARSDDEQSSA heterodimerizes with DKERLARENHSEIERRRRNKMTAYITELS ARNT to induce DMVPTCSALARI<PDI<LTILRMAVSHMI< transcription, all SLRGTGNTSTDGSYKPSFLTDQELKHLIL structured regions, EAADGFLFIVSCETGRWYVSDSVTPVL but only the N- NQPQSEWFGSTLYDQVHPDDVDKLREQ terminal disordered LSTSENALTGRILDLKTGTVKKEGQQSS domain.MRMCMGSRRSFICRMRCGSSSVDPVSV NRLSFVRNRCRNGLGSVKDGEPHFVW HCTGYIKAWPPAGVSLPDDDPEAGQGSKFCLVAIGRLQVTSSPNCTDMSNVCQPTEF ISRHNIEGIFTFVDHRCVATVGYQPQELL GKNIVEFCHPEDQQLLRDSFQQWKLKG QVLS VMFRFRSKNQEWL WMRTS SFTFQ NPYSDEIEYIICTNTNVKNS SQEPRPT IFNG (24- MQDPYVKEAENLKKYFNAGHSDVADN 79 Uniprot: P01579. 156) GTLFLGILKNWKEESDRKIMQSQIVSFYF Human, secreted, KLFKNFKDDQSIQKSVETIKEDMNVKFF type II interferon, NSNKKKRDDFEKLTNYSVTDLNVQRKAI structured secreted HELIQVMAELSPAAKTGKRKRSQ domain only.MST2 (313- MENSDEDELDSHTMVKTSVESVGTMRA 80 Uniprot: Q13188. 437) TSTMSEGAQTMIEHNSTMLESDLGTMVI Human,NSEDEEEEDGTMKRNATSPQVQRPSFMD cytoplasm / nucleus, YFDKQDFKNKSHENCNQNMHEPFPMSK pro-apoptotic kinase, NVFPDNWKVPQDGDF disordered domain. p53 (1-293) MEEPQSDPSVEPPLSQETFSDLWKLLPEN 81 Uniprot: P04637.NVLSPLPSQAMDDLMLSPDDIEQWFTED Human, nucleus, PGPDEAPRMPEAAPPVAPAPAAPTPAAP transcription factor, APAPSWPLSSSVPSQKTYQGSYGFRLGFL N-terminal disordered HSGTAKSVTCTYSPALNKMFCQLAKTCP domain and VQLWVDSTPPPGTRVRAMAIYKQSQHM structured domain. TEWRRCPHHERCSDSDGLAPPQHLIRV EGNLRVEYLDDRNTFRHSVWPYEPPEV GSDCTTIHYNYMCNSSCMGGMNRRPILT IITLEDSSGNLLGRNSFEVRVCACPGRDR RTEEENLRKKG PD-L1 (18- MAFTVTVPKDLYWEYGSNMTIECKFPV 82 Uniprot: Q9NZQ7.W239) EKQLDLAALIVYWEMEDKNIIQFVHGEE Human, cell DLKVQHSSYRQRARLLKDQLSLGNAAL membrane, important QITDVKLQDAGVYRCMISYGGADYKRIT in T-cell activation, VKVNAPYNKINQRILWDPVTSEHELTC extracellular domain QAEGYPKAEVIWTSSDHQVLSGKTTTTN only. SKREEKLFNVTSTLRINTTTNEIFYCTFRR LDPEENHTAELVIPELPLAHPPNERTBax peptide MKKLSECLKRIGDELDS 83 Uniprot: Q07812. (58-72) Human, cytoplasm, mitochondrion membrane, involved in mitochondrialapoptosis, BH3 domain.p53 AD (1- MEEPQSDPSVEPPLSQETFSDLWKLLPEN 84 Uniprot: P04637. 61) NVLSPLPSQAMDDLMLSPDDIEQWFTED Human, nucleus, PGPD transcription factor,N-terminal disordered activation domain. CD3d MEHSTFLSGLVLATLLSQVSPFKIPIEELE 85 Uniprot: P04234.DRVFVNCNTSITWVEGTVGTLLSDITRL Human, cell DLGKRILDPRGIYRCNGTDIYKDKESTVQ membrane, T-cell VHYRMCQSCVELDPATVAGIIVTDVIAT surface receptor, LLLALGVFCFAGHETGRLSGAADTQALL single pass- RNDQVYQPLRDRDDAQYSHLGGNWAR transmembrane NK protein, fully disordered, full length.Myc (17- MPLNVSFTNRNYDLDYDSVQPYFYCDE 86 Uniprot: P01106. 454) EENFYQQQQQSELQPPAPSEDIWKKFEL Human, nucleus, LPTPPLSPSRRSGLCSPSYVAVTPFSLRGD proto oncogene NDGGGGSFSTADQLEMVTELLGGDMVN transcription factor, QSFICDPDDETFIKNIIIQDCMWSGFSAAA intrinsically KL VSEKL ASYQAARKD SGSPNPARGHS V disordered, full CSTSSLYLQDLSAAASECIDPSWFPYPL length. NDSSSPKSCASQDSSAFSPSSDSLLSSTES SPQGSPEPLVLHEETPPTTSSDSEEEQEDE EEIDWSVEKRQAPGKRSESGSPSAGGHS KPPHSPLVLKRCHVSTHQHNYAAPPSTR KDYPAAKRVKLDSVRVLRQISNNRKCTS PRSSDTEENVKRRTHNVLERQRRNELKR SFFALRDQIPELENNEKAPKWILKKATA YILSVQAEEQKLISEEDLLRKRREQLKHK LEQLRNSCA CRBN (47- MINFDTSLPTSHTYLGADMEEFHGRTLH 87 Uniprot: Q96SW2.435) DDD SCQVIPVLPQ VMMILIPGQTLPLQLF Human,HPQEVSMVRNLIQKDRTFAVLAYSNVQE ox cytoplasm / nucleus, REAQFGTTAEIYAYREEQDFGffilVKVKA w E3 ligase, structured IGRQRFKVLELRTQSDGIQQAKVQILPEC domain only.VLPSTMSAVQLESLNKCQIFPSKPVSRED QCSYKWWQKYQKRKFHCANLTSWPRW LYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQET EITTKNEIFSLSLCGPMAAYVNPHGYVHE TLTVYKACNLNLIGRPSTEHSWFPGYAW TVAQCKICASHIGWKFTATKKDMSPQKF WGLTRSALLPTIPDTEDEISP MAX (23- MDKRAHHNALERKRRDHIKDSFHSLRD 88 Uniprot: P61244. 87) SVPSLQGEKASRAQILDKATEYIQYMRR s? Human, nucleus, KNHTHQQDIDDLKRQNALLEQQGEHP transcription factor, intrinsically disordered, DNA binding domain only. PPP1R11 MAEAGAGLSETVTETTVTVTTEPENRSL 89 Uniprot: 060927.TIKLRKRKPEKKVEWTSDTVDNEHMGR Human, cytoplasm, RSSKCCCIYEKPRAFGESSTESDEEEEEG nucleus, E3 ligase, CGHTHCVRGHRKGRRRATLGPTPTTPPQ intrinsically PPDPSQPPPGPMQH disordered, full- length.Alpha MKLFWLLFTIGFCWAQYSSNTQQGRTSI 90 Uniprot: P0DUB6. amylase (1- VHLFEWRWVDIALECERYLAPKGFGGV . Human, secreted, 226) QVSPPNENVAIHNPFRPWWERYQPVSYK glycosyl hydrolase,LCTRSGNEDEFRNMVTRCNNVGVRIYV w inactive fragment. DAVINHMCGNAVSAGTSSTCGSYFNPGS RDFPAVPYSGWDFNDGKCKTGSGDIEN YNDATQVRDCRLSGLLDLALGKDYVRS KIAEYMNHLIDIGVAGFRIDASKHMWPG DIKAIL PMP22 MLLLLLSIIVLHVAVLVLLFVSTIVSQWIV 91 Uniprot: Q01453.GNGHATDLWQNCSTSSSGNVHHCFSSSP Human, cell NEWLQSVQATMILSIIFSILSLFLFFCQLFT membrane, involved LTKGGRFYITGIFQILAGLCVMSAAAIYT in myelination, full VRHPEWHLNSDYSYGFAYILAWVAFPL Il 1 length. ALLSGVIYVILRKRE BAX (42- MIAAVDTDSPREVFFRVAADMFSDGNFN 92 Uniprot: Q07812. 153) WGRWALFYFASKLVLKALCTKVPELIR Human, cytoplasm,TIMGWTLDFLRERLLGWIQDQGGWVRL mitochondrion LKPPHPHHRALTTAPAPPSLPPATPLGPW membrane, involved in mitochondrial apoptosis, isoform gamma.Keapl (321- MAPKVGRLIYTAGGYFRQSLSYLEAYNP 93 Uniprot: Q14145. 611) SDGTWLRLADLQVPRSGLAGCWGGLL Human,YAVGGRNNSPDGNTDSSALDCYNPMTN cytoplasm / nucleus, QWSPCAPMSVPRNRIGVGVIDGHIYAVG E3 ligase, Kelch GSHGCIHHNSVERYEPERDEWHLVAPM domains.LTRRIGVGVAVLNRLLYAVGGFDGTNRL NSAECYYPERNEWRMITAMNTIRSGAG VCVLHNCIYAAGGYDGQDQLNSVERYD VETETWTFVAPMKHRRSALGITVHQGRI YVLGGYDGHTFLDSVECYDPDTDTWSE VTRMTSGRSGVGVAVTME HSPB1 MTERRVPFSLLRGPSWDPFRDWYPHSRL 94 Uniprot: P04792.FDQ AFGLPRLPEE WSQ WLGGS S WPGYV Human, RPLPPAAIESPAVAAPAYSRALSRQLSSG cytoplasm / nucleus, VSEIRHTADRWRVSLDVNHFAPDELTVK molecular chaperone, TKDGWEITGKHEERQDEHGYISRCFTR mostly disordered, KYTLPPGVDPTQVSSSLSPEGTLTVEAPM full length.PKLATQSNEITIPVTFESRAQLGGPEAAK SDETAAK HMGB1 MGKGDPKKPRGKMSSYAFFVQTCREEH 95 Uniprot: P09429.KKKHPDASVNFSEFSKKCSERWKTMSA Human, nucleus, KEKGKFEDMAKADKARYEREMKTYIPPstranscription factor, KGETKKKFKDPNAPKRPPSAFFLFCSEYR partially disordered, PKIKGEHPGLSIGDVAKKLGEMWNNTA full length.ADDKQPYEKKAAKLKEKYEKDIAAYRA KGKPDAAKKGWKAEKSKKKKEEEEDE EDEEDEEEEEDEEDEDEEEDDDDEEXAMPLE 3: Pilot in vitro library PANCS-Binders

[0127] We selected six protein targets to attempt to discover novel binders for, each with varying architectures and degrees of structural order: KRAS4b(G12D), RAF (RBD), Mdm2 (1-188), IFNG, Myc DNA binding domain (DBD), and Sosl disordered domain (FIG. 3A, Table 1). We simply cloned each target into the +AP as a RNAPc fusion and transformed into E. coli host cells with the ZBneg-AP used in our final mock selections (FIG. 15) to prepare the selection materials. We passaged the 108affibody library, which had gone extinct in PACE-based selections, on each E. coli selection strain in culture tubes for 12-hour outgrowths and 5% transfer between passages (FIG. 3 A and FIG. 6). After 4 rounds of passaging (48 hours total), we measured the titer in each condition. KRAS(G12D), RAF(RBD), IFNG, and Mdm2 had high titers (>108), indicating successful selections, while Sosl and Myc (DBD) selections had titers near the limit of detection (<105), indicating failure to enrich binders (Table 2).

[0128] Table 2. Plaque Assay results for 6-target panel de novo library PANCS (FIG. 3A).On-Target Off-Target1030 Activity Titer Activity ActivityTarget Dependent (PFU / mL)aDependent DependentPlaques'1PlaquesbPlaques0KRAS l*109Yes No No G12DRAF 8*108Yes No No Mdm2 l*1010Yes No No IFNG l*1010Yes No No Myc DBD <LODeNofNofNofSosl <LODeNofNofNofaActivity independent titer determined from counting plaques using 1030-1059.bPresence of plaques when a plaque assay was performed using the strain used in PANCS. Tresence of plaques when a plaque assay was performed using an off-target hSUMOl selection strain +AP.dPresence of plaques when a plaque assay was performed using 1030 (no giii present in the E. coli).eLimit of detection (LOD) of 1 PFU was equal to 5*104PFU / mL.fBecause the titer was below the LOD, the observation of no activity dependent plaques is of limited information as no phage were likely added to the quadrant in the activity dependent plaque assay.

[0129] We performed next generation sequencing (NGS) on the four selections with a high titer (FIG. 3B), which revealed that the selections on KRAS(G12D), RAF (RBD), and IFNG each converged onto a single sequence (i.e., >80% of reads belonged to that sequence). The most dominant Mdm2 variant comprised only 2.6% of the population; in retrospect, this lack of convergence is unsurprising as Mdm2 binds an FXXXWF / Y motif common to ~3% of library variants, and therefore, many variants were enriched. For KRAS G12D, RAF (RBD), and IFNG selections, we also sequenced the library, passage 2, and passage 3, which revealed that the relative ratio between active variants is set by passage 2 (FIG. 16), in line with our Mock PANCS results. Finally, we used AlphaFold3 to predict the binding interface for each hit, which, as expected, showed the randomized region of the affibody at the predicted interaction interface (FIG. 3 A and FIG. 17).

[0130] We subcloned the top variants from each selection (those >1% of reads in KRAS G12D, RAF (RBD), and IFNG selections and 4 random variants from the Mdm2 selection) into an expression plasmid (Lux-N) for measuring binding in a previously established E. coliluciferase assay (FIG. 3A)17,19,21. Reconstitution of the proximity dependent split RNAP, measured by the production of luminescence, was observed to be induced following coexpression of each affibody variant and its respective selection target. This indicated variant Target binding in E. coli (FIG. 3B). Positive binding controls, previously published binders discovered by ribosome display (Z(RAF322)32, phage display (NS1 Monobody)31, and rational engineering (C12 12.1 A)33, produced comparable signal to the newly selected affibodies for RAF (RBD), KRAS G12D, and Mdm2, respectively. We confirmed specificity of binding by assaying for binding between one binder from each successful selection with the four targets that gave hits (FIG. 18), which revealed high specificity of each binder (only S-VVD had off-target binding to Mdm2). We purified each of the top binders (FIG. 19) and performed surface-plasmon resonance (SPR) binding assays, revealing binding between top variants and their target of interest with in vitro affinities between 176 and 635 nM (FIG. 3B and FIG. 20). These results confirmed that the selections successfully enriched binder variants.

