PROTEIN SCAFFOLDS FOR DISORDERED REGIONS

CROSS REFERENCE

[001] The present application claims priority to and benefit from U.S. Provisional Application No.: 63/379,648, filed on October 14, 2022, the entire contents of which is herein incorporated by reference.

FIELD

[002] The present invention relates to protein scaffolds that specifically bind to a disordered region of a target protein, and methods for making, screening, and using such protein scaffolds.

BACKGROUND

[003] The targeting of proteins based on their three-dimensional structure, determined experimentally or modeled computationally, has become one of the foundational techniques in modem drug discovery. This has led to a great deal of focus on the secondary structures (e.g., alpha-helices or beta-sheets) for targeting interaction with the protein based on those secondary structures. However, while most proteins or segments thereof fold in a defined three-dimensional structure, studies over the last few decades have discovered that there are numerous segments of polypeptides that do not fold into a defined structure. In fact, some proteins can still carry out their function in an unstructured/disordered state. Utilization of these disordered regions of a target protein have been underutilized in drug development. Thus, there is a need for targeted protein discovery that focuses on these disordered regions. The embodiments described herein satisfy this need and provide related advantages.

SUMMARY

[004] The present disclosure provides engineered scaffold proteins, as well as fusion proteins comprising such scaffolds, that bind a specific target protein or peptide of interest, as well as various uses of the engineered scaffold proteins and fusion proteins.

[005] Provided herein is an engineered scaffold protein that can specifically bind to a disordered region in a target peptide; wherein the engineered scaffold protein comprises: one or more binding domain comprising one or more binding units and one or more hinge units, wherein: the one or more binding units is capable of binding to the disordered region of the target peptide and comprises one or more amino acid alterations relative to a wildtype (WT) counterpart, and the one or more hinge units is capable of stabilizing the structure of the binding domain for binding; wherein binding activity of the target peptide by the engineered scaffold protein is increased relative to a WT counterpart of the engineered scaffold. Also provided herein are engineered scaffold proteins wherein the increase of binding activity comprises an increase in binding frequency, binding rapidity, binding duration, binding affinity, or any combination thereof. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises: decreased immunogenicity relative to a corresponding WT;

1

SUBSTITUTE SHEET (RULE 26) increased solubility relative to a corresponding WT; increased stability relative to a corresponding WT; increased or descreased hydrophobicity relative to a corresponding WT; increased or descreased hydrophilicity relative to a corresponding WT; increased or descreased surface charge relative to a corresponding WT; or any combination of the foregoing. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the disordered region is located in an internal loop, C-terminal tail or N-terminal tail of a target peptide. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the disordered region in the target peptide comprises a linear epitope which is bound by the binding unit. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the linear epitope comprises about 4 to about 40, about 4 to about 30, or about 4 to about 25 amino acids in length. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in a polypeptide, protein, or protein complex. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, and wherein the polypeptide, protein, or one protein within the protein complex is greater than about 30 amino acids in length. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in a polypeptide, protein, or protein complex, wherein the polypeptide, protein, or one protein within the protein complex is greater than about 100 daltons in weight. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in an extracellular protein. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in a membrane protein. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in an extracellular protein or a membrane protein comprising a receptor, an ion channel, or a secreted protein. Also provided herein are engineered scaffold proteins that can specifically bind to disordered regions in target peptides, wherein the target peptide is comprised in extracellular protein comprising a receptor. Also provided herein are engineered scaffold proteins, wherein the binding unit comprises an elongated configuration. Also provided herein are engineered scaffold proteins, wherein upon being folded in its tertiary conformation, the binding unit comprises a three-dimensional conformation comprising one or more amino acids which are anti-sense to one or more amino acids of the disordered region of the target peptide as determined by sense-antisense amino acid pairing. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises two binding units. Also provided herein are engineered scaffold proteins, wherein each of the one or more binding units comprises about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. Also provided herein are engineered scaffold proteins, wherein the one or more binding units each comprises one or more helices comprising alpha helices, 3.10 helices, and/or pi helices. Also provided herein are engineered scaffold proteins, wherein the one or more binding units each comprises one or more alpha helices. Also provided herein are engineered scaffold proteins, wherein the one or more binding units each comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid alterations relative to a WT counterpart. Also provided herein are engineered scaffold proteins, wherein each of the one or more hinge units comprises about 40 to about 200 amino acids, about 60 to about 150 amino acids, or about 80 to about 100 amino acids. Also provided herein are engineered scaffold proteins, wherein the one or more hinge units comprises a concave, or partially concave, configuration. Also provided herein are engineered scaffold proteins, wherein the one or more hinge units comprise a partially flexible conformation that can conform to the disordered region of the target peptide when the disordered region is bound by the engineered scaffold protein. Also provided herein are engineered scaffold proteins, wherein the one or more hinge units comprises one or more beta sheet strands, linear peptides, covalent interactions, non-covalent interactions, chemical agents, or any combination thereof. Also provided herein are engineered scaffold proteins, wherein the hinge unit comprises about 3 to about 12 beta sheet protein strands, or about 6 to about 10 beta sheet protein strands which form one or more beta sheets. Also provided herein are engineered scaffold proteins, wherein the hinge unit comprises one or two beta sheets. Also provided herein are engineered scaffold proteins, wherein the one or more hinge units are covalently attached or linked by one or more linking units to the one or more binding units, or combinations thereof. Also provided herein are engineered scaffold proteins, wherein the one or more linking units are one or more linkers. Also provided herein are engineered scaffold proteins, wherein the one or more linkers comprise a peptide linker. Also provided herein are engineered scaffold proteins, wherein the one or more binding units and one or more hinge units are connected as a monomer. Also provided herein are engineered scaffold proteins, wherein the one or more hinge unit is attached to the N terminus or C terminus of the one or more binding units. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises two binding units, and one hinge unit, wherein the first binding unit is attached to the N terminus of the hinge unit and the second binding unit is attached to the C terminus of the hinge unit. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises two binding units, and one hinge unit, wherein the first binding unit is attached to the N terminus of the second binding unit and the hinge unit is attached to the C terminus of the second binding unit. Also provided herein are engineered scaffold proteins, wherein the two or more binding units are in an anti-parallel configuration. Also provided herein are engineered scaffold proteins, wherein the two or more binding units are semi-symmetrical. Also provided herein are engineered scaffold proteins, wherein the one or more binding units and one or more hinge units are multimeric wherein the one or more binding units and one or more hinge units form as a binding domain in the presence of the target peptide. Also provided herein are engineered scaffold proteins, wherein the binding domain is derived from any one of the scaffold proteins set forth in TABLE 1. Also provided herein are engineered scaffold proteins, wherein the binding domain is in a binding groove architecture. Also provided herein are engineered scaffold proteins, wherein the binding domain comprises a Clamshell structure. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein further comprises one or more immunoglobulin units. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises two immunoglobulin units. Also provided herein are engineered scaffold proteins, wherein the one or more immunoglobulin units is covalently attached or attached by a linking unit to the binding domain, hinge unit, binding unit, or combination thereof. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein does not comprise an immunoglobulin unit. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an isoelectric point of 3.5 to 9, 4 to 8.5, or 4.5 to 8, when measured in an electrophoresis assay. Also provided herein are engineered scaffold proteins, wherein the binding unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in TABLE 4. Also provided herein are engineered scaffold proteins, wherein the hinge unit comprises an amino acid sequence that is at least 65% identical to any one of the sequences in TABLE 5. Also provided herein are engineered scaffold proteins, wherein the disordered region in a target peptide comprises an amino acid sequence selected from the list of bound peptide sequences listed in TABLE 3 or variant thereof having one, two, three, four, five, six, seven, eight, nine, or ten amino acid alterations, or more. Also provided herein are engineered scaffold proteins, wherein said engineered protein scaffold is conjugated to a heterologous agent for extending the half-life of the engineered scaffold. Also provided herein are engineered scaffold proteins, wherein said heterologous agent is selected from the group consisting of polyethylene glycol (PEG), human serum albumin (HSA), and a variant Fc region of an antibody. Also provided herein are engineered scaffold proteins, wherein the half-life of the engineered protein scaffold is extended relative to a WT counterpart. Also provided herein are engineered scaffold proteins, wherein the disordered region in a target peptide comprises an amino acid sequence selected from the list of bound peptide sequences listed in SEQ ID NO: 922-925 or variant thereof having one, two, three, four, five, six, seven, eight, nine, or ten amino acid alterations, or more. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises a functional classification selected from the list of functional classification of human protein scaffolds listed in TABLE 2. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 65% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 75% identical to any one of the sequences set forth in SEQ ID NO: 636-824 Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 80% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 85% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 95% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 97% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 98% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is at least 99% identical to any one of the sequences set forth in SEQ ID NO: 636-824. Also provided herein are engineered scaffold proteins, wherein the engineered scaffold protein comprises an amino acid sequence that is identical to any one of the sequences set forth in SEQ ID NO: 636-824.

[006] Provided herein are fusion proteins comprising an engineered scaffold provided herein and a fusion partner. Also provided herein are fusion proteins, wherein the fusion partner is an enzyme. Also provided herein are fusion proteins, wherein the enzyme catalyzes ubiquitination, post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, nucleic acid modifying proteins, ATPase or GTPase activity. Also provided herein are fusion proteins, wherein the fusion partner selectively binds to a specific region on a target protein. Also provided herein are fusion proteins, wherein the specific region on a target protein is not a disordered region of said target protein.

[007] Provided herein are methods of binding a disordered region of a target protein comprising contacting a target protein comprising said disordered region with the engineered scaffold protein provided herein.

[008] Provided herein are methods of binding a disordered region of a target protein comprising contacting a target protein comprising said disordered region with the fusion protein provided herein.

[009] Provided herein are methods of changing the conformation of a protein comprising a target peptide, the method comprising contacting said target peptide with the engineered scaffold protein provided herein.

[0010] Provided herein are methods of changing the conformation of a protein comprising a target peptide, the method comprising contacting said target peptide with the fusion protein provided herein. [0011] Provided herein are methods of treating a disease comprising administering to a subject an effective amount of the engineered scaffold protein provided herein.

[0012] Provided herein are methods of treating a disease comprising administering to a subject an effective amount of the fusion protein provided herein.

[0013] Provided herein are methods of inducing an immune response in a subject in need thereof, said method comprising administering to said subject the engineered scaffold protein provided herein. [0014] Provided herein are methods of inducing an immune response in a subject in need thereof, said method comprising administering to said subject the fusion protein provided herein.

[0015] Provided herein are methods of detecting a target protein comprising contacting a sample suspected of comprising a target protein with at least the engineered scaffold protein provided herein.

[0016] Provided herein are methods of detecting a target protein comprising contacting a sample suspected of comprising a target protein with at least the fusion protein provided herein.

[0017] Provided herein are kits comprising at least the engineered scaffold protein provided herein.

[0018] Provided herein are kits comprising at least the fusion protein provided herein.

[0019] Provided herein are devices comprising at least the engineered scaffold protein provided herein.

[0020] Provided herein are devices comprising at least the fusion protein provided herein.

[0021] Provided herein are methods of detecting a compound in a sample, said method comprising contacting said sample with the engineered scaffold protein provided herein.

[0022] Provided herein are methods of detecting a compound in a sample, said method comprising contacting said sample with the fusion protein provided herein.

[0023] Provided herein are pharmaceutical compositions comprising the engineered scaffold protein provided herein and a pharmaceutically acceptable excipient.

[0024] Provided herein are pharmaceutical compositions comprising the fusion protein provided herein and a pharmaceutically acceptable excipient.

[0025] Provided herein are methods of preventing, treating, or managing a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition provided herein.

[0026] Provided herein are methods of preventing, treating, or managing a disease in a subject in need thereof by administering an effective amount of the pharmaceutical composition provided herein.

[0027] Provided herein are isolated nucleic acid molecules encoding the engineered scaffold protein provided herein.

[0028] Provided herein are isolated nucleic acid molecules encoding the fusion protein provided herein.

[0029] Provided herein are expression vectors operably linked to the nucleic acid provided herein.

[0030] Provided herein are expression vectors operably linked to the nucleic acid provided herein.

[0031] Provided herein are host cells comprising the vector provided herein.

[0032] Provided herein are host cells comprising the vector provided herein.

[0033] Provided herein are polypeptide display libraries comprising the engineered scaffold protein provided herein.

[0034] Provided herein are polypeptide display libraries comprising the fusion protein provided herein. Also provided herein are polypeptide display libraries, wherein said engineered scaffold protein is displayed on the surface of a virus, or yeast, or displayed as a ribsome or RNA conjugated protein molecule. Also provided herein are polypeptide display libraries, wherein said fusion protein is displayed on the surface of a virus, or yeast, or displayed as a ribsome or RNA conjugated protein molecule.

[0035] Provided herein are collections of isolated nucleic acid molecules encoding the libraries provided herein.

[0036] Provided herein are methods of obtaining an engineered scaffold that binds to a target, said method comprising (a) contacting a target ligand with a library provided herein under conditions that allow an engineered scaffold proto in: target ligand complex to form, and (b) obtaining from the complex, the scaffold that binds the target ligand.

[0037] Provided herein are methods of obtaining a fusion protein that binds to a target, said method comprising (a) contacting a target ligand with a fusion protein library provided herein under conditions that allow a fusion proteimtarget ligand complex to form, and (b) obtaining from the complex, the fusion protein that binds the target ligand.

[0038] Provided herein are methods of obtaining at least two engineered scaffold proteins that bind to a target, said method comprising (a) contacting a target ligand with a library provided herein under conditions that allow an engineered scaffold :target ligand complex to form, (b) engaging said complex with a crosslinking agent wherein the crosslinking of said complex elicits a detectable response and (c) obtaining from the complex, said engineered scaffold proteins that bind the target. Also provided herein are methods of obtaining at least two engineered scaffold proteins that bind to a target, wherein said engineered scaffold proteins recognize the same epitope. Also provided herein are methods of obtaining at least two engineered scaffold proteins that bind to a target, wherein said engineered scaffold proteins recognize distinct epitopes. Also provided herein are methods of obtaining at least two engineered scaffold proteins that bind to a target, wherein said crosslinking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

[0039] Provided herein are methods of obtaining at least two fusion proteins that bind to a target, said method comprising (a) contacting a target ligand with a fusion protein library provided herein under conditions that allow a fusion proto in: target ligand complex to form, (b) engaging said complex with a crosslinking agent wherein the crosslinking of said complex elicits a detectable response and (c) obtaining from the complex, said fusion proteins that bind the target. Also provided herein are methods of obtaining at least two fusion proteins that bind to a target, wherein said fusion proteins recognize the same epitope. Also provided herein are methods of obtaining at least two fusion proteins that bind to a target, wherein said fusion proteins recognize distinct epitopes. Also provided herein are methods of obtaining at least two fusion proteins that bind to a target, wherein said crosslinking agent is selected from the group consisting of an antibody, an antibody fragment, a binding peptide, or an epitope tag.

[0040] Provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, the method comprising: selecting one or more scaffold protein sequences; evaluating the one or more scaffold protein sequences for one or more desired characterics comprising: ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, or any combination thereof; engineering an amino acid sequence of an engineered scaffold protein based on the evaluation of the one or more scaffold protein sequences; wherein the engineered scaffold protein is predicted to have enhancement or improvement of the one or more desired characteristics relative to the one or more scaffold protein counterpart. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein engineering an amino acid sequence of an engineered scaffold protein comprises engineering the amino acid sequence to bind to a linear epitope of a target peptide. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein evaluating the one or more scaffold protein sequences for one or more desired characteristics comprises weighing one or more factors of the one or more scaffold protein sequences relevant to one or more desired characteristics, wherein the one or more weighed factors comprises: three-dimensional conformation, protein domain(s), amino acid sequence, amino acid charge, amino acid polarity, amino acid hydrophobicity/hydrophilicity, amino acid acidity/baseness, or any combination thereof. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein weighing one or more factors comprises: assigning a value to the one or more weighed factors based on an estimated probability of enhancing one or more desired characteristics; assigning a value to the one or more weighed factors, measuring the deviation of said value relative to a target value or to a value assigned to said factor of a second scaffold protein; or both of the foregoing. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein the method further comprises predicting: whether an engineered amino acid sequence represents an engineered scaffold protein exhibiting an enhanced characteristic; whether one or more alteration of the one or more weighed factors improves said value relative to a target value or threshold or to a value assigned to such a factor of a second scaffold protein; or both of the foregoing. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein the one or more alteration comprises one or more amino acid alteration. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein the engineered amino acid sequence of the engineered scaffold protein comprises one or more alterations relative to a counterpart scaffold protein. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein evaluating the one or more scaffold protein sequences comprises manually evaluating the amino acid sequences of the one or more scaffold protein sequences. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide wherein the method further comprises a second or more iteration of: evaluating one more desired characteristics of the engineered amino acid sequence of the engineered scaffold protein; weighing one or more weighed factors of the engineered amino acid sequence of the engineered scaffold protein; or both of the foregoing; and informing further engineering of the amino acid sequences of the of engineered scaffold proteins. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein the method comprises generating the engineered scaffold protein. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein the method comprises assaying the engineered scaffold protein. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein assaying comprises one or more in vitro assay or in vivo assay. Also provided herein are methods of generating a sequence of an engineered scaffold protein that can bind to a disordered region of a target peptide, wherein assaying the generated engineered scaffold protein comprises obtaining data and informing further generation of engineered scaffold proteins.

[0041] Provided herein are systems comprising instructions capable of performing the methods provided herein.

[0042] Provided herein are engineered scaffold proteins comprising an amino acid sequence generated by the methods provided herein or the systems provided herein.

[0043] Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows a schematic of an engineered scaffold or fusion protein thereof acting as an inhibitor on a target mutant protein having a disordered region. (A) An engineered scaffold or fusion protein thereof that masks the mutant region with high specificity and binds to the domain for protein C. (B) An interacting protein that may be wild-type or mutant, and whose interaction causes disease. (C) A protein that preferentially interacts with the engineered scaffold or fusion protein thereof (A) once it is bound to the disordered region and does not cause disease.

[0045] FIG. 2 shows a schematic of a disease target protein without an engineered scaffold or fusion protein thereof vs. with the engineered scaffold or fusion protein thereof acting as an inhibitor. The engineered scaffold or fusion protein thereof (A) masks the mutant region (B) with high specificity and inhibits the interaction.

[0046] FIG. 3 shows a schematic of an engineered scaffold or fusion protein thereof (A) modulating DNA/RNA binding via binding to the disordered region of a disease target, thereby rendering the DNA/RNA unable to bind to the target.

[0047] FIG. 4 shows a schematic of a macromolecule with a disordered region that unable to bind to a target vs. a macromolecule that is able to bind to a target following binding of an engineered scaffold or fusion protein thereof (A). Binding of the engineered scaffold or fusion protein thereof to the disordered region modulates DNA/RNA binding.

[0048] FIG. 5 shows a schematic of a fusion protein having an engineered scaffold fused to a fusion partner that catalyzes ubiquitination. The scaffold-E3 ligase fusion binds to the disease target having a disordered region and ubiquitinates the target, marking it for degradation.

[0049] FIG. 6 shows a schematic of a fusion protein having an engineered scaffold fused to a fusion partner that possesses certain enzymatic activity. The engineered scaffold or fusion protein thereof (A) binds a disease target having a disordered region, and the enzyme portion of the fusion protein modulates biological activity of the target or other proteins in the vicinity of the binding. Such enzymatic activity includes post-translational modification, proteolytic cleavage, dephosphorylation, trans-cis isomerization, protein chaperone activity, catalyzation of nucleic acid modifying proteins, ATPase or GTPase activity, etc.