[0131] Table 3: Summary of all SPR data model fits (FIG. 19, FIG.33, and FIG.36). Target Binder Model Chi2Ka (1 / Ms) kd(1 / s) Ka (nM) KRAS Ab-N- 1 : 1 binding 1.44E+01 4.76E+04 1.80E-02 378 G12D VCD1 : 1 binding 1.99E+01 1.55E+04 6.25E-03 403 Average 3.16E+04 1.21E-02 384 KRAS Ab- Y- 1 : 1 binding 2.19E-01 1.47E+04 8.87E-04 60.3 G12D LHD1 : 1 binding 3.40E-01 1.86E+04 7.99E-04 43.0 Average 1.67E+04 8.43E-04 50.6 KRAS Ab- Y- 1 : 1 binding 4.31E-01 1.14E+04 1.35E-04 11.8 G12D LHY1 : 1 binding 4.17E-01 1.15E+04 1.54E-04 13.4 Average 1.15E+04 1.45E-04 12.6 KRAS Ab-N- 1 : 1 binding 0.2075.14E+00 1.59E+05 3.29E-05G12D LHY1 : 1 binding 5.85E+00 1.72E+05 6.89E-06 0.040 1 : 1 binding 3.84E+00 1.16E+05 5.25E-05 0.453 Average 1.49E+05 3.08E-05 0.206 KRAS Ab- S- 1 : 1 binding 9.98E-02 2.11E+04 1.21E-04 5.7 G12D LCS1 : 1 binding 1.33E-01 8.33E+04 1.73E-04 2.1Average 5.22E+04 1.47E-04 2.8 KRAS Ab-N- 1 : 1 binding 3.601.94E+00 6.58E+04 2.37E-04G12D wss1 : 1 binding 8.10E-01 1.05E+05 1.67E-04 1.59 1 : 1 binding 3.67E+00 1.08E+05 1.66E-04 1.54Average 9.29E+04 1.90E-04 2.04RAF Ab- T- 1 : 1 binding 1.13E+01 6.71E+04 1.79E-02 VPN 2671 : 1 binding 2.36E+01 5.11E+04 1.36E-02 266Average 5.91E+04 1.58E-02 267 RAF Ab-N- 1 : 1 binding 90484.24E+00 1.68E+02 1.52E-03 LFN1 : 1 binding 1.38E+00 1.01E+03 2.08E-03 2059Average 5.89E+02 1.80E-03 3056 RAF Ab- Y- 1 : 1 binding 3.54E+00 2.87E+04 5.75E-02 2004LCS1 : 1 binding 3.59E+00 2.60E+04 3.90E-02 1500Average 2.74E+04 4.83E-02 1764 RAF Ab-N- 1 : 1 binding 4.29E-01 3.87E+03 3.19E-04 82.4WCN1 : 1 binding 7.14E-01 1.15E+04 3.66E-04 31.8Average 7.69E+03 3.43E-04 44.6 RAF Ab- Y- 1 : 1 binding 1.75E+00 7.80E+05 5.86E-03 7.5WID1 : 1 binding 2.06E+00 8.99E+05 5.10E-03 5.7Average 8.40E+05 5.48E-03 6.5 IFNG Ab- S- 1 : 1 binding 3.39E-01 6.89E+02 1.10E-03 1597VVD1 : 1 binding 5.05E+00 1.84E+03 5.06E-04 275Average 1.26E+03 8.03E-04 635 IFNG Ab- S- 1 : 1 binding 1.07E+01 3.12E+02 1.95E-03 6250VCD1 : 1 binding 5.73E+00 7.67E+02 1.75E-03 2282Average 5.40E+02 1.85E-03 3429 Mdm2 Ab-S- 1 : 1 binding 9.21E-01 1.06E+04 1.40E-03 132WFY1 : 1 binding 8.51E-01 3.75E+03 1.13E-03 301Average 7.18E+03 1.27E-03 176 Mdm2 Ab-S- 1 : 1 binding 2.07E+00 2.57E+04 6.47E-03 252WYS1 : 1 binding 1.24E+00 2.20E+04 4.90E-03 223Average 2.39E+04 5.69E-03 238 Mdm2 Ab-T- 1 : 1 binding 2.29E-01 6.44E+04 4.01E-02 623WDN1 : 1 binding 2.88E+00 7.13E+02 8.77E-04 1230Average 3.26E+04 2.05E-02 629 Mdm2 Ab-Y- 1 : 1 binding 4.13E-01 6.73E+03 3.84E-03 571WTT1 : 1 binding 1.56E+00 2.99E+04 6.98E-03 233Average 1.83E+04 5.41E-03 295 Mdm2 Ab-Y- 1 : 1 binding 7.16E-01 7.87E+04 6.69E-04 8.5WCTA46T 1 : 1 binding 9.40E-01 8.54E+04 7.16E-04 8.4Average 8.21E+04 6.93E-04 8.4Mdm2 Ab-Y- 1 : 1 binding 2.29E-01 4.52E+04 9.17E-04 20.3 WCTA46T 1 : 1 binding 6.78E-01 4.05E+04 1.29E-03 31.9 L51FAverage 4.29E+04 1.10E-03 25.8

[0132] To assess reproducibility of the selections, we repeated the entire 6-target selection four additional times in parallel several months later. This yielded highly consistent results in terms of the extinction events and endpoint phage titers (FIG. 21). We performed NGS on each of the replicate selections and observed high reproducibility (r = 0.72; average of each pairwise Pearson’s Correlation for variants >0.1% of NGS reads; variants enriched >5% were identical) between biological replicates (FIG. 3C) and within parallel replicates (FIG. 22; r = 0.95). These results demonstrate that PANCS-Binders can rapidly - in just 48 hours, comprehensively, and reproducibly screen and isolate binder variants from in vitro libraries without the need for replicates or additional screening.EXAMPLE 4: High-throughput PANCS

[0133] We next sought to challenge the PANCS-Binders technology in a multiplexed high-throughput selection by attempting to simultaneously identify binders for a large panel of diverse protein targets in 96-well plate format (FIG. 4A). In addition to scaling down the selection volumes to 1 mL for plate compatibility, we made three additional adjustments for this selection: we extended the linker length between the target and RNAPc to ensure that the position and orientation of the binder was not constrained (FIG. 23), we reduced the selection stringency from 5% to 10% transfer, and we created a second ~108phage library based on an affitin scaffold to have two different scaffold libraries to compare to one another (FIG. 24). We simply cloned each protein of interest (a total of 95 targets) into +APs without additional optimization, as well as a negative control no-fusion +AP consisting of a start codon followed by the 60-amino acid GS linker and RNAPc, to establish a 96-well plate of target selection strains. The 95 targets (Table 1) vary in origin (mammalian, bacterial, or viral), localization (secreted, extracellular domains of membrane proteins, membrane proteins, cytosolic, or nuclear), function, and structure (fully ordered, significant disordered regions, fully disordered). We reasoned that such a diverse target panel should assess the capability for performing PANCS-Binders in a high-throughput manner and provide a realistic estimate of the expected hit rate for 108libraries. To confirm that the selection +APs were functional, we performed an amplification assay (FIG. 25) with phage encoding RNAPN WT, which is notproximity dependent (recombines with RNAPc independent of target and binder interacting), with the -AP (demonstrating sufficient counterselection to prevent non-selective replication) and without the -AP (indicating sufficient target expression for binding induced replication; only four targets did not amplify >10-fold). Notably, this panel has many targets that are difficult to purify from E. coli, which highlights the superior properties of targets expression plasmids over in vitro selection methods.

[0134] After preparing the selection cells, we performed the 4 passage PANCS-Binders over the course of 48 hours and collected endpoint titers using qPCR to identify preliminary hits - titers >107PFU / mL (FIG. 26 and Table 4; see Note below for a detailed discussion of how this threshold was selected, including FIGS. 27-31). For all preliminary hit wells, we collected NGS, performed AlphaFold2 multimer predictions for the top variants (NGS and AlphaFold compiled in FIG. 27), and subcloned variants from passage 4 phage and collected luciferase binding assay for the top variant(s) cloned (FIG. 28). Overall, we validated 79 new binders to 52 targets (FIG. 4B). These results further demonstrate the high correlation between endpoint titer and binder enrichment. We used 16 of these binders to investigate the specificity of our selected binders (FIG. 4C), identifying only one off-target interaction between GAB ARAP and our LC3B binder which is not surprising given the high sequence similarity between GAB ARAP and LC3B (both of which bind LIR domains). With this dataset of 288 pairwise binding measurements, we sought to evaluate the ability of AlphaFold335to identify binding vs non-binding pairs; iPTM values were not well correlated with binding (FIG. 29).

[0135] Table 4. Titers from passage 4 of the 96-target panel PANCS with affibody and affitin libraries (FIG.26). Titers measured using qPCR.Affibody Affitin Affibody Affitin Titer Titer Titer Titer Target (PFU / mL) (PFU / mL) Target (PFU / mL) (PFU / mL) No Fusion 1.45E+05 1.55E+06 Myc (full length) 3.63E+05 6.61E+04 MST2 (linker) 2.88E+03 3.72E+06 KRAS wt 4.27E+05 9.55E+07 Parkin 8.91E+03 SasG (1060- 8.95E+06 1140) 4.27E+05 3.02E+06 PD-L1 9.55E+03 3.55E+06 PCNA-AF 1.02E+06 2.09E+06 CD3d 1.05E+04 9.55E+05 MST2 3.39E+06 4.17E+08 DltD (e / ) 1.66E+04 2.82E+08 HlgA toxin 1.10E+07 1.70E+08 KRAS (G12V) 1.95E+04 3.31E+08 TNFR2 1.51E+07 2.04E+06 BTK (PHdomain) 2.00E+04 2.75E+06 ZB 1.74E+07 2.57E+08 PIK3CA 2.04E+04 3.72E+06 LC3B 1.23E+08 3.55E+08MXD1 2.09E+04 9.12E+05 KRAS (G12D) 1.45E+08 8.13E+08FKBP 2.75E+04 4.17E+06 SortaseA 1.48E+08 1.78E+09 HSPB1 (fulllength) 3.02E+04 2.24E+06 DltD (strep) 2.19E+08 4.57E+08Calprotectin (het- HMGB1 6.03E+04 2.00E+06 12) 2.45E+08 7.08E+08 Sosl (IDR) 7.41E+04 2.40E+06 GAB ARAP 2.82E+08 5.50E+08CalprotectinCDKN1A 7.94E+04 2.34E+06 (sl00a8) 3.63E+08 9.33E+08 PD-1 8.32E+04 1.91E+08 Beclin 5.01E+08 2.14E+08 EIF4A 8.51E+04 1.41E+06 LM02 5.01E+08 3.98E+08 PP2A-B' 9.33E+04 1.86E+06 IFNG 5.01E+08 1.29E+06 PMP22 9.55E+04 7.59E+04 LEV 5.37E+08 1.55E+09 HEWL 1.02E+05 2.19E+08 FRB 5.50E+08 5.75E+08 p53 (full length) 1.15E+05 2.88E+06 Bax (peptide) 6.31E+08 4.07E+08 CRBN 1.26E+05 1.07E+05 HIFla 7.08E+08 4.47E+08 CD3z 1.29E+05 2.19E+06 NRAS 9.12E+08 5.75E+08 EUFlb 1.29E+05 3.16E+06 SH3BP5 1.05E+09 1.32E+09 PilQ 1.32E+05 2.63E+06 HRAS 1.17E+09 7.41E+08 PPP1R11 1.41E+05 9.77E+04 MBP 1.20E+09 3.98E+08 MAX 1.45E+05 1.00E+09 Sas A 1.32E+09 7.41E+08 Fibronectin 1.48E+05 7.76E+08 BRD4 1.35E+09 5.75E+08 TEV 1.51E+05 2.14E+07 SasG (930-1060) 1.38E+09 5.89E+08 IBTK 1.55E+05 6.92E+08 KIX 1.48E+09 6.92E+08 ALKBH5 1.58E+05 2.95E+06 Bcl2 1.51E+09 4.79E+06 BAX 1.62E+05 4.37E+04 HPV-pE7 1.70E+09 2.17E+07 KRAS G12C 1.82E+05 3.47E+08 RBI 1.86E+09 2.95E+08FGFRlc domainVHL 1.91E+05 1.58E+06 D2 1.95E+09 1.55E+09Calprotectin (het- PINK 1.95E+05 3.31E+06 6) 2.19E+09 1.45E+09 PP2A-B 2.00E+05 3.63E+06 mCherry 2.24E+09 8.32E+08 OmpA (ec) 2.00E+05 2.24E+06 IsdB 2.29E+09 3.09E+08 OmpF (ec) 2.04E+05 1.07E+08 p65 2.34E+09 1.51E+09 PP2A-B" 2.09E+05 1.20E+09 14-3-3z 2.51E+09 1.23E+09 OTUBI 2.09E+05 2.40E+07 RAF 2.82E+09 4.47E+08 HSPB1(structured) 2.09E+05 4.68E+08 Mdm2 2.88E+09 1.05E+09CalprotectinAlpha amylase 2.14E+05 2.24E+05 (sl00a9) 3.39E+09 8.13E+08 TRIM 2.29E+05 4.27E+06 PAL 5.13E+09 4.79E+08 NIX 2.29E+05 3.55E+06 TCIM 5.25E+09 8.71E+08 Keapl 2.29E+05 3.98E+08 Myc (DBD) 5.37E+09 2.88E+08 G3BP 2.88E+05 3.24E+06 FnbpA 5.37E+09 6.76E+08 p53 (AD) 2.88E+05 1.95E+05 PIK3CB 6.03E+09 1.58E+09GTF2I 3.39E+05 1.26E+05 PDE6D 8.91E+09 1.45E+09Note: Setting the criteria for a hit based on titer, NGS, and AlphaFold predictions.

[0136] We sought to establish criteria based on titer, NGS, and AlphaFold predictions to identifying hits and reduce false positives. We used the no-fusion endpoint titers as a preliminary cutoff: any well with an endpoint titer >10x the no-fusion endpoint titer was considered a preliminary hit and sequenced (cutoffs of 1.5* 106PFU / mL for affibody and 1.6*107PFU / mL for affitin), yielding 44 affibody and 56 affitin preliminary hits. We compiled the sequences that made up >1% of NGS reads and performed AlphaFol d2 Multimer predictions for the top four hits (excluding truncations, FIG. 27). We collected E. coli luciferase assay data for dominant variants from each of these preliminary hits that did not have a truncation dominate (>10% of NGS reads; FIG. 28). The NGS generally matched what we observed in our initial 6-panel PPI-PANCS: an average of 4.5 variants with >1% of reads (range = 1-24), the most dominant sequence from the selection had on average 57% of reads (range = 3-96%), and convergence was a frequent occurrence with 58% of hits converging onto >50% on one variant; only 17% of hits had a dominant variant with < 20% of reads (FIG. 29). We observed a background level (-0.1-1%) of truncation variants in the NGS results indicating incomplete de-enrichment and confirmed that a subset of selections below our threshold contained high levels of truncations (FIG. 30). Additionally, we showed that with an additional day of passaging (2 rounds), these no-fusion and failed selections do continue to de-enrich to extinction (FIG. 31). We conclude from these NGS data that a lower cutoff would increase our false positive rate by including samples that had not de-enriched; however, with additional passaging, a much lower threshold could have been used as extinctions and successful enrichment become more separated.

[0137] Several hits had dominant variants that were truncations, low convergence, or variants that were enriched on multiple targets. We investigated each of these hits further by luciferase assay (FIG. 28). Of the 143 variants that had >10% of reads within a hit, there were 12 truncations across 10 targets. Each of these targets had a hit in the other library; 8 out of 10 top variants on these targets did have binders (only Bax peptide and SasG did not, however, the truncated variant did bind SasG) within their hits suggesting that enrichment of a truncation as the top variant in one library was not a strong indication of false positives in the other library’s hit. Of the 131 variants that had >10% of reads within a hit (excluding truncations), there were 126 unique variants - 5 variants enriched in two target selections. KRAS G12D and KRAS G12C enriched the same affitin variant, KRAS WT and KRAS G12V enriched the same affitin variant, KIX and NRAS enriched the same affibody variant, RB 1 and UEV enriched the same affitin variant, and the dominant RAF affibody variant was also the 2nd most common affibody variant on BAX peptide. The common KRAS bindingvariants are unsurprising, but we subcloned the hits for these other 6 targets: UEV and KIX bound their top variants, RAF, NRAS, Bax peptide, and RBI did not (FIG. 28).

[0138] Lastly, we hoped to use AlphaFold2 or AlphaFold3 predictions to assist in identifying false positives. We saw a wide distribution of confidence in AlphaFold2 multimer prediction confidences for the top variant from each hit (ipTM of 0.11-0.92; FIG. 29) Additionally, we observed with a panel of binders and targets, all tested in luciferase assay for binding (18x16), that AlphaFold3 was not able to identify the binding pairs (FIG. 32). Lastly, we decided to compare the ipTM to in vitro Kd for all of our measured interactions and saw only a weak correlation (FIG. 37). We conclude from these results that we could not use low ipTM values as a filter. Based on our examination of several features in the NGS data, ignoring the AlphaFold predictions, we conclude that a titer greater than our threshold (lOx the No Fusion) and a dominant variant that is not a truncation are the criteria for identifying a hit; based on this we have 42 affibody hits and 46 affitin hits across 59 targets. Six target tested in luciferase (FIG. 28) failed to have a binder (at least 2x lux signal) across both libraries - Bax peptide, NRAS, FGFR, TEV, PP2A-B”, and KRAS G12V.EXAMPLE 5:Larger libraries, affinity maturation, and mammalian applications of PANCS-Binders hits

[0139] While the initial 55% hit rate and 100s of nM Kd showcase the viability of PANCS-Binders, we wanted to assess whether we could improve the hit rate and identify higher affinity binders by simply using larger libraries in PANCS-Binders. We cloned IO10variant libraries for both the affibody and affitin scaffolds using parallel transformations (FIG. 5 A) - a 100-fold improvement of our initial library size (Table 5).