[0050] FIG. 7 shows a schematic of a functional protein (left), a misfolded protein having at least one disordered region (middle), and a restored functional protein having two ordered domains (right) following binding of a fusion protein having two engineered scaffold proteins (A) fused together or a fusion protein having at least one engineered scaffold protein and a chaperone protein. A functional protein and a resulting restored functional protein are also displayed for reference.

[0051] FIG. 8 shows a schematic of local protein editing by reversing post-translational modifications (PTMs) in a misfolded protein (center) following binding of a fusion protein having an engineered scaffold protein and a fusion partner having enzymatic activity (A). A functional protein (left) and a resulting restored functional protein (right) are also displayed for reference.

[0052] FIG. 9 shows a schematic of local protein editing by sequence specific degradation in a misfolded protein using a fusion protein having an engineered scaffold protein and a fusion partner having degradation activity (A) on the misfolded protein (center). A functional protein (left) and the resulting restored functional protein (right) are also displayed for reference.

[0053] FIG. 10 shows a schematic of various forms an engineered scaffold protein that can be used for therapeutic developments, including a fusion protein having an engineered scaffold protein and a fusion partner, a PEG conjugated engineered scaffold protein, and a multispecific scaffold construct.

[0054] FIG. 11 shows a diagram of an exemplary engineered scaffold protein engineering process.

[0055] FIG. 12 shows a diagram overview of an exemplary stepwise engineered scaffold protein optimization process.

[0056] FIG. 13 shows a diagram of an exemplary engineered scaffold protein development pipeline.

[0057] FIG. 14 shows a schematic of a fusion protein having an engineered scaffold fused to a fusion partner that comprises an Fc region of an antibody. The engineered scaffold binds a target protein having a disordered region that is expressed on the surface of a target cell or binds a target antigen having a disordered region, and the Fc portion of the fusion protein activates a cascade of events resulting in the killing of the target cell and/or destruction of the target antigen.

[0058] FIG. 15A shows a representative SDS PAGE gel of an engineered scaffold protein or fusion protein thereof which is ~22 kDa and runs at the expected size on the PAGE gel. Engineered scaffold proteins range in size from ~ 20 kDa to ~ 50 kDa.

[0059] FIG. 15B shows a size exclusion chromatogram of an engineered scaffold protein, which shows that much of the protein generated runs as a monomer (tallest peak) on the column, while a smaller fraction is split between dimers and multimer aggregates. X-axis represents the volume (mL) and y- axis represents absorbance (Abs, mAu).

[0060] FIG. 16A shows a graph depicting the binding affinity between an engineered protein scaffold (S005, S007, S090) and no scaffold control and target, CD74. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0061] FIG. 16B shows a graph depicting the binding affinity between an engineered protein scaffold (S005, S007, S090) and no scaffold control and target, KAAG1. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0062] FIG. 16C shows a graph depicting the binding affinity between an engineered protein scaffold (S005, S007, S090) and no scaffold control and target, CysLT2. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0063] FIG. 16D shows a graph depicting the binding affinity between an engineered protein scaffold (S005, S007, S090) and no scaffold control and target, CRACM. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0064] FIG. 16E shows a graph depicting the binding affinity between an engineered protein scaffold (S005, S007, S090) and no scaffold control and target, Kvl.5. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0065] FIG. 17A shows a graph depicting the improvement in target binding after scaffold optimization, the points represent no scaffold control, and three versions of engineered scaffold proteins. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0066] FIG. 17B shows a bar graph depicting the improvement in affinity after scaffold optimization. Each bar represents no scaffold control, and three versions of engineered scaffold proteins, respectively and y-axis represents relative binding.

[0067] FIG. 17C shows a bar graph depicting the improvement in relative binding after scaffold optimization. Each bar represents no scaffold control, and three versions of engineered scaffold proteins, respectively and y-axis represents fold change.

[0068] FIG. 17D shows a bar graph depicting the improvement in specificity after scaffold optimization. Each bar represents an engineered scaffold protein, and y-axis represents fold change. [0069] FIG. 18A shows a graph depicting the binding affinity between an engineered protein scaffold (S058, S065, S090) and no scaffold control and target, CD74. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0070] FIG. 18B shows a graph depicting the binding affinity between an engineered protein scaffold (S058, S065, S090) and no scaffold control and target, KAAG1. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0071] FIG. 18C shows a graph depicting the binding affinity between an engineered protein scaffold (S058, S065, S090) and no scaffold control and target, CysUT2r. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0072] FIG. 18D shows a graph depicting the binding affinity between an engineered protein scaffold (S058, S065, S090) and no scaffold control and target, CRACM. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0073] FIG. 18E shows a graph depicting the binding affinity between an engineered protein scaffold (S058, S065, S090) and no scaffold control and target, Kvl.5. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0074] FIG. 19A shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S 164, S 171) and no scaffold control and target, CD74. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0075] FIG. 19B shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S164, S171) and no scaffold control and target, KAAG1. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0076] FIG. 19C shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S164, S 171) and no scaffold control and target, CysUTR2. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0077] FIG. 19D shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S164, S 171) and no scaffold control and target, CRACM. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0078] FIG. 19E shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S164, S 171) and no scaffold control and target, CRACM. X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0079] FIG. 20A shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S177) and no scaffold control and target, MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927). X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0080] FIG. 20A shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S177) and no scaffold control and target, MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927). X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU). [0081] FIG. 20B shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S177) and no scaffold control and target, SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928). X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0082] FIG. 20C shows a graph depicting the binding affinity between an engineered protein scaffold (S090, S177) and no scaffold control and target, TFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929). X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0083] FIG. 20D shows a diagram of a schematic to identify the epitope on CYSLTR2 that binds to the engineered scaffold, the N-terminal binding region was split into 3 N-terminally biotinylated peptides (Epitope 1 (SEQ ID NO: 927), Epitope 2 (SEQ ID NO: 929), Epitope 3 (SEQ ID NO: 928)) with a length of -21AA and an overlap of -10AA.

[0084] FIG. 21 shows a graph depicting binding affinity improvement between an engineered protein scaffold (S005, S090, S177) and no scaffold control and target, CYSLT2R (SEQ ID NO: 928). X-axis represents concentration of target (nM) and y-axis represents fluorescence (AU).

[0085] FIG. 22A shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S180, S189, S193, S194) and no scaffold control and target, CYSLTR2 Epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)). X -axis represents concentration of CYSLTR2 Peptide (nM) and y-axis represents fluorescence (AU).

[0086] FIG. 22B shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S180, S189, S193, S194) and no scaffold control and target, CYSLTR2 Epitope (GMERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929)). X-axis represents concentration of CYSLTR2 Peptide (nM) and y-axis represents fluorescence (AU).

[0087] FIG. 23A shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, CYSLTR2 Epitope

(SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)). X -axis represents concentration of CYSLTR2 Peptide (nM) and y-axis represents fluorescence (AU).

[0088] FIG. 23B shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, KCNA6 Epitope

(QQQEQQPASGGGGQNGQQAMS (SEQ ID NO: 931)). X -axis represents concentration of KCNA6 Peptide (nM) and y-axis represents fluorescence (AU).

[0089] FIG. 23C shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, TNFRSF13B Epitope

(LPPELRRQRSGEVENNSDNSGRYQ (SEQ ID NO: 932)). X -axis represents concentration of TNFRSF13B Peptide (nM) and y-axis represents fluorescence (AU).

[0090] FIG. 23D shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, CD74 Epitope (PPKPVSKMRMATPLLMQALPMGALP (SEQ ID NO: 933)). X-axis represents concentration of CD74 Peptide (nM) and y-axis represents fluorescence (AU).

[0091] FIG. 23E shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, KAAG1 Epitope

(PGAAAAHLPRWPPPQLAASRREA (SEQ ID NO: 934)). X-axis represents concentration of KAAG1 Peptide (nM) and y-axis represents fluorescence (AU).

[0092] FIG. 23F shows a graph depicting the binding affinity between an engineered protein scaffold (S177, S193, S194) and no scaffold control and target, CD40EG Epitope

(NKEETKKENSFEMQKGDQNPQIAAH (SEQ ID NO: 935)). X-axis represents concentration of CD40LG Peptide (nM) and y-axis represents fluorescence (AU).

[0093] FIG. 24A shows a graph depicting binding kinetics between an engineered protein scaffold (S070) and target, an 18 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[0094] FIG. 24B shows a graph depicting binding kinetics between an engineered protein scaffold (S090) and target, an 18 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis time (s).

[0095] FIG. 25 shows a graph depicting binding kinetics between an engineered protein scaffold (S090) engineered to target an 18 amino acid epitope of CLIP and targets, an 18 amino acid epitope of CLIP and a 25 amino acid epitope of CLIP. Y -axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[0096] FIG. 26 shows a graph depicting binding kinetics between an engineered protein scaffold (S005) and target, a 25 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[0097] FIG. 27A shows a graph depicting binding kinetics between an engineered protein scaffold (S098) and target, an 18 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[0098] FIG. 27B shows a graph depicting binding kinetics between an engineered protein scaffold (S098) and target, a 25 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[0099] FIG. 28 shows a graph depicting binding kinetics between an engineered protein scaffold (S058) and target, an 18 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[00100] FIG. 29A shows a graph depicting binding kinetics between an engineered protein scaffold (S068) and target, an 18 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[00101] FIG. 29B shows a graph depicting binding kinetics between an engineered protein scaffold (S068) and target, a 25 amino acid epitope of CLIP. Y-axis is the shift of interference pattern and is proportional to the number of CLIP Peptide molecules the engineered scaffold is binding (nm). X-axis is time (s).

[00102] FIG. 30A shows a graph depicting the binding affinity between between an engineered protein scaffold (vl.O is S007, vl. l is S005, and vl.2 is S090) and no scaffold control and target, GPCR1 (CYSLTR2) (MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP) (SEQ ID NO: 926)). X-axis represents concentration of the GPCR1 Peptide (nM) and y-axis represents fluorescence (AU).

[00103] FIG. 30B shows a bar graph depicting the improvement in relative binding after scaffold optimization. Each bar represents no scaffold control, and three versions of engineered scaffold proteins (vl.O is S007, vl. l is S005, and vl.2 is S090), respectively and y-axis represents relative binding.

[00104] FIG. 31 shows a graph depicting the binding affinity between an engineered protein scaffold (vl.O is S007, vl.l is S005, and vl.2 is S090) and two GPCR targets, GPCR1 (CYSLTR2) (MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 926)) and GPCR2 (Urotensin II) (MALTPESPS SFPGLAATGS SVPEPPGGPNATLNS SWASPTEPS SLEDLVATGTI (SEQ ID NO: 972)). X-axis represents concentration of the GPCR Peptide (nM) and y-axis represents relative fluorescent units (AU).

[00105] FIG. 32 demonstrates occlusion or inhibitor activity of an engineered scaffold protein described herein. In the present figure, Leukotriene C4’s access to CysLTR2’s binding site decreases after binding by an engineered scaffold protein provided herein.

[00106] FIG. 33 demonstrates top view and side view of a three-dimensional rendering of an engineered scaffold protein comprising a binding groove architecture as described herein.

[00107] FIG. 34 demonstrates cross-section views of a three-dimensional rendering of an engineered scaffold protein comprising a binding groove architecture as described herein.

DETAILED DESCRIPTION

[00108] Disclosed herein are compositions and methods to precisely bind a known sequence of amino acids. Such compositions and methods rely upon an engineered scaffold protein disclosed herein. Also disclosed herein are compositions and methods to bind disordered regions of varying lengths found in target proteins. Such compositions and methods bind to a protein in a sequence -specific manner based on an engineered scaffold protein. Still further disclosed herein are methods to locally edit protein structure or function in a sequence-specific manner using such an engineered scaffold protein. Yet further disclosed herein are methods to modulate protein-macromolecule interaction networks using an engineered scaffold protein disclosed herein. These engineered scaffold proteins can be used in methods to sense and detect bioanalytes, to induce local conformational changes, to specifically bind two or more targets simultaneously by fusing and linking multiple binding moieties together, and the like, to the compositions and methods described herein can be used to bind macromolecular targets in the extracellular matrix and the intracellular environment. Further disclosed herein are kits, reagents, and instruments that can detect macromolecules in situ by using an engineered scaffold protein described. Such kits, reagents, and instruments can be used to detect proteins/peptides, such as those present on a bead or chip surface, for protein fingerprinting, protein sequencing, or protein detection.

[00109] As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”

[00110] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[00111] The terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, as used herein, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.

[00112] The terms, “bind,” “binding,” “interact” and “interacting,” as used herein, refer to a non- covalent interaction between macromolecules (e.g. , between two polypeptides or between a polypeptide and a nucleic acid). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequencespecific.

[00113] The term “binding unit,” as used herein, refers to one or more sites on a binding domain which non-covalently interacts with a target peptide so that the target peptide is associated with or bound to an engineered scaffold protein. In some instances, the portion of the target peptide which interacts with a binding unit is a disordered region. In some instances, the portion of the disordered region that interacts with a binding unit is a linear epitope. In some instances, once bound by an engineered scaffold protein, the bound portion is referred to as a “bound peptide”. [00114] The term “binding groove architecture,” as used herein, refers to a three-dimensional configuration of a protein domain which is characterized by a concave interface in a polypeptide lobe or at least two connected polypeptide lobes with the concave interface between the two lobes, wherein binding of a target peptide induces a conformational change such that the lobe(s) fold, at least partially, together, and bury the bound portion of the target peptide in the protein.

[00115] The term “clamshell structure,” as used herein, refers to the three-dimensional architecture characteristic of a binding monomer or multimer which comprises two semi-symmetrical helices supported by a beta sheet. A clamshell structure is an example of a three-dimensional architecture characteristic having a binding groove architecture as described herein.

[00116] The term “disordered region,” as used herein, refers to a segment of a protein that assumes a linear conformation at least some of the time and/or under certain conditions. The linear segment of a protein may be a part of a larger protein that may have stable three-dimensional conformation under physiological conditions. A disordered region may also refer to segments that lack a unique 3- dimensional structure at least some of the time and/or under certain conditions. A few features associated with intrinsically disordered regions (IDRs) include, but are not limited to: higher percentage of polar or charged amino acids; known to have lower number of hydrophobic residues that prevent cooperative folding; sample a variety of 3-dimensional states that are in dynamic equilibrium under physiological conditions; and may exist in ordered states under certain physiological conditions, or in association with binding partners including proteins, nucleic acids and lipids. See, e.g. , M. Madan Babu, The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease. Biochemical Society Transactions (2016). 44: 1185-1200. MTOR, for instance, is a kinase that has a predicted structure consisting of alpha helices along with some regions that do not have a predicted structure (i.e. , disordered regions); this is an example of a protein with an intrinsically disordered region (IDR). SDC-1 is another protein that is considered mostly “unstructured”, with a structure predicted for only a short region, and is also an example of an “intrinsically disordered protein” (IDP), whereby most or all residues do not fold into a rigid structure.

[00117] The term, “fused,” as used herein, refers to at least two sequences that are connected together, such as by a covalent bond (e.g., an amide bond or a phosphodiester bond) or by a linker. The covalent bond can be formed by a conjugation (e.g., chemical conjugation or enzymatic conjugation) reaction.

[00118] The term “fusion partner,” as used herein, refers to a molecule, an agent, a compound, a macromolecule (e.g., a protein) that is fused to an engineered scaffold protein as described herein. In some instances, the fusion partner can impart some function or activity to the fusion protein that is not provided by the engineered scaffold protein.

[00119] The term “fusion protein,” as used herein, refers to a protein comprising at least two heterologous polypeptides. In some instances, a fusion protein comprises one or more engineered scaffold proteins and fusion partners. [00120] The term “hinge unit,” as used herein, refers to a component of an engineered scaffold protein which stabilizes the structural conformation of an engineered scaffold protein described herein and/or stabilizes the binding interaction between the target peptide and an engineered scaffold protein described herein. A hinge unit can also bind, at least partially, to a target peptide described herein.

[00121] The term “immunoglobulin unit,” as used herein, refers to an extracellular membrane- proximal peptide which is capable of recognizing antigens, supporting a binding domain, and/or to enhance structural integrity of the tertiary conformation of an engineered scaffold protein as described herein.

[00122] The term “linking unit,” as used herein, refers to a flexible peptide that connects one or more component of the binding domain provided herein and allows for the change in structural conformation of an engineered scaffold protein when a binding domain of the scaffold binds a target peptide.

[00123] The term “target peptide,” as used herein, refers to a peptide, a polypeptide, a protein domain, two or more protein domains, a peptide on the surface of a protein complex, or any combination thereof. The target peptide can be unmodified (without post-translational modifications) or modified either endogenously or exogenously.

[00124] The term “wild-type counterpart” or “WT counterpart,” as used herein, refers to a protein or portion of the protein from which an engineered protein is derived from. For example, the WT counterpart of an engineered scaffold protein described herein can be the WT protein the engineered scaffold protein is derived from prior to modification for use in accordance with the present disclosure. In another example, the WT counterpart of a binding domain described herein can be the domain or region of the WT protein the binding domain is derived from prior to modification for use in accordance with the present invention, which may be separate and/or different from the WT counterpart of an engineered scaffold protein described herein.

Engineered Scaffold Proteins

[00125] Provided herein are compositions and methods comprising an engineered scaffold protein or a use thereof. An engineered scaffold protein provided herein can bind a known sequence of amino acids. A known sequence of amino acids, in some embodiments, may be an disordered region in a target peptide as described herein. Such a target peptide can have one or more disorder regions as described herein in combination with one or more ordered regions. Binding may be precise, specific, sequencespecific, or any combination thereof. Binding of a target peptide with an engineered scaffold protein described herein may induce one or more activity in vivo and/or in vitro. For example, engineered scaffold proteins and compositions comprising the same can locally edit protein structure or function in a sequence-specific manner, modulate protein-macromolecule interaction networks, sense and detect bioanalytes, induce local conformational changes, specifically bind two or more targets simultaneously by fusing and linking multiple binding moieties together, bind macromolecular targets in the extracellular matrix and the intracellular environment, sense and detect biomolecules, and the like. [00126] In some embodiments, an engineered scaffold protein provided herein can be derived from a scaffold protein described herein. Scaffold proteins disclosed herein demonstrate binding affinity to a specific peptide sequence that can be contained in a disordered region and can be used as a starting point for designing the engineered scaffold proteins disclosed herein based on requirements of therapeutic modality, such as the size of binder, sequence to be bound, and the physiochemical properties such as solubility and temperature sensitivity. Thus, a “scaffold protein” refers to the protein used as a starting point for generation of an “engineered scaffold protein.” Such an engineered scaffold protein has modified binding properties over the starting scaffold protein, including specificity for a disordered region of a target protein. For example, scaffold proteins may be identified from any of the proteins listed in TABLE 1, based on the peptide sequence the scaffold protein is known to bind, such as those described in TABLE 3, and the disordered region of the target protein, and may be modified or engineered to provide for specific binding (e.g., high affinity binding) of the disordered region, the desired size of the engineered scaffold protein or a fusion protein thereof, or the desired specific physiochemical properties of the engineered scaffold protein or a fusion protein thereof (e.g., solubility and temperature). See, e.g., Martins, P.M., Santos, L.H., Mariano, D. et al. Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm. BMC Bioinformatics 22, 1 (2021) for further examples of such proteins that have been known to complex with a peptide sequence, and protein classes that may serve as starting points for the scaffolds disclosed herein.