[0140] Table 5. Library size estimation. Cloning of large ~1O9'10variant affibody and affitin libraries (referred to as 1010libraries). Estimated number of variants is based on the average of 40- and 80-minute timepoints. Titers determined using activity independent plaque assays. # indicates different transformations that were kept separate.Library # 40 minute 80 minute 120 Estimated Amplified Total Total minute Number of Titer Titer Titer Total Titer Variants (PFU / mL) Affibody 1 3.75E+08 1.90E+09 1.30E+09 1.14E+09 2.50E+11 Affibody 2 2.05E+08 2.30E+09 2.10E+09 1.25E+09 1.95E+11 Affibody 3 4.95E+08 3.20E+09 2.50E+09 1.85E+09 1.15E+11 Affibody 4 2.00E+08 3.25E+09 3.50E+09 1.73E+09 7.75E+10Affibody 5 3.10E+08 2.80E+09 1.80E+09 1.56E+09 1.18E+11Affibody 6 2.20E+08 2.90E+09 1.65E+09 1.56E+09 1.43E+11 Affibody 7 1.85E+08 2.60E+09 1.10E+10 1.39E+09 1.78E+11 Affibody 8 1.70E+08 2.15E+09 1.90E+09 1.16E+09 1.20E+11Total 1.16E+10Affitin 1 3.50E+07 5.50E+08 5.50E+08 2.93E+08 1.58E+11 Affitin 2 7.50E+07 6.50E+08 1.20E+09 3.63E+08 1.05E+11 Affitin 3 3.50E+07 6.00E+08 1.15E+09 3.18E+08 2.23E+11 Affitin 4 2.50E+07 4.50E+08 1.20E+09 2.38E+08 6.75E+10 Affitin 5 3.50E+07 6.00E+08 1.30E+09 3.18E+08 1.78E+11 Affitin 6 2.00E+07 6.50E+08 1.15E+09 3.35E+08 1.38E+11 Affitin 7 5.00E+07 5.50E+08 1.40E+09 3.00E+08 1.20E+11 Affitin 8 2.00E+08 5.00E+08 1.45E+09 3.50E+08 8.25E+10Total 2.51E+09

[0141] We selected 8 targets that did not give hits in our initial screens, including VHL, TRIM, and PNCA, and 4 targets that previously generated hits, including KRAS G12D and RAF, and performed a large library PANCS-Binders screen (FIG. 5 A) with 6 rounds of passaging over 72 hours (see Methods for additional details and protocol differences). In addition to getting new hits for the targets that also initially gave hits with smaller libraries, 3 out of the 8 previously failed targets now also yielded hits, as confirmed by the E. coli luciferase assay (38%; FIG. 5A, Table 6). We analyzed these hits by NGS (FIG. 33) and compared the new hits for KRAS G12D both using the E. coli luciferase assay and in vitro (FIG. 5B, binders for other targets shown in FIG. 34 and FIG. 36): the affinity of the best binder obtained from this selection improved from 384 nM to 0.2 nM, representing a ~2000x improvement in affinity. These results confirm that PANCS-Binders is capable of quickly mining IO10variant libraries for novel binders, improving our hit rate (predicted from 55% to 72%), and identifying higher affinity binders, all within a 72-hour selection.

[0142] Table 6. Endpoint titers of large library selections. Passage 6 endpoint phage titers from PANCS with large, IO10, libraries; assessed using activity independent plaque assay. Bold indicates this library -target pair was successful at 108scale. Underline indicates confirmed binders were enriched.Target Titer Titer Target Titer Titer (PFU / mL) (PFU / mL) (PFU / mL) for (PFU / mL) for Affibody for Affitin Affibody for Affitin Library Library Library Library KRAS 1.42E+11 2.38E+09 TRIM21 2.20E+08 <1.0E+05 G12DHR. AS 1.65E+09 4.44E+11 ALKBH5 5.30E+06 <1.0E+05 RAF 2.43E+10 1.45E+10 PINK <1.0E+05 <1.0E+05IFNG 1.93E+10 1.63E+10 PIK3CA 5.00E+07 <1.0E+05VHL 1.03E+10 2.75E+09 BAX <1.0E+05 3.00E+07PCNA 9.25E+09 6.00E+09 NIX 4.00E+07 <1.0E+05

[0143] One advantage of the PANCS-Binders technology is that the same selection strains and phage can quickly be adapted to PACE by transforming the selection strain with a mutagenesis plasmid to allow mutations to accrue during a directed evolution campaign. To test this, we performed PACE-based directed evolution on the passage 4 phage from our initial Mdm2-affibody selection (FIG. 3B) using adaptations of our previous method21. First, we identified higher stringency positive APs with reduced propagation of passage 4 phage (as measured by activity dependent plaque assays). Then we used these two strains to perform PACE over the course of 60 hours of evolution, after which the phage populations converged (6 / 8 subclones sequenced) on an affibody variant Y-WCT with an additional A46T mutation and then an additional L51F mutation (in 1 / 8 with A46T; FIG. 5C). Assessment using the A. coli luciferase binding assays showed the evolved variants have improved affinity for their targets (FIG. 5D). We confirmed this in vitro'. Kd = 8.4 nM for A46T, and 26 nM for A46T / L51F (FIG. 5D and FIG. 36) compared with 176 nM for the highest affinity Mdm2 binder identified from the initial PANCS (a >20x improvement). Critically, the mutations arising from the supplemental PACE are not in the initial randomization sites, nor are they predicted to make direct contacts with the target protein (FIG. 5C). This is not altogether surprising; the power of unbiased directed evolution via PACE for optimizing existing function through non-intuitive mutations is well-established.

[0144] Lastly, we sought to test whether the novel binders could be taken directly from PANCS-Binders into mammalian cells and bind their targets in a functionally relevant manner. We cloned the KRAS G12D and KRAS binders Ab-N-VCD (FIG. 3B) and Ab-N-LHY (FIG. 5B) into a split nano-luciferase complementation assay37and demonstrated robust binding in HEK293T cells (FIG. 5E). Next, we sought to convert Ab-N-LHY into a KRAS degrader by fusing it to an LIR (LC3B interacting region) domain for targeted protein degradation through authophagy20(FIG. 5F). We observed robust degradation of endogenous KRAS in U2OS as assessed by WB in a binderOdependent manner (FIG. 5G and FIG. 38). We then demonstrate that the high affinity Mdm2 binder (Ab-Y-WCT A46T) co-localizes with Mdm2 in the nucleus (FIG. 5H and FIG. 39), confirming binding in mammalian cells. We then overexpressed AB-Y-WCT A46T and several other Mdm2 binders in U2OS cells to see if the binders could inhibit the MDM2-p53 interaction, by monitoring expression levels of Mdm2 and p21, which are both transcriptionally regulated by p53. We observed a stronginduction of both targets upon MDM2 binder expression, which indicates robust inhibition of Mdm2 and activation of p53 (FIG. 51 and FIG. 40). These results demonstrate that PANCS-Binders can produce binder variants with functional binding activity in mammalian cells.EXAMPLE 6:Nanobody library generation and screen

[0145] A nanobody library was designed according to procedures described herein, on which PANCS-Binders was performed.

[0146] DNA sequence of the library (codons are shown):CAAGTTCAGCTAGTAGAATCAGGAGGGGGATTGGTTCAGGCGGGCGGCAGCTTA CGCCTGTCGGCGGCTGCGAGCGGTTTCCCGGTGNNYNNYNNYHHYATGNWYTG GTATCGTCAAGCNCCAGGNAAAGAAAGAGAATGGGTNGCNGSNATTNNYTCTNN YGGTNNYHHYACTNWYTATGCNGATTCNGTNAAAGGAAGATTTACNATTTCNAG AGATAATGCNAAAAATACNGTNTATCTGCAGATGAATTCNTTAAAACCAGAAGA TACNGCNGTNTATTATRYHNWYGTAHHYGTAGGTNNYHHYTACNNBGGTCAAG GTACGCAGGTTACTGTGTCCTCC (SEQ ID NO: 901).

[0147] Amino acid sequence of the library (to show where randomization happens):QVQLVESGGGLVQAGGSLRLSAAASGFPVXXXXMXWYRQAPGKEREWVAXIXSX GXXTXYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYXXVXVGXXYXGQG TQVTVSS (SEQ ID NO: 902).

[0148] 21 binders were found against 16 different targets, including TCIM, KIX, calprotectin monomer sl00a8, Myc DNA-binding domain, BRD4, IBTK, sortase A, mCherry, OTUB1, DltD from Streptococcus sp., HSPB1 structured domain, MBP, FRB, LM02, LC3B, and DltD (ef). Binding assay results are shown in FIG. 41, and binder sequences are shown in Table 7.

[0149] Table 7. Nanobody binder sequences.ID Binder Sequence SEQ ID NO:B8 QVQLVESGGGLVQAGGSLRLSCAASGFPVSYFHMNWYRQAPGKEREW VAAINSPGTITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYT 903 VVHVGFLYSGQGTQVTVSSB10 QVQLVESGGGLVQAGGSLRLSCAASGFPVDDNPMHWYRQAPGKEREW VAAIFSDGNHTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY 904 AL VIVGGF YFGQGTQ VTVS SB23 QVQLVESGGGLVQAGGSLRLSCAASGFPVPYYHMNWYRQAPGKEREW VAAIDSSGCITDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYI 905 HVFVGCNYDGQGTQVTVSSB35 QVQLVESGGGLVQAGGSLRLSCAASGFPVNHYYMIWYRQAPGKEREW VAAIHSDGSITHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYT 906 DVFVGRYYNGQGTQVTVSSB212 QVQLVESGGGLVQAGGSLRLSCAASGFPVRIAPMYWYRQAPGKEREWV 907 AGIIS YVYYID VIVGNYYIGQGTQ VT VS SB214 QVQLVESGGGLVQAGGSLRLSCAASGFPVLCSHMYWYRQAPGKEREW 908 VVKPEDTAVYYIFVSVGIFYB215 QVQLVESGGGLVQAGGSLRLSCAASGFPVHYRSMDWYRQAPGKEREW VAAICSVGDITLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYT 909 IVHVGHLYWGQGTQVTVSSB216 QVQLVESGGGLVQAGGSLRLSCAASGFPVTFNSMYWYRQAPGKEREW VAGICSIGHTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYV 910 D VI VGS S YLGQGTQ VT VS SB217 QVQLVESGGGLVQAGGSLRLSCAASGFPVSLSTMLWYRQAPGKEREWV AGICSDGPSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYID 911 VI VGHHYLGQGTQ VT VS SB218 QVQLVESGGGLVQAGGSLRLSCAASGFPVSYFHMNWYRQAPGKEREW VAAINSPGTITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYT 912 VVHVGFLYSGQGTQVTVSSB219 QVQLVESGGGLVQAGGSLRLSCAASGFPVSHHHMNWYRQAPGKEREW VAGINSSGSTTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYT 913 NVS VGDF YAGQGTQ VT VS SB221 QVQLVESGGGLVQAGGSLRLSCAASGFPVDDNPMHWYRQAPGKEREW VAAIFSDGNHTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY 914 AL VIVGGF YFGQGTQ VTVS SB223 QVQLVESGGGLVQAGGSLRLSCAASGFPVTYPFMLWYRQAPGKEREW 915 VAGINSGGHYTFYADSFTLVKVRRLLCPPB224 QVQLVESGGGLVQAGGSLRLSCAASGFPVAYRIMFWYRQAPGKEREWV 916 AAIPSFVHVGFFYWGQGTQVTVSSB226 QVQLVESGGGLVQAGGSLRLSCAASGFPVPYYHMNWYRQAPGKEREW VAAIDSSGCITDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYI 917 HVFVGCNYDGQGTQVTVSSB228 QVQLVESGGGLVQAGGSLRLSCAASGFPVRITYMNWYRQAPGKEREW 918 VAAIHSNGSKTRRYGGILCLRICRB229 QVQLVESGGGLVQAGGSLRLSCAASGFPVYTGLMIRRIRR*FLQGSRYA 919 GYCVLLMEIFNMLPQSVECRPYB232 QVQLVESGGGLVQAGGSLRLSCAASGFPVSNTFMVWYRQAPGKEREW VAAIFSNGVNTYYADSVKGRFTISRDNAKNTVYLQINVSVGFIYRGQGT 920 QVTVSSB234 QVQLVESGGGLVQAGGSLRLSCAASGFPVPNPLMLWYRQAPGKEREW VAGICSCGNTTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYV 921 IVP VGNI YRGQGTQ VT VS SB236 QVQLVESGGGLVQAGGSLRLSCAASGFPVDPSIMDWYRQAPGKEREWV AGINSTGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYA 922 NVHVGCIYTGQGTQVTVSSB237 QVQLVESGGGLVQAGGSLRLSCAASGFPVPAHPMHWYRQAPGKEREW 923 VAGIDSHGLYIMFMYTMETHODSCloning and Bacterial Strain Handling

[0150] All plasmids and phage (Table 8.1-Table 8.7) were cloned by Gibson Assembly (GA) of PCR fragments generated using Q5 DNA polymerase (NEB). All primers (Table 9) were ordered from IDT. For plasmids, GA mixtures were transformed into chemically competent DH10P E. coli and after a 1 h outgrowth in 2xYT media, were plated on antibiotic selective agar plates to isolate individual clones. For phage, GA mixtures were transformed into chemically competent S1030-1059 E. coli, and after a 2 h outgrowth, a plaque assay was performed to isolate individual phage clones. All plasmids and phage were confirmed by Sanger sequencing. All plasmid maps with annotations of key features are available in Table 8.1-Table 8.7. For constructing selection (+AP / -AP) and A. coli luciferase (2-22 / N-lux / C-lux) strains, SI 030 E. coli was made chemically competent and then single or double transformations were used (and then repeated as needed until all plasmids were incorporated). E. coli strains was grown on agar plates static at 37 °C or in solution at 37 °C with 200 rpm shaking with Luria Broth (LB) supplemented with the appropriate antibiotic unless otherwise indicated. Antibiotics were used at standard concentrations: kanamycin (40 pg / mL), chloramphenicol (33 pg / mL), and carbenicillin (100 pg / mL).

[0151] Table 8.1. Plasmids (Phage).BinderAA SEQIDID Description Binder AA Sequence NO: Plasmid mapVO- Gen 2 RNAPN-RAF SKTSNTIRVFLPNKQRTVVNVRNGMSL 96 benchling . com / s / seq- VO (WT) HDCLMKALKVRGLQPECCAVFRLLHEH JJql S2iNyzsJmd6xiEdv?m KGKKARLDWNTDAASLIGEELQVDFL =slm- aRoQFIVy6IMYBSH2fwfJ vo- Gen 2 RNAPN-RAF SKTSNTIRVFLPNKARTVVNVRNGMSL 97 benchling . com / s / seq- VI (Q66A) HDCLMKALKVRGLQPECCAVFRLLHEH rY 6quiRgFHXfckenpJEf?m KGKKARLDWNTDAASLIGEELQVDFL =slm- gCcVfoLclXRHKLDA5uA cvo- Gen 2 RNAPN-RAF SKTSNTIRVFLPNKQRTVVNVRNGMSL 98 benchling . com / s / seq- V2 (K84A) HDCLMAALKVRGLQPECCAVFRLLHEH nSrOgO 1 RqNMC JJ2a4g5L?KGKKARLDWNTDAASLIGEELQVDFL m=slm- xLrFr8p8h2BD919gzg4V vo- Gen 2 RNAPN-RAF SKTSNTIRVFLPNKQRTVVNVRNGMSL 99 benchling . com / s / seq- V3 (R89L) HDCLMKALKVLGLQPECCAVFRLLHEH i!bNxc5abNZF szpmc3xa?KGKKARLDWNTDAASLIGEELQVDFL m=slm- d6r!iU34PHGHWuq48pRh vo- Gen 2 RNAPN- GSVKVKFVVRGEEKEVDTSKIRSVWRG 100 benchling . com / s / seq- V8 Affitin (SasA) GKWVDFSYDDNGKQGYGYVLEKDAPK kGbUlx3dZtABiwGPTOv ELLDMLARAEREKKL T?m=slm- ktQkaNLZ JpvROy VOHF c J VO- Gen 2 RNAPN- GSVSSVPTKLEVVAATPTSLLISWDAPA 101 benchling . com / s / seq- 24 Monobody (KRAS) VTVDYYVITYGETGGNSPVQKFEVPGS OOmrOViWfuJUJs4 VUj 3m KSTATISGLKPGVDYTITVYAWGWHGQ ?m=slm- VYYYMGSPISINYRT ZzNRWIOe8dXJESbPQ4yf vo- Gen 2 RNAPN- VSSVPTKLEVVAATPTSLLISWDAPAVT 102 benchling . com / s / seq- 80 Monobody VDHYVITYGETGGNSPVQEFTVPGSKST BMAv02BGSfvUsKloWo (hSUMOl) ATISGLSPGVDYTITVYAWVSDEYTHG Mq?m=slm- YYSYSPISINYRT k4Z8qtY23ikBzE9EvwAc VO- Gen 2 RNAPN- VDNKFNKEVNLAADEIWLLPNLNNQQ 103 benchling . com / s / seq- 81 Affibody (RAF) AWAFITSLKDDPSQSANLLAEAKKLND jlkztBeV91rkgW31BhwO?m AQAPK =slm- HWw4VrRT6Gkt6WjA4nR 075- Gen 2 RNAPN- VDNKFNKEPRAARLEITVLPNLNREQG 104 benchling . com / s / seq- 103 Affibody (PDL1) GAFIVSLWDDPSQSANLLAEAKKLNDA w0kNAOKhSETKaNFpG3QAPK r2?m=slm- ZXJq3YteiKU513VNQVsV 73- Gen 2 RNAPN- GSVKVKF* * *GEEKEVDTSKIRS VWRGG 105 benchling . com / s / seq- 9 Affitin (SasA) 3x KWVDFSYDDNGKQGYGYVLEKDAPKE tdkTtlPlgVCtBPQclQDf?m STOP LLDMLARAEREKKL =slm- lg2FWZ0yPhLVlZC5Uuyn Lib Gen 2 RNAPN- VDNKFXKEXXXAXXXIXXLPNLNXXQ 106 benchling . com / s / seq- 5 Affibody Library XXAFXXSLXXDPSQSANLLAEAKKLND 02e90z0Dmx8QenF nZSty ?AQAPK m=slm- OkP6P9eDWs34LeLC4wR XLib Gen 2 RNAPN- GSVKVKFXXXGEEKEVDTSKIXXVXRX 10V benchling . com / s / seq- 8 Affitin Library GKXVXFXYDDNGKXGXGXVXEKDAP lTpmkq9ednWVnGyHVr59KELLDMLARAEREKKL ?m=shn- nwE4qG2wm!sMLJcMUD pjVO- Genl RNAPN (WT)- MQDPYVKEAENLKKYFNAGHSDVADN 108 benchling . com / s / seq- 63 IFNG (not PPI GTLFLGILKNWKEESDRKIMQSQIVSFY SUVQimpl V6bYvocVzZex dependent) FKLFKNFKDDQSIQKSVETIKEDMNVKF ?m=slm- FNSNKKKRDDFEKLTNYSVTDLNVQRK JcqT YIMgGMwxBlz 1 DvS AIHELIQVMAELSPAAKTGKRKRSQ X VO- Gen 2 RNAPN- VDNKFNKELHYAIHEIGNLPNLNVYQII 924 benchling . com / s / seq- 76 Nanobody (GFP) AFVNSLDNDPSQSANLLAEAKKLNDAQ QlKprPEEsxCRlESiSryT?APK m=slm-iZ961TwJBfLyGKy4Mgrk