[00127] In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is less than about 100 amino acids, about 100 amino acids to about 500 amino acids, or more than about 500 amino acids. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, or greater than 500 amino acids. In some embodiments, engineered scaffold proteins provided herein comprise an amino acid sequence that is at least about 160 amino acids, about 180 amino acids, about 200 amino acids, about 220 amino acids, about 240 amino acids, about 260 amino acids, about 280 amino acids, about 300 amino acids, about 320 amino acids, about 340 amino acids, about 360 amino acids, about 380 amino acids, about 400 amino acids, about 420 amino acids, or about 440 amino acids. In certain embodiments, an engineered scaffold protein described herein is a size suitable for packaging into a vector, such as a viral vector. For example, in some embodiments, an engineered scaffold protein described herein is less than 250 amino acids.

[00128] In some embodiments, engineered scaffold proteins provided herein comprise a protein Isoelectric (pl) point of less than about 4 pl, about 4 pl to about 8 pl, or greater than 8 pl. In some embodiments, engineered scaffold proteins provided herein comprise a pl point of about 4 pl, about 4.2 pl, about 4.4 pl, about 4.6 pl, about 4.8 pl, about 5 pl, about 5.2 pl, about 5.4 pl, about 5.6 pl, about 5.8 pl, about 6 pl, about 6.2 pl, about 6.4 pl, about 6.6 pl, about 6.8 pl, about 7 pl, about 7.2 pl, about 7.4 pl, about 7.6 pl, about 7.8 pl, or about 8 pl. A person of ordinary skill in the art would understand how to determine the pl point of an engineered scaffold protein described herein.

[00129] Binding of an engineered scaffold protein or therapeutic molecule thereof may be characterized by describing its affinity for a target peptide. The affinity of an engineered scaffold protein or fusion protein thereof as described herein for its target protein, in some embodiments, is less than 5pM, less than IpM, less than 500nM, less than 200nM, less than lOOnM, less than 50nM, less than 20nM, less than lOnM, less than InM, less than lOOpM, less than lOpM, less than IpM, less than lOOfM, or less than laM. Such affinities can be achieved by the engineered scaffold protein or fusion protein thereof being specific for a particular disordered region of a target protein.

[00130] In some embodiments, an engineered scaffold protein provided herein comprises one or more amino acid alterations relative to a WT counterpart. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more, relative to a WT counterpart.

[00131] In some embodiments, the one or more amino acid alterations is one or more amino acid substitutions, deletions, insertations, or any combination thereof. In some embodiments, the one or more amino acid substitution is one or more conservative substitution, one or more non-conservative substitution, or combinations thereof. In some embodiments, the one or more amino acid alterations are one or more conservative substitutions. A conservative substitution as described herein, refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, the term “non-conservative substitution” as used herein refers to the replacement of one amino acid residue for another that does not have a related side chain. Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe

(F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly

(G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic -hydroxyl; Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gin (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M). In some embodiments, the one or more amino acid alterations are one or more non-conservative substitutions. In some embodiments, the one or more amino acid alterations are substitutions of one or more hydrophobic residues with one or more charged residues or polar residues, or combinations thereof. For example, one or more of a Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), or Tyr (Y) may be substituted with one or more of Asp (D), Glu (E), Lys (K), Arg (R), His (H), Asn (N), Gin (Q), Ser (S), Thr (T), or any combination thereof.

[00132] In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing relative to a corresponding WT. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in a decrease of immunogenicity when administered to a subject relative to a corresponding WT. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase of solubility relative to a corresponding WT. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase of stability relative to a corresponding WT. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in modification of hydrophobicity relative to a corresponding WT. Modification of hydrophobicity relative to a corresponding WT can be an increase in hydrophobicity or a decrease in hydrophobicity relative to a corresponding WT depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in modification of hydrophilicity relative to a corresponding WT. Modification of hydrophilicity relative to a corresponding WT can be an increase in hydrophilicity or a decrease in hydrophilicity relative to a corresponding WT depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in a modification of surface charge relative to a corresponding WT. Modification of surface charge relative to a corresponding WT can be an increase in surface charge or a decrease in surface charge relative to a corresponding WT depending on the desired activity of the engineered scaffold protein. Desired activity can be related to binding activity, solubility, and the like as described herein.

[00133] In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein provided herein comprises an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in SEQ ID NO: 636-824.

[00134] In some embodiments, an engineered scaffold protein provided herein comprises one or more amino acid alterations relative to any one of the amino acid sequences set forth in SEQ ID NO: 636- 824. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more relative to any one of the amino acid sequences set forth in SEQ ID NO: 636-824. In some embodiments, an engineered scaffold protein comprising one or more amino acid alterations is a variant of an engineered scaffold protein described herein. It is understood that reference to an engineered scaffold protein also refers to an engineered scaffold protein variant as described herein.

[00135] In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or some combination of the foregoing relative to any one of the amino acid sequences set forth in TABLE 1. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in a decrease of immunogenicity when administered to a subject relative to any one of the amino acid sequences set forth in TABLE 1. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase of solubility relative to any one of the amino acid sequences set forth in TABLE 1. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in an increase of stability relative to any one of the amino acid sequences set forth in TABLE 1. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in modification of hydrophobicity relative to any one of the amino acid sequences set forth in TABLE 1. Modification of hydrophobicity can be an increase in hydrophobicity or a decrease in hydrophobicity relative to any one of the amino acid sequences set forth in TABLE 1 depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in modification of hydrophilicity relative to any one of the amino acid sequences set forth in TABLE 1. Modification of hydrophilicity can be an increase in hydrophilicity or a decrease in hydrophilicity relative to any one of the amino acid sequences set forth in TABLE 1 depending on the desired activity of the engineered scaffold protein. In some embodiments, one or more amino acid alterations in an engineered scaffold protein provided herein can result in a modification of surface charge relative to any one of the amino acid sequences set forth in TABLE 1. Modification of surface charge can be an increase in surface charge or a decrease in surface charge relative to any one of the amino acid sequences set forth in TABLE 1 depending on the desired activity of the engineered scaffold protein.

[00136] In some embodiments, compositions and methods described herein comprise the use of one or more engineered scaffold protein. In such embodiments, the engineered scaffold protein may interact or bind with one target peptide or more than one target peptide. In some embodiments, compositions and methods described herein comprise the use of two, three, or four or more engineered scaffold protein, wherein the engineered scaffold proteins target one, two, three, four, or more target peptides. [00137] In some embodiments, engineered scaffold proteins provided herein comprise one or more of a: binding domain, immunoglobulin unit, linker, fusion partner, or any combination thereof.

[00138] In some embodiments, engineered scaffold proteins provided herein are also engineered to reduce immunogenicity of said engineered scaffold protein. For example, in some embodiments, a scaffold protein as described herein is selected by determining prevalent genes, such as prevalent HLA alleles, as the scaffold protein from which engineered scaffold proteins are derived from. In another example, where an engineered scaffold protein comprises one or more amino acid alterations, in some embodiments, such alterations are of amio acids that can be buried or distal from the surface of the binding domain, so that the binding unit can be potent and selective to a target peptide described herein. In a further example, an engineered scaffold protein can exclude one or more immunoglobulin units which can function to deter direct binding and trigger an immune response.

Binding Domain

[00139] Provided herein are compositions and methods comprising an engineered scaffold proteins or a use thereof, wherein the engineered scaffold protein comprises one or more binding domains. In some embodiments, a binding domain described herein can be engineered to bind to a target peptide as described herein. In some embodiments, a binding domain described herein can be engineered to bind to a disordered region in a target peptide as described herein.

[00140] In some embodiments, an engineered scaffold protein described herein comprises 1, 2, 3, 4 or more binding domains. In some embodiments, where an engineered scaffold protein comprises 2 or more binding domains, such binding domains may be covalently attached, attached by one or more linkers, or combinations thereof. Examples of organization of binding domains in constructs described herein are set forth in Example 4.

[00141] In some embodiments, a binding domain comprises one or more: binding units, hinge units, linking units, linkers, or combinations thereof.

Binding Unit

[00142] In some embodiments, an engineered scaffold protein provided herein comprises a binding domain comprising one or more binding units. In some embodiments, a binding unit non-covalently binds to a target peptide. In some embodiments, a binding unit non-covalently binds to one or more target peptides. In some embodiments, a binding unit binds to a disordered region in a target peptide. In some embodiments, a binding unit binds to a target peptide in a sequence-specific manner.

[00143] In some embodiments, a binding unit provided herein comprises one or more amino acids that are capable of binding to one or more amino acids of a target peptide. In some embodiments, a binding unit described herein comprises one or more amino acids that are capable of binding to one or more amino acids of a disordered region in a target peptide. In some embodiments, a binding unit described herein comprises one or more amino acids that are capable of binding to one or more amino acids of a disordered region in a target peptide in a sequence-specific manner. It is understood that not all of the one or more amino acid residues that are capable of binding to a target peptide need to bound by the one or more amino acids of the disordered region of the target peptide for sequence -specificity to occur. In some embodiments, the one or more amino acid residues that are capable of binding to a target peptide may not be contiguous in the primary structure of an engineered scaffold protein described herein, but rather become comprised in the binding unit of the engineered scaffold protein once the engineered scaffold protein is in its tertiary structure. In some embodiments, the one or more amino acids of the binding unit that are capable of binding to a target peptide are oriented in the binding unit to optimize binding ability to the disordered region in the target peptide. In some embodiments, the one or more amino acids of the binding unit that are capable of binding to a target peptide are antisense to one or more amino acids of a disordered region in a target peptide as determined by corresponding sense-antisense amino acid pairing. Such amino acid pairing is described in Stambuk et al., Theory in Biosciences, 123(4):265-275 (2005). In some embodiments, the one or more amino acids of the binding unit that are capable of binding to a target peptide form at least one non-covalent interaction with one or more amino acids of a disordered region in a target peptide as determined by corresponding sense- antisense amino acid pairing. [00144] In some embodiments, upon folding into its tertiary structure, the binding unit can comprise a three-dimensional comformation comprising one or more amino acids which are antisense to one or more amino acids of a disordered region in a target peptide as determined by corresponding sense- antisense amio acid pairing. In some embodiments, upon folding into its tertiary structure, the binding unit can comprise a three-dimensional comformation comprising one or more amino acids that are capable of forming at least one non-covalent interaction with one or more amino acids of a disordered region in a target peptide as determined by corresponding sense-antisense amino acid pairing.

[00145] In some embodiments, binding unit provided herein comprises an elongated conformation. In some embodiments, a binding unit provided herein comprises one or more secondary structural motifs. In some embodiments, a binding unit comprises one or more helices. Suitable helices include alpha helices, 3.10 helcies and pi helices. In some embodiments, a binding unit provided herein comprises one or more helices. In some embodiments, a binding unit provided herein comprises one or more, two or more, three or more, four to more, or five or more helices. In some embodiments, a binding domain described herein comprises two binding units wherein each binding unit is a helix. In some embodiments, a binding domain described herein comprises two helices. In some embodiments, a binding domain described herein comprises two helices, wherein the two helices are semi-symmetrical. In some embodiments where a binding domain comprises two helices, the helices run anti-parallel in the three-dimensional conformation of the binding domain.

[00146] In some embodiments, a binding unit provided herein comprises one or more alpha helices. In some embodiments, a binding unit provided herein comprises one or more, two or more, three or more, four to more, or five or more alpha helices. In some embodiments, a binding domain described herein comprises two binding units wherein each binding unit is an alpha helix. In some embodiments, a binding domain described herein comprises two alpha helices. In some embodiments where a binding domain comprises two alpha helices, the alpha helices run anti-parallel in the three-dimensional conformation of the binding domain.

[00147] In some embodiments, a binding unit comprises an amino acid sequence which is less than 40 amino acids, between 40 and 200 amino acids, or greater than 200 amino acids. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is about 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, or 200 amino acids in length. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is about 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 105 amino acids, or about 110 amino acids.

[00148] In some embodiments, a binding unit provided herein comprises one or more amino acid alterations relative to a WT counterpart. In some embodiments, a binding unit comprising one or more amino acid alterations is a variant of a binding unit described herein. It is understood that reference to a binding unit also refers to a binding unit variant as described herein. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more relative to a WT counterpart. In some embodiments, one or more amino acid alterations in a binding unit provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing relative to a WT counterpart.

[00149] In some embodiments, a binding unit comprises an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any one of the sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, a binding unit provided herein comprises an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in TABLE 4.

[00150] In some embodiments, a binding unit provided herein comprises one or more amino acid alterations relative to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more relative to any one of the amino acid sequences set forth in TABLE 4. In some embodiments, one or more amino acid alterations in a binding unit provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing relative to to any one of the amino acid sequences set forth in TABLE 4

Hinge Unit

[00151] In some embodiments, an engineered scaffold protein provided herein comprises a binding domain comprising one or more hinge units. In some embodiments, the hinge unit stabilizes the structural conformation of an open-unbound engineered scaffold protein, open-bound engineered scaffold protein, closed-unbound engineered scaffold protein, closed-bound engineered scaffold protein, partially bound engineered scaffold protein, or any combination thereof. A hinge unit can also bind to one or more target peptides. In some embodiments, a hinge unit can also be engineered to non- covalently bind to one or more target peptides. In some embodiments, a hinge unit can bind to a disordered region in a target peptide. In some embodiments, a hinge unit can bind to a target peptide in a sequence-specific manner. In some embodiments, a hinge unit and a binding unit can work together coopertively, collaboratively, and/or synergistically to bind to a target peptide.

[00152] In some embodiments, a binding domain provided herein comprises one or more hinge units. In some embodiments, a binding domain provided herein comprises 1, 2, 3, 4, 5, or more hinge units as described herein. In some embodiments, a hinge unit comprises a linear, or a partially linear, three- dimensional conformation. In some embodiments, a hinge unit comprises a concave, or a partially concave, three-dimensional conformation. In some embodiments, a hinge unit comprises a convex, or a partially convex, three-dimensional conformation. In some embodiments, a hinge unit comprises a flat, or partially flat, three-dimensional conformation. In some embodiments, a hinge unit described herein comprises a complementary, or partially complementary, conformation to a disordered region in a target peptide as determined by corresponding sense-antisense amino acid pairing. In some embodiments, a hinge unit described herein comprises a flexible, or partially flexible, configuration to conform to the three-dimensional structure of the disordered region in a target peptide described herein. In some embodiments, the hinge unit comprises non-covalent surface forces or charges that can drive binding of the target peptide to the binding domain. Non-covalent surface forces or charges include hydrophobicity, hydrophilicity, and polarity of the surface of the hinge unit.

[00153] In some embodiments, a hinge unit comprises one or more linear peptide strands, secondary structural motifs (e.g., beta sheets), one or more covalent interactions (e.g., disulfide bonds), one or more non-covalent interactions (e.g. , hydrogen bonds, salt bridges, etc. ), chemical agents (e.g. , DMSO, PEG, starch, etc.), or combinations thereof. [00154] In some embodiments, one or more hinge unit is connected to the one or more binding unit. In some embodiments, a binding domain comprises two binding units and one hinge unit, wherein the two binding units are connected by the hinge unit.

[00155] In some embodiments, a hinge unit provided herein comprises one or more secondary structural motifs. In some embodiments, a hinge unit provided herein comprises one or more P-sheet proteins. In some embodiments, the one or more P-sheet proteins form one or more P-sheets. In some embodiments, the one or more P-sheets each comprise about 2 to about 20 or more strands of P-sheet proteins. In some embodiments, the one or more P-sheets each comprise about 4 strands, 5 strands, 6 strands, 7 strands, 8 strands, 9 strands, 10 strands, 11 strands, or about 12 strands of P-sheet proteins. In some embodiments, the one or more P-sheets each comprise about 6 to about 10 strands of P-sheet proteins. In some embodiments, a hinge unit provided herein comprises one or more, two or more, three or more, four to more, or five or more P-sheets.

[00156] In some embodiments, a hinge unit comprises an amino acid sequence which is less than 40 amino acids, between 40 and 200 amino acids, or greater than 200 amino acids. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is about 40 amino acids, 60 amino acids, 80 amino acids, 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, or 200 amino acids in length. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is about 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 105 amino acids, or about 110 amino acids.

[00157] In some embodiments, a hinge unit provided herein comprises one or more amino acid alterations relative to a WT counterpart. In some embodiments, a hinge unit comprising one or more amino acid alterations is a variant of a hinge unit described herein. It is understood that reference to a hinge unit also refers to a hinge unit variant as described herein. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more relative to a WT counterpart. In some embodiments, one or more amino acid alterations in a hinge unit provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing relative to a WT counterpart.

[00158] In some embodiments, a hinge unit comprises an amino acid sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any one of the sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 60% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 65% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 70% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 75% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 80% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 85% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 90% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is at least about 95% identical to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, a hinge unit provided herein comprises an amino acid sequence that is 100% identical to any one of the amino acid sequences set forth in TABLE 5.

[00159] In some embodiments, a hinge unit provided herein comprises one or more amino acid alterations relative to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, the one or more amino acid alterations comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations, 11 amino acid alterations, 12 amino acid alterations, 13 amino acid alterations, 14 amino acid alterations, 15 amino acid alterations, 16 amino acid alterations, 17 amino acid alterations, 18 amino acid alterations, 19 amino acid alterations, 20 amino acid alterations, or more relative to any one of the amino acid sequences set forth in TABLE 5. In some embodiments, one or more amino acid alterations in a hinge unit provided herein can result in an increase binding activity comprising binding more selectively, binding more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing relative to to any one of the amino acid sequences set forth in TABLE 5

[00160] In some embodiments, the one or more binding units and one or more hinge units may be covalently connected, connected by a linking unit, or linked by a linker as described herein. In some embodiments, the one or more binding units and one or more hinge units is a monomer. In some embodiments, the hinge unit is connected or attached to the N terminus of the binding unit. In some embodiments, the one or more binding units and one or more hinge units are connected as a monomer. In some embodiments, the one or more binding units and one or more hinge units are multimeric. In some embodiments, the one or more binding units and one or more hinge units are multimeric wherein the one or more binding units and one or more hinge units form as a binding domain in the presence of a target peptide.

Linking Units

[00161] In some embodiments, an engineered scaffold protein provided herein comprises a binding domain comprising one or more linking units, some embodiments, a linking unit comprises one or more of a beta-sheet strand, an alpha helix, a linear peptide, or combinations thereof. In some embodiments, a linking unit described herein connects a set of binding domain components. For example, one or more linking unit connects a set each of binding domain components comprising a binding unit and a hinge unit where the one or more linking unit is located in between the set. In some embodiments, an engineered scaffold provided herein comprises 1, 2, 3, 4, 5, or more linking units. Linking units can also be referred to as linkers as described in detail herein.

Immunoglobulin Unit

[00162] In some embodiments, an engineered scaffold protein provided herein comprises a binding domain comprising one or more immunoglobulin units. In some embodiments, an engineered scaffold comprising an immunoglobulin unit described herein may be useful in compositions or methods for inducing an immune response. In some embodiments, an engineered scaffold provided herein comprises one or two immunoglobulin units. An immunoglobulin unit described herein can be connected to one or more of a binding unit and/or a hinge unit covalently, by one or more linking unit, or by one or more linker as described herein.

[00163] In some embodiments, the immunogenicity of an engineered scaffold protein described herein is improved by removing the one or more immunoglobulin units, thereby reducing the risk of an autoimmune response.

Binding Groove Architecture

[00164] In some embodiments, a binding domain comprises a three-dimensional structure referred to herein as a binding -groove architecture. In some embodiments, a binding -groove architecture comprises one or more polypeptide lobes and a binding groove. In some embodiments, the polypeptide lobe of the binding-groove architecture each comprise one or more binding units as provided herein. In some embodiments, the binding groove of the binding -groove architecture comprises one or more hinge units as described herein. In some embodiments, the lobes, binding groove, or components thereof are connected by one or more linking units and/or linkers as described herein. Accordingly, in some embodiments, an engineered scaffold comprising a binding-groove architecture provided herein comprises one or more of: binding units, hinge units, linking units, linkers, or any combination thereof. Three-dimensional rendering of a binding groove architecture can be seen in FIGS. 33 and 34.