[0152] Table 8.2. Plasmids (+APs).CGG RNAPC+APPositive selectionprot&in (POI)ID Target ORI, RBS (PcGG) / (Pkat) 1 Plasmid Map6 AA Linker Sequence: TSGGSG (SEQ ID NO: 109)31-69 KRAS (WT) pl5a, SD8 / SD8; 6 AA benchling. com / s / seq-QorElY6vvP0bDTiVgCUN?m=slm- linker kgoT 1 gDlDSgp3nNcP5H531-70 KRAS (WT) pSClOl, SD8 / SD8; 6 benchling. com / s / seq-uoeGETX8o6z6qTEIVTPn?m=slm- AA linker p6KSgwAx4P8nB3U49dHb67- KRAS pl5a, SD8 / SD8; 6 AA benchling. com / s / seq-poKVGTKsSekoJzfDpLBD?m=slm- 130 (G12D) linker aeOcCRLcCJpblzrrXo7166-5 Mdm2 (1- pl5a, SD8 / SD8; 6 AA benchling. com / s / seq-SRsejdlxiEms9jFC0ibj?m=slm- 188) linker 1 aB34M8HQwvzADSw7hCs66-6 IFNG pl5a, SD8 / SD8; 6 AA benchling . com / s / seq-cE41 F 90kMq2nnML 3 QDax?m= slm- linker YQGlywLAKMJQDZGARKrH67-49 Sosl pl5a, SD8 / SD8; 6 AA benchling. com / s / seq-331uLfC8RYnwpA6Vxqyd?m=slm- linker KviINCCSUzlRAfU78r3A8-37 Myc DBD pl5a, SD8 / SD8; 6 AA benchling. com / s / seq-GCrOfOjlC7ES7b8401Z6?m=slm- linker kgth2LUXb87ZnDX7f8Yt73-40 RAF RED pl5a, SD8 / SD8; 6 AA benchling .com / s / seq-LhbbW3RW sk8tX V 9MpSbw?m= slm- linker gOUPJK3YD23OUQX50WoG73-66 RAF RED pSClOl, SD8 / SD8; 6 benchling. com / s / seq-zPDFzGI2710ZcxBfVXmC?m=slm- AA linker 3KED3hx4BloSKvG0qJ6s73-67 Mdm2 (1- pSClOl, SD8 / SD8; 6 benchling. com / s / seq-1 lPJ9yjPBiCrT3E7ogok?m=slm- 188) AA linker GPlAbKKKKlgUj sNy61wK73-65 Mdm2 (1- pl5a, SD8 / sd5; 6 AA benchling . com / s / seq-BF CFEgkEqQQSgZ v vdTKn?m= slm- 188) linker ujGzZ2gWiXd2OUxBREli79- IFNG pSClOl, SD8 / SD8; 6 benchling .com / s / seq-HAHkYMDpp4nMn9B Yp7vr?m= slm- 128 AA linker TLI4ukXqGrZkvPFhwpEI73-63 IFNG pl5a, SD8 / sd5; 6 AA benchling. com / s / seq-jjYgcBHgpvQ3j5IxrSXU?m=slm- linker CDjEcS 1 ZXe44iDHPe6jN20 AA Linker Sequence: TSGGSGGSGSSGSGGTSGSG (SEQ ID NO: 110) 73-31 KRAS pl5a, SD8 / SD8; 20 AA benchling. com / s / seq-5wpJUNYgrOkT5qjh8mhV?m=slm- (G12D) linker fgsFkeIA99Ur5wTWEFob73-41 RAF RED pl5a, SD8 / SD8; 20 AA benchling. com / s / seq-QrfAd2dknEcOGXwKmIHj ?m=slm- linker cbzOGyOXpZlaQMONHU373-34 ZB pl5a, SD8 / SD8; 20 AA benchling. com / s / seq-M0fP6w4gtYx81aJwX4D3 ?m=slm- linker MW3QPL7xWaJz78SXsTz640 AA Linker Sequence: TSGGSGGSGSSGSGGTSGSGGSGSGTGSGSSGSGGTSGSG (SEQ ID NO: 111) 73-32 KRAS pl5a, SD8 / SD8; 40 AA benchling. com / s / seq-Xb AD0KtbE3nhoaX664OO?m=slm- (G12D) linker uvktqlpE8pMiLJJ5FwcN73-42 RAFRBD pl5a, SD8 / SD8; 40 AA benchling. com / s / seq-6w AbVdVuh Wk9PosRBmHH?m=slm- linker L9XAD432ChIUuwL6q35O73-35 ZB pl5a, SD8 / SD8; 40 AA benchling. com / s / seq-vFjmVRA3rWP6JOKIWCOs?m=slm- linker INqCQEzSnM4x7w84aAR960 AA Linker Sequence: TSGGSGGSGSSGSGGTSGSGGSGTGTGTGSSGSGGTSGSGGSGSGTGSGSSGSGGTSGSG (SEQ ID NO: 112) 75- No Fusion pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-OAkkU SRN 3 T JhdoLilhRD?m= slm- 154 linker bEfoe3bp4 IFrsbQWj GA973-21 KRAS pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-Zr3IVL Y C sltuB wW aWx Jr?m= slm- (G12D) linker wRhiE YUzN 1 wIXpbqdbih73-36 ZB pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-B3VpJwTHB JNtDB96TRU3?m=slm- linker iQ706YAxBZMMwGKFHJVF73-39 GFP pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- v5EQQGCh486eb Y JK J 51 r?m= slm-linker ypcPwaGL9gxg6AgQIkhn- p65 (17- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-cdSliWqWg0tdHYgBTdOP?m=slm- 0 293) linker pEDkDzXE0Z4F8vYUSiit- FGFRlc pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-Uzm5jeJGfrrqaKG4tbn6?m=slm- 5 domain D2 linker Oanxy dY CDzy fpqszed47- TCIM pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-6IddRZlHDqTN3MCPSaK8?m=slm- 6 linker Ls84NSQmfDGHZLtK9WM8- TRIM21 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-oEa6R5xeroyTLQdBFxcy?m=slm- 7 (277-475) linker qT4xnij 61aqu2uwpCMdM- PDE6D pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-DN JTqsoqchb6Ifc 8LmkH?m= slm- 8 linker S7qdc6giMN9TxTm03COK- RAF pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-653ffOYStxIyWyOcrwUG?m=slm- 2 linker popOBqO 1 ASaK3UElPBLr- NRAS pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-rB JDOntLGgEuAuq 1 kSED?m= slm- 4 linker y shqst61 vCj ixpRdgNb Y-29 Beclin (175- pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-bKebT eEQKLuRZBaobE 1 r?m= slm- 267) linker 2EpZm0zHwRtbAbAu6Dx8- Mdm2 (1- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-PxdMGjVyvDqAJDmdvnLQ?m=slm- 8 188) linker QGrbcwpi2958fCkylPHM- Calpro het pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-hj EXy 4EpozczGXmnCBaw?m= slm- 9 12aa linker kaXmkr7hxqA6SVFKn2FS- HlgA toxin pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-Et7y9HSeppcxczv2YEmK?m=slm- 0 linker cIebjjpTK98RJnKIl 9EK- Bcl2 pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-RMXbBBMr4cQa8y uBErNu?m= slm- 1 linker pheOA6NggSSQHM8pYvx9- Calpro mon pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-ACHXVmlJqRuo6vu5yxVR?m=slm- 2 sl00a9 linker qEU7Hbl2g9DTVirxt3e9- KIX pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-75hSB7CK7Ss5mdiXwroT?m=slm- 3 linker dFHu3 Sze Y s6wCnenE2hQ- HRAS pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-GOe4zj T11 Mdf4PRb310 S?m= slm- 5 linker aVc4rYGqhQtzmXMU7JX5- KRAS wt pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-ZeZ4aYcPMa4jt6kE0JA8?m=slm- 6 linker urQbtWwArEqCnNkcetGl- G3BP pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-RmH YPArmkMpqTnNprOOP?m= slm- 7 linker 2kBpsjPaVT6hPhPNxF xx- NIX pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-r3 wj G4NZ4y LcCRmEG7 Y d?m= slm- 3 linker P4Nzro5JL8BBBdOORv5r- PP2A-B pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-18IkmHloviCvDSJqO8dh?m=slm- 0 linker I67s5YoyoAAaUOnnArDm- Calpro mon pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-ldbycwm28J9gHToTdejh?m=slm- 1 sl00a8 linker STtWIVscpGItdu8VEBy3- FOXP3 pl5a, SD8 / SD8; 60 AA benchling . com / s / seq- 5RUKlkWIsN Y OKRF xp9H8?m= slm- 2 linker 5TvJ2jmfkNogUtIie2Rr- KRAS pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-GFtPQ2Xf6XG5izoUTCsA?m=slm- 8 (G12V) linker CxBk737fChjA24RXaPyO- Sosl pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- w80QM JY 61 wsy X3 exlMz4?m= slm- 6 linker kRREHX73IefmCI7iWAO6- PP2A-B' pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-TwI5xEyskYV7Sw9GsjRh?m=slm- 7 linker TzAhfOORng5ajuOAwFgi- mCherry pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-SoOZUDzTf8WRPuUM19tO?m=slm- 8 linker bnWoNtGNBoNtU3YjwZPc- Myc (short) pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-EnqwDf6RRDLp9dlxJV4T?m=slm- 9 linker V2L8jbdYZITnjwyPReYe- PP2A-B" pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-4gOERZifGohryh49vugB?m=slm- 2 linker zclfZd87C5VyT8hlOZ68- MBP pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-CTYBnSJpKhNMPCGe49xt?m=slm- 3 linker Yy5Ydwaik5tUJ7PRdDW7- Fibronectin pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-zdsltF3MW4Ugw2tCggjl?m=slm-5 linker p Y7ugHj 1 aXcbiLaCPTIR- VHL pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-qb7tgAd3oKFG9Z6EqSdo?m=slm- 6 linker jGDEhoC2D3a3kO81 tTL6- BTK (PH) pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-5ALCwW2eLTMX311VJet3?m=slm- 7 linker sB Y39ws0zDeS7oLoG5 SH- Calpro het pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-CBtjqRnxx4ayrkRAR6XS?m=slm- 0 6aa linker yZoAl 3Q4kBtyBDHr8w9R- GAB ARAP pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-CU3BT33LpDQcqNz52AYW?m=slm- 1 linker pM8ur5mMQ5m8gMYj fW 4x- GTF2I pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-D6ZmbBc9m2P7cdeZfjkI?m=slm- 4 linker wbxKJl T7nHnx8u0dFvP3- OmpF (ec) pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-bpykRa7MihJDhXglLuqN?m=slm- 5 linker pgYxnvoDyuSMfeGKks8P- 14-3-3z pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-IWbQqj RmQzcj UY tigy we?m= slm- 6 Monomeric linker yt4r5Yag09nYxefCzHFJ(L12Q,R18E andS58E)- PCNA-AF pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-X JUWLK9eW ZX7HA W auHcu?m= slm- 7 linker oDXOs 1 RnucKQ JcBeHREu- TEV pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-81ffIJaX0kRAI6VLInsY?m=slm- 9 linker galMvGSadT65JKoFySYK- FnbpA pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-GjkFFN 12 siUB skbdkmDc?m= slm- 0 linker ASm8m8Enw4i3jjTBtBmf- IBTK pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-Fy laT64LESd9RoHJb4yy?m=slm- 1 linker nfQUj cy qVNACboxbxfRP- PAL pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-ecoHsMaIhmdTYwSoQV5i?m=slm- 2 linker c8zDVquNiy8OOq62bqlL- PIK3CB (3- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-7A7cpLNdlIJM7KkPgZaS?m=slm- 3 115) linker Y9jqMOLRNBhwlpGpLJOH- SortaseA pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-jGkTemzmKXVsPiaVzTmO?m=slm- 4 (25-206) linker jMbvmE2g0pp 16T VTSZOc- PilQ (468- pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-khnm I Imrdsh Md7IJ I'h A 7 fe?m= slm- 6 549) linker vsPUDoswrA 1 dUAEs8dIZ- DltD (ef) pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-pr9kZE87jy877Ze V21wM?m=slm- 7 linker 01 vwtAKq8spWRMl IWVcC- TNFR2 (33- pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-mAyN SH YPq2EBro 3MaKM7?m= slm- 8 205) linker x8FVA5aJGLiVHCDET5Li- CDKN1A pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-PQ5 irZBkcDj h3R4UHBKN?m= slm- 9 linker cFdFMaHd2Gl 1 r6awo41 C- SH3BP5 pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- A vc 55NRDP6F 4mj V qeGl V?m= slm- 0 linker o4BfkBdFLzii 1 YkDe JOP- OTUBI (1- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-O82hpMpccs8FoUNuFrfY?m=slm- 1 102) linker DGRFy YlG6QHybT Y31 P3b- DltD (strep) pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-CECchAZI4HL7qEmLC3np?m=slm- 2 linker tgKN7aPE8KlKr01PaaS7- PINK pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-qPq6x39UehzUtZRClaOI?m=slm- 0 linker yHzeA88dr7KzF6ILdl 8b- RBI pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-22kbw64bDo512vL4T6J3?m=slm- 5 linker IGZoUdtzriYriAAKfviD- SasG (1060- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-vlqOK4ok3Nnz2RAIsmeV?m=slm- 6 1140) linker TGfDQbkCbhmIYbt8kl cU- SasG (930- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-iLLWrGfiClMGYArCPXsI?m=slm- 7 1060) linker Fhe4Uyj9NfoC8CywBOKA- FKBP pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-x40cA81pEv7xirBM9uxs?m=slm- 8 linker asEJgRJgltfptOrbcjjj- BRD4 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-uWLE77Emo9YGUtV2nx5 v?m=slm- 0 linker WmFAuRn21QPoZsF4hFHX- IsdB (125- pl5a, SD8 / SD8; 60 AA benchling . com / s / seq- SzEr41Z JNcB JmUB 8QO2?m= slm-1 269) linker seJsEbcdhbgolFAfQwpN-50 HEWL pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-CxlSVSbklEqF3Syw042I?m=slm- linker H3KNC82Pha4FHZB37MbH- HSPB1 (90- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-PJZGJ4mun3IKcOJErxeO?m=slm- 3 171) linker Xs5wlOr4SfvbdgRUM6Ed- KRAS pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-2tvdweDEql Y 05 sPUNA V Y?m= slm- 4 G12C linker csLi8d4weLPbhqzzlYOU-94 Mst2 linker pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-BemPhamuT 5 sy wqWLa5h5 ?m= slm- linker YmellP0qdFuIUAm0Sf6u-51 LMO2 (28- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-NXc2ysSollG8dClPXNgL?m=slm- 150) linker YfpX 1 lOeij crk5tYWpZ8-52 HIFla pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-LrW4a6u72I2kro ID lNiK?m=slm- linker F o74FDHKJbx8z2PrQe0z-23 MXD1 pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-bBeNxj omj gPQRSF iW GOn?m= slm- linker QyDn8Obf66PvZrYt6iKI- OmpA (ec, pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-qGyYe5Xj4xBsdfY7kAAg?m=slm- 5 84-224) linker 3Pw3 eZnnYOG 1 s 1 rfDqRN-40 CD3z pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-fF2AIC8XFYpLI5dEVeJk?m=slm- linker qUnT9qGpKKdfn3OOS 1 p 1-41 PIK3CA (1- pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- Aned8XSfCRP 1 MoDFp Y wR?m= slm- 105) linker 9Q9yuEB 1 ZIsZuz Yop Y4X-44 UEV pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- WTDkd5R V746q VT qvZF sw?m= slm- linker HU9FTb2LkTO29rWI5Wp-46 FRB pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-mtFkW0QpGYU17MfoCaHn?m=slm- linker P9kNCCZeZbWASii3O9Bx-47 PD-1 pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-bKsW CH YU Y tEDZhay fEFE?m= slm- linker kkprEYggLqVT9u5bZgwT-49 EIF4A pl5a, SD8 / SD8; 60 AA benchling . com / s / seq- 8mGcOba7W oGg vwy Sy429?m= slm- linker CzuPCOp2kSy8ZxM6VD7V- SasA pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-6BAA9bssJHU2rrzvFfnQ?m=slm- 3 linker BeLryRJBSo2jGsp6ZaQ2- LC3B pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-BVnxoGkEIF8WqzyciCqM?m=slm- 6 linker zsHlH0xb2vn5ws0dScqE- ALKBH5 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-4J5UYICjPsrfCcBQexOw?m=shn- 7 linker 62da3X8otCUcHupJT2yw- HIFlb (1- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-ao4jpxAORmJ8nMHYWkdF?m=slm- 4 474) linker TRnAWFqxP 1 nEEQ8iCra6- IFNG (24- pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-KsZIMX JQ7LRRHC7GSqti?m= slm- 8 156) linker y!qgGLjFLyxIbwwJ2he8- MST2 (313- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-jtOStbjKLwrI2hWHuPye?m=slm- 4 437) linker z79ntiBqlr4xrktdn7Qj- p53 (1-293) pl5a, SD8 / SD8; 60 AA benchling.com / s / seq-xaOF9j7eTve5wPY7OSzA?m=slm- 5 linker XUsSlY215Zor6alp6ry-48 PD-L1 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-B74sRi7ocrSyHvdQiQVZ?m=shn- linker xLrtaQpvUHFhWyCJ84mv-1 Bax peptide pl5a, SD8 / SD8; 60 AA benchling .com / s / seq- 8 J7TAMAqN4ZLESQkEQ4G?m= slm- linker SFiX8Vx8PpbEJ9s39bMJ- p53 AD (1- pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-20LFYp0870jPugBjKupY?m=slm- 1 61) linker YlQqlGdSkzxOrHiTQfkZ-93 CD3d pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-4hR2jNV|iLOXJPDLGbX8B?m=slm- linker P33HEy8NnH2bAUbX7wbM- Myc (long) pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-hendvxf!hlwtdR4OwfQ v?m= slm- 9 linker w8EH9I6dGWql6qgNJvYL- CRBN pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-7mvwe76RtMPWca9NQqpC?m=slm- 2 linker oL Sx4D VQMj 0Mh58fhIgZ- MAX pl5a, SD8 / SD8; 60 AA benchling .com / s / seq-rtp66xstwB 8HFXFEQ Vj e?m= slm- 3 linker duRSy Ovzl 1 gABrDn4rus- PPP1R11 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-MbPMAihvvKbWdKD6JtgO?m=slm-8 linker D92oVzXfQpLUeLExAAve5- Alpha pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-5emdqxm8ALcLLo7uNPJ6?m=slm- 75 amylase linker hi6YfuFD8Uc84mYewk3Y5- PMP22 pl5a, SD8 / SD8; 60 AA benchling . com / s / seq- sPUg wnqHHCrprRrHXZk7?m= slm- 83 linker JomY sxil wgtlrgWU8Fai5- BAX pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-dv8svMLWo7jRgBNANgao?m=slm- 89 linker 0XX5aejmRwEcR\Y IgMrll6-42 Keapl pl5a, SD8 / SD8; 60 AA benchling. com / s / seq-EdPHe3rCNlvOvTwxeQCZ?m=slm- linker KCWjIy09bMTDnKUY92o06-43 HSPB1 pl5a, SD8 / SD8; 60 AA benchling . com / s / seq-IfKi5 eLDKox 1 xmAmAeah?m= slm- linker CwP7gFgtQY7tnrVkHMt56-45 HMGB1 pl5a, SD8 / SD8; 60 AA benchling. com / s / seq- 5hGuCN41oBaLzdNTzHz2?m=slm-linker ZdlLMfeR7j QGkp5RVitC

[0153] Table 8.3. Plasmids (-APs).ID Description Plasmid mapRBS for PT? / RBS for PkatAA Sequence: MASEQLEKELQALEKELAQLKWKNQALEKKLAQ (SEQ ID NO: 113)0-06 ZBneg, 6 AA linker, sd5 / sd2 benchling. com / s / seq-nHRnXEU6UGqfIjfZ9C0L?m=slm- GVJXRV43bBgZm751qBm70-05 6 AA linker, sd5 / sd5 benchling .com / s / seq- sR9z YNKCh7TIIPkbpZAf?m= slm- EMUTvNHfcWsCxBdhwxRZ0-01 6 AA linker, sd8 / sd8 benchling. com / s / seq- VAE5JvugbkmYxlAYTWDf?m=slm- sI2hSzJ94ZnivxzSP6753-79 60 AA linker, sd8 / sd8 benchling .com / s / seq-qptnks4 V 9 vC AkgQsGtmf?m= slm-0SHHTsCgDhaxvivYq6yY

[0154] Table 8.4. Plasmids for luciferase assay.P ’ J23114Lux~NSEQ ID ID Description AA Sequence NO: Plasmid map -22 PT? driven LUXAB onpSClOl benchling . com / s / seq- Udyz6dlESk91OHBzq fsO?m=slm- pPVnaTxvld7HcIZ8In VO7- Lux-N VDNKFNKEPRAARLEITVLPNLNREQGGAFIVSLWDDPS 114 benchling . com / s / seq- 53 Affibody QSANLLAEAKKLNDAQAPK 4Hat7AliLPOjVB5Sf (PDL1) uyL?m=slm- kG731uQP41mO9klo5 XkO7- Lux-N GSVKVKFVVRGEEKEVDTSKIRSVWRGGKWVDFSYDD 115 benchling . com / s / seq- 44 Affitin NGKQGYGYVLEKDAPKELLDMLARAEREKKL MzeK19F5W14KL6Vc (SasA) GZvu?m=slm- EWzvy4QEH0p4MplFX39677- Lux-N VSSVPTKLEVVAATPTSLLISWDAPAVTVDHYVITYGET 116 benchling . com / s / seq- 147 Monobody GGNSPVQEFTVPGSKSTATISGLSPGVDYUTVYAWVSDE DIRqB 3 iHFxxrwaJ30 (hSUMOl) YTHGYYSYSPISINYRT gE7?m=slm- ZXarjzcklDUgI2snLz HR77- Lux-N VDNKFNKEVNLAADEIWLLPNLNNQQAWAFITSLKDDP 117 benchling . com / s / seq- 154 Affibody SQSANLLAEAKKLNDAQAPK BbfsAlyrXYrH5iGF0 (RAF) TXP?m=slm- 8PKvilso0qMW70ijQ h4W77- Lux-N GSVSSVPTKLEVVAATPTSLLISWDAPAVTVDYYVITYG 118 benchling . com / s / seq- 134 Monobody ETGGNSPVQKFEVPGSKSTATISGLKPGVDYTITVYAWG wbjyt230LVnMaXfV9 (KRAS; WHGQVYYYMGSPISINYRT pml?m=shn- NS1) YRG6N9pCEBxRhtv mJQ4P77- Lux-N C12 SSVEKKPEGVNTGAGDRHNLKTEWPELVGKSVEEAKKV 119 benchling . com / s / seq- 155 (Mdm2) ILQDKPEAQIIVLPVGTMPRFMDYWEGLNRIDRVRLFVD LneOyfLblsQ 1 SqHsor KLDNIAEVPRVG aL?m=slm- TN5991HPKseJUorED h3LLux- Lux-N VDNKF?KE???A???I??LPNLN??Q??AF??SL??DPSQSANLL 120 benchling . com / s / seq- NLib Affibody AEAKKLNDAQAPK mVSKERuVdRGIeQ7 5 Library j4OKo?m=shn- Variant Hk4Rjrf5ZYCffF4uTr bZLux- Lux-N GSVKVKF???GEEKEVDTSKI??V?R?GK?V?F?YDDNGK?G 121 benchling . com / s / seq- NLib Affitin ?G?V?EKDAPKELLDMLARAEREKKL nS183aPslWFrsSEeCr 8 Library pe?m=shn- Variant Uzf6HPcmoEH5NHE LKy7K65-50 Lux-N MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNW 122 benchling . com / s / seq- RNAPN(WT)- KEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKED z6k7WcWzlkehV2RIf IFNG (not MNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELI Esz?m=shn- PPI QVMAELSPAAKTGKRKRSQ nF8asOivpVyPfh7mAXFBdependent