Scaffold Proteins

[00165] An engineered scaffold protein provided herein, or a component thereof, can be derived from a scaffold protein. In some embodiments, each component of an engineered scaffold protein provided herein is independently derived from a scaffold protein. Therefore, each of a binding domain, binding unit, hinge unit, linking unit, immunoglobulin unit, and the like, is derived from a scaffold protein or a component thereof. Such a scaffold protein, or a component thereof, would serve as a wild-type counterpart or an experimental control for the corresponding component when describing comparable metrics herein.

[00166] In some embodiments, the engineered scaffold protein provided herein, or a component thereof, is derived from a scaffold protein described in TABLE 1. In some embodiments, the engineered scaffold protein is an engineered MHC II monomer able to bind to disordered regions in intact proteins. In some embodiments, the engineered scaffold protein is an engineered MHC I molecule able to bind disordered regions in intact proteins. In some embodiments, the engineered scaffold protein is an MHC- like protein engineered to bind disordered regions in intact proteins. In some embodiments, the engineered scaffold protein is an engineered zinc finger binding protein able to bind linear epitopes in intact proteins. In some embodiments, the engineered scaffold protein is an engineered caspase protein able to bind linear epitopes in intact proteins. In some embodiments, the engineered scaffold protein is an engineered protein ligase, including biotin ligase, sortase, subtilisin derived enzyme, etc. In some embodiments, the engineered scaffold protein is an engineered protease from TEV, enterokinase, thrombin, factor Xa, etc. In some embodiments, the engineered scaffold protein is an engineered ACT domain protein. In some embodiments, the engineered scaffold protein is an engineered kinase and/or phosphatase. In some embodiments, the engineered scaffold protein is an engineered chaperone protein.

Genesis and Design

[00167] In some embodiments, an engineered scaffold protein is designed to bind to a target peptide described herein. As described herein, a scaffold protein may be selected as a binding protein, from which an engineered scaffold protein is derived from. Selection of the scaffold may be based on the type of molecule bound (e.g., DNA, RNA, or protein), known interactions (e.g., binding selectivity, strength, affinity, etc. ) with the molecule, immunogenicity of the scaffold, function or biological activity of the scaffold, and the like. Upon selection of the scaffold protein, an amino acid sequence of an engineered scaffold protein may be generated to bind to a target peptide.

[00168] Accordingly, in some embodiments, also provided herein is a method of generating an amino acid sequence of an engineered scaffold protein which binds to a disordered region of a target peptide. In some embodiments, the method comprises selecting one or more scaffold protein sequences. Selection of one or more scaffold protein sequences may be through any suitable technique, such as mining from a metagenomic database. In some embodiments, to select more than one scaffold protein, a sequence homology search may be performed (e.g. , protein domain searches, pairwise or HMM-based alignment).

[00169] In some embodiments, the method comprises evaluating the one or more scaffold protein sequences for a desired function, activity and/or characteristic. In some embodiments, evaluating the one or more scaffold protein sequences for a desired function, activity and/or characteristic comprises weighing factors relevant to ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or a combination thereof, of the scaffold protein(s) to generate amino acid sequence of an engineered scaffold protein predicted to have one or more enhanced or improved desired characteristic comprising: ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or combination thereof. Examples of such weighed factors comprises: the three-dimensional conformation, the individual protein domains, amino acid sequence, charge, polarity, hydrophobicity/hydrophilicity, and/or the acidity/baseness of certain amino acid residues (e.g. , amino acid residues that interact or bind with the ligand), and the like, or any combination thereof, of the scaffold protein(s). In some embodiments, weighing one or more factors of a scaffold protein comprises: assigning a value to the one or more weighed factors based on an estimated probability of enhancing one or more desired characteristic, assigning a value to the one or more weighed factors and measuring the deviation of said value relative to a target value or threshold or to a value assigned to such a factor of a differing scaffold protein, or both. In some embodiments, the method further comprises predicting whether a generated amino acid sequence represents an engineered scaffold protein exhibiting an enhanced characteristic; whether one or more alteration of the one or more weighed factors improves said value relative to a target value or threshold or to a value assigned to such a factor of a differing scaffold protein; or both. A differing scaffold protein can be a naturally occuring scaffold protein, a WT counterpart, or a second scaffold protein where more than one scaffold protein is selected in methods described herein. In some embodiments, one or more alteration comprises one or more amino acid alteration.

[00170] In some embodiments, the method comprises selecting and evaluating one or more scaffold protein sequences as described herein, and engineering the scaffold protein sequence to bind to a linear epitope of a disordered region of a target peptide. In certain embodiments, an engineered scaffold protein sequence, such as the binding unit, or in some embodiments, the hinge unit, or both, of an engineered scaffold protein, is also engineered to have based on the estimated probability of having enhancements of one or more desired characteristics e.g., enhanced ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or a combination thereof). Engineering of sequences of engineered scaffold proteins can be through suitable methods, such as computational, rational or directed evolution methods. Examples include: site directed mutagenesis, rational design, domain swapping, ancestral sequence reconstruction, relative to the corresponding scaffold protein(s). Accordingly, methods described herein generate engineered scaffold proteins which comprise one or more alterations or one or more amino acid alterations relative to a WT counterpart and/or the corresponding scaffold protein. [00171] In some embodiments, the method comprising engineering and/or generating the amino acid sequence(s) of engineered scaffold protein(s) by performing the methods described herein. In some embodiments, the method further comprises engineering and/or generating an amino acid sequence based on the selected scaffold protein(s), the generated amino acid sequence representing an engineered scaffold protein predicted to have enhanced or improved ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or a combination thereof.

[00172] In some embodiments, evaluating the one or more scaffold protein sequences comprises manually evaluating the amino acid sequences of the one or more scaffold protein sequences.

[00173] In some embodiments, the method further comprises: a second iteration of evaluating one or more desired characteristics (e.g., ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or a combination thereof), of the generated engineered scaffold protein; weighing the one or more weighed factors (e.g., three- dimensional conformation, individual protein domains, charge, polarity, hydrophobicity/hydrophilicity, and/or the acidity/baseness of certain amino acid residues (e.g. , amino acid residues that interact or bind with the ligand), and the like, or a combination thereof), of the generated engineered scaffold protein; or both; to inform further generation of engineered scaffold proteins. In some embodiments, the aforementioned method steps may be repeated more than once or multiple times.

[00174] In some embodiments, the method further comprises assaying the generated engineered scaffold protein. In some embodiments, the method further comprises assaying the generated engineered scaffold protein in an in vitro or in vivo assay, obtaining data on ligand binding, immunogenicity, binding selectivity, binding frequency, binding speed, binding affinity, binding duration, function or biological activity, resistance to proteolytic cleavage, solubility, stability, half-life, and the like, or a combination thereof, and weighing the data against the prior predicted outcomes of the same.

[00175] Accordingly, in some embodiments, a method of obtaining an engineered scaffold protein or fusion protein thereof that binds to a target peptide is described herein. In some embodiments, said method comprises contacting a target peptide with an engineered scaffold protein, fusion protein thereof, nucleic acid encoding the same, host cell expressing the same, or library comprising any of the foregoing under conditions that allow an engineered scaffold proteimtarget peptide complex to form. In some embodiments, methods futher comprise obtaining from the engineered scaffold proteimtarget peptide complex, the engineered scaffold protein scaffold that binds the target peptide, and preferably the engineered scaffold protein scaffold that has improved binding activity on the target peptide relative to the scaffold protein counterpart. [00176] In some embodiments, a method of obtaining at least two engineered scaffold proteins that bind to one or more target peptides. In some embodiments, said method comprises contacting one or more target peptide with an engineered scaffold protein, fusion protein thereof, nucleic acid encoding the same, host cell expressing the same, or library comprising any of the foregoing under conditions that allow an engineered scaffold protein :target peptide complex to form. In some embodiments, the method further comprises engaging said engineered scaffold proteimtarget peptide complex with a crosslinking agent wherein the crosslinking of said engineered scaffold proteimtarget peptide complex elicits a detectable response. Examples of crosslinking agents include an antibody, an antibody fragment, a binding peptide, or an epitope tag. In some embodiments, the method further comprises obtaining from the complex, said engineered scaffold proteins that bind the one or more target peptide. In some embodiments, the two or more engineered scaffold proteins bind to the same epitope of a target peptide or to distinct epitopes of the target peptide. In some embodiments, methods of evaluating and/or methods of assaying described herein further comprise the evaluation of an engineered scaffold protein or analysis of an engineered scaffold protein as fused to a fusion partner and/or heterologous agent. Fusion partners and heterologous agents are further described herein.

[00177] In some embodiments, the results from in vitro and/or in vivo assays can be used herein to inform further or other evaluation of scaffold protein(s) and/or generating future iterations of engineered scaffold proteins.

[00178] In some embodiments, the above method can be performed in a computer readable medium. Accordingly, in some embodiments, also described herein are systems for performing a method of generating an amino acid sequence of an engineered scaffold protein which binds to a disordered region of a target peptide. Such a system can comprise a computer readable medium comprising a computer readable memory that is capable of storing instructions for performing the methods described herein. Computer readable memory can be local (e.g., a hard drive) or online (e.g., cloud). For example, the computer readable memory can comprise instructions for evaluating scaffold protein(s), generating amino acid sequences of engineered scaffold protein(s), or both. In some embodiments, systems described herein are computer systems utilizing the computer readable medium as described herein, wherein the system further comprises a processor operatively coupled to the computer readable medium. In some embodiments, the processor is configured to execute the instructions to perform a method as described herein. The system can further comrpise means for user input and output, such as a keyboard, monitor, and mouse.

[00179] In some embodiments, a system described herein can be configured to access a database, such as a local or online {e.g. cloud). Exemplary databases include protein structure databases, protein sequence databases, homology databases, nucleic acid sequence databases, and the like.

[00180] Upon execution of a method described herein, a system can further comprise information obtained by executing a method described herein. For example, upon execution of a method described herein, a system can comprise predicted outcomes from evaluating scaffold protein(s) and/or engineered scaffold protein(s). A system can comprise the amino acid sequences of engineered scaffold proteins generated from the performed methods as described herein. Further, a system can comprise the data and/or results generated from the in vitro and/or in vivo assays as described herein. Such systems can include means for transferring the information obtained by the methods described herein. In some instances, the systems can include means to transmit information obtained by the methods described herein into an external database (e.g. a local database or an online database).

Fusion Partners

[00181] In some embodiments, compositions and methods provided herein further comprise a fusion partner or a use thereof. Also provided herein is a fusion protein having an engineered scaffold protein as described herein and a fusion partner. In some embodiments, a fusion protein comprising multiple engineered scaffold proteins described herein is multispecific (e.g., bispecific). Such a multispecific fusion protein can include two, three, four, or more engineered scaffold proteins as well as one, two or more fusion partners.

[00182] Fusion partners provided herein comprise one or more of: a protein or a functional fragment thereof, an enzyme or a functional fragment thereof, an antibody or a functional fragment thereof, a compound or molecule which increases half-life of an engineered scaffold protein, a cytotoxic agent, a therapeutic agent, an immunologic agent, a detection agent, more than one of the foregoing, or any combination thereof. Examples of a fusion partner comprising a protein or a functional fragment thereof include a human serum albumin (HSA) or a domain thereof (e.g. , a HSA binding domain), a fibrinogen or a domain thereof (e.g., a fibrinogen binding domain), complement component Iq (Clq), transferrin or a domain thereof (e.g., a transferrin binding domain), a cell penetrating peptide (CPP), and the like. Examples of a fusion partner comprising an enzyme or a functional fragment thereof include a ubiquitin ligase, trypsin, kinase, phosphatase, chaperone, and the like. Examples of a fusion partner comprising an antibody or a functional fragment thereof include a Fc region of an antibody. Examples of a fusion partner comprising a compound or molecule which increases half-life of an engineered scaffold protein include polymers (e.g., polyethylene glycol or PEGylation), carbohydrates, post-translational modifications (e.g., N-terminal glycosylation, and/or polymer mimetics), HSA binding domain, variant Fc domain, transferrin binding domain, fibrinogen binding domain, and the like. See e.g., Examples of a fusion partner comprising a cytotoxic agent include a Fc region of an antibody, complement component Iq, a chemotherapeutic agent, and the like. Examples of a fusion partner comprising a therapeutic agent or therapeutic partner described herein include anti-cancer drugs, anti-inflammatories, anti-bacterials, anti-virals, cytokines, toxins, enzymes, neuroprotective agents, soluble factor traps, and the like. Examples of a fusion partner comprising an immunologic agent include a foreign antigen and the like. Examples of a fusion partner comprising a detection agent include a fluorescent molecule, a radioisotope, a reporter molecule, a detectable signal and the like. Other examples of suitable fusion partners are described in Zaman R, et. al., Current strategies in extending half-lives of therapeutic proteins. J Control Release. 2019 May 10;301 : 176-189. doi: 10.1016/j.jconrel.2019.02.016. Epub 2019 Mar 5. PMID: 30849445; and in Silver, et. al., Engineered antibody fusion proteins for targeted disease therapy, Trends in Pharmacological Sciences, Volume 42, Issue 12, 2021, Pages 1064-1081, ISSN 0165-6147, doi.org/10.1016/j.tips.2021.09.009.

(sciencedirect.com/science/article/pii/SO 165614721001899).

[00183] Exemplary fusion partners are set forth in TABLE 10.

[00184] Binding of the engineered scaffold protein or fusion protein thereof can modify specific biology/biochemistry of the target peptide, intracellular microenvironment comprising the target peptide, a cell expressing the target peptide, or extracellular environment the target peptide is localized to.

[00185] In some embodiments, the engineered scaffold protein or a fusion protein thereof as described herein, once bound to a target protein, acts as antagonist or inhibitor. An inhibitor as described herein, when used in reference to an engineered scaffold protein or fusion protein thereof refers to a protein that can hinder, restrain, or prevent the activity of a target protein once it binds to the target protein. Such an engineered scaffold protein or fusion protein thereof can function in various therapeutic modalities as described herein. In some embodiments, the engineered scaffold protein or fusion protein thereof, once bound to a target protein, acts as a degrader of the target protein by ubiquitination of the target protein. In some embodiments, the engineered scaffold protein or fusion protein thereof is conjugated to a drug that, once bound to a target protein, provides for the delivery of the drug to a cell expressing the target protein. In some embodiments, also provided herein is a fusion protein comprising an engineered scaffold protein as described herein and an Fc region of an antibody that, once bound to a target protein expressed by a cell, facilitates antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, also provided herein is an engineered scaffold protein or a fusion protein thereof as described herein that, once bound to a target protein expressed by a cell, prevents ADCC. In some embodiments, also provided herein is a fusion protein comprising an engineered scaffold protein as described herein and a fusion partner that can bind complement component Iq (Clq) that, once bound to a target protein expressed by a cell, facilitates complement-dependent cytotoxicity (CDC). In some embodiments, also provided herein is an engineered scaffold protein or a fusion protein thereof as described herein that, once bound to a target protein expressed by a cell, prevents CDC. In some embodiments, also provided herein is an engineered scaffold protein or a fusion protein thereof as described herein that provides for the presentation of an antigen on an antigen presenting cell, thereby generating an immune response. Such an engineered scaffold protein or fusion protein thereof can be used as an infectious vaccine, a neurodegeneration vaccine, or a cancer neoepitope vaccine.

[00186] In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target specific marker for use in the detection of a diagnostic marker of a disease. This includes the generation of or use of a panel of engineered scaffold proteins or fusion protein thereof for autoimmune diseases, different oncology indicators, and neurodegenerative diseases, etc. In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target specific binder and/or reagent for in situ and alt-immunofluorescence detection. In some embodiments, the engineered scaffold protein or fusion protein thereof provides a target specific binder and/or reagent for protein fingerprinting and sequencing.

[00187] In some embodiments, an engineered scaffold protein or fusion protein thereof as provided herein is inhibitor or comprises inhibition activity, wherein the engineered scaffold protein or fusion protein thereof can function as an inhibitor of a target protein once it binds to the target protein and prevents a biological activity of that target protein from occurring. Such biological activity may include protein-protein interaction, protein-nucleic acid interaction, protein isomerization, protein post translational modification (e.g., phosphorylation, glycosylation, acetylation, ubiquitination, proteolytic cleavage, etc. ), protein refolding, and protein aggregation, etc. Biological activity may also include a mechanism whereby one interaction partner may be inhibited from binding, thereby facilitating a different interacting partner to bind more efficiently and triggering a different biological activity. In this scenario, the therapeutic target can be a protein other than a target protein, either in the extracellular matrix or in the intracellular environment. By preventing biological activity, cell growth may be prevented, for example, by disruption of certain signaling pathways involved with apoptosis. By binding to targets that prevent immune cell activity, the immune system itself may destroy a diseased cell (e.g., a cancer cell) or a pathogenic cell.

[00188] In some embodiments, an engineered scaffold protein or a fusion protein thereof as provided herein, comprises an activity that is provided by a fusion partner as described herein.

[00189] In some embodiments, the engineered scaffold protein is fused to a trypsin enzyme and selectively binds to a specific disordered region on a target protein. Once bound, any Lysines or Arginines in the vicinity of the binding interphase of the target protein is cleaved by trypsin, thereby destroying the target protein. In some embodiments, the engineered scaffold protein is fused to a kinase or a phosphatase, and selectively binds to a specific disordered region on a target protein. Once bound, the kinase or phosphatase then preferentially phosphorylates residues in the vicinity of the binding site (in the case of a kinase) or de-phosphorylate (in the case of phosphatase). In some embodiments, the engineered scaffold protein is fused to an E3 ligase and selectively binds to a specific disordered region on a target protein. Once bound, the E3 ligase subsequently ubiquitinates the target protein in the vicinity of the binding site, thereby targeting the protein of interest for degradation via the proteasome. In some embodiments, the engineered scaffold protein is fused to an Fc region of an antibody, which acts as a fusion antibody molecule, where the engineered scaffold protein can serve the purpose of the antibody Fab domain that specifically binds to a known epitope of an antigen (i.e. , the disordered region that the binder recognizes specifically). In each of the embodiments, the engineered scaffold protein acts as a homing agent that specifically targets a protein of interest and effects a biological change or activity in the protein of interest through the action of the fusion partner.

[00190] In some embodiments, provided herein is a fusion protein having an engineered scaffold protein as described herein and a fusion partner, wherein the fusion partner is a chaperone protein. Here, the engineered scaffold protein targets a specific disordered region of a misfolded protein, and the chaperone molecule acts in a directed manner to refold the misfolded protein. In emphysema, for instance, al -antitrypsin is a protease inhibitor that is misfolded and accumulates in hepatocytes, al- antitrypsin has several disordered loops in its predicted structure, one of which is ALVNYIFFKGK (SEQ ID NO: 925). By designing a binder to this disordered region and fusing a chaperone enzyme (e.g., Hsp70 or Hsp90), misfolded al -anti -trypsin can be specifically targeted by the binder and the chaperone can fold the protein correctly.

[00191] In some embodiments, an engineered scaffold protein provided herein is a universal scaffold or a universal scaffold protein, wherein the engineered scaffold protein comprises a binding framework that encompasses a structural basis for designing and engineering multiple different artificial binding sites. The binding sites for such scaffold proteins can be loops or rigid structural domains in the scaffold protein. Such universal scaffold proteins may consist of a single polypeptide chain. Additionally, the universal scaffold protein can possess an intrinsic conformational stability and thermodynamic stability and be amenable to humanization. A universal scaffold protein can also be engineered to bind multiple different disordered regions in different proteins with high affinity and specificity. In other words, a universal scaffold protein can be modified to result in different engineered scaffold proteins with different binding properties (e.g., sequence specificity and/or affinity).