[0155] Table 8.5. Plasmids (Lux-C).PBAD^ T7 RNAPCLux-CPositive selectionprotein (POI)Lux-C Target Lux-C Map6 AA Linker Sequence: TSGGSG (SEQ ID NO: 109)12-48 KRAS G12D benchling. com / s / seq-uiSpEZdsBlTbxgExQKzA?m=slm-tjVz7COoGbkxxZsU8hwQ12-64 RAF RED benchling.com / s / seq-QdDkS0EM8CzsfT20bokO?m=slm-syqroCCI8jVvkll9MEUK 72-83 Mdm2 (1-188) benchling. com / s / seq-qN74BV6NcLVA40q7sWOG?m=slm-tYPlL76qLlDmjmQSlgnI 72-76 IFNG benchling. com / s / seq-9BsSkfIQelRzlI17Eynm?m=slm-buZIVDFvFMxriNzCASlF60 AA Linker Sequence: TSGGSGGSGSSGSGGTSGSGGSGTGTGTGSSGSGGTSGSGGSGSGTGSGSSGSGGTSGSG (SEQ ID NO: 112) 77-59 No Fusion benchling. com / s / seq-yExBSN9d57SyhGQvdX8k?m=slm-4cQuASRmUdkXWzuNG9ol 73-21 KRAS (G12D) benchling. com / s / seq-bHUdEMQyJEyYaNnCzWYo?m=slm-kqR0hl614DqdUkezQdiP 73-22 ZB benchling. com / s / seq-cUHUKYXlIfvrpiwQffeK?m=slm-f810TByalR6o360d414873-30 GFP benchling. com / s / seq-jW7Zau5g2UgKlso86pGd?m=slm-b8yxDRGFVA9FnKMtqaMv 76-152 p65 (17-293) benchling. com / s / seq-MFerrPNUPyioanle5CPY?m=slm-7aRHx4Zp6Q72hCtJElod76-105 FGFRlc domain D2 benchling. com / s / seq-9ExlW51AuuNbWmXbwFtM?m=slm-EVPbFTSOCZimmw58YBEK73-167 TCIM benchling. com / s / seq-nTnoMwlXCUOjQnnlZXIE?m=slm-BYn2HKhFztBOk5msXYea-173 TRIM21 (277-475) benchling.com / s / seq-qCMpyJNRnolctgFACU7q?m=slm-pOqLvGfB2rU01wy0zrm3 -88 PDE6D benchling.com / s / seq-8pMSWqjiCrQuri2DX3Kl?m=slm-3knkHTbWEwJnUEY4iboI -23 RAF benchling.com / s / seq-O0XHCpWbTjdtbr6hzREO?m=slm-6HSgWO74Jclt0JNGMmUi -97 NRAS benchling.com / s / seq-MNEcNbqEA5bJjd5OxWp4?m=slm-kItXGR5rmeQg46qZz5G8 -35 Beclin (175-267) benchling.com / s / seq-5yo7IVzcSnXLhI6MHSI?m=slm-lRI2HFD37sHjuktcnQJv -74 Mdm2 (1-188) benchling.com / s / seq-lTuETCqeoOd8g8H8z2Xm?m=slm-dYuzgB8XONA4AEyrfbsV -149 Calpro het 12aa benchling.com / s / seq-tkeIuocLYFD41AhUPhFY?m=slm-rfu4gtDjm6WRHHhjYiO -145 HlgA toxin benchling.com / s / seq-4rYld71VjIo3hrfWFvjX?m=slm-DNEtvtTEscs8BIdOOOJV -99 Bcl2 benchling.com / s / seq-XXBGkJPjCBIubOQuCogn?m=slm-DRPjkgVNKUODowo7PDjX -75 Calpro mon sl00a9 benchling.com / s / seq-Pr7YIp6rzdeTHHdAgiKE?m=slm-kCzo82h7fupB5DXEgs47 -80 KIX benchling.com / s / seq-mkPkHaYFfPfnW81wRvlf?m=slm-FalsloRZmTpa36tOT2Ey -98 HRAS benchling.com / s / seq-pgTuUgg65DUpRlfn20uo?m=slm-zeyRxnr9T8TrgjagcSCj -96 KRAS wt benchling.com / s / seq-YNF06s3yMNWhoWHHQZm?m=slm-pOpYJTkPYEfYd25kMJqP -106 G3BP benchling.com / s / seq-p6tyxpHvpRKNiw5c2wfR?m=slm-YTOOFgotSluGRC3VgMAY -154 NIX benchling.com / s / seq-xefyGFfkOtoGCYsSEXtH?m=slm-pKPjhEMHVIswdlrsarhV -175 PP2A-B benchling.com / s / seq-NUTSz9eO3JOJlfiwyrE5?m=slm-yAu6yfORB4nt6Pzc8qrU -58 Calpro mon sl00a8 benchling.com / s / seq-41mAWyETIGyDdPQXoXmY?m=slm-RqgYH6D5qrjU2ZN5jgBx FOXP3-95 KRAS (G12V) benchling.com / s / seq-2mdc5SUWhQmRJX7ImMwk?m=slm-3YNe0gdVMwQA84eFeMZJ -169 Sosl benchling.com / s / seq-BetRKoyZuNBdlnUuMrp6?m=slm-dYRwQhH10u5AfxcmmTU9 -176 PP2A-B' benchling.com / s / seq-vMWqBFiKweSC31bfvTet?m=slm-C3zToe5IZgJanvOwg5Jb -150 mCherry benchling.com / s / seq-kYMKyHRGdy2oTZvt2IU3?m=slm-xRUOuQEeTy6oTcayxgBM -73 Myc (short) benchling.com / s / seq-s86vJaylws2nFE8d2svR?m=slm-SlShPTCEI2HTtX0nl5If -166 PP2A-B" benchling.com / s / seq-BY4emeWMOUzOfwxXcfvt?m=slm-tcINDmZcOgsImkBKTvfx -151 MBP benchling.com / s / seq-RP3yWG9oCdfyKJMPJdi7?m=slm-bdiXkPhY9MVs3uDPfc3c -146 Fibronectin benchling.com / s / seq-ZzKvhno6Q626q81EjKld?m=slm-qlkowg5flGJeA9VklDI0 -78 VHL benchling.com / s / seq-e5ttFNgV64jbsyNZQ3PY?m=slm-jnoBlNbfrsKSaq2Ep8E9 -159 BTK (PH) benchling.com / s / seq-cZ9gtCN49KI6B2RBmoyP?m=slm-qIKp2sQWKCw2RvVRqMlX -148 Calpro het 6aa benchling.com / s / seq-Vrfr5UGSgFAO7gMuIx6F?m=slm-UXGody3pDClwTz535g2a -77 GAB ARAP benchling.com / s / seq-QWrUgcltA0dgKyKnI5fN?m=slm-TRc3OoEsRBgRJn0jMb2z -160 GTF2I benchling.com / s / seq-wbAjXeo3iMuzOgsGCymv?m=slm-HZwpAi3kcEaOrU708mZN -85 OmpF (ec) benchling.com / s / seq-PCc30ARPOrkMJ7TZhvKw?m=slm-pWneGyvjBIBOYNQrZtnA -101 14-3-3z Monomeric benchling.com / s / seq-rYnWfjvOKjhc9ak4KfD7?m=slm-P6aEDJAlglnAvJWgnMIF (L12Q, R18E andS58E)-165 PCNA-AF benchling.com / s / seq-NyXUSZ8TBJmIT3RRKy2Q?m=slm-QIapA5oxPUqOlkuZv6V -175 TEV benchling.com / s / seq-gtv7gSu5b2FGnMJJbnlD?m=slm-4fv9YIXApwoWPXu8mhkd -172 FnbpA benchling.com / s / seq-IheXG0iYJlZxW30gE3Y0?m=slm-XvsmEjdyVf3S8|iErjM0E -161 IBTK benchling.com / s / seq-duQlBjHihWV9fAG3GRnc?m=slm-EEq4y4tZ8zUtm72XAPUG -162 PAL benchling.com / s / seq-DARw5POmwzvIJ9uWmeGO?m=slm-sDNz3BY9REpDtN6tyjXy -171 PIK3CB (3-115) benchling.com / s / seq-M7YP15R5qVffKYHqggRo?m=slm-kcbPs3fDDZT63IINpyc4 -173 SortaseA (25-206) benchling.com / s / seq-Ru7jLju6GyoLCTdaSbsn?m=slm-XLo9CRmzkKxn4yGdNwQk -163 PilQ (468-549) benchling.com / s / seq-Ru7jLju6GyoLCTdaSbsn?m=slm-XLo9CRmzkKxn4yGdNwQk -164 DltD (ef) benchling.com / s / seq-i5tvCTZlzMwFyoDZtVIu?m=slm-MdE8CotxvlvH2bozEaZ2 -104 TNFR2 (33-205) benchling.com / s / seq-IvN8nTYDCjqopYDlVWkE?m=slm-r3CzTPlvJvIKRsO8WfZb -166 CDKN1A benchling.com / s / seq-istCEzGSqnP9JetiQCuN?m=slm-RISOjKarN6Ff2X2KVkaH -81 SH3BP5 benchling.com / s / seq-Ilmxq7TOjVqvnYaXbq69?m=slm-S9kmHpbmaAehQlCif04r -82 OTUBI (1-102) benchling.com / s / seq-yBlNWiwMPwIP08fY6rTE?m=slm-Ek7dlP6R9a8umhclHnGY -165 DltD (strep) benchling.com / s / seq-EbBpShA9x0Ode6p8PHAA?m=slm-KY2U9M0jhl6wfoGTp8OG -155 PINK benchling.com / s / seq-A2zauOogFx5uYT7qmcT0?m=slm-llbMTuobOlB89f2kHsSu -83 RBI benchling.com / s / seq-Xpy5OSXeQ51g6hi8WiGl?m=slm-28UkfqgISqwlC5vZBdBU -86 SasG (1060-1140) benchling.com / s / seq-w5s9EZlNQkLdhYruHFzm?m=slm-13EcEXHVKY71NMyzHFw8 -171 SasG (930-1060) benchling.com / s / seq-eAt4uQ54Hllpbx756DmO?m=slm-OMUDgEmYp8pQsQsHP17a -179 FKBP benchling.com / s / seq-EjiHC9vGIVQExzl6BYzG?m=slm-FlerSN3uC2FpJhXgIewN -180 BRD4 benchling.com / s / seq-bHjjllDx3IogSnCjPck3?m=slm-qsp748T6DRU2YsMWrx3g -87 IsdB (125-269) benchling.com / s / seq-BX4NIEpvXhRHIAns94D?m=slm-4mQ9Te3U6StSI7HIN3Ny -102 HEWL benchling.com / s / seq-vfQnr94eWESHUc62TMCm?m=slm-xUAlKr8fRnMdlcavpdlO-164 HSPB1 (90-171) benchling.com / s / seq-GUv6WsRBpj4gz6bKvn3W?m=slm-iXkRj7y0gZgfLlPG29pI73-181 KRAS G12C benchling. com / s / seq-fSgcR78NvYNOyJjJ3DmN?m=slm-HBlw62dQtsObif5QH6NI Mst2 linker76-103 LMO2 (28-150) benchling. com / s / seq-isKCMFIWJoigsZ18JsEJ?m=slm-5kBZFRPsk6xqvvslyub2 76-178 HIFla benchling.com / s / seq-yEjxiXRm7O8Cvwc7ygFu?m=slm-jY0kKsGMQjCrXnlAumaJ 76-158 MXD1 benchling. com / s / seq-SXFtHNpq5CNf3YlGqBzz?m=slm-yAz9909ZIqdM8yxl3RbS 76-84 OmpA (ec, 84-224) benchling. com / s / seq-gEac4hfWY7YSGwDZ2tlK?m=slm-FHCEGTHZ6TtM3BR38qLT 73-168 CD3z benchling.com / s / seq-zhEuJgddfbTM13LhVoeE?m=slm-xkjuBuJDolZVinKxxInh 73-172 PIK3CA (1-105) benchling.com / s / seq-uarUD9PQsIuP4MamV15m?m=slm-N3NhFTD6CqskaShokbqy 73-176 UEV benchling.com / s / seq-lx3VYe3Rn6C12xHsf2My?m=slm-uBwK3p8e3BRgYpSLxbYu 73-178 FRB benchling.com / s / seq-P2jkD6cGA69xaoxjgGiV?m=slm-cpQ3pvLLLgDsEorNynV0 73-182 PD-1 benchling. com / s / seq-yQzKolJh4I6HZq5rVG2V?m=slm-14baz2qEaANS3WMAKP5E 73-187 EIF4A benchling.com / s / seq-zvEJ48QR8qUPsd40SF8M?m=slm-FVmn8NH5S01MEdwUVnYh 76-71 SasA benchling.com / s / seq-Gx9NNJ91hTJzPumE9pZ7?m=slm-LlRumENXQNADsFgxn9T5 76-79 LC3B benchling. com / s / seq-rlt5IzSBDshWhl9Rb2fu?m=slm-SpL56BpmJVPlNz5nKQcW 76-156 ALKBH5 benchling.com / s / seq-s6c48MGHowq9jMME85fF?m=slm-23tUDuPOmBliHJnMrSFO 76-177 HIFlb (1-474) benchling. com / s / seq-4qbTb9Y8HldXECOCqQxH?m=slm-bH3t5YRzVtbYHKm7XGF 76-69 IFNG (24-156) benchling. com / s / seq-Rxb7OuSxhwC 1 KxJykaPR?m=slm-qgj WG0 Jvg76X7yQpLdCb 77-54 MST2 (313-437) benchling. com / s / seq-3QXRIY0pRUN97KNELCgH?m=slm-eJW6pYZYT851VaVwyHhC 76-144 p53 (1-293) benchling. com / s / seq-Fu83Td8HuPIQwC2nRt64?m=slm-SWkrs4aEcLewx5C6BloC 73-183 PD-L1 benchling. com / s / seq-RvhD3N2PUlhj77T4fwoo?m=slm-FWNH2UNTgw5H7MTcHQFl 77-60 Bax peptide benchling. com / s / seq-aINRsklXSYSJcJv8aURp?m=slm-zKz2j49bk5rlv2mVWmMc 76-68 p53 AD (1-61) benchling. com / s / seq-oboHu3wQGGrOxVZXR8mT?m=slm-CL2SXiwFRno7endZDLq8 73-169 CD3d benchling. com / s / seq-WcQwZtijlEoLBlKU2yBt?m=slm-XAGmLlpF0bAJlqi2L7qA 76-147 Myc (long) benchling. com / s / seq-KWoSoJbUvTLOHeElTIut?m=slm-2LNV6oSuS8u6YcLQCfKx 76-153 CRBN benchling. com / s / seq-B APh6kwfucKj YDmmPXFp?m=slm-iO 1 HyK2nie JmwBXY 5n6z 76-100 MAX benchling. com / s / seq-GKeMoRGv4qd77TtfORFX?m=slm-i9MBGOae66SnGxHPZgvF 73-170 PPP1R11 benchling. com / s / seq-XHGhEcm7nvFEbuDNURBH?m=slm-SRIlvBiOGmF6ybMdXEbZ 76-76 Alpha amylase benchling. com / s / seq-SBXDewfrSaxr7UHdZPKi?m=slm-xPkWa4IgjbTC38snLtEc 73-174 PMP22 benchling. com / s / seq-AFHDnBiEoYvLILOGoita?m=slm-Bi5fpdTxyr84R5wTOuow 77-53 BAX benchling. com / s / seq-SxhwkmdaNisoVdvFR6IP?m=slm-pbu3gAKdt55C3yvTXplM 73-186 Keapl benchling. com / s / seq-c4pJiEFPzHNjGtrojl30?m=slm-VCVLymlr9ip2BtvQwbWY 73-163 HSPB1 benchling. com / s / seq-sOzF6JEREPkrjU54uVFv?m=slm-vhYiNPEDQpHkYFGytVSr73-177 HMGB1 benchling. com / s / seq-Hme2Y2BCw59yBZQevApH?m=slm-00Z5hzluKqBGFduBmlC0

[0156] Table 8.6. pET plasmids for protein purification. Description Plasmid mapKRAS G12D 1-169 pET (HIS tag) benchling. com / s / seq- 1 S W QgCORLPELFTGwT nCV?m=slm- bIm9FfhwnFWabV0jgDcxRAF (RBD) pET (His tag) benchling.com / s / seq-D3VuadJQQRyylcSsMwAN?m=slm- DpvKaocf8aFnOcZS3uWvMdm2 (1-188) pET (His tag) benchling. com / s / seq-o AWBkEqTG3 gFhdhtOXgG?m=slm- d7xeIGRU5PZIm4ooKwTrIFNGpET (His tag) benchling.com / s / seq-bJuJ9eGchlO8XJPvCn7f?m=slm- sF68NxIg9TzwSyTlADkm3xFLAG-GST-Affibody binder benchling. com / s / seq-2h4RsRW rou5PCfY x4 A9S?m=shn-PbduezfmBQcWVeCQ025

[0157] Table 8.7. CMV plasmids.Description Plasmid MapTarget split NanoLuc benchling.com / s / seq-irhIGNg6FDqyVXUYSpRO?m=slm- tRQlv6Im796pYvLSLXDBBinder split NanoLuc benchling.com / s / seq-OoyonZHpjwnrXIcd9qDI?m=slm- ewQylhZqd9FbB6Axnbs6Binder-LIR fusion benchling. com / s / seq-oEPOngFLZMXyy vU 9NUIh?m=slm-yHYpECMuMIUp7G2Y5EIPBinder only benchling.com / s / seq-a42TroNswKf69IzCp9oA?m=slm- KgcTScaWOEOcWOHPsAfhMdm2-mCherry benchling. com / s / seq-apmlnageLF s9kyBL SZqi?m=slm- FSLivJllYDoy6UhLVwMUBinder-GFP benchling.com / s / seq-jsSL7OyGOMNORbAGnD7U?m=slm-gwZEdWQQKi2Z8uw IBjdn

[0158] Table 9. Primers.ID Description Sequence SEQ (“n” represents any nucleotide) ID NO:VC- Forward primer for qPCR ggttcagcaaggtgatgctt 123 525VC- Reverse primer for qPCR accgcctcacctctgtttta 124 526MS- Forward primer for Affibody CATCATCATGCTAGCGTAGATAATAAATTTWM 125 783 Library YAAAGAAKKGNNYDMYGCADHYNNYSARATT NNYNNYCTGCCGAACTTAAACSNSNNYCAGVD RNNYGCCTTCVTHNNYAGCCTGNNYRAYGACC CGTCCCAAAGCGCTAATTTGCTGGCGGAG MS- Reverse primer for Affibody CATCATgctagcGCCAGAGCCAGAGCCACCCTTGT 126 618 Library AGACMS- Forward primer for Affitin Library CATCATgctagcTCGTGCGGAACGTGAGAAGAAAC 127 624 TGTAATGGAGMS- Reverse primer for Affitin Library CATCATGCTAGCATATCTAATAATTCTTTCGGTG 128 799 CATCTTTTTCRNNAACRNNACCRNNACCRNNTT TACCATTATCATCATARNNAAARNNAACRNNTT TACCRNNACGRNNAACRNNRNNAATTTTAGAA GTATCAACTTCTTTTTCTTCACCRNNRNNRNNA AACTTGACTTTAACTGATCCGCTCGAGCCAGAGGeneral Forward for insert for tggctctggctctggctcgagcnnnnnnnnnnnnn 129 cloning scaffold into phage or Lux- NGeneral Reverse for insert for cgattgagggagcatgttgaaaatctccattannnnnnnnnnnn 130 cloning scaffold into phageGeneral Reverse for insert for gctgaggagtgccgttaattaagtttannnnnnnnnnnn 131 cloning scaffold into Lux-NBR- Forward for vector for cloning tggagattttcaacatgctccctcaatcg 132 305 scaffold into phageMS- Forward for vector for cloning taaacttaattaacggcactcctcagcaaatataatgacc 133 40 scaffold into Lux-NKJ- Reverse for vector for cloning CTCTGGCTCTGGCTCGAGC 134 14 scaffold into phage or Lux-NGeneral Forward for insert for ggatccctcgaaaggaggaaaaaaaaATGnnnnnnnnnnnn 135 cloning target into +APGeneral Forward for insert for gtctgacataaatgaccgctATGnnnnnnnnnnnn 136 cloning target into Lux-CGeneral Reverse for insert for ctttaccgcttccacccgacgtnnnimnnnnnnn 137 cloning target into +AP / Lux-CKJ- Forward for vector for cloning ACGTCGGGTGGAAGCGGT 138 19 target into +AP or Lux-CMS- Reverse for vector for cloning ttttttttcctcctttcgagGGATCCtaggtag 139 46 target into +APBR- Reverse for vector for cloning ACAACTCAAGTCTGACATAAATGACCGCT 140 116 scaffold into Lux-CMS- Forward for insert for subcloning cacgtctacaagGGTGGCTCTG 141659 library variants into Lux-NJD- Reverse for insert for subcloning tcaatcggttgaatgtcgccctt 142 856 library variants into Lux-NMS- Forward for vector for subcloning aatcggttgaatgtcgcccttacttaattaacggcactcctcagc 143 660 library variants into Lux-NMS- Reverse for vector for subcloning cacgtctacaagGGTGGCTCT 144 661 library variants into Lux-NMS- Forward primer for extending 6 aa cgggtggaagcggtGGTAGCGGGTCGTCGGGAAGTGG 145 829 linker to 20 aa TGGTACGTCCGGGTCCGGTaaagcatttatgcaagttgtcgag gcMS- Reverse primer for extending 6 aa CGTCGGGTGGAAGCGGT 146 298 linker to 20 aa or 40 aaMS- Forward primer for extending 6 aa cgggtggaagcggtGGCTCGGGCTCATCAGGTTCTGGT 147 830 linker to 40 aa GGGACTAGTGGGTCCGGAGGATCAGGATCGGG GACCGGAAGTGGCTCCTCTGGTTCTGGCGGAAC TTCCGGTTCAGGAaaagcatttatgcaagttgtcgaggcMS- Forward primer for extending 6 aa CAGGCACAGGGACCGGAAGTAGCGGATCGGGT 148 831 linker to 60 aa GGCACAAGTGGGTCTGGCGGCTCTGGATCAGGC ACCGGCAGTGGGTCTTCGGGGAGTGGAGGTAC CTCAGGCTCAGGAaaagcatttatgcaagttgtcgaggcMS- Reverse primer for extending 6 aa CGTCGGGTGGAAGCGGTGGTTCAGGGTCCTCAG 149 832 linker to 60 aa GTTCGGGTGGAACTAGTGGCTCTGGTGGGTCGG GGACAGGCACAGGGACC MS- Reverse primer for Amplicon-EZ gactggagttcagacgtgtgctcttecgatctgggcgacattcaaccgattgag 150 1156 NGS for all barcodes (underline is ggadaptor, remainder is primingregion)Forward primer for Amplicon-EZ 151 NGS Barcode 1 (underline isMS- adaptor, bold is barcode, remainder acactctttccctacacgacgctcttccgatctAAGGTTgcgcgtagggc 1144 is priming region) acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctTAGGATgcgcgtagggc 152 1145 NGS Barcode 2 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctTGAGATgcgcgtagggc 153 1146 NGS Barcode 3 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAGTGATgcgcgtagggc 154 1147 NGS Barcode 4 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAAGTGTgcgcgtagggc 155 1148 NGS Barcode 5 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAAGTTGgcgcgtagggc 156 1149 NGS Barcode 6 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAGATGTgcgcgtagggc 157 1150 NGS Barcode 7 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAGTAGTgcgcgtagggc 158 1151 NGS Barcode 8 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctGAATTGgcgcgtagggc 159 1152 NGS Barcode 9 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctGATATGgcgcgtagggc 160 1153 NGS Barcode 10 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctGTAATGgcgcgtagggc 161 1154 NGS Barcode 11 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctGTATAGgcgcgtagggc 162 1155 NGS Barcode 12 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAACCTTgcgcgtagggc 163 1346 NGS Barcode 13 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctTAC C ATgcgcgtagggc 164 1347 NGS Barcode 14 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctTCACATgcgcgtagggc 1651348 NGS Barcode 15 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctACTCATgcgcgtagggc 166 1349 NGS Barcode 16 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAACTCTgcgcgtagggc 167 1350 NGS Barcode 17 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctAACTTCgcgcgtagggc 168 1351 NGS Barcode 18 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctACATCTgcgcgtagggc 169 1352 NGS Barcode 19 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctACTACTgcgcgtagggc 170 1353 NGS Barcode 20 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctCAATTCgcgcgtagggc 171 1354 NGS Barcode 21 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctCATATCgcgcgtagggc 172 1355 NGS Barcode 22 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctC TAATCgcgcgtagggc 173 1356 NGS Barcode 23 acgtctacaagMS- Forward primer for Amplicon-EZ acactctttccctacacgacgctcttccgatctC TATACgcgcgtagggc 174 1357 NGS Barcode 24 acgtctacaagForward primer for cloning the CATCATCATctgcagATGAATTCNTTAAAACCAGA 925 nanobody library AGATACNGCNGTNTATTATRYHNWYGTAHHYG MS- TAGGTNNYHHYTACNNBGGTCAAGGTACGCAG 802 GTTACTGTGTCCTCCTAAtggagReverse primer for cloning the CATCATCATctgcagATANACNGTATTTTTNGCATT 926 nanobody library ATCTCTNGAAATNGTAAATCTTCCTTTNACNGA ATCNGCATARWNAGTRDDRNNACCRNNAGARN NAATNSCNGCNACCCATTCTCTTTCTTTNCCTGG MS- NGCTTGACGATACCARWNCATRDDRNNRNNRN803 NCACCGGGAAACCGCTCGCAGCCPlaque Assays