[00192] The half-lives of the engineered scaffold proteins or fusion proteins thereof disclosed herein may be extended by several techniques. In some embodiments, the half-lives of engineered scaffold proteins or fusion proteins thereof disclosed herein may be extended by fusion to a compound or molecule as described herein. For instance, the half-life of an engineered scaffold protein or a fusion protein thereof may be extended by chemically conjugating it to polymers, carbohydrates, post- translational modifications that include N-terminal glycosylation, and/or polymer mimetics, the overall protein size can be increased, thereby helping reduce renal clearance and prolonging the bioavailability of the biologic or protein. Such chemical modifications may also aid in preventing the action of other molecules or proteins that would otherwise degrade the engineered scaffold or fusion protein thereof.

[00193] Another strategy to improve half-life involves fusing the engineered scaffold or fusion protein thereof to an HSA binding domain, variant Fc domain, Transferrin binding domain, or fibrinogen domain. Fusion to any of these domains can increase the size of the protein, thereby also reducing the rate of renal clearance. Additionally, these fusion partners can all bind to the Fc-Rn receptor or CTLA and be subjected to receptor mediated endocytosis, therefore also increasing the half-life. See, e.g., Al- Qahatani et al (2019), Biomedicine & Pharmacotherapy, Zaman et al (2019), Journal of Controlled Release. Half-life may be also increased by fusion to an antibody ScFv region or PEGylation of the engineered scaffold or fusion protein thereof.

[00194] Multiple engineered scaffold proteins and/or fusion partners can be fused or linked together to make a multi-specific drug (e.g., a fusion protein). A multi-specific scaffold construct as described herein can refer to a construct encoding or containing an engineered scaffold protein described herein or a fusion protein thereof, which can target multiple proteins and inhibit all of the target proteins as an antagonist or work as an agonist for all of the target proteins or be an antagonist for a subset of target proteins and an agonist for a subset of the target proteins. Protein domains can be fused or linked together to generate an engineered scaffold protein. For example, one or more binding unit, hinge unit, immunoglobulin unit, and/or linking unit may be fused or linked together. In another example, one or more alpha helix, beta sheet, immunoglobulin unit, or combinations thereof can be fused or linked together.

Linkers

[00195] In some embodiments, an engineered scaffold protein described herein comprises one or more linkers. The linkers disclosed herein can be flexible, cleavable, or rigid, depending on the nature of the application. Further linkers can be used to modulate solubility, increase expression, improve biological activity, enable targeting, or alter Pk. See, e.g., Chen et al., Adv Drug Deliv Rev., 65(10): 1357-69 (2013).

[00196] In some embodiments, an engineered scaffold protein provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more linkers. In some embodiments, an engineeered scaffold protein provided herein does not comprise any linkers. In some embodiments, a peptide linker comprises 3-25 amino acids in length. In some embodiments, a linking unit described herein can be replaced by a linker as provided herein.

[00197] In some embodiments, a linker may be a peptide linker or a non-peptide linker. In some embodiments, a peptide linker comprises 1 to 30 amino acids in length. In some embodiments, a peptide linker comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids in length or more.

[00198] Protein domains can be linked together by short peptide linkers that can be any one of several, but not limited to, the following: LE; (GGS)n n =1,2, 3, 4, 5; (GGGGS)n n=l,2,3,4,5- (SEQ ID NO: 973); (SSG)n n = 1,2, 3, 4, 5; GGGGGG (SEQ ID NO: 974); GGGGGGGG (SEQ ID NO: 975); and Peptide 1- nucleic acid-Peptide2. In some embodiments, Peptide 1 is any peptide sequence of 5 or more amino acids with a functional group at the N terminus. In some embodiments, Peptide2 is any peptide sequence of 5 or more amino acids with a functional group at the C terminus of the peptide. In some embodiments, the two peptides are conjugated to each other by a nucleic acid.

[00199] The constructs disclosed herein can be assembled in multiple ways. In some embodiments, the construct is Nterm-Binderl-Linkerl-Binder2-Linker2-Binder3-Linker3 -Linker(n-l)- Binder(n)-Cterm. Here, “Nterm” denotes the N terminus of the biologic (e.g., therapeutic protein), ’’Cterm” denotes the C terminus of the molecule, “Binder” denotes an engineered scaffold protein or other binding protein, but at least one engineered scaffold protein is present in the molecule. In some embodiments, the fusion protein containing two engineered scaffold proteins is bispecific. A bispecific is a particular example of two engineered scaffold proteins linked together in the above construct. Each linker can be any one of the linkers disclosed herein and any combination of linkers can be used to fuse the binder moieties together.

[00200] In some embodiments, described herein is a multi-specific scaffold construct or a use thereof. A multi-specific scaffold construct can target multiple proteins and inhibit all of the target proteins as an antagonist, work as an agonist for all of the target proteins or be an antagonist for a subset of target proteins and an agonist for a subset of the target proteins. A single domain or a subset of domains in the multispecific scaffold construct can also be used as a means of homing in on the target cell or target proteins, and the other domains in the therapeutic can work as inhibitors or agonists, or a combination of inhibitors and agonists.

[00201] A multi-specific scaffold construct can disrupt protein-protein interactions by simultaneously binding to multiple partners of the protein interaction network and disrupt critical cellular processes necessary for cellular growth and survival. A multi-specific therapeutic can also function to bring together several proteins together that may not be able to come together on their own and facilitate protein-protein interactions between all the binding partners by being a hub.

[00202] Exemplary linkers are set forth in TABLE 9.

Vectors

[00203] Compositions and methods described herein comprise one or more vectors or uses thereof. In some embodiments, one or more vectors used herein comprises one or more nucleotide sequences encoding one or more components of a composition or method described herein. In some embodiments, one or more vectors used herein is conjugated to or otherwise attached to one or more components of a composition or method described herein. In some embodiments, one or more components comprise one or more engineered scaffold proteins, fusion partners, linkers, fusion proteins, target peptides, or functional fragments thereof. In some embodiments, where more than one vector is used, compositions and methods described herein can comprise a library of vectors, each vector encoding or conjugated to one or more component of a composition or method as described herein. In some embodiments, components described herein are encoded by or conjugated to the same vector, or each component is encoded by or conjugated to a different vector, or combinations thereof. [00204] In some embodiments, vectors described herein comprise or encode one or more regulatory elements. Such regulatory elements can be operably linked to a nucleotide sequence encoding an engineered scaffold protein, fusion partner, linker, fusion protein, target peptide, or functional fragment thereof, or more than of the foregoing. Regulatory elements can include transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide. In some embodiments, a vector comprises or encodes for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), and selectable markers. In some embodiments, a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites. In general, vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of engineered scaffold proteins, fusion partners, fusion proteins, target peptides or functional fragments thereof as described herein.

[00205] Vectors can be expression vectors. Vectors can be a viral vector, such as an AAV vector or a lentiviral vector, or a non-viral vector, such as a lipid, lipid particle, cell-penetrating peptide, or mRNA. [00206] The engineered scaffold or fusion protein thereof disclosed herein may be used for therapeutic purposes by delivery of the engineered scaffold or fusion protein thereof to a target cell. Intracellular delivery of such therapeutics can be by AAV vector, lentiviral vector, cell-penetrating peptide or as mRNA.

[00207] The engineered scaffold protein or fusion protein thereof may be used for a polypeptide display library. In some embodiments, a polypeptide display library provided herein comprises an engineered scaffold protein or fusion thereof expressed and displayed by a vector described herein. In some embodiments, the engineered scaffold protein or fusion protein or fusion thereof is comprised in an expression vector and delivered to a host cell for expression. Accordingly, in some embodiments, provided herein is a host cell comprising an engineered scaffold protein or fusion protein described herein. In some embodiments, a polypeptide display library provided herein displays an engineered scaffold protein or fusion thereof on the surface of a virus or yeast, or displayed as a ribosome or RNA conjugated protein molecule.

[00208] In some embodiments, also provided herein are isolated nucleic acid molecules encoding an engineered scaffold protein described herein, a fusion thereof, one or more components of the following, or combinations thereof. In some embodiments, an expression vector can be operably linked to an isolated nucleic acid molecule provided herein. Accordingly, provided herein are compositions or methods comprising an isolated nucleic acid molecule encoding an engineered scaffold protein described herein, a fusion thereof, one or more components of the following, or combinations thereof, and in some embodiments, as operably linked to an expression vector. Target Peptides

[00209] Disclosed herein are compositions and methods that comprise a target peptide or a use thereof. In some embodiments, an engineered scaffold protein or fusion protein thereof as described herein interacts with or binds to a target peptide. In some embodiments, interacting or binding with a target peptide results in the detection of the target peptide or results in inducing a biological or therapeutic activity. Accordingly, also disclosed herein are compositions and methods for detecting a target peptide or inducing a biological or therapeutic effect.

[00210] A target peptide described herein can be part of a polypeptide, larger protein or protein complex. Accordingly, when referring to a target peptide that is comprised in or comprised with a polypeptide, protein or protein complex, reference is also made to the target polypeptide, target protein, and/or target protein complex. In some embodiments, reference to a target peptide also refers to a nucleic acid (i.e., a DNA or RNA molecule) which encodes the target peptide. Accordingly, in some embodiments, compositions and methods described herein comprise a nucleic acid encoding a target peptide as described herein. In some embodiments, a target peptide may be referred to herein as a target ligand.

[00211] In some embodiments, a target peptide comprises 2 to 50, 3 to 40, or 4 to 30 amino acids in length. In some embodiments, a target peptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50amino acids. In some embodiments, a target peptide comprises at least 4, at least 15, or at least 30amino acids. [00212] In some embodiments, a target peptide is comprised in a polypeptide, protein, or protein complex. In some embodiments, the polypeptide, protein or one protein within a protein complex comprising the target peptide can be greater than about 30 amino acids in length. In some embodiments, the target peptide is comprised in a polypeptide, protein, or protein complex, wherein the polypeptide, protein, or one protein within a protein complex is greater than about 30 amino acids in length. In some embodiments, the polypeptide, protein, or one protein of a protein complex comprising a target peptide is about 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 130 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 220 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, 270 amino acids, 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, 330 amino acids, 340 amino acids, 350 amino acids, 360 amino acids, 370 amino acids, 380 amino acids, 390 amino acids, 400 amino acids, 410 amino acids, 420 amino acids, 430 amino acids, 440 amino acids, 450 amino acids, 460 amino acids, 470 amino acids, 480 amino acids, 490 amino acids, 500 amino acids, or greater, in length.

[00213] In some embodiments, the polypeptide, protein or one protein of a protein complex comprising the target peptide can be greater than about 100 daltons in weight. In some embodiments, the target peptide is comprised in a polypeptide, protein, or protein complex, wherein the polypeptide, protein, or one protein of a protein complex is greater than about 100 daltons, 1000 daltons, 2000 daltons, 4000 daltons, 6000 daltons, 8000 daltons, 10000 daltons, 12000 daltons, 14000 daltons, 16000 daltons, 18000 daltons, 20000 daltons, 22000 daltons, 24000 daltons, 26000 daltons, 28000 daltons, 30000 daltons, 32000 daltons, 34000 daltons, 36000 daltons, 38000 daltons, 40000 daltons, 42000 daltons, 44000 daltons, 46000 daltons, 48000 daltons, 50000 daltons, 52000 daltons, 54000 daltons, 56000 daltons, 58000 daltons, 60000 daltons, 62000 daltons, 64000 daltons, 66000 daltons, 68000 daltons, 70000 daltons, 72000 daltons, 74000 daltons, 76000 daltons, 78000 daltons, 80000 daltons, 82000 daltons, 84000 daltons, 86000 daltons, 88000 daltons, 90000 daltons, 92000 daltons, 94000 daltons, 96000 daltons, 98000 daltons, 100000 daltons, or greater in weight.

[00214] In some embodiments, where a target peptide is comprised in a polypeptide, protein or protein complex, an engineered scaffold protein described hereni can be engineered to be multispecific (e.g., bispecific or trispecific) - binding to multiple regions of the polypeptide, protein or protein complex. Regions can be disordered, ordered, or combinations thereof.

[00215] In some embodiments, a target peptide described herein is comprised in a cell. In some embodiments, a target peptide described herein is comprised in a population of cells. In some embodiments, a target peptide described herein is comprised in a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, a non-naturally occurring cell, or any combination thereof. In some embodiments, a cell comprising a target peptide described herein is comprised in a eukaryotic cell. In some embodiments, a target peptide described herein is comprised in a population of: naturally-occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, cells of an invertebrate, fly cells, cells of vertebrate, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non-living cells, modified cells, derived cells, non-naturally occurring cells, or any combination thereof. In some embodiments, a cell comprising a target peptide described herein is comprised in a population of eukaryotic cells.

[00216] In some embodiments, a target peptide described herein is isolated from a cell. In some embodiments, a target peptide described herein is isolated from any one of: a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, and a non-naturally occurring cell. In some embodiments, the target peptide is isolated from a population of cells. In some embodiments, a target peptide described herein is isolated from a population of: naturally-occurring cells, eukaryotic cells, prokaryotic cells, plant cells, fungal cells, animal cells, cells of an invertebrate, fly cells, cells of vertebrate, mammalian cells, primate cells, non-human primate cells, human cells, living cells, non- living cells, modified cells, derived cells, non-naturally occurring cells, or any combination thereof. In some embodiments, a target peptide described herein is comprised in a sample. In some embodiments, a target peptide described herein is comprised in a sample obtained or harvested from an organism. In some embodiments, a target peptide described herein is comprised in a sample obtained or harvested from: a eukaryotic organism, a prokaryotic organism, a plant, a fungal organism, a bacterium, a virus, an animal, an invertebrate, a fly, a vertebrate, a mammalian organism, a mouse, a primate, a non-human primate, a human.

[00217] In some embodiments, a target peptide described herein, or a cell or organism comprising the target peptide, is comprised in a system for detecting target peptides, such as a kit as described herein. In some embodiments, a target peptide described herein is comprised in a device for detecting target peptides. In some embodiments, a target peptide is naturally-occurring or comprises one or more modifications, for example modifications for use in a system or method described herein. Such modifications include fusion or conjugation to a detection agent as sdescribed herein.

[00218] In some embodiments, a target peptide described herein is comprised in an or is an extracellular protein. In some embodiments, a target peptide described herein is membrane protein. In some embodiments, a target peptide described herein is comprised in a or is a G protein-coupled receptor (GPCR), an ion channel, or a secreted protein. In some embodiments, a target peptide described herein is comprised in or is any of the proteins set forth in SEQ ID NO: 922-925, or a portion thereof. [00219] In some embodiments, a target peptide described herein is associated with a disease or disorder. In some embodiments, a target peptide described herein is encoded by a nucleic acid that is associated with a disease or disorder. In some embodiments, a target peptide described herein is comprised in a cell that is associated with a disease or disorder. In some embodiments, a target peptide described herein is comprised in an organism that is afflicted with a disease or disorder. In some embodiments, a disease or disorder described herein is a genetic disease or disorder, pathogeneic disease or disorder, infectious disease or disorder, communicable disease or disorder, immunologic disease or disorder, a mutagenic disease or disorder, or any combination thereof. In some embodiments, a disease or disorder described herein is associated with any of the target proteins set forth in SEQ ID NO: 922- 925. Also disclosed herein are methods of treating, preventing, or inhibiting a disease or disorder.

[00220] In some embodiments, an engineered scaffold protein or fusion protein thereof as described herein can interact with or bind a disordered region of a target peptide. In some embodiments, an engineered scaffold protein or fusion protein thereof as described herein interacts with or binds a linear epitope present in a disordered region of a target peptide. In some embodiments, a target peptide can be an intrisically disordered peptide. In some embodiments, a target peptide may comprise an ordered structure, but still also comprise a disordered region.

[00221] In some embodiments, an engineered scaffold protein or fusion protein thereof as provided herein can interact with or bind to an ordered region of a target peptide. In some embodiments, an engineered scaffold protein or fusion protein thereof as provided herein can interact with or bind to an ordered region and a disordered region of a target peptide, either at the same binding domain or in different binding domains. Accordingly, engineered scaffold proteins or fusion proteins described herein that can be engineered as described herein to bind to disordered regions can also be engineered to bind to ordered regions.

Disordered Region

[00222] In some embodiments, an engineered scaffold protein described herein can be engineered to interact with or bind to a disordered region of a target peptide as described herein. In some embodiments, the disordered region can be fully disordered or partially disordered. Once a target peptide is selected, disordered regions can be determined from Uniprot, or another sequencing database, and a disordered epitope comprising the complete region, or a subset of that disordered region, may be used for engineered scaffold protein development in accordance with any of the methods and examples disclosed herein.

[00223] In some embodiments, a disordered region of a target peptide can have an extended conformation that an engineered scaffold protein described herein can bind to. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a disordered region of a target peptide comprising an extended conformation. In some embodiments, an extended conformation of a disordered region is a linear conformation or linear sequence.

[00224] In some embodiments, a disordered region is comprised in a loop, a C terminal tail, or an N terminal tail of a target peptide described herein. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a loop, a C terminal tail, or an N terminal tail of a target peptide described herein. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a disordered region comprised in a loop, a C terminal tail, or an N terminal tail of a target peptide described herein. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a linear sequence of a disordered region comprised in a loop, a C terminal tail, or an N terminal tail of a target peptide described herein.

[00225] Disordered regions of a target peptide described herein can have known biological functions. In some embodiments, an engineered scaffold protein described herein is designed to bind to a target peptide thereby inducing, altering or inhibiting the biological functions of the disordered region.

[00226] In some embodiments, a disordered region of a target peptide described herein can function as a recognition site for enzyme active sites, or as a ligand site which is recognized by the binding surface of a protein partner or partners. A protein partner(s) as described herein refers to a polypeptide which can recognize and interact with a ligand site of a target peptide. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a disordered region that functions as a recognition site for enzyme active sites, or as a ligand site which is recognized by the binding surface of a protein partner or partners. In some embodiments, binding of an engineered scaffold protein to a disordered region functioning as a recognition site can induce structural modification, proteolytic cleavage and post-translational modification removal or addition of the target peptide as described in further detail herein. In some embodiments, binding of an engineered scaffold protein to a disordered region functioning as a ligand site can induce complex promoting, docking, and targeting or trafficking of the protein partner(s) as described in further detail herein. In some embodiments, binding of an engineered scaffold protein to a target peptide can activate a disordered region functioning as a recognition site or as a ligand site thereby inducing the aforementioned activity. In some embodiments, binding of an engineered scaffold protein to a target peptide can occlude a disordered region functioning as a recognition site or as a ligand site thereby inhibiting the aforementioned activity.

[00227] In some embodiments, a disordered region of a target peptide can function as a promoter of protein-protein interactions. In some embodiments, an engineered scaffold protein described herein is engineered to interact with or bind to a disordered region that functions as a promoter of protein-protein interactions. In some embodiments, such disordered regions comprise preformed structural elements (PSEs), molecular recognition features (MoRFs), molecular recognition (MoREs), or prestructured motifs (PreSMos). In some embodiments, disordered regions comprising MoRFs undergo disorder-to- order transitions upon binding their interaction partners (i.e., folding upon binding). An interaction partner can be any protein that binds to a disordered region that is functioning as a promoter of proteinprotein interactions. In some embodiments, disordered regions comprising PSEs or MoRFs can function as initial contact points for interaction events, wherein conformational selection of preformed elements and induced folding occur. In some embodiments, binding of an engineered scaffold protein to a target peptide can activate a disordered region functioning as a promoter of protein-protein interactions thereby inducing the aforementioned activity. In some embodiments, binding of an engineered scaffold protein to a target peptide can occlude a disordered region functioning as a promoter of protein-protein interactions thereby inhibiting the aforementioned activity.

[00228] In some embodiments, a disordered region of a target peptide can function as a DNA binder, RNA binder, or protein binder. In some embodiments, binding of an engineered scaffold protein to a target peptide can activate a disordered region functioning as a DNA, RNA, or protein binder thereby inducing the aforementioned activity. In some embodiments, binding of an engineered scaffold protein to a target peptide can occlude a disordered region functioning as a DNA, RNA, or protein binder thereby inhibiting the aforementioned activity.

[00229] In some embodiments, a disordered region comprises consecutive amino acids that comprise the complete sequence or a subset of the sequences enumerated that are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, or at least 50 consecutive amino acids of a target peptide.

[00230] From the selected target peptide sequences, disordered regions can be determined from Uniprot and a disordered epitope comprising the complete region, or a subset of that disordered region, may be used for engineered scaffold protein development in accordance with any of the methods and examples disclosed herein. Development of such an engineered scaffold protein may also be utilized for any of the applications disclosed herein, including vaccine development, therapeutic development, immunomodulation/suppression, detection, and diagnostics.

[00231] In embodiments where more than one engineered scaffold protein or fusion protein thereof is used, such engineered scaffold protein or fusion protein can recognize the same linear epitope or distinct linear epitopes.

[00232] Non-limiting examples of linear epitopes and disordered regions oftarget peptides are set forth in SEQ ID NO: 922-925

Pharmaceutical Compositions

[00233] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising an engineered scaffold protein described herein and a carrier thereof for administration in a subject.

[00234] In certain embodiments, the pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of an engineered scaffold protein, formulated together with one or more pharmaceutically acceptable: carriers (additives) and/or diluents. In some embodiments, when an engineered scaffold protein herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99%, or 10 to 30% of an engineered scaffold protein in combination with a pharmaceutically acceptable carrier.

[00235] A pharmaceutical composition of the present disclosure can be delivered, e.g. , subcutaneously or intravenously with a standard needle and syringe or a pen delivery device. The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. The injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying an engineered scaffold protein herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be fdled in an appropriate ampoule. [00236] Compositions of the present disclosure can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The amount of an engineered scaffold protein disclosed herein contained can be about 5 to about 500 mg per dosage form in a unit dose. In one embodiment, an engineered scaffold protein can be contained in about in about 5 to about 100 mg, for example for a parental dosage form. In other embodiments, an engineered scaffold protein can be contained in about 10 to about 250 mg for the other dosage forms.

[00237] For example, oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets can be used as solid dosage forms. These can be prepared, for example, by mixing the engineered scaffold protein, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[00238] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. In some embodiments, pharmaceutical formulations and medicaments may be prepared as liquid suspensions or aqueous solutions, for example, using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, or any combination of these. In some embodiments, pharmaceutical compositions can be prepared in a lyophilized form. The lyophilized preparations can comprise a cryoprotectant known in the art. The term “cryoprotectants” as used herein generally includes agents, which provide stability to the protein from freezing-induced stresses. Examples of cryoprotectants include polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants also contribute to the tonicity of the formulations. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or par- enteral administration. [00239] As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations. [00240] For nasal administration, the pharmaceutical formulations and medicaments may be a spray or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bio- availability modifiers or any combination of these. A propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

[00241] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which can be prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer’s solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. In some embodiments, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono- , di- or tri -glycerides.

[00242] For injection, the pharmaceutical formulation and/ or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers or any combination of these.

[00243] For rectal administration, the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum. Rectal suppositories are prepared by mixing one or more compounds herein with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories. Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.

[00244] The concentration of an engineered scaffold protein in these compositions can vary widely, e.g., from less than about 10%, least about 25% to as much as 75% or 90% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

[00245] In some embodiments, pharmaceutical compositions comprising an engineered scaffold protein described herein can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. [00246] Pharmaceutical compositions are optionally manufactured such as, by way of example only, by means of mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[00247] In certain embodiments, compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. In other embodiments, compositions may also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[00248] In some embodiments, sustained-release preparations can be used. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an engineered scaffold protein of the present disclosure, in which the matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)- 3 -hydroxybutyric acid. While polymers such as ethylene -vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thiodisulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose. [00249] In some embodiments, an engineered scaffold protein can be administered with one or more agents capable of promoting penetration of the engineered scaffold protein across the blood-brain barrier. In some embodiments, an engineered scaffold protein can be linked with a viral vector, e.g. , to render an engineered scaffold protein more effective or increase transport across the blood-brain barrier. For example, delivery of agents can be by administration of an adenovirus vector to motor neurons in muscle tissue. Delivery of vectors directly to the brain, include but are not limited to the striatum, the thalamus, the hippocampus, or the substantia nigra.

[00250] In embodiments, an engineered scaffold protein can be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In some embodiments, an engineered scaffold protein can be coupled to a substance that promotes penetration or transport across the bloodbrain barrier, e.g., an antibody to the transferrin receptor. In some embodiments, osmotic blood brain barrier disruption can be assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. In some embodiments, the composition can be encapsulated in glucose- coated polymeric nanocarriers.

[00251] The compositions herein may be administered alone or in combination with another therapeutic. The additional therapeutic may be administered prior, concurrently, consecutively, or subsequently to the administration of the composition.

[00252] The compositions disclosed herein, comprising an engineered scaffold protein, described herein, can also contain more than one active agent as necessary for the particular indication being treated, such as those with complementary activities that do not adversely affect each other. For example, the composition can further comprise an anti-inflammatory, a therapeutic protein, a steroid, an analgesic, a non-steroidal anti-inflammatory, a corticosteroid, an immune system modulator, an additional engineered scaffold protein, more than one of the foregoing, or any combination thereof.

Methods of Use

[00253] Provided herein are compositions and methods for binding a target peptide comprising the use of an engineered scaffold protein or a fusion protein thereof described herein. Binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein is capable of inducing a biological or therapeutic activity. For example, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can result in: activation of a binding site; inhibition or occlusion of a binding site; induction of conformational biasing; prevention of nuclear localization; disruption of membrane binding; modulation of macromolecule interactions; occlusion or sequestering of post-translational modifications (PTMs); prevention of pro-protein processing; induction of cell degradation; blockade of substrate recruitment; and the like; or any combination thereof.

[00254] Also provided herein are compositions comprising an engineered scaffold protein or a fusion protein thereof described herein, wherein the engineered scaffold protein or the fusion protein thereof is bound, fused, conjugated, or otherwise attached to a therapeutic partner. Therefore, in some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can deliver or localize the therapeutic partner to the target peptide, a cellular microenvironment the target peptide is comprised in, a cell comprising the target peptide, or an extracellular environment which the target peptide is adjacent to or comprised in. Accordingly, in some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein is capable of inducing a biological or therapeutic activity effected by a therapeutic partner described herein. For example, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can result in: inducing cell death; inducing proteolytic cleavage; modulation of PTMs; inducing de-phosphorylation; inducing protein isomerization; inducing protein disaggregation and/or refolding; modulation of ATPase and/or GTPase activity; inducing a immune response; and the like; or any combination thereof.

[00255] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can activate a binding site. In some embodiments, activation of a binding site comprises activation of a signaling pathway. In some embodiments, the signaling pathway that can be activated by an engineered scaffold protein or a fusion protein thereof can include GPCRs, Ion-Channels, Protein Pumps, Enzyme-linked receptors, Single-pass membrane proteins, or secreted proteins. Examples of signaling pathways that can be activated by an enginereed scaffold protein described herein include MAPK-PK, RAS/RAF, RHO, FAK1, MEK/MAPK, MAK, MKK, AKT, PI3K- AKT, EGF receptor, Her2 receptor, Her3 receptor, Her4 receptor, p38, NF-Kp, NGF, NT-3, NT-4, BDNF, JNK, neurotrophin, PLC-yl, estrogen receptors, progesterone receptors, androgen receptors, GPER30, PIK3/PTEN, VEGF receptor pathway inhibitors, cell adhesion, TGFbeta/SMAD, WNT, Hedgehog/GLI, HIF1 alpha, JAK/STAT, Notch, CD95/ApolL, mesolimbic dopamine, mesocortical dopamine, nigrostriatal dopamine, nigrostriatal dopamine, serotonergic pathways, acetylcholine, GAB Anergic, glutamatergic, control of Gl/S transition, DNA damage control, and apoptosis. In some embodiments, the signaling pathways include other signaling pathways as disclosed herein. Accordingly, in some embodiments provided herein is a method of activation of a signaling pathway comprising the use of an engineered scaffold described herein.

[00256] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can inhibit or occlude a binding site. In some embodiments, inhibition or occlusion of the binding site comprises disruption of the protein binding to one or more binding partners. In some embodiments, the engineered scaffold protein or the fusion protein thereof described herein can form an intramolecular interaction with the target peptide. In some embodiments, the engineered scaffold protein or the fusion protein thereof described herein can form an intermolecular interaction with the target peptide. In some embodiments, the intramolecular and/or intermolecular interactions with the target peptide results in blocking a protein comprising the target peptide. In some embodiments, the intramolecular and/or intermolecular interactions can sterically occlude the target protein binding site. In some embodiments, the engineered scaffold protein or the fusion protein thereof may disrupt the protein binding activity by blocking of active site, hydrophobic pocket, or causing steric hinderance on the surface of the target. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can partially or fully blocks, occludes, inhibits, or neutralizes a biological activity of the protein. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can reduce access to the protein’s binding site. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can block access to the protein’s binding site. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can inhibit with signaling of the protein. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can block signaling pathways of the protein. In some embodiments, binding of the target peptide on the protein by the engineered scaffold protein or the fusion protein thereof described herein can inhibit binding of signaling molecules. Accordingly, in some embodiments provided herein is a method of occluding the binding of a protein to one or more binding partners or inhibiting a biological activity of the protein comprising the use of an engineered scaffold described herein. A schematic model of occlusion by an engineered scaffold protein described herein or a fusion protein thereof can be seen in FIG. 33.

[00257] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce conformational biasing. In some embodiments, induction of conformational biasing comprises stabilizing or locking a protein comprising the target peptide in a conformational state. In some embodiments, the conformation state is an active conformational state. In some embodiments, the conformational state is a partially active conformational state. In some embodiments, the conformational state is an inactive conformational state. In some embodiments, the engineered scaffold protein or the fusion protein thereof described herein selectively binds to the target peptide comprised in the protein, the binding of which may not affect the function of the protein comprising the target peptide. Accordingly, in some embodiments, use of the engineered scaffold protein or the fusion protein thereof described herein can stabilize a protein comprising the target peptide in a functional conformational state. In some embodiments, the functional conformation state is an active, a basal, a partially active, or an inactive conformational state. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can correct misfolded proteins. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can correct misfolded proteins. In some embodiments, correction of misfolded proteins can restore protein biological activity or function. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can prevent protein dissociation. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can prevent degradation of one or more proteins. Accordingly, in some embodiments provided herein is a method of stabilizing or locking a protein comprising a target peptide comprising the use of an engineered scaffold described herein.

[00258] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can prevent nuclear localization. In some embodiments, prevention of nuclear localization comprises blocking the transportation of a protein comprising the target peptide into the nucleus. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can block a nuclear localization signal of the protein. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can block a nuclear export signal of a protein. In some embodiments, prevention of nuclear localization by binding of the target peptide by the engineered protein described herein can occur in the presence of a nuclear localization signal. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can prevent the protein from one or more protein-protein interactions. In some embodiments, the protein-protein interactions can result in localization to the nucleus in the form of a complex. Thus, in some embodiments, the binding of the target peptide by the engineered protein or the fusion protein thereof described herein can prevent the protein from forming the complex and translocating to the nucleus. Accordingly, in some embodiments provided herein is a method of preventing nuclear localization comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00259] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can disrupt membrane binding. In some embodiments, disruption of membrane binding comprises reducing or preventing an attachment or insertion of a protein comprising the target peptide into the cell membrane. In some embodiments, the attachment can be prevented by blocking access to the protein’s hydrophobic residues. In some embodiments, disruption of membrane binding can comprise an increase in soluble protein. In some embodiments, disruption of membrane binding can lead to aberrant targeting of the protein to a cell’s cytoplasm and/or intracellular membrane in vitro. In some embodiments, disruption of membrane binding can lead to aberrant targeting of the protein to a cell’s cytoplasm and/or intracellular membrane in vivo. In some embodiments, abberant targeting of the protein can result in impaired access to pathways. In some embodiments, impaired access to pathways can prevent membrane binding partner molecules from signalling normally. Accordingly, in some embodiments provided herein is a method of disrupting membrane binding comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00260] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can modulate macromolecule interactions. In some embodiments, the macromolecule interactions comprise multivalent binding, dynamic binding, and coupled folding and binding interactions. In some embodiments, modulation of macromolecule interactions can include any suitable macromolecule. Non-limiting examples of macromolecules include disaccharides, polysaccharides, proteins, lipids, fatty acids, nucleic acids, crosslinked or non-crosslinked polymers, organometallic compounds, and other macromolecular scaffolds. In some embodiments, the macromolecule comprises the aggregation of two or more macromolecules. In some embodiments, the macromolecule interactions comprise protein-protein interactions, protein-DNA interactions, and protein-RNA interactions. In some embodiments, modulation of macromolecule involves a macromolecule binding to a protein. In some embodiments, the macromolecule that binds to the protein can be a protein binding partner, an antigen, an epitope, a peptide, a ligand, a receptor, a carbohydrate, a chemical, a small molecule, or an inhibitor. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can result in increased protein binding to macromolecule. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can result in decreased protein binding to macromolecule. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can be used to inhibit one or more macromolecule from binding the protein. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof increases binding, reduces binding, or changing the conditions under which the protein binds to a macromolecule. Accordingly, in some embodiments provided herein is a method of modulating macromolecule interations comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00261] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can occlude or sequester post-translational modifications (PTMs). In some embodiments, occlusion or sequesteration of PTMs can involve one or more PTMs. Examples of PTMs can include phosphorylation, acetylation, methylation, N-linked glycosylation with different sugars, O-linked glycosylation with different sugars, citrullination or deimination, crotonylation, butyrylation, ubiquitination, C-mannosylation, methionine oxidation, sulfation, amidation, sumoylation, S-nitrosylation or nitrosylation, neddylation, deimination, OclcNAc, ADP-ribosylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, formylation, carboxylation, hydroxylation, and Proline cis-trans isomerization. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can be used to partially inhibit access to PTMs on a protein. In some embodiments, the protein can include, but is not limited to, chaperone proteins, effector proteins, assembler proteins, scavenger proteins, degredation proteins, or enzymes. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can be used to inhibit access to PTMs on the protein. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can be used to block and/or sequester PTMs from effector proteins. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof can be used to block PTMs from enzymes that catalyze the removal of PTMs. In some embodiments, blocking PTMs can decrease the functional state in which the protein can exist in the cell. In some embodiments, blocking PTMs can decrease the activity of the protein. Accordingly, in some embodiments provided herein is a method of occluding or sequestering PTMs comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00262] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can modulate PTMs. Accordingly in some embodiments, modulation of PTMs comprises a method of stimulating the activity of an endogenous or exogenous PTM enzyme capable of producing and/or removing PTMs. In some embodiments, the endogeneous or exogenous PTM enzyme can result in a PTM alteration. In some embodiments, the PTM alteration comprsies phosphorylation, acetylation, methylation, N-linked glycosylation with different sugars, O-linked glycosylation with different sugars, citrullination or deimination, crotonylation, butyrylation, ubiquitination, C-mannosylation, methionine oxidation, sulfation, amidation, sumoylation, S- nitrosylation or nitrosylation, neddylation, deimination, OclcNAc, ADP-ribosylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, formylation, carboxylation, hydroxylation, proline cis-trans isomerizationdeubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OclcNAc, de-ADP-ribosylation, demethylation, de -hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof. Examples of the endogenous or exogenous PTM enzymes include carboxylate-amine ligases, cyclases, dehydrogenases, cyclodehydratase decarboxylases, epimerases, hydroxylases, peptidases, dehydratases, transferases, esterases, oxygenases and isomerases, ubiquitin ligase, SUMO transferase, methyltransferase, demethylase, acetyltransferase, glycosyltransferase, palmitoyltransferase and/or related hydrolase, lanthionine bond forming enzymes, cytolysin forming enzymes, cyanobactin forming enzymes, thiopeptide forming enzymes, conopeptide forming enzymes, microviridin forming enzymes, cyclotide forming enzymes, bacteriocin forming enzymes and subtilosin forming enzymes. In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof increased a biological activity of an endogenous or exogenous PTM enzyme. In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof decreased the biological activity of an endogenous or exogenous PTM enzyme. Accordingly, in some embodiments provided herein is a method of modulating PTMs comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00263] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce de-phosphorylation. In some embodiments, induction of dephosphorylation comprises increasing enzymatic activity of phosphatase enzymes. In some embodiments, the engineered scaffold protein or the fusion protein thereof increases the binding of phosphatase to a target protein compring the target peptide. Examples of phosphatase enzymes include, but is not limited to, bacterial alkaline phosphatases, mammalian alkaline phosphatases, plant acid phosphatases, mammalian acid phosphatases and alkaline phosphatase conjugates. Accordingly, in some embodiments provided herein is a method of inducing de-phosphorylation comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00264] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce protein isomerization. In some embodiments, induction of protein isomerization involves the activation of an isomerase enzyme. In some embodiments, activation of the isomerase enzyme results in increase racemization of a target protein. In some embodiments, activation of the isomerase enzyme results in increase epimerization of a target protein. In some embodiments, the isomerase enzyme can be aldose atriose phosphate isomerase, bisphosphoglycerate mutase, triosephosphate isomerase, phosphomutases, epimerases, racemases, and carbon-skeleton mutases, or photoisomerase. In some embodiments, the engineered scaffold protein orthe fusion protein thereof described herein can induce isomerization of an amino acid residue. In some embodiments, the amino acid residure is proline. In some embodiments, binding of the target peptide by the engineered scaffold protein or a fusion protein thereof described herein can increase the isomerization of proline. In some embodiments, the isomerization of proline is catalyzed by a proline isomerase. In some embodiments, binding of the target peptide by an engineered scaffold protein or the fusion protein thereof described herein increases the biological activity of the proline isomerase resulting in one or more conformations of proline. In some embodiments, the one or more conformations of proline include cis conformation and trans conformation. In some embodiments, binding of the target peptide by an engineered scaffold protein or the fusion protein thereof described herein increases cis-trans isomerization of proline. Accordingly, in some embodiments provided herein is a method of inducing protein isomerization comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00265] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can modulate ATPase/GTPase activity. In some embodiments, modulation of ATPase/GTPase activity comprises activating the ATPase/GTPase. In some embodiments, modulation of ATPase/GTPase activity comprises inhibiting the ATPase/GTPase. In some embodiments, binding of the target peptide by an engineered scaffold protein or the fusion protein thereof described herein increases ATP hydrolysis. In some embodiments, binding of the target peptide by an engineered scaffold protein or the fusion protein thereof described herein increases GTP hydrolysis. In some embodmients, modulation of ATPase activity can be used to treat a disease. In some embodiments, the disease comprises aberrant ATPase activity. In some embodmients, modulation of GTPase activity can be used to treat a disease. In some embodiments, the disease comprises aberrant GTPase activity. Examples of a disease with aberrant ATPase/GTPase activity include, but are not limited to, stroke, cardiovascular diseases (e.g., angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertensive heart disease, pulmonary heart disease, valvular heart disease, rheumatic fever, rheumatic heart disease, endocarditis, mitral valve prolapse, aortic valve stenosis, congenital heart disease, valvular and vascular obstructive lesions, atrial or ventricular septal defect, patent ductus arteriosus, and myocardial disease), Wilson disease, Menkes syndrome, kidney disorders (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, systemic lupus erythematosus, Goodpasture’s syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, chronic inflammatory diseases, acute tubular necrosis, acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, renal tubular acidosis, tubulointerstitial diseases, acute and rapidly progressive renal failure, chronic renal failure, and nephrolithiasis), hypotension, hypertension, ischemic injury, neurological disorders (e.g., Alzheimer’s disease and Parkinson’s disease), muscle disorders, pulmonary disorders (e.g., emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture’s syndrome, idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonitis, chronic interstitial pneumonia, fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener’s granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), hepatic disorders (e.g., hepatic vein thrombosis, portal vein obstruction, thrombosis, hepatitis, and cirrhosis), proliferative disorders (e.g., neoplasms or tumors such as carcinomas, sarcomas, adenomas, and myeloid leukemia), and disorders in which a positive ionotropic effect is desired. Accordingly, in some embodiments provided herein is a method of modulating ATPase/GTPase activity comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00266] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can prevent pro-protein processing. In some embodiments, prevention of pro-protein processing comprises blocking proteolytic cleavage. In some embodiments, blocking proteolytic cleavage involves binding, partial binding, blocking, or burying the catalytic site from one or more proteases. In some embodiments, the one or more proteases can include proprotein convertases, serine proteases, metalloproteases, cysteine proteases, threonine proteases and aspartic acid. In some embodiments, prevention of pro-protein processing can result in a reduction in the number of bioactive peptides. In some embodiments, prevention of pro-protein processing can result in a reduction in the number of bioactive proteins. In some embodiments, prevention of pro-protein processing can result in a reduction in the number of active enzymes. Accordingly, in some embodiments provided herein is a method of preventing pro-protein processing comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00267] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce protein disaggregation and/or refolding. In some embodiments, induction of protein disaggregation and/or refolding involves the activation of chaperone enzymes. Examples of chaperone enzymes can be Hsp90 family of proteins, Hsp70 family of proteins, immunophilin family of proteins, peptidase C56 family of proteins, Pplase family of proteins, 14-3-3 family of proteins, small heat-shock protein group of proteins, small GTPase family of proteins, Hsp40 (DnaJ) family of proteins, and clusterin, Grpl70, calreticulin, Hspl05, Hsp70-interacting protein (CHIP), alpha-crystallin or any of the combinations thereof. In some aspects, the activation of chaperone enzymes results in an increase in the number of refolded proteins. In some aspects, the activation of chaperone enzymes results in an increase in the number of disaggregated proteins. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can increase the refolding of a multimeric protein. In some embodiments, the multimeric protein includes, but is not limited to, dimers, trimers, tetramers, pentamers, hexamers, septamers, octamers, or nonamer. The multimeric protein subunits can be identical (homodimer, etc.) or one or more can differ within the native protein (heterodimers, etc.). Accordingly, in some embodiments provided herein is a method inducing protein disaggregation and/or refolding comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00268] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce proteolytic cleavage. In some embodiments, induction of proteolytic cleavage comprises increasing proteolytic cleavage. In some embodiments, increasing proteolytic cleavage comprises an increase in accessibility to a protein to a catalytic enzyme. In some embodiments, the catalytic enzyme is a protease. In some embodiments, increasing proteolytic cleavage involves binding, partial binding, stabilizing, or exposing a catalytic site in the protein to one or more proteases. In some embodiments, the one or more proteases can include proprotein convertases, serine proteases, metalloproteases, cysteine proteases, threonine proteases and aspartic acid. In some embodiments, induction of proteolytic cleavage can result in an increase in the number of bioactive peptides. In some embodiments, induction of proteolytic cleavage can result in an increase in the number of bioactive proteins. In some embodiments, induction of proetolytic cleavage can result in an increase in the number of active enzymes. Accordingly, in some embodiments provided herein is a method of inducing proteolyic cleavage comprising the use of an engineered scaffold protein or a fusion protein thereof described herein. [00269] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce cell degradation. In some embodiments, induction of cell degradation can induce cell death. In some embodiments, induction of cell degradation and/or cell death comprises activating apoptotic pathways, autophagic cell death pathways, necrotic pathways, complement dependent cytotoxicity, or antibody-dependent cellular cytotoxicity. In some embodiments, the therapeutic methods as disclosed herein can comprises a pharmacological drug conjugated to the engineered scaffold or fusion protein thereof and delivered to a specific cell as a therapeutic. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in a therapeutic method wherein the induction of apoptosis is therapeutically desirable. In some embodiments, the therapeutic method comprises increase in apoptosis. In some embodiments, the increase in apoptosis can be used to selective kill a desired target cell. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can induce greater apoptosis in the desired target cell than a non-target cell. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in a therapeutic method wherein the induction of autophagy is therapeutically desirable. In some embodiments, the therapeutic method comprises increase in autophagy. In some embodiments, the increase in autophagy can be used to selective kill a desired target cell. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can induce greater autophagy in the desired target cell than a non-target cell. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in a therapeutic method wherein the induction of necrosis is therapeutically desirable. In some embodiments, the therapeutic method comprises increase in necrosis. In some embodiments, the increase in necrosis can be used to selective kill a desired target cell. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can induce greater necrosis in the desired target cell than a non-target cell. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in a therapeutic method wherein the induction of complement dependent cytotoxicity is therapeutically desirable. In some embodiments, the therapeutic method comprises increase in complement dependent cytotoxicity. In some embodiments, the increase in complement dependent cytotoxicity can be used to selective kill a desired target cell. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can induce greater complement dependent cytotoxicity in the desired target cell than a non-target cell. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in a therapeutic method wherein the induction of antibodydependent cellular cytotoxicity is therapeutically desirable. In some embodiments, the therapeutic method comprises increase in antibody-dependent cellular cytotoxicity. In some embodiments, the increase in antibody-dependent cellular cytotoxicity can be used to selective kill a desired target cell. In some embodiments, the binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can induce greater antibody-dependent cellular cytotoxicity in the desired target cell than a non-target cell. In some embodiments, the therapeutic methods as described herein can be used to treat a disease. In some embodiments, the disease includes, but is not limited to, cancer, infectious disease, neurodegenerative disease, cardiovascular disease, pulmonary disease, muscular disease, hepatic disease, autoimmune and/or inflammatory diseases, and proteinopathies. In some embodiments, the therapeutic methods described herein can be used to selectively kill cancer cells that are resistent to other therapies. Accordingly, in some embodiments provided herein is a method of inducing cell degredation comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00270] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can block substrate recruitment. In some embodiments, blocking of substrate recruitment inhibits a protein-protein interaction. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein prevents protein-substrate binding. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can restrict active site access. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can block essential substrate recruitmennt sites. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can block catalytic sites. In some embodiments, the engineered scaffold protein or the fusion protein thereof can physically block substrate binding. Accordingly, in some embodiments provided herein is a method of blocking substrate recruitment comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