[0159] Activity independent plaque assays can be used to determine the phage titer via plaque counting. Activity dependent plaque assays can be used to check for robust phage replication on a given strain. For activity independent plaque assays, an S1030-1059 E. coli culture (1059 plasmid encodes gill expressed from the phage shock promoter to produce gill after phage infection), is grown to stationary phase in LB with carbenicillin, subcultured 1 / 10 in fresh LB with antibiotic to an OD600 of 0.4-0.6, and then used as the selection strain in the plaque assay. Similarly, for activity dependent strains, S1030 with a +AP (and -AP) were grown similarly for use in the plaque assay. For the plaque assay, an initial dilution of the stock can be added based on the expected titer, but generally, 2 pL of a phage stock (or diluted stock) is added to 100 pL of subculture, mixed, and then serially diluted (2 pL into 100 pL) to create 4 dilutions. 750 pL of 50 °C top agar (7 g / L agar, 25 g / L LB) was added to each dilution and then transferred in its entirety to one quadrant of a bottom agar plate (15 g / L agar, 15 g / L LB). After 10-16 h of incubation at 37 °C, plaques become visible and were counted in the quadrant with 10-200 plaque forming units (PFU).Phage Amplification Rates

[0160] To determine phage amplification rate, the titer of a phage stock is determined using an activity independent plaque assay. Based on this titer, a diluted stock is made that should be 500 PFU / mL (the titer of this diluted stock is confirmed using an activity independent plaque assay). The activity dependent strain (+AP / -AP) is grown to stationary phase in LB with carbenicillin and kanamycin, subcultured 1 / 10 in fresh LB with antibiotic to an OD600 of 0.4-0.6. 2 pL, 1000 PFU, are added to 1 mL of this subculture and then incubated at 37 °C with shaking for 12 h. The cells are then pelleted and the cell-free supernatant collected for use in an activity independent plaque assay to determine the titer at the end of the amplification assay. The endpoint titer is divided by the starting titer (1000 PFU / mL) to determine the amplification rate.PACS / PACEGeneral procedures for continuous flow experiments

[0161] PACS19and PACE17were performed as previously described. All tubing, chemostat bottles, and lagoon flasks were bleached thoroughly, rinsed with DI water, and then autoclaved to ensure sterility. 10 L carboys of Davis Rich media were prepared as described previously17. Inlet lines consist of short needles unable to reach the culture and outlet lines consist of long needles able to reach the culture. Each chemostat had an inlet line for fresh media, an inlet line with a sterile filter for airflow, an outlet line for waste, and an outlet line for each lagoon. Each lagoon had an inlet line from the chemostat, an inlet line with a sterile filter for airflow, and an outlet line for waste. For PACE lagoons, each lagoon also has an inlet line for arabinose. Each chemostat and lagoon has a magnetic stir bar.Colonies of the selection strain were used to inoculate a 5 mL culture in the relevant media (see below) and grown to stationary phase. This culture was then used to inoculate a 200 mL chemostat (250 mL bottle). This culture was stirred in a 37 °C cabinet until an OD600 of ~0.5 and then fresh Davis Rich media was flowed into the chemostat at -1 vol / h. The chemostat was monitored for 4 h to ensure that the flow rate maintained a stable OD600 of -0.5. Phage was then added to each lagoon and then culture was flowed into the lagoon to a volume of 20-25 mL and let incubate for 1 h prior to beginning flow of 1 vol / h. Samples from the lagoons were collected from the waste lines at various timepoints.PACE with libraries and with PANGS output

[0162] PACE was performed as describe previously17in line with standard PACE protocols38. For PACE with RAF and IFNG with the affibody library (Figure S2), two selection strength strains were used for 36 h each with a 12 h mixing step (60 h total). +APs (ori, glll / RNAPc RBS strength): pl5 SD8 / SD8 to pSClOl SD8 / SD8 for RAF and from pSClOl SD8 / SD8 to pl 5 SD8 / sd5 for IFNG. In addition to the +AP, each strain had a ZBneg-AP (20-1) and MP6 (see Table 8.1-Table 8.7). The initial selection strain supported activity dependent plaques of the affibody binders isolated from PANCS of the affibody library, confirming that binders capable of propagating on the initial selection strain exist in the library. One lagoon was used for each chemostat, initially seeded with IO10PFU of library phage and arabinose began flowing during the 1 h of incubation of phage with culture in the lagoon prior to beginning flow at 1 vol / h. Samples were collected prior to beginning the mixed strain phase (24 h) and after completion of the second strain (60 h) and titers were assessed by activity independent plaque assay. For the PACE with Mdm2 (FIG. 5A) starting from the final passage of PANCS with the Affibody library (FIG. 3A), the same protocol was followed with the selection strengths of the +AP being pSClOl SD8 / SD8 to p 15 SD8 / sd5 (both strains produced small activity dependent plaques using the passage 4 phage and large activity dependent plaques at the 60 h timepoint of PACE).PACS with mock libraries

[0163] PACS was performed as describe previously19. The selection strain (S 1030 / 31-69 / 20-6) was prepared by double transformation into chemically competent E. coli. Mock libraries were composed of 1010PFU inactive phage (Affitin (SasA)) and varying amounts (0 (negative control), 10, 102, 103, 104, or 105PFU) of active phage (RAF WT). In addition, a lagoon was seeded with only 103active phage as a positive control. Each of these was done in duplicate lagoons / chemostats. Samples were taken at 12, 24, and 48 h and the titer was determined by activity independent plaque assay (FIG. 8).PANCSGeneral protocol for PANCS

[0164] For each passage, a selection strain (+AP / -AP) is grown to stationary phase in LB with carbenicillin and kanamycin, subcultured 1 / 10 in fresh LB with carbenicillin and kanamycin to an OD600 of 0.4-0.6 prior to adding phage. For passage 1, stock phage are added to the subculture and incubated at 37 °C with shaking (200 rpm) for 12 h, thencentrifuged to pellet the cells and collect the cell-free supernatant (referred to as passage 1 phage). For subsequent passages, some fraction of the cell-free supernatant from the prior passage is added to the subculture and incubated at 37 °C with shaking (200 rpm) for 12 h, then centrifuged to pellet the cells and collect the cell-free supernatant (referred to as passage # phage). Titers of each passage or just the final passage were then determined using activity independent plaque assays, or for the 96-target panel, using qPCR (see below).Details for specific PANCS

[0165] For PANCS development, a variety of culture volumes, transfer rates, and number of passages were used. For the Mock PANCS (FIG. 2E) and the 6-target panel PANCS (FIG. 3A, Table 2), we performed 4-passage PANCS with 5 mL cultures, initially seeded with IO10PFU for passage 1, and seeded with 250 pL of prior passage for passages 2-4 (5% transfer). For the 96-target panel PANCS (FIG. 4A, Table 4), we performed 4-passage PANCS with 1 mL cultures (2 mL deep 96-well plates), initially seeded with 2*109PFU for passage 1, and seeded with 100 pL of prior passage for passages 2-4 (10% transfer). For additional passaging of this PANCS, we did 2% transfer for two passages (FIG. 31). For the 1010library PANCS (FIG. 5A), 6 passage PANCS was performed using either a 2% (RAF, IFNG, KRAS G12D, and HRAS) or 5% (PCNA, ALKBH5, PINK, PIK3CA, TRIM21, VHL, NIX, and BAX) transfer rate between passages and passage 1 was seeded with 5*1010PFU; however, unlike previous PANCS, the volume of each passage was changed as well: 500 mL for passage 1, 125 mL for passage 2, 25 mL for passage 3, and 5 mL for passage 4-6.Split-RNAP E. coli Luciferase Assays

[0166] We followed a slightly modified version of our previously reported assay17. For each target, a two-plasmid strain (S1030 / 2-22 / C-lux) was made chemically competent and each binder and non-binder N-Lux plasmid was transformed to make the three-plasmid luciferase strain. Colonies were picked for each binder and non-binder for each strain to inoculate 1 mL of LB with kanamycin, chloramphenicol, and carbenicillin and grown at 37 °C with shaking (200 rpm) for 12-16 h. Strains were then subcultured 7.5 pL into 143 pL of LB with kanamycin, chloramphenicol, carbenicillin, and L-arabinose (2 mg / mL final concentration) in white side, clear bottom 96-well assay plates (Coming 3610) and incubated at 37 °C with shaking (200 rpm) for 3.5 h prior to reading the OD600 and luminescence signal on a BioTek Synergy Neo2 plate reader. Luminescence signal is first divided by theOD600 to normalize luminescence to cell growth. Then the luminescence / OD600 is normalized for all strains with the same C-Lux plasmid (target-RNAPc expression plasmid) are divided by the non-binder signal (non-binders set equal to 1). Due to differences in expression levels of each target and differences in how binding impacts expression level of a target, we do not believe direct comparisons in fold change over non-binder can be made across different targets, and therefore, we plot all binders for an RNAPc expression plasmid separately from other RNAPc expression plasmids.NGS of PANCS Hits

[0167] We used the Amplicon EZ service provided by Genewiz (Azenta) for Illumina sequencing of each of our hits, which provides 50,000+ paired end reads per sample. We used primers to install Illumina partial adaptors and barcodes to PCR products extending from the linker to after the stop codon of our scaffold in phage - see Table 9. PCR was performed with Q5 DNAP polymerase (NEB) directly from phage (1 pL) in a 25 pL PCR reaction; the initial denaturation step was 10 min at 98 °C to release the ssDNA from the phage particle, a 63 °C Ta, and a 40 second extension time were used with 30 cycles. PCR products are confirmed by gel (5 pL), and the remaining 20 pL of PCR product is pooled with other barcoded PCR products and column purified (Zymo DCC5). Qubit dsDNA High Sensitivity kit is used to determine an accurate concentration of the sample prior to dilution and submission for sequencing. For the NGS data in FIG. 3, FIG. 6, FIG. 16, and FIG. 22, we used 7 barcodes / sequencing sample yielding >15,000 reads per condition (library or PANCS passage). For the NGS data in FIG. 27, FIG. 32, and FIG. 33, we used 24 barcodes / sequencing sample yielding >1000 reads for nearly all PANCS samples (reads listed in tables for each library -target pairing in each figure). BB Merge was used to merge each paired end reads (jgi.doe.gov / data-and-tools / software-tools / bbtools / bb-tools-user-guide / bbmerge-guide / ) and then MatLab was used to separate reads by barcode and to translate reads using modified scripts as described previously39.AlphaFold predictions

[0168] As a preliminary estimate of how our binders interact with their target, we used AlphaFold2 multimer collab40,41for predicting the interaction between binder and target for the top 4 variants above 1% of reads (FIG. 27). AlphaFold3 was released after this analysis, and we subsequently used AlphaFold3 (golgi.sandbox.google.com)35to predict binding interactions for each top variants from our 6-target panel PANCS (FIG. 17), our IO10libraryPANCS (FIG. 33), and for all of the binder-target pairs examined in FIG. 4C (FIG. 32). We implore readers to utilize these predictions only for hypothesis generation rather than as data indicative of an actual interaction.qPCR to estimate phage titers

[0169] qPCR was tested across several primers that prime to M13 phage genes for linear response of a phage serial dilution. Primers VC-525 and VC-526 were chosen (Table 9). Power Up SYBR mix was used with the following PCR protocol: 10 minutes at 95 °C (to denature phage particle and release ssDNA); 40 cycles of 20 seconds at 95 °C, 20 seconds at 60 °C, and 20 seconds at 72 °C; then 10 seconds at 95 °C and 60 seconds at 65 °C. qPCR was run on a QuantStudio6Pro. Each run includes a standard curve for which the titer is assessed using activity independent plaque assay.Library ConstructionGeneral Protocol

[0170] Libraries were designed based on previously published randomizations26,42.Randomization was installed into a template phage using primers with degenerate codons (IDT; see Table 9). We optimized each step of this protocol to maximize the number of clones obtained. PCR conditions were optimized for each library to produce robust PCR product at 25 cycles and then tested for production at lower cycles to reduce amplification bias (18 or fewer cycles were used for each library reported here). PCR was then scaled up to produce 20-100 mg of PCR product. PCR products were concentrated using the Wizard Kit (Promega) and then digested using Dpnl and Nhel-HF (NEB) using a multidose cycle: for -20-50 mg of PCR product in 300-400 mL, and then digested with Dpnl and Nhel, purified using a Zymo Gel Extraction kit, and then ligated with T4 DNA Ligase (NEB). Ligated products were then electroporated into 1059 E. coll cells. The cells were then recovered in 50 mL of 37 °C SOC media and incubated for 2 h at 37 °C with shaking - samples were collected throughout this time for determining the titer by plaque assay. At 2 h, the cells were pelleted and the cell-free supernatant was collected. 1030-1059 (activity independent replication strain) was grown overnight and then subcultured 1 : 10 to and OD600 of 0.6 at 37 °C with shaking (200 rpm). The phage (cell-free supernatant) was then amplified by adding the phage to this subculture for 8-10 h. At the conclusion of this outgrowth, cells were pelleted and the cell-free supernatant was sterile filtered to create the final library stock (titer determined by activity independent plaque assay).Affibody Library

[0171] The affibody library (FIG. 6) was cloned from the Affibody (PDL1) phage (Table 8.1-Table 8.7) using MS-783 and MS-618 (Table 9) by Q5 DNAP (NEB) with a Taof 68 °C. For generating the 108size library, the E. coli strain used for the electroporation was SS320 (a highly electrocompetent strain that is capable of phage replication) rather than 10b. The 108library size was generated with a single transformation of 3 mg ligation product. For the IO10library size, eight transformations of 10 mg ligation product were performed (Table 5).Affitin Library

[0172] The affitin library (FIG. 24) was cloned from either Affitin (SasA), 1010library, or a version of the Affitin (SasA) phage with three stop codons inserted into a region randomized by the primers (Table 8.1-Table 8.7), 108library, using MS-624 and MS-799 primers (Table 9) by Q5 DNAP with GC enhancer with a Taof 68 °C. Both the 108and 1010libraries were generated following the general protocol with a single 4 mg and eight 8 mg transformations, respectively (Table 5).Nanobody Library