[00271] In some embodiments, binding of a target peptide by an engineered scaffold protein or a fusion protein thereof described herein can induce an immune response. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can stimulate an immune response in vivo. In some embodiments, stimulating the immune response results in the upregulation of a gene that encodes a cytokine, chemokine or lymphokine. Examples of such genes include alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL 0 12, IL-18, MHC, CD80, CD86 and IL- 15. In some embodiments, binding of the target peptide by the engineered scaffold protein or the fusion protein thereof described herein can be used in the form of a vaccine. In some embodiments, the vaccine comprises inactivated vaccines, live -attenuated vaccines, messenger RNA (mRNA) vaccines, subunit, recombinant, polysaccharide, and conjugate vaccines, neoepitope vaccine, neurodegeneration vaccine, infectious disease vaccine, toxoid vaccines or viral vector vaccines. Accordingly, the vaccines can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means to deliver the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, a live-attenuated vaccine or a killed vaccine. Accordingly, in some embodiments provided herein is a method of inducing an immune response comprising the use of an engineered scaffold protein or a fusion protein thereof described herein.

Therapeutic Uses

[00272] Accordingly, the engineered scaffold proteins or fusion proteins thereof disclosed herein may be developed as therapeutic molecules. In some embodiments, the therapeutic molecule can bind to any protein on the cell surface, a secreted protein in the extracellular space, or a protein in the intracellular compartment. The protein target of the therapeutic molecule may be a receptor, cell surface marker, a membrane-bound protein, an enzyme, intracellular protein, a signaling pathway component, or other proteins. The therapeutic molecule may act as an inhibitor where the therapeutic molecule binds to the target protein and inhibits a critical biological activity. It can also act to modulate protein-protein interactions either as an antagonist or agonist. The therapeutic molecule may also modulate the enzymatic activity of the target protein or another protein that interacts with said target protein. For example, by fusing a protease enzyme with the engineered scaffold, the therapeutic molecule may be engineered to bind specifically to a target protein and site cleave the target protein. In some embodiments, the modality prevents the aggregation of proteins by binding to the target protein and thereby changing its biophysical properties. As an example, a-Synuclein is a precursor for plaque formation in many neurodegenerative diseases. The plaques form due to aggregation of a-Synuclein; by binding to the target protein and preventing its aggregation, it may be possible to treat neurodegenerative diseases. Similar to an antibody-drug conjugate, a pharmacological drug may be conjugated to the engineered scaffold or fusion protein thereof and delivered to a specific cell as a therapeutic.

[00273] Accordingly, disclosed herein are methods of prophylaxis/treatment of a disease or disorder assocaited with a target peptide. Further disclosed herein are methods of treatment comprising administering to a subject in need thereof an effective amount of an engineered scaffold protein, a fusion protein thereof, or a pharmaceutical composition comprising the same. A subject in need thereof can be a subject who is suffering from or is at a risk of developing a disease or disorder associated with a target peptide.

Devices and Diagnostics

[00274] Provided herein are compositions and methods for detecting the presence or absence of a bioanalyte comprising the use of a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein. A bioanalyte as described herein refers to a peptide, small molecule/metabolite (e.g., dNTP), nucleic acid (e.g., double stranded DNA, single stranded DNA, RNA, circular DNA, circular RNA, amino acid, etc.), or a whole cell, cellular compartment/organelle, antibody, serum component, hemoglobin, etc.

[00275] Detecting the presence or absence of a bioanalyte with the detection agent comprising the engineered scaffold protein or the fusion protein thereof described herein involves binding and labeling the bioanalyte. For example, labeling the bioanalyte by an engineered scaffold protein or a fusion protein thereof described herein includes a specific tag conjugated to the engineered scaffold protein or the fusion protein thereof described herein. Examples of the specifc tag include, an imaging agent, a polypeptide linked to an imaging agent, a chelating agent, a fluorescnet label, a luciferase enzyme, a self-labeling protein, a peptide tag (e.g., ALFA-tag, AviTag, FLAG-tag, HA-tag, iCapTag, etc.) and a Biotin Carboxyl Carrier Protein. By labeling the engineered scaffold protein or the fusion protein thereof with the specific tag, the presence or absence of a bioanalyte in a sample can be determined by interaction between the engineered scaffold protein or fusion protein thereof and the bioanalyte. Such interaction can identify the specific bioanalyte that the engineered scaffold or fusion protein interacts with. Similarly, a peptide/protein sequence encoding a post-translational modification (PTM) may be identified by engineering an engineered scaffold or fusion protein to a specific peptide sequence that encodes the PTM. The detection may be for protein sequencing, protein fingerprinting, protein quantification or protein identification. In some embodiments, a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein can be used to detect a bioanalyte or a biological molecule. In some embodiments, the bioanalyte or the biological molecule includes, but is not limited to, small molecule/metabolite (e.g., dNTP), nucleic acid (e.g., double stranded DNA, single stranded DNA, RNA, circular DNA, circular RNA, amino acid, etc.), glucose, potassium, lactate, uric acid, sodium, chloride, proteins (e.g., receptors, channels, soluble protein, etc.), lipids, and cells. An example of detecting such a bioanalyte with the any of the engineered scaffold proteins disclosed herein is to generate a diverse set of engineered scaffold proteins that bind an orthogonal set of peptide sequences serving as a peptide barcode.

[00276] In some embodiments, a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein is provided in a form of a device. In some embodiments, the device comprises a solid support, and a detection agent holding part. In some embodiments, the detection agent holding part includes a site on the device to which a liquid sample is added. Examples of the liquid sample includes, urine, blood, sweat, mucus, interstitial fluid, chyme, saliva, bile, semen, cerebrospinal fluid, phlegm, pus, breast milk, and synovial fluid. By adding the liquid sample containing the bioanalyte and/or biological molecule to the device, through the detection agent holding part of the device, the detection agent comprising the engineered scaffold protein or the fusion protein thereof described herein can bind to the bioanalyte and/or biological molecule to be measured or quantified. In some embodiments, the device can comprise a solid support such as a porous membrane or microfluidic chip. In some embodiments, the device is a microfluidic chip. Microfluidic chips can be manufactured by methods known in the art, for example, can be manufactured by preparing a flow channel having a mixing section and a reaction section, one or more inlets, and a waste liquid storage section, on a small piece of glass or plastic. The inlet is used for injecting a liquid sample containing the substance to be measured, and an inlet for injecting a washing liquid and/or an enzyme substrate solution into the channel may be provided separately, as necessary. In some embodiments, the device is formed by a microfluidic chip which includes a device having only a bioanalyte and/or biological molecule capturing part. In some embodiments, the bioanalyte and/or biological molecule capturing part comprises an engineered scaffold protein or a fusion protein thereof described herein. In some embodiments, the device can be a kit that comprises a detection agent comprising an engineered scaffold protein or a fusion protein thereof described herein.

[00277] In some embodiments, the compositions and methods for detecting the presence or absence of a bioanalyte comprises an assay system in which a kit comprising a detection agent comprising the engineered scaffold protein or the fusion protein described herein and a measurement apparatus capable of measuring a signal generated from a reporter substance of the detection agent. In some embodiments, the detection agent is held in a a detection agent holding part. In some embodiments, the detection agent holding part is combined with the measurement apparatus. In some embodiments, the signal generated can include fluorescence, luminescence, color, turbidity, radiation, or signal on a spectrometer. In some embodiments, the spectrometer includes, but is not limited to, an optical absorption spectrometer, an optical emission spectrometer, an electron spectroscopy, a mass spectrometer, a time-of-flight spectrometer, and a magnetic spectrometer. In some embodiments, the measurement apparatus can comprise a liquid pump. In some embodiments, the liquid feed pump can be used to control the speed at which the liquid sample containing the bioanalytle and/or biological molecule moves on the device. [00278] In some embodiments, a device comprising a detection agent includes an engineered scaffold protein or a fusion protein thereof that is immobilized on a solid support in a state where the three- dimensional structure of the engineered scaffold protein or the fusion protein is retained. In some embodiments, the liquid sample is added to the device comprising the immobilized engineered scaffold protein or fusion protein thereof. In some embodiments, the immobilized engineered scaffold protein or fusion protein thereof forms a complex with a bioanalyte or biological molecule in the liquid sample, and a signal is generated upon binding. In some embodiments, the signal generated can be used to create a standard cure or a dose-effect curve. In some embodiments, a signal intensity measured when a liquid sample containing an unknown concentration of a bioanalyte or a biomolecule can be inserted into the stardard curve to determine the amount of the bioanalyte or biomolecule. A biological molecule as described herein, refers to a protein (e.g. , receptors, channels, soluble protein, membrane bound protein, post-translationally modified proteins), a lipid, a carbohydrate, etc.

[00279] In some embodiments, described herein assay method for detecting, monitoring, or measuring a given ligand, such as a target ligand, in a sample which comprises inducing an expression vector described herein to express an engineered scaffold protein or a fusion protein thereof and then contacting the engineered scaffold protein or a fusion protein thereof with the sample and observing whether the engineered scaffold protein or a fusion protein thereof interacts with the target ligand. In such embodiments, methods comprise the use of a polypeptide display system.

[00280] Further details and examples can be found in the Examples provided herein.

SEQUENCES AND TABLES

TABLE 1 : Exemplary Human Protein Scaffolds and Associated Sequences

Ill

TABLE 2: Functional Classification of Exemplary Human Protein Scaffolds

[00281] The following table provides information about the peptide known to bind to each human protein scaffold (i.e., “bound peptide”).

TABLE 3: Bound Peptide Information

[00282] The following table provides exemplary sequences for use in a binding unit in a binding domain of an engineered scaffold protein as described herein.

TABLE 4: Exemplary Binding unit Sequences

[00284] The following table provides exemplary sequences comprised in a disordered region as described herein.

TABLE 6: Exemplary Engineered Scaffold Sequences

[00285] The following table provides exemplary sequences comprised in a disordered region as described herein.

TABLE 7: Disordered region amino acid sequences

601/PRO

[00286] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES

Example 1. Detecting a bioanalyte using engineered scaffold protein

[00287] An engineered scaffold protein generated from the scaffolds disclosed herein can target a specific protein that is present on only one type of cells’ information about the type of cells, and quantitative measures of cell counts and relative ratio of different types of cells can be determined. For example, by identifying the population of T cells and their relative counts compared to B cells, an engineered scaffold is designed for CD28 (CTLA) present on T cells, but not on B Cells, and CD22, which is present on B cells and not T cells. Each engineered scaffold is labeled with a different fluorophore or a different peptide tag that binds to a fluorescent antibody. Labeled binders are incubated with a population of T cells and B cells and are subsequently washed to be rid of free binders. Flow cytometry is then used to sort and separate out the cells with two different colors. A quantitative assessment on the different types of cells may then follow.

[00288] Similar assays can also be set up to interrogate the presence of other bioanalytes using fluorescently labeled engineered scaffolds or engineered scaffolds with peptide tags that can bind to fluorescent antibodies. For example, an engineered scaffold for a particular target protein expressed by am mRNA, which may be a biomarker for a cell, can be designed, and the presence of particular cells can be determined in a population of cells. This can be made quantitative by combining the assay with single molecule microscopy.

Example 2. Scaffold Engineering

[00289] The following non-limiting example demonstrates the development of an engineered scaffold that is designed to target a specific epitope.

[00290] C-myc is an oncogene that is a well validated cancer target, which is very difficult to drug because of its intrinsic disorder. To target this disordered region, the sequence EPLVLHE that is present in C-myc in its disordered region is identified as the target epitope.

[00291] TEV protease that lacks its catalytic activity (C151A) is chosen as the scaffold for the binder development. Library of variants (up to a theoretical maximum of 10 ⁸ ) is generated using Twist Biosciences’ SOLD approach of spreading the variants across the full length of the enzyme sequence, maintaining the C151A mutation.

[00292] The library of variants are cloned into a yeast vector and fused to Agal protein at either the N-terminus or at the C-terminus along with a 6xHistidine tag at the C-terminal end of the Agal-TEV variant fusion, and transfected in yeast cells.

[00293] Functionally expressed and displayed variants are incubated with magnetic beads carrying the recognition peptide EPLVLHE and selected for binding. Binders from this selection were washed out and labeled with the tag containing protein and subjected to FACS sorting to separate binders from nonbinders.

[00294] From the first round of selection, positive binders and negative binders are separated out and yeast cells are subjected to NGS sequencing to identify the binders and non-binders. The binders from the first round of selection are grown up and further subjected to successive rounds of screening with more stringent binding conditions to arrive at a potent binder for the target sequence.

[00295] Data generated from each round of selection are used to generate a database of engineered scaffold proteins and their relative strengths for the target. A flow-chart demonstrating the scaffold engineering process can be seen in FIG. 11.

Example 3. Binder Development Pipeline

[00296] A non-limiting example of the binder development pipeline is displayed in FIG. 13. Yeast display of cell surface protein scaffolds is performed using well known methods such as those described in Angelini et al 2015, Methods in molecular Biology, ‘Protein Engineering and Selection Using Yeast Surface Display’. This procedure involves starting with the design of engineered scaffold libraries for each scaffold protein. The yeast strain with Agal gene stably integrated into the chromosome is chosen. The nucleic acids encoding engineered scaffold proteins, fused with the Aga2 gene, is cloned into a circular yeast display vector using Gibson assembly and further established protocols. The yeast library is transformed into competent cells and selected using a URA3 complementation method. The yeast library cells are initially grown in YPD medium, which are subsequently used to inoculate SD-CAA medium. Cells are passaged once and induced, where the induced cells are then used to screen for binders. Screening is done in two steps using a magnetic bead-based screening followed by flow cytometry.

[00297] In the first step, a negative selection for magnetic bead binders is performed to deplete the library of streptavidin coated magnetic bead binders by incubating the library with magnetic beads that do not have the immobilized target. The flow through from the negative selection is then incubated with magnetic beads that have the target immobilized via biotin streptavidin linkage. After stringent washing to remove non-binders, the beads were collected and the binder library was isolated.

[00298] Selected yeast cells from the magnetic beads screening step are further subjected to fluorescence-activated cell sorting (FACS) to enrich for higher affinity binders to target protein. Yeast display vectors are designed to have a tag epitope (His or FLAG) at the C terminus end of the engineered scaffold protein to be able to be labeled with an antibody. The target protein will be labeled with a different tag (His, FLAG, myc). Using a two color FACS sorting, only yeast cells showing engineered scaffold proteins binding to target proteins exhibits dual color and are collected. Repeated cycles of selection and screening yields potent binders to the target protein.

Example 4. Monomeric MHC Designs [00299] The following non-limiting example displays various engineered monomeric MHC constructs and designs for an engineered scaffold protein derived from MHC proteins. The engineered scaffold proteins may be generated in accordance with any of the methods and examples disclosed herein.

TABLE 8: MHC Specific Sequences for Chimera 1-4

Bold text indicates an MHC II alpha (a) chain sequence. Underlined text indicates a linker. Italicized text indicates an MHC II beta ( ) sheet sequence. Bold and underlined text indicates an MHC I alpha chain sequence. Underlined and italicized text indicates a sequence for other protein fusions.

[00300] The specific linker sequences used in the MHC constructs are also listed below.

TABLE 9: Specific Linker Sequences

[00301] The (GGGGS)n linkers are flexible linkers and are believed to increase solubility and stability. The LE linker is believed to increase PK in an antibody (e.g., acts as a dipeptide linker). QSEAGSH (SEQ ID NO: 976) is an MHC I linker from the crystal structure of 2XPG. This is the linker between the alpha helix and the second part of the beta sheet.

[00302] The specific fusion partner sequences used in the MHC constructs disclosed herein are also listed below.

TABLE 10: Fusion Sequences

Constructs for Chimera 1 -4

[00303] The constructs for each of chimera 1-4 listed in TABLE 8 are described as follows.

[00304] Chimera 1. The construct for chimera 1 is shown below, where domain 1 and domain 3 can be a part of MHC II a chain or MHC II chain, and domain 2 can be a part of an MHC I a chain. [00305] Chimera 2. The construct for chimera 2 is shown below, where domain 1 can be a part of an MHC II a chain or MHC II p chain, and domain 2 can likewise be a part of a MHC II a chain or a MHC II chain. The two domains are connected by a short peptide linker.

[00306] Chimera 3. The construct for chimera 3 is shown below, where domain 1 or domain 3 can be part of an MHC I a chain, while domains 2 and 4 can be part of an MHC II a/p chain and domains 2 and 3 are connected by a short peptide linker

[00307] Chimera 4. The construct for chimera 4 is shown below, where domain 1 can be part of an MHC I a chain or an MHC II a/p chain and similarly for domain 2.

[00308] All of the above constructs can have C or N terminal fusions that can include purification tags such as a His tag (HHHHHH), FLAG tag, Myc Tag, GST tag, a solubility tag like SUMO, a protease cleavage tag such as TEV protease cleavage tag (ENLYFS) or Thrombin cleavage tag, a serum albumin binding domain or transferrin domain or transferrin binding domain.

Example 5. Engineered Scaffold Protein production from inclusion bodies

[001] The present example describes methods for generating engineered scaffold proteins as described herein. E. coli pET28 vectors or pD451 vectors encoding engineered scaffold proteins of interest were transformed into BL21 DE3 cells. Overnight starter cultures were grown from single colonies, which were then used to seed IL cultures with appropriate antibiotics and incubated at 37C. When the optical density reached 0.6-0.8, protein production was induced by addition of ImM IPTG and the flasks were maintained at 37C with shaking for 16 hours. Subsequently the cells were harvested by centrifugation at 8000xg for 10 minutes, resuspended in Lysis buffer (50mM Tris pH 8, 500mM NaCl, lOmM Imidazole) and lysed using a sonicator. The sonicator was set to pulse mode with 15s on time and 45 s off time for a total of 10 cycles, while the cells were maintained in an ice bucket.

[00309] Cell lysate was subjected to centrifugation at 18000xg for 30 minutes to clarify the solution and the cell pellet containing the protein of interest in inclusion bodies was preserved while the supernatant was discarded. Pellets were resuspended in lOmL of Urea Buffer (50mM Tris pH 8, 500mM NaCl, lOmM Imidazole, 6M Urea) and allowed to incubate on a rotator at room temperature overnight to re-solubilize the protein of interest from inclusion bodies. The resolubilized solution was clarified once again to remove aggregates by centrifuging at 18000xg for 30 minutes and supernatant was saved. The re -solubilized protein was applied to a Nickel column that had previously been equilibrated with Uysis Buffer and allowed to bind under gravity flow. The column was then washed extensively with a wash buffer(50mM Tris pH 8, 500mM NaCl, 25mM Imidazole) to remove Urea and refold the protein on the column. Protein of interest was eluted with an elution buffer(50mM Tris pH 8, 500mM NaCl, 250mM Imidazole). Eluted protein was buffer exchanged into PBS, flash frozen and stored.

[00310] Engineered scaffold proteins generated by the above method include S001, S005-S007, S031, S043-S044, S049, S055-S060, S065-S069, S077-S078, S082, S090, S095-S096, S098-S102, S107, S117-S118, S132-S134, S139-S140, S143-S145, S161-S167, S169-S180, S183-S215, S227, S232, S236, S240, S242, S249, S258, and SEQ ID NO: 914.

Example 6: Characterizing Engineered Scaffold Proteins

[00311] The present example characterizes generated engineered scaffold proteins as described herein. The molecular weight of engineered scaffold proteins generated from the method of Example 5 range from about 20kDato about 50kDa. SDS PAGE was run on an engineered scaffold protein (S177 (SEQ ID NO: 827) which confirmed the expected size (21.92 kDa) on the gel and can be produced in soluable form as demonstrated by FIG. 15A.

[00312] To analyze the soluble proteins produced, an engineered scaffold protein (S090 (SEQ ID NO: 740)) were run on a Superdex 200 Increase 10/300 column by Cytiva, using an AKTA FPLC. The UV absorbance at 280nM was recorded as a function of elution volume. Three peaks at different elution volumes were recorded for S090 and as seen in FIG. 15B. The results in FIG. 15B indicates that the engineered scaffold proteins are generated as a mixture of monomers, dimers and multimers with at least 50% of the protein existing as monomers.

Example 7. Modified Sandwich ELISA assay for Protein-Peptide Interactions

[00313] Disordered epitopes derived from target proteins with a biotin were synthesized either chemically or expressed in E. coli and subsequently labeled with a biotin using a sortase mediated ligation of a short biotinylated peptide.

[00314] Binder proteins were coated on the surface of a high binding capacity 96 well plate by incubating lOOpL of protein at 5pg/mL in a coating solution (lOmM Tris pH 8.8, 50mM NaCl), overnight at 4C. The next morning, the coating solution was aspirated out and wells were washed once with 300pL ELISA wash buffer (lx PBS, 0.05% Tween-20) and solution was aspirated out. The wells were blocked with 200pL of a blocking buffer (IxPBS, 0.05% Tween-20, 3% BSA) at room temperature for 1 hour. After one hour, the blocking solution was aspirated out and lOOpL of biotinylated polypeptide in a binding buffer (lx PBS, 0.05% Tween-20, 0.5% BSA) at various concentrations was added to the appropriate well and incubated for 20 minutes. Peptide solution was aspirated out after 20 minutes and wells were washed 5 times with 300pL of ELISA wash buffer. To detect the biotinylated peptide, lOOpL of streptavidin-HRP (50 ng/mL) was added to each well and incubated for 1 hour. The wells were subsequently washed 5 times with 300pL of ELISA wash buffer. lOOpL of QuantaBlu Fluorogenic Peroxidase substrate solution was added to each well, incubated for 30 minutes and reaction stopped by adding lOOpL of stop solution. Fluorescent signal from each well was measured using a Biotek Cytation5 plate reader by exciting the fluorophore at 325 nM and measuring emission at 420nM. Fluorescent signals were plotted as a function of polypeptide concentration.

[00315] Engineered protein scaffolds S007, S005 and S090 were derived from MHC II molecules and the binding activities of these engineered scaffolds was measured in an ELISA assay as described herein. Results for engineered protein scaffolds S007, S005 and S090 binding increasing amounts of disordered regions of CD74, KAAG1, CysLT2, CRACM, and Kvl.5 relative to a negative control are shown in FIGS. 16A-E. In human cells, every MHC II based molecule is associated with a CLIP peptide derived from CD74 and accordingly, CD74 binding is used as a positive control. The data indicates that the engineered scaffold proteins can bind to long disordered polypeptides derived from extracellular disease target proteins covering GPCRs (e.g., CysLT2), ion channels (e.g., CRACM, Kvl.5) and membrane proteins (e.g., CD74, KAAG1).

[00316] The results in FIG. 17A demonstrate that scaffold optimization and rational design increases binding of a target peptide, which is CysLT2 in this assay. In FIG. 17A, scaffold vl.O refers to S007 which was derived from MHC II and comprises a binding unit (comprising an N-terminal alpha chain of 82 amino acids), a hinge unit (comprising an N-terminal beta chain of 98 amino acids) and an immunoglobulin unit (i.e., a beta-globulin domain), and includes a binding groove architecture as described herein. The binding unit is fused to the N terminal of the hinge unit, which is fused at its C terminal to the immunoglobulin unit. Scaffold vl. l refers to S005, which is derived from S007 by removing the immunoglobulin unit. Scaffold vl.2 refers to S090, which was rationally designed based on vl.l (S005), and includes amino acid alterations to increase binding efficiency of the target peptide without compromising the binding groove architecture.

[00317] Binding affinity, relative binding and binding specificity relative to the negative control was measured at 10,000 nM of the target peptide, results of which can be seen in FIG. 17B-D. The fluorescent binding signals were normalized to controls, which consisted of binding reactions without any scaffold. As such, data was derived from the binding isotherms by dividing the fluorescent signal at that particular target concentration for a particular engineered scaffold protein by the fluorescent signal for a control reaction which does not contain any scaffold protein. FIG. 17B indicates that scaffold optimization and rational design increases binding affinity of an engineered scaffold protein to a target peptide (CYSLTR2). Likewise, FIG. 17C and 17D indicate that scaffold optimization and rational design increases relative binding and binding specificity of an engineered scaffold protein to a target peptide (CYSLTR2), respectively.

[00318] Engineered scaffolds S058 and S065 were derived from MHC I proteins. The binding activities of these engineered scaffolds were measured in an ELISA assay as described herein, relative to S090, which was derived from MHC II proteins, and relative to a negative control. Results for binding of CD74 (positive control), KAAG1, CRACM, CysLTR2, and Kvl.5 can be seen in FIGS. 18A-18E.

[00319] Engineered scaffolds S164 and S171 were computationally derived as described herein and the binding activities of these engineered scaffolds were measured in an ELISA assay as described herein relative to S090 and a negative control. Results for binding of CD74 (positive control), KAAG1, CRACM, CysLTR2, and Kvl.5 can be seen in FIG. 19A-19E.

[00320] TABLE 11 summarizes the results of the assays described herein and shows that the engineered scaffold proteins bind to target peptides with selectivity, and even if more than one target peptide can be bound, binding strength differs. FlhG is a non-specific hexamer protein, used as a positive control to demonstrate that it does not bind the target of interest.

TABLE 11: Targets vs Engineered Scaffolds

Key

Example 8. Selective Binding of Linear Epitopes

[00321] Next, ELISA assays as described in Example 7 were undertaken to determine linear epitopes of disordered regions of targets of interest.

MERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 926) is the N- terminal disordered region of CYSLTR2, a target of interest. Naturally occuring MHC molecules usually bind to peptides that range in size from 8-20 amino acids. However, certain linear epitopes may be greater than 20 amino acids in length, accordingly, narrowing the targeted linear epitope is beneficial to enhance binding activity. In the present assay, the ~42 amino acids N-terminal disordered region was split into 3 N-terminally biotinylated peptides with a length of -21AA and an overlap of -10AA, MERKFMSLQPSISVSEMEPNG (SEQ ID NO: 927), SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928), and TFSNNNSRNCTIENFKREFFP (SEQ ID NO: 929).

[00322] Results of S090 and SI 77 binding to the three separate linear epitopes relative to a negative control can be seen in FIGS. 20A-20D. Results indicate that targeting SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928) results in the most increased amount of binding. S 177 was computationally derived from S005 by methods described herein. Upon targeting the optimized linear epitope of CYSLT2R, SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928), binding improvement can be seen with S005, S090 and SI 77 relative to a negative control in FIG. 21.

[00323] A similar assay was undertaken to compare binding of a short linear epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)) against a longer amino acid sequence of the disordered region of CYSLTR2 (GMERKFMSLQPSISVSEMEPNGTFSNNNSRNCTIENFKREFFP (SEQ ID NO: 930)). Assayed scaffolds include: S177 a computationally engineered variant, S189 which is derived from SI 77 with an extra dicysteine, SI 80 which is a wild-type, SI 93 which is a engineered scaffold protein linked to an N terminal low affinity class Il-associated invariant chain peptide (CLIP peptide) with short linker, SI 94 which is an engineered scaffold protein with a low affinity CLIP peptide with linker in middle of the construct. Results can be seen compared to a negative control in FIGS. 22A-22B.

Example 9: Engineered Scaffold protein binding selectivity

[00324] Next, binding selectivity of the short linear epitope (SISVSEMEPNGTFSNNNSRNC (SEQ ID NO: 928)) of CYSLTR2 was measured against the short linear epitopes for KCNA6, TNFRSF13B, KAAG1, CD40LG, and CD74 (positive control). Results can be seen in FIGS. 23A-23F. Selective binding was only found for CD74 (positive control) and CYSLTR2.

[00325] Likewise, TABLE 12 summarizes the results of the assays described herein, and represents a heat map of engineered scaffold proteins and binding selectivity to disordered regions of target peptides as measured by fluorescent signal strength. TABLE 12 shows that engineered scaffold proteins bind to target peptides with selectivity, and even if more than one target peptide can be bound, binding strength differs.

TABLE 12: Targets vs Scaffolds

Key

Example 10: BLI Assay using OCTET RED

[00326] Disordered epitopes of target proteins with a biotin were synthesized either chemically or expressed in E. coli and subsequently labeled with a biotin using a sortase mediated ligation of a short biotinylated peptide.

[00327] Streptavidin sensor tips for the BLI instrument were equilibrated in an equilibration buffer (IxPBS, 0.05% Tween 20, 0.5% BSA) for 5 minutes. Biotinylated polypeptide in the equilibration buffer at 0.5pM was loaded on the streptavidin sensor over 60s. After substrate loading, the sensor tips were washed in the equilibrating buffer for 5 minutes to block non-specific binding and to obtain a baseline reading. Peptide loaded and blocked tips were then introduced into wells containing polypeptides in the equilibrating buffer at varying concentrations. The binding and dissociation data are fit using the OCTET software to obtain k _on , k _O ff and consequently KD, the binding affinity.

[00328] Results for S078 and S090 targeting an 18 amino acid linear epitope of CLIP can be seen in FIG. 24A and 24B. FIGs. 25-29 demonstrates that engineered scaffold proteins, although engineered to target an 18 amino acid epitope of CLIP (CD74), can bind an 18 amino acid epitope and a 25 amino acid epitope. This effect is unexpected as native MHC II molecules are only known to bind to short peptides that are about 18-20 amino acids in length. Moreover, by comparing the binding of S090 vs. S005 to an 25AA long peptide sequence, binding by S090 (which is the iterative version of S005) to the target peptide is seen to be improved over binding by S005 (vl . 1) demonstrating that optimizing the scaffold and rational design the scaffold has improved binding affinity (FIG. 25 and FIG. 26).

Example 11: Binding of GPCRs

[00329] Engineered scaffold proteins were designed by rational engineering to enhance binding affinity to selected target peptides in the GPCR family. Engineered scaffolds included vl .0 (S007), vl .1 (S005), vl.2 (S090). The engineered scaffold proteins were generated as described in Example 5 and ELISA assays were run as described in Example 7. FIGS. 30A and 30B demonstrates that vl .2 exhibits the best performance and demonstrates that the engineered scaffold proteins bind the target peptide with >65-fold higher affinity compared to a non-specific protein binder. The best performing engineered scaffold was assayed to establish selectivity of the GPCR 1 target over a related GPCR2 target. Results in FIG. 31 demonstrates that vl.2 exhibits binding selectivity for the targeted GPCR over a related GPCR.

Example 12: Engineered Scaffold Proteins derived from non-MHC proteins

[00330] Caspases are proteases that bind to and cleave tetrapeptide sequences after an aspartate residue at the C-terminal end of the substrate sequence. For the proteolytic activity, a specific cysteine residue in the catalytic core is necessary. Caspase-derived engineered scaffold proteins are generated by mutating the cysteine in the enzyme active site to an alanine or other residue. By doing so, the proteolytic activity of caspases is abrogated. Moreover, residues in the binding unit of the caspase- derived engineered scaffold proteins are altered to increase specificity to particular sequences of target peptides and with a high binding affinity. This same approach can be employed to any class of proteins that bind to short polypeptide sequences from 4-50 amino acids, such as those described herein.

Example 13: Engineered Scaffold Fusion Proteins

[00331] An E3 ligase is fused to an engineered scaffold protein at its C terminal end. The engineered scaffold protein is used to specifically bind to a disordered region on a target peptide with high affinity and selectivity. When the fusion protein binds to the target protein, the C-terminally fused E3 ligase is brought in close contact with the target peptide and can ubiquitinate the target peptide . The ubiquitinated protein is then sequestered and shuffled to a degradation pathway by natural cellular mechanisms, e.g., as described. This same approach can be used for modulating post translational modifications on the target peptide by fusing for example a kinase, protein phosphatase, etc.

[00332] Steps include producing the fusion protein of interest in soluble form, and separately, engineered scaffold protein and the fusion partner (e.g., E3 Ligase). Specificity of engineered scaffold protein-Ligase fusion to target peptide of interest is established using ELISA. Once the specificity is established, activity of the fusion protein is validated with a cell free ubiquitination assay. To establish activity inside cells, the fusion protein is encoded in a mammalian expression plasmid and transfected into mammalian cells (e.g., HEK293) and expression of the fusion protein is verified by Western blot analysis. Targeted degradation of the target peptide or protein is determined and validated by transfection and western blot analysis. The expected outcome is that in the presence of the fusion protein, the target protein is ubiquitinated whereas in the absence of the fusion protein, the target protein is not ubiquitinated. [00333] From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[00334] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[00335] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.