[0173] The nanobody library was cloned from Nanobody (gfp) phage (Table 8.1-Table 8.7), using MS-802 and MS-803 primers (Table 9) by Q5 DNAP with GC enhancer with a Taof 68 °C. A 108library was generated following the general protocol (except with Pstl-HF was the restriction enzyme rather than Nhel-HF) with one 3 mg and one 5 mg transformation, pooled together for the library outgrowth to make a single nanobody library.Protein PurificationGeneral Protocol for Tarset Proteins

[0174] Each target protein (KRAS G12D (1-169), RAF, IFNG, and Mdm2 was cloned into a pET28 vector with a C-terminal 6xHis tag and transformed into BL21 E. coli (Table 8.1-Table 8.7). Cells were grown to an OD600 of 0.8 (37 °C with shaking), chilled on ice, induced with 1 mM IPTG, and then incubated with shaking at 16 °C overnight. Cells were pelleted and resuspended in a lysis buffer (25 mM Tris (pH 7.8), 10% glycerol, 200 mM NaCl). Prior to lysing by sonication, cells were treated with PMSF. The soluble fraction of the lysate was incubated with Ni2+resin, washed with lysis buffer containing 50 mMimidazole, then eluted in lysis buffer containing 250 mM imidazole, and finally buffer exchanged into lysis buffer and concentrated.General Protocol for Binder Proteins

[0175] Each binder variant was cloned into a pET30 vector with an N-terminal 3xFLAG and GST tag and transformed into BL21 E. coli (Table 8.1-Table 8.7). Cells were grown to an OD600 of 0.8 (37 °C with shaking), chilled on ice, induced with 1 mM IPTG, and then incubated with shaking at 16 °C overnight. Cells were pelleted and resuspended in a lysis buffer (25 mM Tris (pH 7.8), 10% glycerol, 100 mM NaCl). The soluble fraction of the lysate was incubated with GST resin, washed with lysis buffer, then eluted in lysis buffer containing 10 mM L-glutathione, and finally buffer exchanged into lysis buffer and concentrated.Purified binders shown in FIG. 19.Surface Plasmon Resonance

[0176] Surface Plasmon Resonance was performed on a Biacore 8000 using a NTA chip for immobilizing the His-tagged target proteins. Target concentrations were optimized to elicit a response of -50-100 RU (180 s of 5 uL / s) and then a range of binder concentrations were tested to identify concentrations that produced robust binding (90 s of 30 uL / s). All SPR conducted at 10 °C to maintain slow dissociation of the His-tagged immobilized protein. All dose-responses were fit to a kinetic model for 1:1 binding using the Biacore evaluation software - all fits passed the quality checks in this software (Table 3).Split Nano-Luciferase Assay

[0177] 62.5 ng of the N-terminus of Nano-Luciferase-binder fusion plasmid and 62.5 ng of the KRas(G12D)-C-terminus of Nano-Luciferase fusion plasmid were co-transfected into HEK293T cells using 500 ng of PEI in 96-well glass bottom plate (Cellvis, P96-1-N).Transfection was performed in triplicate. After 36 hours, the Nano-luciferase activity was measured using Nano-Gio® Live Cell Assay System (Promega, N2011).Endogenous KRAS Degradation Assay

[0178] 1000 ng of binder-LIR fusion plasmids were transfected into U2OS cells by 0.3 uL of Lipofectamine 3000 in a 24-well plate. After 4 h, the media was replaced. After 48 h, the cells were collected and subjected to western blot analysis with the appropriate antibodies.Mdm2 binder-Mdm2 Co-Localization Assay

[0179] 125 ng of the GFP-binder fusion plasmid and 125 ng of the mCherry-Mdm2 fusion plasmid were co-transfected into HEK293T cells using 0.075 pL of Xfect™ Transfection Reagent (Takara Bio, 631317) in 96-well glass bottom plate (Cellvis, P96-1-N). After 4 h, the media was replaced. After 36 h, cells were imaged with a Leica fluorescence microscope.Mdm2-p53 Inhibition Assay

[0180] 1000 ng of Mdm2 binder plasmids were transfected into U2OS cells by 0.3 uL of Xfect™ Transfection Reagent (Takara Bio, 631317) in a 24-well plate. After 4 h, the media was replaced. After 48 h, the cells were collected and subjected to western blot analysis with the appropriate antibodies.List of references incorporated by reference herein:1. Bandrowski, A., Pairish, M., Eckmann, P., Grethe, J. & Martone, M. E. The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res 51, D358-D367, doi:10.1093 / nar / gkac927 (2023).2. Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, doi:10.1126 / science.aao5902 (2018).3. Park, M. Surface Display Technology for Biosensor Applications: A Review. Sensors (Basel) 20, doi:10.3390 / s20102775 (2020).4. Carter, P. J. & Lazar, G. A. Next generation antibody drugs: pursuit of the 'high- hanging fruit'. Nat Rev Drug Discov 17, 197-223, doi:10.1038 / nrd.2017.227 (2018).5. Ayoubi, R. et al. Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. Elife 12,doi: 10.7554 / eLife.91645 (2023).6. Laustsen, A. EL, Greiff, V., Karatt-Vellatt, A., Muyldermans, S. & Jenkins, T. P.Animal Immunization, in vitro Display Technologies, and Machine Learning for Antibody Discovery. Trends Biotechnol 39 , 1263-1273,doi : 10.1016 / j .tibtech.2021.03.003 (2021 ).7. Sidhu, S. S., Lowman, H. B., Cunningham, B. C. & Wells, J. A. Phage display for selection of novel binding peptides. Methods Enzymol 328, 333-363,doi : 10.1016 / s0076-6879(00)28406- 1 (2000).8. Xie, V. C., Styles, M. J. & Dickinson, B. C. Methods for the directed evolution of biomolecular interactions. Trends Biochem Sci 47, 403-416,doi: 10.1016 / j .tibs.2022.01.001 (2022).9. Wellner, A. et al. Rapid generation of potent antibodies by autonomous hypermutation in yeast. Nat Chem Biol 17, 1057-1064, doi:10.1038 / s41589-021- 00832-4 (2021).10. Philpott, D. N. etal. Rapid On-Cell Selection of High-Performance Human Antibodies. ACS Cent Sci 8, 102-109, doi:10.1021 / acscentsci.lc01205 (2022).Lopez-Morales, J. et al. Protein Engineering and High-Throughput Screening by Yeast Surface Display: Survey of Current Methods. Small Science 3,doi: 10.1002 / smsc.202300095 (2023).Porebski, B. T. et al. Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening. Nat Biomed Eng 8, 214-232, doi:10.1038 / s41551-023-01093-3 (2024).McConnell, A., Batten, S. L. & Hackel, B. J. Determinants of Developability and Evolvability of Synthetic Miniproteins as Ligand Scaffolds. J Mol Biol 435, 168339, doi:10.1016 / j.jmb.2023.168339 (2023).Kordon, S. P. et al. Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3 / Latrophilin3 by a synthetic binder. 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ATNC: Versatile Nanobody Chimeras for Autophagic Degradation of Intracellular Unligandable and Undruggable Proteins. J Am Chem Soc 145, 24785-24795, doi:10.1021 / jacs.3c08843 (2023).Xie, V. C., Pu, J., Metzger, B. P., Thornton, J. W. & Dickinson, B. C. Contingency and chance erase necessity in the experimental evolution of ancestral proteins. Elife 10, doi: 10.7554 / eLife.67336 (2021).Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol 38, 471-481, doi:10.1038 / s41587-020-0412-8 (2020).Hubbard, B. P. et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nat Methods 12, 939-942, doi:10.1038 / nmeth.3515 (2015).Packer, M. S., Rees, H. A. & Liu, D. R. Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat Commun 8, 956, doi:10.1038 / s41467-017-01055-9 (2017).Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat Biotechnol 37, 1070-1079,doi : 10.1038 / s41587-019-0193 -0 (2019).Woldring, D. R., Holec, P. V., Stern, L. A., Du, Y. & Hackel, B. J. A Gradient of Sitewise Diversity Promotes Evolutionary Fitness for Binder Discovery in a Three-Helix Bundle Protein Scaffold. Biochemistry 56, 1656-1671 (2017).Behar, G. et al. Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications. Biotechnol Bioeng 116, 1844-1855, doi:10.1002 / bit.26989 (2019).Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57-63, doi:10.1038 / nature26155 (2018).Dewey, J. A., Azizi, S. A., Lu, V. & Dickinson, B. C. A System for the Evolution of Protein-Protein Interaction Inducers. ACS Synth Biol 10, 2096-2110,doi : 10.1021 / acssynbio.1 c00276 (2021 ).Block, C., Janknecht, R., Herrmann, C., Nassar, N. & Wittinghofer, A. Quantitative structure-activity analysis correlating Ras / Raf interaction in vitro to Raf activation in vitro. Nat Struct Biol 3, 244-251, doi:10.1038 / nsb0396-244 (1996).Spencer- Smith, R. et al. Inhibition of RAS function through targeting an allosteric regulatory site. Nature chemical biology 13, 62-68, doi: 10.1038 / nchembio.2231 (2017).Grimm, S., Salahshour, S. & Nygren, P. A. Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity. N Biotechnol 29, 534-542, doi: 10.1016 / j.nbt.2011.10.008 (2012).Karlsson, G. B. et al. Activation of p53 by scaffold-stabilised expression of Mdm2-binding peptides: visualisation of reporter gene induction at the single-cell level. Br J Cancer 91, 1488-1494, doi: 10.1038 / sj bjc.6602143 (2004).Jing, L. et al. 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Claims

CLAIMSWhat is claimed is:

1. A method of identifying a binder of a target peptide or protein, the method comprising:(a) engineering a host cell to express a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase (RNAP);(b) exposing the host cell to a high diversity library of phage-encoded protein variants each tagged with a second half of the proximity-dependent split RNA polymerase, wherein the phage library is replication-deficient, and wherein the phage-encoded protein variants are expressed within the host cell;(c) reconstituting the proximity-dependent split RNA polymerase upon binding of a phage-encoded protein variant to the target peptide or protein, whereby the reconstituted RNAP initiates expression of a phage gene in the phage encoding the binding protein variant to initiate replication of the phage encoding the binding protein variant; and(d) isolating the replicated phage encoding the binding protein variant to identify the binding protein variant as the binder of the target peptide or protein.

2. The method of claim 1, wherein the high diversity library comprises 105- 1012phage-encoded protein variants.

3. The method of claim 1 or claim 2, wherein step (d) further comprises serially passaging the library to de-enrich phage that do not encode the binding protein variant and enrich phage encoding the binding protein variant before isolating the replicated phage encoding the binding protein variant.

4. The method of any one of claims 1-3, wherein the host cell is E. coli.

5. The method of any one of claims 1-4, wherein the phage is an Ml 3 phage.

6. The method of any one of claims 1-5, wherein the binder is a diagnostic agent.

7. The method of any one of claims 1-6, wherein the binder is a therapeutic agent.

8. The method of claim 7, wherein the binder is a part of an antibody-drug conjugate.

9. The method of any one of claims 1-8, wherein the binder is a peptide inhibitor or a protein inhibitor.

10. The method of any one of claims 1-9, wherein the binder promotes degradation of the protein to which it binds or of some other biomolecule.

11. The method of any one of claims 1-10, wherein the binder regulates enzymatic activity.

12. The method of any one of claims 1-11, wherein the binder affects cellular signaling.

13. The method of any one of claims 1-12, wherein the binder alters interactions between the target peptide or protein and its endogenous binding partners.

14. The method of claim 13, wherein the binder inhibits interactions between the target peptide or protein and its endogenous binding partners.

15. The method of claim 13, wherein the binder promotes interactions between the target peptide or protein and its endogenous binding partners.

16. The method of any one of claims 1-15, wherein the target peptide or protein is disordered or lacks secondary structure.

17. The method of any one of claims 1-16, wherein the binder is part of a targeted delivery technology.

18. The method of any one of claims 1-17, wherein the binder is part of an engineered cell surface receptor.

19. The method of any one of claims 1-18, wherein the binder is part of an engineered virus.

20. A composition for identifying a binder of a target peptide or protein, comprising: (a) a host cell comprising an expression vector encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase; and(b) a high diversity, replication-deficient phage library, wherein each phage of the library encodes a different protein variant tagged with a second half of the proximitydependent split RNA polymerase,wherein the phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, andwherein the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to initiate replication of the phage-encoded protein variant.

21. The method of claim 20, wherein the high diversity library comprises 105'12phage-encoded molecular glue peptide candidates.

22. The composition of claim 20 or claim 21, wherein the host cell is E. coli.

23. The composition of any one of claims 20-22, wherein the phage is an M13 phage.

24. The composition of any one of claims 20-23, wherein the binder is a diagnostic agent.

25. The composition of any one of claims 20-24, wherein the binder is a therapeutic agent.

26. The composition of claim 25, wherein the binder is a part of an antibody-drug conjugate.

27. The composition of any one of claims 20-26, wherein the binder is a peptide inhibitor or a protein inhibitor.

28. The composition of any one of claims 20-27, wherein the binder promotes degradation of the protein to which it binds or of some other biomolecule.

29. The composition of any one of claims 20-28, wherein the binder regulates enzymatic activity.

30. The composition of any one of claims 20-29, wherein the binder affects cellular signaling.

31. The composition of any one of claims 20-30, wherein the binder alters interactions between the target peptide or protein and its endogenous (natural) binding partners.

32. The composition of claim 31, wherein the binder inhibits interactions between the target peptide or protein and its endogenous (natural) binding partners.

33. The composition of claim 31, wherein the binder promotes interactions (gluing) between the target peptide or protein and its endogenous (natural) binding partners.

34. The composition of any one of claims 20-33, wherein the target peptide or protein is disordered or lacks secondary structure.

35. The composition of any one of claims 20-34, wherein the binder is part of a targeted delivery technology.

36. The composition of any one of claims 20-35, wherein the binder is part of an engineered cell surface receptor.

37. The composition of any one of claims 20-36, wherein the binder is part of an engineered virus.

38. A system for identifying a binder of a target peptide or protein, the system comprising:(a) a host cell comprising an expression vector encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase; and(b) a high diversity, replication-deficient phage library, wherein each phage of the library encodes a different protein variant tagged with a second half of the proximitydependent split RNA polymerase,wherein the phage library is configured to express the phage-encoded protein variants within the host cell upon introduction into the host cell, andwherein the proximity-dependent split RNA polymerase is configured to reconstitute upon the binding of a phage-encoded protein variant to the target peptide or protein and initiate expression of a phage gene to induce replication of the phage-encoded protein variant.

39. The system of claim 38, wherein the phage library comprises 105- 1012phage-encoded protein variants.

40. The system of claim 38 or claim 39, wherein the host cell is E. coli.

41. The system of any one of claims 38-40, wherein the phage is an M13 phage.

42. The system of any one of claims 38-41, wherein the binder is a diagnostic agent.

43. The system of any one of claims 38-42, wherein the binder is a therapeutic agent.

44. The system of claim 43, wherein the binder is a part of an antibody-drug conjugate.

45. The system of any one of claims 38-44, wherein the binder is a peptide inhibitor or a protein inhibitor.

46. The system of any one of claims 38-45, wherein the binder promotes degradation of the protein to which it binds, or of some other biomolecule.

47. The system of any one of claims 38-46, wherein the binder regulates enzymatic activity.

48. The system of any one of claims 38-47, wherein the binder affects cellular signaling.

49. The system of any one of claims 38-48, wherein the binder alters interactions between the target peptide or protein and its endogenous (natural) binding partners.

50. The system of claim 49, wherein the binder inhibits interactions between the target peptide or protein and its endogenous (natural) binding partners.

51. The system of claim 49, wherein the binder promotes interactions (gluing) between the target peptide or protein and its endogenous (natural) binding partners.

52. The system of any one of claims 38-51, wherein the target peptide or protein is disordered or lacks secondary structure.

53. The system of any one of claims 38-52, wherein the binder is part of a targeted delivery technology.

54. The system of any one of claims 38-53, wherein the binder is part of an engineered cell surface receptor.

55. The system of any one of claims 38-54, wherein the binder is part of an engineered virus.

56. An expression vector for use in a method of identifying a binder of a target peptide or protein encoding a target peptide or protein tagged with a first half of a proximity-dependent split RNA polymerase.

57. A kit for use in a method of identifying a binder of a target peptide or protein, comprising a host cell, an expression vector, and a high diversity library of phage-encoded protein variants.

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